Identification and characterization of proteolytic activity of Enterococcus spp. isolated from milk and Roncal and Idiazábal cheese

International Journal of Food Microbiology 38 (1997) 17–24

Identification and characterization of proteolytic activity of ´ Enterococcus spp. isolated from milk and Roncal and Idiazabal cheese C. Arizcun*, Y. Barcina, P. Torre ´ y Bromatologıa ´ , Escuela Tecnica ´ ´ ´ Area de Nutricion Superior de Ingenieros Agronomos , Universidad Publica de Navarra, ´ , 31006 Pamplona, Spain Campus Arrosadıa Received 19 September 1996; received in revised form 28 April 1997; accepted 20 July 1997

Abstract ´ Roncal and Idiazabal cheeses are manufactured from ewe’s milk in the Autonomous Region of Navarre in Spain. Levels of enterococci are high in these cheeses and in other types of ewe’s-milk cheeses. The present study has identified ´ enterococci present in the milk used and in the Roncal and Idiazabal cheeses after 120 days of ripening. A total of 282 strains were isolated and identified, and the cytoplasmic and extracellular enzymatic activities of some of the strains were assessed. The dominating species were Enterococcus faecalis, which accounted for 85% of the total both in the milk as well as in the two types of cheese, and Enterococcus faecium, Enterococcus durans, and Enterococcus avium which were present in lower proportions. Aminopeptidase and proteinase activity levels in enterococci were low, and considerable variation between strains of the same species was recorded, highlighting the need to study individual strains when selecting the most suitable bacteria as a starter culture. Aminopeptidase activity levels for the enterococci were appreciably higher at pH 7 than at pH 5.5, hence aminopeptidase activity by enterococci would appear to be less significant in the normal manufacturing conditions of the two cheeses.  1997 Elsevier Science B.V. Keywords: Ewe’s-milk cheese; Enterococci; Identification; Enzymatic activity

1. Introduction ´ The cheeses, Roncal and Idiazabal, are made from raw ewe’s milk in Navarre, a region in the north of Spain; both are regulated by Appellations of Origin. Knowledge of the microorganisms present in cheese, and their contributions to cheese maturation is an *Corresponding author. Tel.: 1 34 48 169091; fax: 1 34 48 169187

essential basis for developing an appropriate manufacturing technology (Hegazi and Abo-Elnaga, 1990) and a first step for selection of specific indigenous starter cultures (Ramos et al., 1981; Requena et al., 1992). Enterococci are a new genus erected from the former genus Streptococcus (Schleifer and Kilpper¨ 1984) accepted in the latest edition of Bergey’s Balz, Manual of Determinative Bacteriology (1994). Enterococci have been used as hygiene indicators,

0168-1605 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved. PII S0168-1605( 97 )00091-3

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C. Arizcun et al. / International Journal of Food Microbiology 38 (1997) 17 – 24

mainly in waters (Vanos, 1991; Tsakalidou et al., 1993). Nowadays it is known that these organisms are not only found in man and animal intestine, but also in soil, plants, insects and birds (Mundt, 1982; Vanos, 1991). They also occur in several dairy products (Wessels et al., 1988) without constituting a health hazard to man (Varnam and Evans, 1991). On the other hand, it has been mentioned that they may ˜ contribute to the formation of biogenic amines (Inigo et al., 1986). Bacteria of this genus have been reported at different stages of ripening in many different cheese varieties. Their levels tend to remain rather stable, probably because of their ability to grow under ´ adverse conditions (Suarez et al., 1983). They are ´ de also capable of surviving pasteurization (Garcıa Fernando et al., 1992). They are extremely resistant to broad ranges of salt concentration (Poullet et al., 1993) and they are not affected by smoking, often ´ applied in the manufacture of Idiazabal cheese ´ (Perez-Elortondo et al., 1993b). Counts of enterococci tend to be particularly high (i.e. 10 4 to 10 7 cfu / g) in ewe’s-milk cheeses: i.e., Manchego (Ramos et al., 1981); La Serena ´ (Fernandez del Pozo et al., 1988); Torta del Casar (Poullet et al., 1993); Feta (Litopoulou-Tzanetaki et ´ al., 1993). In Idiazabal cheese, this genus is the most abundant after lactococci, lactobacilli and Leuconos´ toc spp. (Perez-Elortondo et al., 1993a). The high levels observed make it likely that their proteolytic and lipolytic activities are important for ´ aroma formation (Trovatelli et al. (1987); Gonzalez de Llano et al., 1992). The effect on cheese proteolysis by adding enterococci has been investigated in a number of studies (Wessels et al., 1990; Litopoulou-Tzanetaki et al., 1993). The objectives of the present study were to identify the enterococcal species present in the milk ´ and in Roncal and Idiazabal cheeses after 120 days of ripening and to establish their enzymatic activities.

regulations approved by the Regulatory Boards of ´ the Appellations of Origin for Idiazabal cheese ´ (Ministerio de Agricultura, Pesca y Alimentacion, 1993) and Roncal cheese (Ministerio de Agricultura, ´ 1991), respectively. Pesca y Alimentacion, For each batch, samples of the milk and two cheeses after 120 days of ripening were collected and analysed. Samples were transported to the laboratory at 48C and analysed that same day. Sampling was performed according to the procedures of the International Commission on Microbiological Specifications for Foods (ICMSF, 1982). Enterococci were determined and purified on KF Streptococcus Agar (Difco Laboratories, Detroit, MI, USA) supplemented with 1% (v / v) Bacto TTC Solution (Difco). Eight colonies were picked from each sample at random. Strains were stored for up to 30 days at 48C in Elliker Broth (Difco).

2.2. Identification of strains Bacteria were identified to genus level according to the criteria of Sharpe (1979). The strains were assigned to the genus Enterococcus following the ¨ (1984). classification by Schleifer and Kilpper-Balz Further identification was according to the criteria of ´ ´ Orvin (1986) and Hernandez Haba and Dubon (1992). The biochemical tests employed have been described by Gross et al. (1975) and Gireaud-Galzy (1985). Not all the strains fully conformed to the type strain for each species and strains were assigned to a species when no more than two of their test results differed from the type strain.

2.3. Statistical treatment of the data

2. Materials and methods

Statistical processing of the data was performed using the SPSS-X Statistical Package (SPSS Inc., 1988). Since the attributes were qualitative in nature, a x 2 -test was applied. To ensure that the test results were sufficiently reliable, rows and columns were pooled or discarded when the number of entries with theoretical frequencies lower than 5 made up more than 20% of the data matrix.

2.1. Isolation of strains

2.4. Measurement of enzymatic activity

´ Nine batches of Idiazabal cheese and nine batches of Roncal cheese were studied. The cheeses were made from raw ewe’s milk in accordance with the

Proteinase activity and aminopeptidase activity were measured for 13 enterococcal strains from 120day-old cheeses. They were selected among 40

C. Arizcun et al. / International Journal of Food Microbiology 38 (1997) 17 – 24

strains for which acid production had been measured ´ initially according to the method of Martınez Moreno (1976) and enzymatic activity had been tested using the Api-zym system (Api System SA, Montalieu Vercieu, France). The 13 selected strains were the strains with the highest acid production and enzymatic activity indicator values. Activity levels were measured in cell-free extracts (cytoplasmic activity) and in intact cells (extracellular activity). Intact cells were obtained following the method of Requena et al. (1991), and cell-free extract was prepared from ruptured cells according to a modified version of the method of Tsakalidou and Kalantzopoulos (1992). The procedure can be briefly described as follows. Cells in the logarithmic growth phase were centrifuged at 12 000 3 g at 48C for 15 min. The pellet thus obtained was washed twice with 0.9% (w / v) NaCl solution, resuspended in 50 mM / l of pH 7 Tris–HCl buffer, and then sonicated for 8 min (at 10-s intervals). The cell-free extract was the supernatant obtained by re-centrifuging at 12 000 3 g at 48C for 15 min. The method employed for determination of aminopeptidase was a modified version of the technique described by Requena et al. (1991). A solution of 15.5 mM of the two substrates, L-leucine p-nitroanilide and L-lysine p-nitroanilide (Sigma Chemical Co., St. Louis, MO 63178, USA) in 0.05 ml of methanol was prepared. An amount of 200 ml of each of these solutions was pipetted into two tubes for each strain to be tested. A total of 0.4 ml of intact cells or cell-free extract was added along with 3.6 ml of pH 7 or pH 5.5 50-mM sodium phosphate buffer. The solution was incubated by shaking in a warmwater bath at 308C for 4 h, and the reaction was then quenched by adding 1 ml of 30% (v / v) acetic acid. The content of the tubes was then filtered through Whatman Cat. No. 1440 090 filter paper (Whatman

19

International Limited, Springfield Mill, Maidstone, Kent, UK). The amount of p-nitroaniline released was measured using a spectrophotometer at 410 nm. The results have been expressed in nanomoles of substrate degraded per mg of protein per hour. Protein concentration was determined according to Lowry et al. (1951). The method employed (Church et al., 1983) was based on reaction of o-phthaldialdehyde (OPA) (Sigma) and b-mercaptoethanol with the amino groups released by proteolysis of casein. Lyophilized casein from sheep milk (Sigma) was dissolved in pH 8 sodium phosphate buffer (2 mg / ml), and 2.7 ml of solution were placed in each of two test tubes. An amount of 0.3 ml of intact cells or cell-free extract was added to each of the tubes, which were then incubated by shaking in a warm-water bath at 378C for 4 h. Next, 4 ml of OPA reagent prepared according to Church et al. (1983) was added to 200 ml of the solution, and after 2 min absorbance was measured at 340 nm. The results have been expressed in mg of leucine released per mg of protein per hour.

3. Results and discussion

3.1. Identification of strains Enterococcal counts were 3.660.4 log 10 cfu / ml in milk and 5.160.2 log 10 cfu / g in cheese. A total of 282 isolates of enterococci were identified. Four species in this genus were isolated: Enterococcus faecalis, Enterococcus faecium, Enterococcus durans and Enterococcus avium (Table 1). There were no significant differences in the species distributions between the two cheeses at the 95% level of significance, Enterococcus faecalis

Table 1 ´ Identification of 282 Enterococci spp. isolated from milk and Roncal and Idiazabal cheese after 120 days of ripening Species

´ Idiazabal

Roncal Milk

Enterococcus faecalis Enterococcus faecium Enterococcus durans Enterococcus avium

Cheese

Milk

Cheese

No.

%

No.

%

No.

%

No.

%

35 1 6 0

83.3 2.4 14.3 0

87 7 0 6

87 7 0 6

36 0 2 4

85.7 0 4.8 9.5

84 10 1 3

85.7 10.2 1.0 3.1

No., number of strains; %, percentage of total strains in each cheese.

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C. Arizcun et al. / International Journal of Food Microbiology 38 (1997) 17 – 24

being the dominating species present, constituting more than 85% of the total number of isolates. Strains of Enterococcus faecalis may play an important role in the ripening of these cheeses. As reported by Trovatelli et al. (1987) they produce acetaldehyde and diacetyl, two substances that contribute appreciably to the organoleptic properties of cheeses. Enterococcus avium accounted for approximately 5% of the strains that were isolated. This species does not reduce methylene blue and does not use pyruvate as an energy source (Orvin, 1986). On the other hand, it is capable of fermenting carbohydrates, namely, arabinose, melibiose, mannitol, and melezitose, but not inulin. Enterococcus durans is commonly isolated from milk and milk products (Colman et al., 1992), hence its presence in the two cheeses considered here is not surprising. Predominance of Enterococcus faecalis over other enterococcal species and the occurrence of Enterococcus faecium and Enterococcus durans in lower levels have been reported for milk samples (Batish and Ranganathan, 1984) and various kinds of ´ cheese, i.e., Gamonedo (Gonzalez de Llano et al., 1992), Torta del Casar (Poullet et al., 1993), An-

thotyro (Kalogridou-Vassiliadou et al., 1994) and Mozzarella (Gatti et al., 1993). In addition, Enterococcus durans was reported to be the main ´ (Suarez ´ enterococcal species in Mahon et al., 1983) and in Feta (Tzanetakis and Litopoulou-Tzanetaki, 1992) cheese. On comparing the species of enterococci in the raw milk and in the cheeses after 4 months of ripening, the proportions of Enterococcus faecalis were similar both in the milk and in the two types of cheese considered. However, the minor species present all showed significant differences (Table 1).

3.2. Enzymatic activity levels Table 2 sets out the extracellular enzymatic activity values for the 13 enterococcal strains with the highest activity indicator values selected from among the 40 strains tested using the Api-zym system. On the whole the enterococci exhibited low levels of extracellular enzymatic activity. Only one enzyme, leucine arylamidase, was able to hydrolyze more than 30 nm of the substrate L-leucyl-2-naphthylamide and was the most active enzyme in six of

Table 2 Proteolytic and lipolytic enzymatic activity values measured using the Api-zym system (nanomoles of substrate hydrolyzed) for 13 enterococcal strains a selected from among 40 strains tested on the basis of high enzymatic activity indicator values Enzyme

Alkaline phosphatase Esterase Esterase lipase Lipase Leucine arylamidase Valine arylamidase Cysteine arylamidase Trypsin Chymotrypsin Acid phosphatase Naphthol-AS-BI phosphohydrolase a-Galactosidase b-Galactosidase b-Glucuronidase a-Glucosidase b-Glucosidase N-Acetyl-b-glucosaminidase a-Mannosidase a-Fucosidase a

Strain no. 1

2

3

4

5

6

7

8

9

10

11

12

13

0 10 20 0 30 0 5 0 0 5 0 0 . 40 0 0 0 0 0 0

5 10 20 0 20 5 10 0 10 20 5 0 30 0 30 0 0 0 0

0 10 20 0 30 20 5 0 0 5 5 0 20 0 10 0 5 0 0

5 10 20 0 30 5 30 0 5 30 . 40 0 5 0 5 0 0 0 0

5 10 10 0 . 40 . 40 20 0 10 20 5 0 . 40 0 10 5 10 0 5

0 20 20 0 . 40 10 5 0 5 5 5 0 5 0 5 0 0 0 0

0 5 20 0 20 10 5 0 0 5 0 0 30 0 5 5 0 0 0

0 5 5 0 . 40 30 5 0 0 10 5 20 . 40 5 . 40 30 0 0 0

5 10 20 0 . 40 5 . 40 0 5 30 10 0 5 0 . 40 5 0 0 0

5 5 10 5 40 . 40 5 0 0 10 10 0 20 0 10 0 20 0 0

0 5 5 0 30 30 5 0 0 10 5 10 . 40 5 20 30 0 0 0

5 0 20 0 30 5 5 0 10 20 5 0 10 0 30 0 0 0 0

0 10 10 0 . 40 5 10 0 5 10 5 0 5 0 30 0 0 0 0

Strain nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 were identified as Enterococcus faecalis; strain nos. 11 and 12 as Enterococcus faecium; and strain no. 13 as Enterococcus avium.

C. Arizcun et al. / International Journal of Food Microbiology 38 (1997) 17 – 24

the 13 strains tested. Quantitatively, the next most active enzymes were b-galactosidase, a-glucosidase, valine arylamidase, acid phosphatase and esterase lipase. None of the remaining 13 enzymes tested exceeded a mean value of 10 nm of substrate hydrolyzed. The strains displayed considerable variation, even though 10 of the strains belonged to the same species (Enterococcus faecalis). Such often appreciable fluctuations have also been reported for bacteria in the genera Lactobacillus (Bouton et al., 1994) and Lactococcus (Olivares et al., 1993). This finding highlights the importance of studying each bacterial strain individually before it can be selected for use in the cheese manufacturing industry, as

21

already pointed out by certain workers (Bhowmik and Marth, 1988). Table 3 presents the values for acid production by the 13 selected enterococcal strains in skim milk. According to the classification developed by Accolas and Auclair (1970), the strains tested in this study can be considered slow acid producers, except for ´ strain 5, which attained a value of 31.68 Dornic, in the range of moderately fast acid producers (30–358 ´ Dornic). These findings agree with those reported by ´ Martınez Moreno (1976). Table 4 presents extracellular and cytoplasmic aminopeptidase activity values for the 13 selected enterococcal strains tested at pH 5.5 and pH 7. To

Table 3 ´ Acid production after 6 h at 308C (measured in degrees Dornic) by the 13 enterococcal strains selected on the basis of the Api-Zym test results a,b Strain no.

Acid production

1

2

3

4

5

6

7

8

9

10

11

12

13

27.2

24.6

29.5

26.8

31.6

29.4

27.9

29.2

24.6

22.4

29.2

29.0

24.7

a

Strain nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 were identified as Enterococcus faecalis; strain nos. 11 and 12 as Enterococcus faecium; and strain no. 13 as Enterococcus avium. b Each value is the mean of four analyses; coefficient of variation value was less than 7% for each strain.

Table 4 Extracellular and cytoplasmic aminopeptidase activity a,b on the substrates L-leucine-p-nitroanilide ( p-Leu) and L-lysine-p-nitroanilide ( p-Lys) at pH 5.5 and pH 7 in the 13 selected enterococcal strains c Strain no.

Extracellular activity p-Leu

1 2 3 4 5 6 7 8 9 10 11 12 13 a

Cytoplasmic activity p-Lys

p-Leu

p-Lys

pH 5.5

pH 7

pH 5.5

pH 7

pH 5.5

pH 7

pH 5.5

pH 7

6 37 3 32 239 126 42 24 2 180 232 39 12

12 42 8 39 446 307 43 28 9 306 479 114 19

15 42 5 11 101 66 38 17 0.7 97 95 20 7

16 34 7 51 457 236 50 52 15 269 459 118 35

2 32 8 28 1120 511 4 91 8 388 575 424 2

4 49 48 25 1784 710 11 76 37 1164 651 1359 6

3 37 12 40 981 498 5 117 11 319 332 361 4

3 48 16 63 1405 695 3 214 23 1568 634 808 6

Expressed as nanomoles of substrate hydrolyzed per mg of protein per hour. Each value is the mean of four replications; coefficient of variation value for each strain did not exceed 13% and was lower than 5% in 67% of the cases. c Strain nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 were identified as Enterococcus faecalis; strain nos. 11 and 12 as Enterococcus faecium; and strain no. 13 as Enterococcus avium. b

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C. Arizcun et al. / International Journal of Food Microbiology 38 (1997) 17 – 24

the extent possible the test substrates employed were chosen for their specificity, hence, as pointed out by Crow et al. (1994), they may be taken to be indicative of the enzymatic activities tested The results highlight the high variability in enzymatic activity values recorded between strains of the same species, which in the case of cytoplasmic leucine aminopeptidase at pH 7 attained levels of 1784 to 4 nm of substrate hydrolyzed per mg of protein per hour. These disparities were similar to the differences between species, indicating that species cannot be differentiated on the basis of aminopeptidase activity. Variability between strains of the same species has also been reported by a number of other investigators (Tsakalidou et al., 1993; Schmidt et al., 1994). Cytoplasmic aminopeptidase activity values in the enterococcal strains tested were higher than the extracellular activity values in eight of the 13 strains considered. Strains nos. 1, 7, and 13 displayed higher extracellular activity values, while activity levels in strain no. 2 did not show significant differences and in strain no. 4 varied depending upon pH level and substrate type. Preference of enterococcal strains for leucine or lysine showed variability depending on the strain, pH and the location of the aminopeptidases in or on the cell. Peptidase activity in the different strains was not correlated with the acid production values for those same strains. This finding was also reported by Crow et al. (1993), who pointed out that for bacteria to be able to satisfy their nutritional requirements, at least some peptidase activity must be extracellular. For 48 of the 52 tests of enzymatic activity (92.3%), activity for a given enzyme suspension on a given substrate was appreciably higher at pH 7 than at pH 5.5. This finding suggests that in the normal conditions in these two cheeses (pH 5.1–5.8), aminopeptidase activity levels for the enterococci are lower than optimal. These results are in agreement with the results reported for other bacterial genera, such as Lactococcus (Kamaly and Marth, 1988; Visser, 1993). Proteolytic activity by bacteria may be strongly influenced by variations in pH as Giori et al. (1985) have already pointed out. Table 5 shows the extracellular and cytoplasmic proteinase activity values for the 13 selected enterococcal strains. Differences in caseinolytic activity between the different strains were considerable,

Table 5 Extracellular and cytoplasmic caseinolytic proteinase activity a,b in the 13 selected enterococcal strains c Strain no.

Extracellular activity

Cytoplasmic activity

1 2 3 4 5 6 7 8 9 10 11 12 13

50 583 28 148 288 112 156 26 204 20 97 188 157

52 593 44 79 175 135 34 49 23 29 60 76 65

a

Expressed as nanomoles of leucine released per mg of protein per hour. b Each value is the mean of four replications; coefficient of variation value for each strain did not exceed 12% and was lower than 5% in 60% of the cases. c Strain nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 were identified as Enterococcus faecalis; strain nos. 11 and 12 as Enterococcus faecium; and strain no. 13 as Enterococcus avium.

ranging between 20 and 288 nm of leucine released per mg of protein in the extract per hour in 12 of the 13 strains. Strain no. 2 exceeded those values by a broad margin, releasing 583 and 593 nm in the same experimental conditions. The limited data on proteolytic activity in enterococci in the literature corroborate this diversity in proteolytic activity levels (Carrasco de Mendoza et al., 1988). Such variability has also been reported for lactococcal strains (Coolbear et al., 1994) and it has also been verified experimentally during cheese manufacturing (Schmidt et al., 1994). The present study and the previously published reports cited above confirm that proteolytic activity levels need to be measured individually for each strain. Proteinase activity was mainly extracellular in seven of the 13 selected strains tested (strains nos. 4, 5, 7, 9, 11, 12, and 13), mainly cytoplasmic in three (strains nos. 3, 8, and 10), and was similar in both cell locations in the remaining three (strains nos. 1, 2, and 6). Published reports on the distribution of proteinase activity in cells have been variable (Crow et al., 1993; Visser, 1993). Extracellular proteolytic activity in Enterococcus faecalis appears to be associated with a metalloproteinase capable of hy-

C. Arizcun et al. / International Journal of Food Microbiology 38 (1997) 17 – 24

drolyzing the casein in milk (Pritchard and Coolbear, 1993). Addition of that proteinase to the vats during cheese manufacturing increased the amount of peptides and polypeptides released during ripening, and large amounts of that enzyme caused flavour and aroma defects in the cheese (Pritchard and Coolbear, 1993). Consequently, it is also necessary to determine the appropriate dose of enzyme to be added to achieve the desired effects over the course of ´ de Fernando et al., 1992). cheese ripening (Garcıa

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