Article pp.161-175 du Vol.21 n°2 (2001)

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Evaluation of enzymatic activities

of microorganisms isolated from Spanish

dry-cured hams during the commercial shelf-life

Antonia GARCÍA RUIZ^*, María Deseada NÚÑEZ, María Dolores CABEZUDO

RÉSUMÉ Criblage des activités enzymatiques des micro-organismes isolés sur les jambons secs espagnols pendant leur conservation commerciale.

Le rôle des micro-organismes dans le développement des flaveurs caractéris- tiques du jambon sec reste mal connu. Nous avons isolé et identifié les micro- organismes présents sur des jambons espagnols du commerce et nous avons étudié leurs activités enzymatiques au cours de leur conservation en rayon après la période de maturation. Nous avons observé que les jambons présen- taient une large gamme de microcoques et de staphylocoques, avec comme espèces dominantes S. xylosus et S. aureus (coagulase-négatif). Nous avons mis en œuvre différents essais pour identifier les activités enzymatiques. Aucun des isolats ne manifestait d’activité protéolytique. Les activités esterase et esterase lipase de M. halobius, de M. varians et de S. aureus étaient supé- rieures à celles de S. xylosus.

Mots clés : activité enzymatique, micro-organismes, jambon sec, conservation.

SUMMARY

The role of microorganisms in the development of the aroma characteristics of dry-cured ham remains unclear. The microorganisms present in commercially available Spanish hams purchased at stores were isolated and identified, and we study the enzymatic activities of these microorganisms during the commercial shelf-life, after the ripening period. A broad range of micrococci and staphylococci species were present in the samples analysed, the dominant species being S. xylosus and S. aureus (coagulase-negative). Different assays were used to test for enzymatic activities. None of the isolates tested exhibited proteolytic activity. M. halobius, M. varians, and S. aureus had higher esterase and esterase lipase activities than S. xylosus.

Key words: enzymatic activities of microorganisms, dry-cured ham, commer- cial shelf-life.

Department of Analytical Chemistry and Food Technology, Faculty of Chemistry, University of Castilla-La Mancha, Avenida de Camilo José Cela s/n.13071 Ciudad Real, Spain.

* Correspondance.

[email protected]

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1 - INTRODUCTION

Dry-cured hams are traditional food products in Spain and are much appre- ciated for their organoleptic characteristics. Production has undergone conside- rable growth in recent years. The hams are prepared by salting ham haunches of different breeds of white porks, followed by curing during 8-24 months.

During this process a series of physico-chemical and enzymatic changes rela- ted to product aroma and flavour development take place (TOLDRÁet al., 1993;

CÓRDOBAet al., 1994).

Most researchers have reported the enzymes present in the meat to be the only factors responsible for the changes taking place during the dry-curing of hams (TOLDRÁet al., 1993; FLORESet al., 1994). However, there have been some studies on the enzymatic properties of the microorganisms isolated from dry- cured hams, intended to elucidate the possible relationships between their functional characteristics and the development of the typical sensory attributes of dry-cured hams (CÓRDOBAet al., 1994).

CORNEJOand CARRASCOSA(1991), HUERTAet al. (1987, 1988) and FADDAet al. (1999) measured the proteolytic and lipolytic activity of microorganisms iso- lated from dry-cured hams on substrates such as casein, gelatine, and tween 80 and tributyrin, respectively, though their results were not reproducible.

Using a protein substrate obtained from Biceps femoris muscle from which the enzymatic factors and microorganisms naturally present in the meat had been removed, BERMELLet al. (1992) found that some strains of Staphyolococ- cus xylosus, Pediococcus pentosaceus, and Lactobacillus curvatus did not bring about any significant increases in non-protein nitrogen (NPN), with only certain strains of Cryptococcus albidus yielding an increase of 7%.

RODRÍGUEZet al. (1998) considered possible contributions of the proteolytic systems of the different microorganisms isolated from dry-cured Iberian hams (made from the native Iberian pork breed) to the final product characteristics using SDS-PAGE electrophoresis. Those same workers observed that Penici- llium chrysogenum contributed significantly to the proteolysis taking place during the dry-curing process in hams.

Many papers deal with changes in the microflora of Spanish dry-cured ham during the ripening process but no papers mention the role of the microorganisms during the store period.

The aim of the present study was to identify the microorganisms present in commercially available dry-cured hams purchased in the market place and to characterize the residual enzymatic activity of the microflora isolated from stored hams.

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2 - MATERIALS AND METHODS

2.1 Materials

The study was performed using ten hams purchased from various commercial establishments in the province of Ciudad Real (Castilla-La Mancha, Spain).

Samples from the surface of the hams were taken aseptically from the Semi- membranosus muscle. Samples from the interior of the hams were taken from the excised Biceps femoris muscle after it had been flamed with absolute ethyl alcohol for a few seconds

2.2 Analytical methods

2.2.1 Physical and chemical analysis

Dry matter (DM) was determined according to the method described in Iso Standard 1442 (ISO, 1973). Water activity (A_w) was measured using a Decagon Devices CX-2 dew-point hygrometer (Giralt, USA). The pH was recorded using a MicropH 2002 pH-meter (Crison Instruments, Barcelona, Spain) after homogenizing 10 g of sample in 10 mL of distilled water.

2.3 Microbiological assays

2.3.1 Sample preparation and counts

Preparation of the Semimembranosus and Biceps femoris muscle samples and the counts of micrococci (Mannitol Salt Agar, Difco); staphylococci (Baird Parker Agar, Difco); lactic acid bacteria (M17 Agar for lactococci, Leuconostoc Agar for Leuconostoc and Rogosa Agar for lactobacilli, Difco); yeasts (Malt Agar); aerobic psychrotrophic bacteria, halotolerant bacteria (Tryptic soy Agar, Difco) and total aerobic mesophilic bacteria (Standard methods Agar, Difco);

enterococci (m-Enterococcus Agar, Difco); sulfite reducing clostridia (SPS Agar, Difco) and coliforms (Brillant Green Bile Agar, Difco) were carried out according to FRANCISCO et al. (1981) by homogenizing 10 g of sample in 90 mL of 2%

sodium citrate and preparing the corresponding series of decimal dilutions.

2.3.2 Isolation and identification tests

Approximately 20% of the colonies were collected from Leuconostoc Agar (ADSA), Mannitol Salt Agar (Difco), and Baird Parker Agar (Difco) plates according to the criterion of HARRIGANand McCANCE(1976). Colony purity was valida- ted by reseeding on sterile plates containing those same media. Colony appearance and cell morphology were examined and catalase activity was tested for all the isolates (HARRIGANand McCANCE, 1976).

The gram-positive, catalase-positive isolates (Micrococcus and Staphylo- coccus strains) were subjected to the nitrate-reducing test in accordance to APHA(1984) recommendations. The effect of sodium chloride and sodium nitrite concentration on the growth of all nitrate-reducing strains was studied in Brain Heart Infusion (Difco), supplemented separately with 10% sodium chloride or 100 ppm sodium nitrite. Strains that did not produce turbidity and/or sediment

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in the culture test tubes after incubation at 34 °C for 48 h were excluded from further testing.

The gram-positive, catalase-negative isolates were considered as potential lactic acid bacteria and were tested for arginine deamination and CO₂production from glucose after incubation of the plates at 30°C for 7 d and 48 h, respectively in each case, for subsequent assignment to the appropriate genus.

Species identification

A) Micrococcus and Staphylococcus: The API-STAPH Identification System (API System S.A., BioMérieux, Montalieu Vercie, France) was used to identify the selected strains. In separating the strains belonging to the genus Micrococ- cus from those belonging to the genus Staphylococcus, the Furazolidone Agar (Difco) test (RHEINBADEN and HADLOK, 1981) was used instead of the lysosta- phine test. Colony characteristics, pigmentation in P agar (10 g·L^–1 peptone, 5 g·L^–1 yeast extract, 5 g·L^–1 NaCl, 1 g·L^–1 glucose, 12 g·L^–1 agar, pH = 7.0), tolerance to 15% sodium chloride, growth at 45°C, and tests for coagulase and Dnase activity were all employed as means of identification in addition to the API-STAPH system, in accordance with HOLTet al. (1994).

B) Lactic acid bacteria: Identification was based on the method described in HOLTet al. (1994). Fermentation tests were performed at 30°C for 48 h using the API 50 CH Identification System (API System S.A., BioMérieux, Montalieu Ver- cie, France).

The biochemical profiles of Micrococcus, Staphylococcus and lactic acid bacteria obtained were interpreted automatically by means of the APILAB Plus computer-aided identification program (BioMérieux, France). Following identification the strains were stored in MRS broth (Difco) and Brain Heart Infusion (Difco) containing 20% glycerol (Difco) at – 20°C, with subcultures being prepared every 6 months.

Reference strains

Pure cultures from the Spanish Type Culture Collection (CECT) were used as reference: Micrococcus luteus (CECT 245), Staphylococcus aureus (CECT 520), Staphylococcus xylosus (CECT 237), Pediococcus pentosaceus (CECT 923), and Leuconostoc mesenteroides subsp. mesenteroides (CECT 219).

2.3.3 Standard methods for detecting enzymatic activities

A semi-quantitative enzymatic test was applied to selected identified strains.

Each microorganism was grown on P agar (described above) incubated at 37°C for 24 h. The microorganisms were then suspended in sterile distilled water to a final turbidity between MacFarland standards Nos 5 and 6. Three ApiZym strips (API System S.A., BioMérieux, Marcy L’Étoile, France) per microorganism were then inoculated with each of the microbial suspensions (65µL). The strips were incubated at 37°C for 4 h. Colour intensity values ran- ging from 0 to 5 were determined in accordance to the manufacturer’s instruc- tions. Enzymatic activities were assayed on the following substrates:

– Phosphatase alcaline on 2-naphthyl phosphate;

– Esterase (C4) on 2-naphthyl butyrate;

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– Esterase lipase (C8) on 2-naphthyl caprylate;

– Lipase (C14) on 2-naphthyl myristate;

– Leucine arylamidase on L-leucyl-2-naphthylamide;

– Valine arylamidase on L-valyl-2-naphthylamide;

– Cystine arylamidase on L-cystyl-2-naphthylamide;

– Trypsin on N-benzoyl-DL-arginine-2-naphthylamide;

– Chymotrypsin on N-glutaryl-phenylalanine-2-naphthylamide;

– Phosphatase acid on 2-naphthyl phosphate;

– Naphthol-AS-BI-phosphohydrolase on Naphthol-AS-BI-phosphate;

– α-galatosidase on 6-Br-2-naphthyl-αD-galactopyranoside;

– β-galactosidase on 2-naphthyl-βD-galactopyranoside;

– β-glucoronidase on Naphthol-AS-BI- βD-glucoronide;

– α-glucosidase on 2-naphthyl-αD-glucopyranoside;

– β-glucosidase on 6-Br-2-naphthyl-βD-glucopyranoside;

– N-acetyl-β-glucosaminidase on 1-naphthyl-N-acetyl-βD-glucosaminide;

– α-mannosidase on 6-Br-2-naphthyl-αD-mannopyranoside;

– α-fucosidase on 2-naphthyl-αL-fucopyranoside.

3 - RESULTS AND DISCUSSION

3.1 Physico-chemical analyses

Table 1 presents the DM, A_w, and pH values for the samples analysed.

These results are comparable to the findings of ASTIASARÁN et al. (1988) and CARRASCOSAand CORNEJO(1991).

Table 1

Physico-chemical characteristics of the surface (Semimembranosus muscle) and the interior (Biceps femoris muscle) samples from commercially available

Spanish dry-cured hams

Semimembranosus Biceps femoris (surface) (interior) Dry matter (g/100 g ham) 56.66 (± 2.96) 43.66 (± 3.15)

Water activity (Aw) 0.859 (± 0.03) 0.881 (± 0.03)

pH 5.93 (± 0.09) 5.99 (± 0.15)

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3.2 Microorganism counts

Table 2 sets out the microorganism counts for the samples analysed. In most cases surface counts were higher than the interior.

Micrococcus and Staphylococcus, lactococci and leuconostoc, and yeasts were present, in accordance with the findings of CARRASCOSAet al. (1991) and SILLAet al. (1989).

No lactobacilli, aerobic psychrotrophic bacteria, sulfite-reducing clostridia, or coliforms were detectable in any of the samples considered. The action of the salt together with the low temperatures during the processing period could inhibit the growth of those microorganisms. Similar results have been reported for Iberian hams (FRANCISCOet al., 1981), Italian prosciutto (GIOLITTIet al., 1971), and American country-style hams (GRAHAMet al., 1971).

Table 2

Counts of microorganism isolated from commercially available Spanish dry-cured hams (log cfu/g)

Semimembranosus Biceps femoris (surface) (interior)

Micrococci and Staphylococci bacteria 5.00 4.43

Lactic acid bacteria

Lactococci 4.77 4.45

Leuconostoc 4.68 4.54

Lactobacilli nd nd

Yeast 4.29 4.55

Aerobic psychrotrophic bacteria nd nd

Halotolerant bacteria 4.71 4.47

Total aerobic mesophilic bacteria 5.30 8.20

nd: Not detected

3.3 Identification

Figure 1 shows the diagrams of the identification of the 89 isolates conside- red.

A) Micrococcus and Staphylococcus

Of the 89 initial isolates, 78 were catalase-positive. Most of those (N = 68) were able to reduce nitrate and to grow in the presence of NaCl and NaNO₂. The isolates that did not present those two characteristics were discarded, because nitrate-reductase may help accelerate colour development in dry- cured hams (LÜCKEand HELCHEMAN, 1986).

The furazolidone test was applied to the 68 remaining isolates and yielded two major groups, G. Micrococcus (N = 16) and G. Staphylococcus (N = 52).

Table 3 lists the biochemical profiles for the 52 isolates assigned to the genus Staphylococcus. All were able to grow at 45°C and in the presence of 15% NaCl, in agreement with the findings of CAMPANINIet al. (1987) and SILLAet al. (1989).

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Figure 1 Identification diagram for the microorganisms isolated from commercially available Spanish dry-cured hams

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Table 3

Biochemical characteristics of staphylococcus isolated from commercially available Spanish dry-cured hams

Staphylococcus xylosus Staphylococcus aureus

Reference Strains Reference Strains

strain isolated strain isolated

CECT237 (N = 18) CECT 520 (N = 10)

Growth with:

10% NaCl + + + +

15% NaCl + 67(28) – 10(60)

100 ppm sodium nitrite + + + +

45ºC + + + +

Resistance to:

Furazolidone – – – –

Aerobic acid produced from:

D-Glucose + + + +

D-Fructose + + + +

D-Mannose + 95(5) + 60(30)

Maltose + + + 90(10)

Lactose + + + +

D-Trehalose + 95(5) + 70(30)

D-Manitol + 83(6) + [80]

Xylitol – [17] – –

D-Melibiose – [5] – –

Nitrate reduction + + + +

Alkaline Phosphatase + 95 + +

Acetyl-methyl-carbinol + + – +

Raffinose – – – –

Xylose + 78(11) – [80]

Sucrose + + + +

alfa-methyl-glucoside – [6] – –

N-Acetyl-glucosamine + 83(6) + 20(80)

Arginine dihydrolase – – + +

Urease + + + +

Coagulase – – + –

Deoxyribonuclease – – + +

(Dnase agar)

Symbols: +: positive reaction; –: negative reaction; w: weak reaction; 54: 54% of the strains are positive; [28]: 28% of the strains are weak; nd: not determined; N: number of isolates; CECT: Spanish Type Culture Collection.

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Table 3 (continued)

Biochemical characteristics of staphylococcus isolated from commercially available Spanish dry-cured hams

Staphylococcus Staphylococcus Staphylococcus

epidermidis saprophyticus spp.

Strain isolated Strain isolated Strain isolated

(N = 8) (N = 4) (N = 12)

Growth with:

10% NaCl + + +

15% NaCl 12(38) (75) 75(17)

100 ppm sodium nitrite + + +

45ºC + + +

Resistance to:

Furazolidone – – –

Aerobic acid produced from:

D-Glucose + + +

D-Fructose + + +

D-Mannose 12(63) – 42(17)

Maltose + + 83(9)

Lactose + + 67(17)

D-Trehalose 12(38) 75(25) 83

D-Manitol – – 17(17)

Xylitol [12] 25(75) [42]

D-Melibiose – – 8

Nitrate reduction 6 75 83

Alkaline Phosphatase + + 75(17)

Acetyl-methyl-carbinol 88 + 75(8)

Raffinose – – [8]

Xylose [25] – [42]

Sucrose + + +

alfa-methyl-glucoside – – [8]

N-Acetyl-glucosamine 38(62) 75(25) 75(8)

Arginine dihydrolase 62 25 8

Urease + + +

Coagulase – – nd

Deoxyribonuclease – – nd

(Dnase agar)

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These microorganisms help to maintain colour stability in the meat and pre- vent rancidity by lowering the hydrogen peroxide content as a consequence of their catalase activity (ANDRÉS, 1977). Furthermore, the activity of these microbial groups has the effect of reducing microbial contamination, shortening processing time, and increasing the pH (SELGASet al., 1986).

All the S. xylosus isolates were able to convert glucose into acid. That pro- perty was not reported for isolates of this same species identified by RODRÍGUEZ et al. (1994) in Spanish Iberian hams.

All the microorganisms identified as S. aureus were coagulase-negative.

B) Lactic acid bacteria

Table 4 lists the biochemical profiles of the three isolates assigned to the genus Leuconostoc and Pediococcus. Though making up only a small propor- tion of the total, because of their ability to lower the pH, lactic acid bacteria contribute to aroma development in dry-cured hams and at the same time inhibit the growth of undesirable microorganisms (DEMEYERet al., 1986).

3.4 Enzymatic activities

Various isolates were selected from each of the species identified in the pre- ceding section for the enzymatic activity determinations. The results are shown in Table 5.

Table 4

Biochemical characteristics of lactic acid bacteria from commercially available Spanish dry-cured hams

Leuconostoc mesenteroides Pediococcus pentosaceus

CECT 219 (N = 2) CECT 923 (N = 1)

CO₂from glucose + + – –

NH₃from arginine – – w –

Growth at 45ºC nd nd – –

Dextran + + nd nd

Acid produced from:

Glycerol – – – –

Erytritol – – – –

D-Arabinose – 50 – –

L-Arabinose – 50 + +

Ribose – 50 + +

D-Xylose – – – +

L-Xylose – – – –

Adonitol – – – –

Beta-methyl xylose – – – –

Galactose + w + +

D-Glucose + + + +

D-Fructose + + + +

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Table 4 (continued)

Biochemical characteristics of lactic acid bacteria from commercially available Spanish dry-cured hams

Leuconostoc mesenteroides Pediococcus pentosaceus

CECT 219 (N = 2) CECT 923 (N = 1)

D-Mannose + 50(50) + +

L-Sorbose – [50] – –

Rhamnose – – + –

Dulcitol – – – –

Inositol – – – –

Manitol – [50] – w

Sorbitol – – – –

alfa-methyl-D- mannoside – – – –

alfa-methyl-D- glucoside w 50 + +

N-Acetyl-Glucosamine w [50] + +

Amygdalin + [50] + –

Arbutin + – + w

Aesculin + 50 + +

Salicin – [50] + +

Cellobiose + – + +

Maltose + 50 + +

Lactose – + – –

Melibiose – 50 – w

Sucrose + 50 + +

Trehalose + 50 + +

Inulin – – – –

Melezitose – – – –

D-Raffinose – – – –

Starch – – – –

Glucogen – – – –

Xylitol – – – –

Beta-gentibiose – – – –

D-Turanose + 50 + +

D-Lixose – – – –

D-Tagarose + – + –

D-Fucose – – – –

L-Fucose – – – –

D-Arabitol – – – –

L-Arabitol – – – –

Gluconate – 50(50) – w

2-ceto-gluconate – 50 – –

5-ceto-gluconate – – – –

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Table 5

Enzymatic activities of the microorganisms isolated from commercially available dry- cured hams and of the standard strains considered

Enzymatic activity detected Lipolytic Arylamidase Glucolytic

Microorganisms A B C D E F G H I J

Staphylococcus S M T T T L L T T T

xylosus CECT 237

Staphylococcus L L T T T-L T-L T-L-M T T T

xylosus (N = 6)

Staphylococcus S S T T T T T L T T

aureus CECT 520

Staphylococcus S-L M-L T T T T T T T T

aureus (N = 5)

Staphylococcus L-M L-M T T T T T T T T

epidermidis (N = 3)

Staphylococcus L M T T T T T T T T

saprophyticcus (N = 4)

Micrococcus luteus S S T T T L S L T T

CECT 245

Micrococcus M M T T T T-L T T T T

halobius (N = 2)

Micrococcus L-M L-M T T T T T T-L T T

varians (N = 2)

Micrococcus L L T T T T T T T T

kristinae (N = 1)

M. halobius/ L-M L-M T T T T-L T T T T

M. kristinae (N = 2)

Micrococcus spp. L L T T T T T T T T

(N = 1)

L. mesent. subsp. T T L T T M T T T L

mesent. CECT 219

L. mesent subsp. T T T T T T T T T-L T

mesenteroides (N = 3)

Pediococcus T T S S T T T T S S

pentosaceus CECT 923

Pediococcus T T T T T L T T T T

pentosaceus (N = 1)

Colour intensity values: T (< 1, trace); L (1-2, low); M (3, medium); S (4-5, strong).

A: esterase; B: esterase lipase; C: leucine arylamidase; D: valine arylamidase; E: alfa-galactosidase; F:

beta-galactosidase; G: beta-glucoronidase; H: alfa-glucosidase; I: beta-glucosidase; J: N-A-B-glucosaminidase; N: number of isolates.

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None of the isolates tested exhibited proteolytic activity. MOLINAand TOLDRÁ (1992) found absence of endopeptidase activity and a very weak leucine aryla- midase activity on the strains of S. xylosus isolated during the processing of Spanish Serrano dry-cured ham. These authors reported strains of P. pentosa- ceus with a strong leucine and valine arylamidase activities, during the ripening period of Serrano ham.

NÚÑEZet al. (1998) reported strains of S. xylosus with high levels of exocellu- lar aminopeptidase activity on such substrates as L-alanine, L-methionine, and L-valine.

The isolates of M. halobius and M. varians displayed moderate lipolytic acti- vity levels (esterase and esterase lipase). The strains of S. aureus, S.

epidemidis, and S. saprophyticus displayed similar behaviour, while S. xylosus exhibited lower lipolytic activity, which agreed with the findings of DELARRAS (1982).

The isolates of L. mesenteroides subsp. mesenteroides exhibited low or moderate levels of glucosidase activity.

4 - FINAL OBSERVATIONS

A broad variety of microbial species were isolated from commercially available Spanish dry-cured hams. The microorganisms exhibited characteristics well suited to technical processing, such as nitrate reductase activity and an ability to grow in the presence of sodium nitrite, at high salt concentrations and high temperatures.

The enzymatic activities found should be indicative of the microbiological stability of the samples analysed, so meat enzymes could be the main factor responsible for the biochemical changes (proteolysis and lipolysis) that may take place during the commercial shelf-life of dry-cured hams.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial support for this study provi- ded by the company GRUPO NAVIDUL.

Received 25 September 2000, accepted 5 February 2001.

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