Article pp.159-172 du Vol.24 n°2 (2004)

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ARTICLE ORIGINAL ORIGINAL PAPER

Comparison of mycoflora associated to Canestrato Pugliese cheese produced

according three protocols

M. Sinigaglia¹*, M. Albenzio², M. R. Corbo¹, C. Ciccarone³

SUMMARY

The associated mycoflora of the Canestrato Pugliese cheese produced from raw milk, pasteurized milk or by heating the curd in hot whey were com- pared. In order to evaluate the ability to colonize Canestrato Pugliese cheese, the effects of pH, salt content, temperature and nutritional source on growth of some strains were investigated.

The cheese produced according to a traditional protocol harboured a more heterogeneous mycoflora than to modern protocols. However, the species most frequently isolated from our samples (Fusarium moniliforme, Aspergil- lus niger and Aspergillus flavus) were infrequently associated with cheese and indicated a strong contamination of the ripening rooms used for cheese ripening only for a short while. Moreover, since during mould growth a risk of mycotoxin production in the cheese exists, the isolation of mycotoxigenic moulds could represent a health concern. The results obtained emphasize the need to control relative humidity and hygienic conditions of the ripening houses and provide an improved understanding of fungi of Canestrato Pugliese cheese.

Key words

Canestrato Pugliese cheese, pasteurisation, fungi, physiological characteri- zation.

1. Department of Food Science, Agricultural Faculty, University of Foggia, Via Napoli 25, 71100 Foggia, Italy.

2. Department of Livestock, Engineering, Mechanics and Economic Productions, Agricultural Faculty, Uni- versity of Foggia, Via Napoli 25, 71100 Foggia, Italy.

3. Department of Agro-Environmental Science, Chemistry and Plant Defence, Agricultural Faculty, Univer- sity of Foggia, Via Napoli 25, 71100 Foggia, Italy.

* Corresponding author: [email protected].

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1 – INTRODUCTION

Canestrato Pugliese is a DOP (Denomination of Origin Protected) semi-hard Italian cheese with a very large market popularity. It may be produced according to a traditional protocol, which includes heating of the curd in hot whey (ca.

80˚C for 30 s), and from pasteurised or raw milk. Also, starters as thermophilic lactic acid bacteria may be used or not.

The main goal of pasteurisation is, obviously, the elimination of pathogens which may be present in milk; however, milk pasteurisation can exert evident effects on the ripening process as well as on the sensory characteristics of cheese (GRAPPIN and BEUVIER, 1997). Moreover, cheeses produced with raw milk present a very different microflora at the genus level and also in term of species within the same genus (MACEDO et al., 1995).

It could be interesting to determine whether pasteurisation is able to influence also the mycoflora of Canestrato Pugliese cheese. In fact, in the course of ripening, drying causes water activity lowering on the Canestrato surface; thus, a microenvironment is created which enhances growth of fungi that become dominant so that cheese often shows severe visible mould growth. With the exception of soft cheeses with white surfaces and blue veins for which the mould growth is beneficial and characterizing, association between fungi and cheese is regarded as detrimental. In fact, spoilage of cheese by penicillia and aspergilli occurs frequently and can cause economic losses; furthermore mould growth can constitute a health hazard because of mycotoxin production.

In their study on the associated mycoflora of cheese, LUND et al. (1995) have reported that the dominant fungi were Penicillium spp. In particular, Penicillium commune, from which P. camemberti is derived (PITT and HOCKING, 1997), was found as the most occurring (42%) species on cheeses from country in several parts in the world. The other species were infrequent but were found on cheeses produced in various countries indicating that the associated mycoflora is independent of the origin and processing of cheese.

Unwanted fungal growth on cheese may be reduced by strictly hygienic standards but the problem is common and recurring. In order to control growth of unwanted fungi, knowledge about the effects of environmental factors influ- encing fungal development is needed.

In this work we compare the associated mycoflora of the Canestrato Pugliese cheese produced from raw milk, pasteurized milk or by heating the curd in hot whey. Moreover, in order to evaluate the ability to colonize Canes- trato Pugliese cheese, the effects of pH, salt content, temperature and nutritional source on growth of some strains were investigated.

2 – MATERIALS AND METHODS

Cheesemaking and samples. The protocols for the production of the cheeses are reported in figure 1. The three types of Canestrato Pugliese cheese differed with respect to heat treatment: R cheese was produced from raw ewes’

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milk, P cheese from pasteurized milk and T (traditional) cheese by heating the curd in hot whey. Thermophilic lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) were used as starters in R and P cheeses only. Another distinguishing feature of T cheese was that the curd was held at ca. 20˚C instead of 42˚C for 14 h.

Ewes’ milk

Pasteurization (ca. 72˚C for 30 sec) (P^a cheese) – no pasteurization (R and T cheeses)

Heating (R and T cheeses) or cooling (P cheese) of milk to 43˚C

Inoculum of the milk with starter at ca. 6.0 log cfu mL^-1 (Lactobacilus delbrueckii subsp.

bulgaricus and Streptococcus thermophilus) (R and P cheeses) – no inoculum with starters (T cheese)

Milk held at 43˚C for 30 min (R and P cheeses) – lacking phase (T cheese)

Addition of liquid calf rennet (ca. 12 ml L^-1) (all cheeses) Coagulation of milk at 30 –35˚C after ca. 30 min (all cheeses)

Cutting of coagulum (size of the curd after cutting: ca. 0.5 – 1.0 cm) (all cheeses) Removal of the whey (R and P cheeses) – Heating the curd in hot whey (ca. 80˚C for 30 sec) and

subsequent removal of the whey (T cheese)

Curd held at ca. 42˚C for 14 h (R and P cheeses)–Curd held at ca. 20˚C for 14 h (T cheese) Dry-salting of the curd (all cheeses)

Ripening at ca. 10 – 15˚C for 65 days (environmental humidity ca. 95 %) (all cheeses)

aR, raw cheese; P, pasteurized cheese; T, traditional cheese

Figure 1

Protocol of the production of Canestrato Pugliese cheeses.

The three types of Canestrato Pugliese cheese (R, P and T) represented the main types available on the Italian market. All cheeses had a weight of ca.

1.5 kg and were cylindrical in shape.

Two batches of Canestrato Pugliese cheese were produced on different days from milk from the same farms. The cheese samples (totally 36 samples, 6 for each production protocol and for each batch) were taken from two batches, sent to our laboratory under refrigeration (ca. 4˚C) and analyzed immediately in triplicate after ripening of 1, 14, 24, 37, 50 and 64 days.

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Isolation and identification of fungi. For mycological analysis, cheese samples (20 g) were diluted in 180 ml of 0.9% (w/v) sodium chloride solution and homoge- nized in a Stomacher Blender 400 (PBI International, Milan, Italy). Serial dilutions were made in the same solution and plated on the following two media: Malt Extract Agar (MEA, Oxoid Ltd, Basingstoke, UK), used as a general medium for isolation of fungi and Dichloran Rose Bengal Chloramphenicol Agar (DRBCA; PITT

and HOCKING, 1985), used in order to restrict the growth of most of the common Mucoraceous fungi. The plates were incubated for 5-7 days at 25˚C. All the result- ing colony types were inoculated onto the same two media until pure cultures were obtained. Fungi were identified according to the official methods described by PITT and HOCKING (1997). Differential diagnosis at species level was supported by data-bank matching and collection type-strain mating.

Diagnoses were referred to the following taxonomic keys:

Fusarium Link, GERLACH and NIREMBERG (1982);

Aspergillus Fr.: Fr., KOZAKIEWICZ (1989);

Penicillium Link, PITT (1979);

Trichoderma Pers., RIFAI (1969).

Mucor P. Micheli: Fr., identification was achieved by micro and macroscopic inspection on 7-days grown cultures. As to Trichothecium roseum (Pers.) Link, diagnosis was based on conidial onthogenesis (COLE and KENDRICK, 1981).

Analytical methods. NaCl concentration and pH were determined as sug- gested by the INTERNATIONAL DAIRY FEDERATION (1988, 1989). Water activity (a_w) was determined at 25˚C by using an Aqua Lab Decagon (Pullman, WA, USA) instrument.

Temperature, pH and salt content studies. Studies on the effects of temper- ature, pH and NaCl concentration were carried out with fifteen strains of different species isolates from 50- and 64-days ripened R, P and T cheeses. For each isolate a conidial suspension was prepared by washing mould agar slopes with a Tween 80 solution (0.05%).

The growth rate of the different strains was determined at pH values ranging from 3 to 8 (incubation temperature 25˚C) and at 10, 15, 20, 25, 30 and 35˚C.

The medium used was MEA (Oxoid) with a pH of 5.6; the pH was adjusted with citric acid or NaOH (Carlo Erba). Moreover, sodium chloride was added to standard medium in order to achieve final concentration of 2.5, 5, 7.5 and 10%

(w/v) (pH 5.6, incubation temperature 25˚C).

The fungal growth was determined by inoculating the center of agar plates with a loop of conidial suspension (ca. 5✕10³) and measuring daily the colony diameter. The colony diameter data were modelled according to the Gompertz equation as modified by ZWIETERING et al. (1990):

y = A exp ⎨-exp[(µmax e/A)(λ-t)+1]⎬

where y is the colony diameter (cm), A is the maximum colony diameter attained, µmax is the maximal growth rate (∆ cm day^-1), λ is the lag time (days), and t is the time.

The experimental data were modelled through the nonlinear regression pro- cedure of the statistic package Statistica for Windows (Statsoft, Tulsa, USA).

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Growth on semi-synthetic medium. In order to study the influence of nutri- tional source, the fungal growth rate was determined using the semi-synthetic cheese media developed by HANSEN and NIELSEN (1997). The medium had the following composition (g): casein, 100; lactate (90%), 8.3; lactose, 7.9; CaCl₂ 2 H₂O, 7.3; MgSO₄ 7 H₂O, 2.6; agar, 20; FeSO₄ 7 H₂O, 0.025; CuSO₄ 5 H₂O, 0.004; unsalted butter, 250 and water to a total weight of 1 kg.

This medium was found to resemble cheese regarding performance of fungal growth (HANSEN and NIELSEN, 1997). The pH values of 5.6 was obtained by using 1 M NaOH. The incubation temperature was 25˚C. The colony diameter (cm) was measured daily and modelled according to the Gompertz equation modified by ZWIETERING et al. (1990).

3 – RESULTS

3.1 Mycological examination

Table 1 shows the results of mycological examination of the three cheese types analyzed during the ripening. The fungi isolated from P and R cheeses belonged to the genera Fusarium Link, Aspergillus Fr.: Fr., Penicillium Link and Mucor P. Micheli: Fr.. In traditional (T) cheese, besides the above-mentioned fungi, were isolated Trichoderma viride Pers. and Trichothecium roseum (Pers.) Link.

Except 64-days ripened cheeses, strains belonged to the genus Fusarium were found on all samples. Most of isolates belonged to the species F. monili- forme J. Sheld. as typical form and as F. lactis Pirotta and Riboni, nomen confu- sum according to GERLACH and NIRENBERG (1982). Moreover some isolates were presumptively identified as F. culmorum (W. G. Smith) Sacc.

Strains belonging to the genus Penicillium were infrequently isolated, the low frequency of isolation of P. commune, 12% in P and T cheeses and 6% in R cheese (Table 1), does not correspond well with other reports of identification of isolates from cheeses (KIVANC, 1992; LUND et al., 1995). On the contrary, beyond Fusarium spp., Aspergillus niger Tiegh. and A. flavus Link were the most frequently occurring species in the cheeses investigated.

Mucor circinelloides Tiegh. was isolated from the three types of Canestrato Pugliese but only in the samples examined at the 24^th day of ripening.

In T (traditional) Canestrato Pugliese cheese were also isolated:

1) A. restrictus. This species has been isolated from dried foods quite frequently; it does not produce mycotoxins (PITT and HOCKING, 1997).

2) Trichoderma viride 3) Trichothecium roseum

These findings seem to indicate that Canestrato Pugliese cheese, made according to the traditional protocol, presents a greater fungal diversity.

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Table 1

Fungi isolated from three types of Canestrato Pugliese cheese during ripening.

Type of cheese

Fungi isolated No of isolates

Days of ripening

1 14 24 37 50 64 Total %^a

P^b Fusarium moniliforme 6 4 4 4 4 – 22 36

Fusarium spp.^c 2 – – 2 4 – 8 12

Penicillium commune 2 2 – 2 – – 6 12

Aspergillus niger – – 4 2 2 4 12 18

Aspergillus flavus – – 2 2 2 6 12 18

Mucor circinelloides – – 2 – – – 2 4

R Fusarium moniliforme 6 4 4 6 6 – 26 46

Fusarium spp. 2 – 2 – 2 – 6 10

Penicillium commune – – – – – 2 2 2

Aspergillus niger – 2 2 2 2 4 12 20

Aspergillus flavus – – 2 2 – 4 8 14

T Fusarium moniliforme 6 2 4 2 2 – 16 28

Fusarium spp. 2 2 – – – – 4 6

Penicillium commune – 4 – – – 2 6 12

Aspergillus niger – – 2 2 4 4 12 18

Aspergillus flavus 2 – – – 4 4 10 15

Aspergillus restrictus – – – – 2 2 4 6

Trichoderma viride – – – – 2 – 2 3

Trichothecium roseum – – – – 2 2 4 6

a Frequency calculated as (number of isolates of the species over the total number of isolates)✕100;

b R, raw cheese; P, pasteurized cheese and T, traditional cheese; ^c Not identified to species level

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3.2 Physicochemical characteristics

The evolution over time of the pH, a_w and NaCl concentration for the three cheese types is reported in Table 2. It is possible to observe that, relatively to T cheese, the pH decrease was less rapid and values comparable to those observed in P and R cheeses after 1 day of ripening were attained only after 14 days of maturation. Anyway, for all the cheeses, pH decreased until 37 days of ripening, then increased slightly, probably due to the oxidation of lactic acid by surface growing moulds.

As far as water activity concerns, it must be considered that T cheese was characterized by a_w values higher than those observed for P and R cheeses at the same ripening phase. Moreover T cheese also showed the lowest NaCl concentrations. These results could explain the greater heterogeneity of T cheese mycoflora.

Table 2

Mean values^a for the pH, a_w and NaCl concentration of Canestrato Pugliese cheeses during ripening.

Days of ripening P^b R T

pH

1 5.55±0.02 5.62±0.05 6.92±0.03

14 5.53±0.05 5.60±0.10 5.54±0.04

24 5.41±0.10 5.57±0.06 5.53±0.07

37 5.38±0.06 5.56±0.05 5.48±0.09

50 5.49±0.06 5.58±0.02 5.50±0.03

64 5.53±0.12 5.78±0.08 5.53±0.02

a_w

1 0.989±0.01 0.982±0.04 0.994±0.07

14 0.939±0.02 0.935±0.03 0.946±0.01

24 0.903±0.08 0.880±0.02 0.922±0.10

37 0.900±0.02 0.868±0.05 0.917±0.03

50 0.854±0.06 0.860±0.08 0.907±0.02

64 0.842±0.05 0.852±0.02 0.900±0.07

NaCl concentration

1 1.05±0.03 1.23±0.09 1.49±0.01

14 5.54±0.12 4.36±0.08 1.94±0.14

24 5.72±0.21 5.16±0.012 3.09±0.05

37 5.75±0.09 5.43±0.07 3.99±0.09

50 6.30±0.15 5.88±0.09 4.27±0.11

64 8.20±0.22 5.99±0.12 5.12±0.08

a Averaged data from two batches of cheeses analyzed in triplicate.

b R, raw cheese; P, pasteurized cheese; T, traditional cheese.

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3.3 Effect of temperature, pH and salt content on growth rate

Fifteen strains of different species isolated from 50- and 64-days ripened Canestrato Pugliese cheeses were investigated for their ability to grow at different temperatures, pH and NaCl concentrations.

The colony diameter “growth curve” were typical of microbial growth comprising a lag phase and a short accelerating period followed by a linear phase; so the curves were modelled according to the Gompertz equation modified by ZWIETERING et al. (1990). The predicted curves fitted well the experimental points as indicated by the determination coefficients (R²) that ranged between 0.956 and 0.998.

Table 3 shows the growth rates of the strain investigated at different temperatures and pH. It is possible to observe that the growth rate was, in general, 3-4 times lower at 15˚C than at 30˚C. The lowest difference between both temperature was given by A. flavus 84R which was characterized by highest growth rate at 15˚C.

Moreover all strains belonging to the species F. moniliforme as well as A. restrictus 89T, P. commune 87T and T. roseum 98T were unable to grow at 35˚C.

Table 3

Colony growth rate^a of fungi isolated from Canestrato Pugliese cheese at different temperatures and pH

Temperature (°C)^b pH^c

Strains 15 20 25 30 35 3 4 5 6 7 8

Fm68T^d 0,57 1,46 1,54 1,51 –^e 0,79 1,26 1,46 1,58 1,72 1,64

Fm75P 0,41 1,19 1,47 1,76 – 0,71 1,16 1,25 1,67 1,70 1,75

Fm76P 0,65 1,27 1,29 1,52 – 0,96 1,37 1,51 1,66 1,62 1,49

Fm77P 0,38 1,32 1,56 1,97 – 0,83 1,29 1,64 1,69 1,72 1,79

Fm71R 0,53 1,42 1,70 2,09 – 0,71 1,19 1,59 1,67 1,78 1,99

Fm72R 0,55 1,23 1,49 1,97 – 0,72 1,24 1,60 1,63 1,70 1,75

Af80P 0,93 0,92 1,24 1,52 0,71 0,80 0,91 1,04 1,26 1,31 1,26 Af84R 1,06 1,24 1,30 1,38 0,89 0,83 1,22 1,23 1,32 1,45 1,32 Af92T 0,70 0,97 1,25 1,54 1,10 0,75 1,12 1,17 1,26 1,34 0,96 An82P 0,30 0,60 0,68 0,78 0,98 1,00 1,04 0,78 0,71 0,70 0,70

An90R 0,45 070 0,75 0,90 1,01 0,98 1,12 0,94 0,80 0,75 0,72

An88T 0,33 0,58 0,79 0,94 0,96 0,70 0,84 0,81 0,80 0,75 0,76

Ar89T 0,13 0,26 0,51 0,46 – 0,27 0,37 0,39 0,61 0,58 0,46

Pc87T 0,32 0,46 0,58 0,61 – 0,40 0,51 0,57 0,93 0,61 0,59

Tr98T 0,77 1,40 1,61 1,37 – – 0,54 1,57 1,64 1,54 1,77

aµmax (cm per day) calculated according to the Gompertz equation modified by Zwietering et al.

(1990); ^b incubation temperature was 25°C; ^c pH was fixed at 5.6; ^d Fm = Fusarium moniliforme; Af

= Aspergillus flavus; An = Aspergillus niger; Ar = Aspergillus restrictus; Pc = Penicillium commune;

Tr = Trichothecium roseum. Strains are named by numbers and letters which refer to the type of cheeses from they were isolated. (R, raw cheese; P, pasteurized cheese; and T, traditional cheese);

e No growth

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As regard pH, the growth of most strains investigated was little affected by pH ranging from 3 to 8: in fact, at pH 3 T. roseum 98T was the only fungus unable to grow, all the others were only slowed down. These findings agree with the results reported by WHEELER et al. (1991); however, when superimposed on other growth limiting factors, the effect of pH on mould growth may become evident (HORNER and ANAGNOSTOPOULOS, 1973).

A graphic representation of the effect of salt content on the growth is reported in Fig. 2 and 3 that show the influence of NaCl concentration on µmax of F. moniliforme and Aspergillus spp., respectively. In general, the growth rate of F. moniliforme decreased with NaCl concentration increasing; on the contrary as far as A. flavus concerns, low concentrations were stimulating while high concentrations were inhibitory. Instead NaCl concentrations up to 10% showed a positive influence on A. niger: in fact, the highest µmax was observed when 2.5% NaCl was present, but the growth rates in the presence of 5, 7.5 and 10%

salt were higher than those of the control (Fig. 2b).

A. restrictus 89T, P. commune 87T and T. roseum 98T were differently influenced by salt content: A. restrictus 89T grew slowly under all conditions, but its growth was substantially unaffected by NaCl concentration. P. commune 87T showed the maximum development at 2.5%, while T. roseum 98T was strongly slowed down by NaCl concentrations ranging between 5 and 10%

(data not shown).

Figure 2

Effect of NaCl concentration on µmax at 25°C of Fusarium moniliforme strain.

2,4

2,0

1,6

1,2

0,8

0,0 0,4

0,0 2,5 5,0 7,5 10,0

%NaCl

µmax

Fm68T Fm71R Fm72R Fm75P Fm76P Fm77P

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Figure 3

Effect of NaCl concentration on µmax at 25°C of Aspergillus spp. strain.

3.4 Growth on semisynthetic cheese medium

Table 4 shows the colony growth rates of the strains investigated on semi- synthetic cheese medium and MEA at the same temperature and pH level. Only F. moniliforme strains grew on MEA faster than on semi-synthetic medium while the growth rates of all the other strains were on model system higher than on MEA. This could be due to the ability of F. moniliforme species to colonize ecological niche unlike cheese.

2,4

2,0

1,6

1,2

0,8

0,0 0,4

0,0 2,5 5,0 7,5 10,0

%NaCl

Af80P Af84R Af92T An82P An88T An90R

µmax

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Table 4

Comparison between maximal growth rates measured on “cheese medium” and MEA

4 – DISCUSSION AND CONCLUSIONS

These findings seem to indicate that some process operations can exert selection pressure on the mycota. In fact, semi-hard Canestrato Pugliese cheese produced according to a traditional protocol harboured a more heterogeneous mycoflora. However, in general, the species most frequently isolated from our samples were infrequently associated with cheese; in fact, several investigators have reported that the dominant fungi isolated from cheese are Penicillium spp. (NORTHOLT et al., 1980; ARAN and EKE, 1987; TSAI et al., 1988;

LUND et al., 1995). The factors enabling fungi to cause spoilage in cheese are the ability to grow at low temperature, growth in low oxygen concentration, lipolytic activity and growth at reduced water activity. P. roqueforti and

Cheese medium^a MEA

Strains µmax^b R² µmax R²

Fm68T^c 1,34 0,995 1,74 0,996

Fm75P 1,05 0,981 1,48 0,997

Fm76P 1,15 0,986 1,45 0,998

Fm77P 1,18 0,994 1,55 0,997

Fm71R 1,29 0,995 1,66 0,995

Fm72R 1,12 0,992 1,36 0,995

Af80P 2,33 0,994 1,33 0,995

Af84R 1,93 0,990 1,38 0,996

Af92T 1,81 0,998 1,07 0,996

An82P 2,10 0,990 1,96 0,995

An90R 2,23 0,989 1,98 0,994

An88T 2,25 0,992 2,09 0,997

Ar89T 1,01 0,992 0,43 0,983

Pc87T 1,20 0,993 0,66 0,993

Tr98T 1,50 0,998 1,36 0,997

a This medium was developed by Hansen and Nielsen (1997)

b Gompertz equation parameters: µmax=maximal growth rate; R², regression coefficient

c Fm = Fusarium moniliforme; Af = Aspergillus flavus; An = Aspergillus niger;

Ar = Aspergillus restrictus; Pc = Penicillium commune; Tr = Trichothecium roseum.

Strains are named by numbers and letters which refer to the type of cheeses from they were isolated.

(R, raw cheese; P, pasteurized cheese and T, traditional cheese).

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P. commune meet all these criteria and are thus the most successfull spoilage moulds on cheese.

On the contrary, the species of the genus Fusarium appear to be of less importance and are part of cheese mycoflora infrequently.

The high frequency of isolation of F. moniliforme from our cheeses could indicate a too high relative humidity of the ripening room air as well as a strong contamination of wooden shelves in ripening rooms. Besides, the cheese fac- tory was situated in the open country and this could explain the high frequency of Fusarium species widely distributed in soils, particularly cultivated soils, and active in decomposition of cellulosic plant materials (PITT and HOCKING, 1997).

Moreover the ripening rooms had been used for cheese ripening only for a short while.

Beyond F. moniliforme, A. niger and A. flavus were the most frequently occurring species in the cheeses investigated. A. niger prevalent in warmer cli- mates, has been isolated from a large range of foods including different types of cheeses (PITT and HOCKING, 1997). This species is usually regarded as benign fungus; in fact, it is categorized as “generally regard as safe” by U.S.

Government. However, recently, ABARCA et al. (1994) have isolated two strains producing ochratoxin A. Also A. flavus, frequently occurred in cheeses analyzed after 24 days of ripening. This species is considered the main source of aflatoxins. Cheese often contain A. flavus and sometimes aflatoxins in countries where refrigeration is not always available (HASSANIN, 1993; EL SAWI et al., 1994); however, the presence of A. flavus does not necessarily mean the presence of aflatoxin.

Other species isolated were Mucor circinelloides which has been reported to spoil cheese (NORTHOLT et al., 1980) and to grow in media containing 15% (w/v) NaCl (=0.90 a_w) and A. restrictus that has been isolated from dried foods quite frequently (PITT and HOCKING, 1997).

It is interesting to denote that in the 64-days ripened cheeses Fusarium spp.

was not found; while A. niger and, at a minor degree, A. flavus became the dominant fungi. This fact could be linked to the stimulating effect that NaCl exerted on growth rate of these species. Moreover, the decreasing water activity of the cheese surface effects the dominant fungal flora and the impact of changing environmental factors can be determinant on inter-specific interaction between fungi. MARIN et al. (1998) in a study on maize fungi reported that A.

niger was the most competitive species with high growth rates at intermediate a_w levels. On the other hand, Fusarium species were dominant at a_w>0.95 and the dominance was due to their ability to grow rapidly and invasively.

At the end of ripening, Canestrato Pugliese cheese, indifferently from the protocol used for its production, showed severe visible mould growth that fac- tory staff reduced by brushing the cheese surface, However, massive growth could be a serious problem both for consumer and for the workers handling the cheese in dairy due to allergic reactins (PALMAS et al., 1989).

Our results provided some information on mycoflora associated to Canes- trato Pugliese cheese. In particular, Canestrato Pugliese cheese produced according to a traditional protocol showed a more heterogeneous mycoflora probably thanks to the a_w values higher than those observed for P and R cheeses.

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F. moniliforme, A. niger and A. flavus were the most frequently occurring species in cheeses analyzed.

In general, the highest growth rate of the strain investigated were observed at 30˚C, and the effect of NaCl on the growth differed in relation to the mould species.

Besides, the unsuitability of the ripening rooms could also explain the high mould infection and why the most frequently occurring fungi are recognized colonizing ecological niches different from cheese and, presumably, have not the enzymes necessary for good growth on cheese. Moreover, since during mould growth a risk of mycotoxin production in the cheese exists (SCOTT, 1989), the isolation of mycotoxigenic moulds could represent a health concern.

In conclusion, these results emphasize the need to control relative humidity and hygienic conditions of the ripening houses and can provide an improved understanding of fungi of Canestrato Pugliese cheese. However further investi- gation on fungi isolated from cheeses made in other factories is necessary to compare the occurrence and the physiologic characteristics of the species isolated from this kind of cheese.

REFERENCES

ABARCA M. L., BRAGULAT M. R., CAS- TELLA G., CABANES F. J., 1994. Ochra- toxin A production by strains of Aspergillus niger var. niger. Appl. Envi- ron. Microbiol., 60, 2650-2652.

ARAN N., EKE D., 1987. Mould mycoflora of Kasar cheese at the stage of consump- tion. Food Microbiol., 4, 101-104.

COLE G.T., KENDRICK W. B., 1981. Biology of Conidial fungi. Academic Press, New York.

EL-SAWI N. M., EL-MAGRABY O. M. O., MOHRAN H. S., ABO-GHARBIA M. A., 1994. Abnormal contamination of cottage cheese in Egypt. J. Appl. Anim. Res., 6, 81-90.

GERLACH W., NIREMBERG H., 1982. The Genus Fusarium - a Pictorial Atlas. Kom- missionverlag Paul Parey, Berlin.

GRAPPIN R., BEUVIER E., 1997. Possible implications of milk pasteurization on the manufacture and sensory quality of ripened cheese. Int. Dairy J., 7, 751-761.

HANSEN B. V., NIELSEN P. V., 1997. Devel- opment of a semisynthetic cheese medium for fungi using chemometric methods. J. Dairy Sci., 80, 1237-1245.

HASSANIN N. I., 1993. Detection of mycotoxigenic fungi and bacteria in processed cheese in Egypt. Int. Biodeterior. Biode- grad., 31, 15-23.

HORNER K., J., ANAGNOSTOPOULOS G.

D., 1973. Combined effects of water activity, pH and temperature on the growth and spoilage potential of fungi. J. Appl.

Bacteriol., 36, 427-436.

INTERNATIONAL DAIRY FEDERATION, 1988. Determination of salt content.

Standard 12B, International Dairy Fede- ration, Brussels.

INTERNATIONAL DAIRY FEDERATION, 1989. Determination of pH. Standard 115A, International Dairy Federation, Brussels.

KIVANC M., 1992. Fungal contamination of Kashar cheese in Turkey. Die Nahrung, 6, 578-583.

KOZAKIEWICZ Z., 1989. Aspergillus species on stored products. Mycol. Papers, 161, 1-188.

LUND F., FILTENBORG O., FRISVAD J. C., 1995. Associated mycoflora of cheese.

Food Microbiol., 12, 173-180.

(14)

MACEDO A. C., MALCATA F. X., HOGG T.

A., 1995. Microbiological profile in Serra ewe’s cheese during ripening. J. Appl.

Bacteriol., 79, 1-11.

MARIN S., COMPANYS E., SANCHIS V., RAMOS A. J., MAGAN N., 1998. Effect of water activity and temperature on compet- ing abilities of common maize fungi. Mycol.

Res., 120, 959-964.

NORTHOLT M. D., VAN EGMOND H. P., SOENTORO P., DEIJLL E., 1980. Fungal growth and the presence of sterigmato- cystin in hard cheese. J. Assoc. Off. Anal.

Chem., 63, 115-119.

PALMAS F., COSENTINO S., CARDIA P., 1989. Fungal air-borne spores as health risk factors among workers in alimentary industries. Eur. J. Epidemiol., 2, 239-243.

PITT J. I., 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press, Lon- don.

PITT J. I., HOCKING A. D., 1985. Fungi and food spoilage. Academic Press, Sidney.

PITT J. I., HOCKING A. D., 1997. Fungi and food spoilage. Blackie Academic & Pro- fessional, London.

RIFAI M. A., 1969. A revision of the genus Trichoderma. Mycol. Papers, 116, 1-56.

SCOTT P. M., 1989. Mycotoxigenic fungal contamination of cheese and other dairy products. In: Mycotoxins in dairy products. (Ed: H. P. Van Egmond) Elsevier Science Publishers Ltd., Barking Essex, UK, 193-259.

TSAI W. J., LIEWEN M. B., BULLERMAN L. B., 1988. Toxicity and sorbate sensitivity of molds isolated from surplus commodity cheeses. J. Food Prot., 51, 457-462.

WHEELER K. A., HURDMAN B. F., PITT J. I., 1991. Influence of pH on the growth of some toxigenic species of Aspergillus, Penicillium and Fusarium. Int. J. Food Microbiol., 12, 141-150.

ZWIETERING M. H., JONGENBERGEN I., ROMBOUTS F. M., VAN’T RIET K., 1990.

Modelling of the bacterial growth curve.

Appl. Environ. Microbiol., 56, 1875-1881.