Serpa Cheese: Technological, biochemical and microbiological

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Serpa Cheese: Technological, biochemical andmicrobiological characterisation of a PDO ewe’s milk

cheese coagulated with Cynara cardunculus L.Luisa Roseiro, R. Andrew Wilbey, Manuela Barbosa

To cite this version:Luisa Roseiro, R. Andrew Wilbey, Manuela Barbosa. Serpa Cheese: Technological, biochemical andmicrobiological characterisation of a PDO ewe’s milk cheese coagulated with Cynara cardunculus L..Le Lait, INRA Editions, 2003, 83 (6), pp.469-481. <10.1051/lait:2003026>. <hal-00895510>

https://hal.archives-ouvertes.fr/hal-00895510

https://hal.archives-ouvertes.fr

469Lait 83 (2003) 469–481© INRA, EDP Sciences, 2003DOI: 10.1051/lait:2003026

Original article

Serpa cheese: Technological, biochemical and microbiological characterisation of a PDO ewe’s milk cheese coagulated with Cynara cardunculus L.

Luisa B. ROSEIROa,b, R. Andrew WILBEYb, Manuela BARBOSAa*

a INETI – Instituto Nacional de Engenharia e Tecnologia Industrial DTIA, Estrada do Paço do Lumiar, No. 22, 1649-038 Lisboa, Portugal

b School of Food Biosciences, The University of Reading, Reading, RG6 6AP, UK

(Received 25 February 2003; accepted 27 May 2003)

Abstract – Portugal has a strong tradition of cheesemaking from raw ewe’s milk; most of thesecheeses are still made on a traditional farmhouse scale. Their production is protected by ProtectedDesignation of Origin (PDO) but the specific biochemical aspects of the majority still need to becharacterised. Two different cheesemaking procedures, traditional and semi-industrial, werecompared technologically, biochemically and microbiologically. It was observed that, despite thehighly significant difference between artisanal and semi-industrial cheeses (P < 0.001), bothproducts were within the limits of national regulations for most parameters except maturationtemperature, humidity and the value for the maturation index. Although the present study was notfully representative of the region, the results obtained suggest that the specific regulations for Serpacheese should be revised and that other parameters, such as moisture and salt-in-moisture content,which are very much dependent on the cheesemaking process, should be included in order tocharacterise better this traditional cheese.

Serpa cheese characterisation / PDO cheese / ewe’s milk cheese / Cynara cardunculus L. /vegetable coagulant

Résumé – Le fromage Serpa : caractérisation technologique, biochimique et microbiologiqued’un fromage AOP au lait de brebis, coagulé par Cynara cardunculus L. Le Portugal a une fortetradition dans la fabrication de fromage au lait cru de brebis, continuant à être fabriqués pour lamajorité à l’échelle des fermes traditionnelles. La production de quelques-uns de ces fromages estprotégée par l’AOP (Appellation d’Origine Protégée) mais leurs caractéristiques spécifiques restentinconnues. Deux procédés de fabrication (artisanal et semi-industriel) ont été réalisés et les résultatsobtenus ont été comparés. On a pu observer que, malgré la différence significative des deux procé-dés de fabrication (P < 0.001), tous les deux avaient des valeurs comprises dans les limites imposéespar la réglementation. Toutefois, certains paramètres, tels que la température et les conditionsd’humidité pendant la maturation, ainsi que les valeurs proposées pour l’index de maturation,étaient différentes. Cela nous conduit donc à suggérer que certains paramètres pourraient être révi-sés et d’autres tels que l’humidité et le sel dans l’humidité, qui dépendent beaucoup du procédé defabrication, soient dorénavant inclus afin de permettre une meilleure caractérisation de ce fromagetraditionnel.

Caractérisation du fromage Serpa / fromage AOP / fromage au lait de brebis / Cynaracardunculus L. / coagulant végétal

* Corresponding author: [email protected]

470 L.B. Roseiro et al.

1. INTRODUCTION

Portugal has a strong tradition of regionalcheesemaking, the traditional cheese varie-ties being made from raw ewe’s and goat’smilk or from the mixture of both types ofmilk in various proportions. Many of thesecheeses are still made on a farmhouse scaleand are traded directly within their produc-tion area or to cheese dealers. Despite thedominance of “industrial” pasteurised cow’smilk cheese in the market, the farmhouseewe’s and goat’s milk cheeses are the mostrepresentative of the country and are instrong demand by the consumer. The pro-duction of some of these cheeses is nowprotected either by a Protected Designationof Origin (PDO) or by the Protected Geo-graphic Indication (PGI) [5, 9].

Serpa cheese is an example of an arti-sanal regional Portuguese cheeses with thePDO designation. Its region of productionis a demarcated area in Alentejo, the big-gest and driest province of inland Portugal,situated towards the south of the country,between the Tagus and Guadiana rivers.Cheese production plays an important partin the local agricultural economy. Like theother Portuguese PDO cheeses, Serpa cheeseis named after a village, perhaps one of themost beautiful in Portugal, on the left sideof the river Guadiana.

The origin of Serpa cheese is lost intime. It is believed that it was derived fromthe equally famous Serra da Estrela cheese,since it has a similar method of productionbased on the same ingredients, i.e., rawewe’s milk and an aqueous extract of driedcardoon flowers (Cynara cardunculus L.)as coagulant [24, 27]. Furthermore, it hasbeen suggested that this cheese was intro-duced to the Alentejo by shepherds fromSerra da Estrela (a mountainous region inthe north of Portugal) during the tran-shumance periods. The milk for Serra daEstrela cheese is produced by flocks of 40–60 “Bordaleira” ewes under an intensivepasturage exploitation system, the cheesebeing made almost immediately after milk-ing. However, Serpa cheese milk was tradi-

tionally collected from Portuguese “Merino”sheep and flocks could attain more than600 ewes under an extensive exploitationsystem [30]. Also, the extent of milkingtimes and the distance between the milkingand cheesemaking areas would contributeto a different microflora in the milk. This,together with other factors, resulted inorganoleptic characteristics different fromthose of Serra da Estrela cheese.

Serpa cheese is considered to be one ofthe best Portuguese cheeses and is mainlyartisanally produced, although nowadayssome is also produced by a semi-industrialprocedure. The main characteristics thatdistinguish Serpa cheese along with someother PDO Portuguese and Spanish ewe’smilk cheeses, and which differentiate themfrom ewes’s milk cheeses from other coun-tries, is their semi-soft texture [25]. Thistexture is due not only to the type of milkand technology used, but also to the vege-table coagulant, which is highly proteo-lytic. Serpa cheese is appreciated, not onlyin its production region, but also all overPortugal. This is also very important forthe Baixo Alentejo region from the eco-nomical point of view, though only inrecent years has attention been drawn to itsdevelopment and protection by the nationalauthorities, after Portugal’s entrance intothe EC.

Despite Serpa cheese being one of themost sought-after PDO cheeses in Portugal,little has been done to characterise its pro-duction other than a study of the maturationof Serra da Estrela and Serpa cheeses in 1970[31]. The aim of the present work was tocharacterise technologically, biochemicallyand microbiologically two cheesemakingprocedures (artisanal and semi-industrial)and to examine how these products relate tothe regulations for Serpa cheese.

2. MATERIALS AND METHODS

2.1. Cheese dairies

After a thorough search within thedemarcated region of Serpa cheese, we chosetwo different dairies in Baixo Alentejo,

Characterisation of the PDO Serpa cheese 471

which were the most representative of thetraditional and the semi-industrial produc-tion, to perform the experimental work.One was a traditional family business,dairy B, where the cheesemaker had beenmaking Serpa cheese for over 50 years andin which about 100 L of ewe’s milk fromthe Merino breed were transformed intocheese each day, using simple manualtechnology. The other, dairy C, was a rela-tively new dairy that produced Serpacheese using the traditional approach withsome improvements of the technology on asemi-industrial scale, employing six womenas cheesemakers to transform ca. 1000 L ofLacaune ewe’s milk per day.

This investigation consisted of the man-ufacture of nine cheeses in each dairy onthe same day, according to their own meth-odology and maturation conditions. Themanufacture parameters were recorded.This procedure was repeated three times foreach dairy between the months of Januaryand March, corresponding to the peak ofproduction.

2.1.1. Artisanal manufacture

2.1.1.1. Preparation of the coagulant

In the artisanal dairy, the amount ofdried flowers was calculated empirically

by the cheesemaker, e.g. for 50 L milk, ahandful of flowers was used (ca. 30 g).These were then soaked in ca. 0.5 L of coldwater, squeezed, and the wet flowers weremacerated in a mortar for a while. Themacerated flowers were added again to thesame water, mixed well and then squeezedfrom the water that was now of a purple-brownish colour. This aqueous extract wasthen filtered through a cloth and added tothe milk.

2.1.1.2. Cheesemaking process

The milk was collected twice a day bythe cheesemaker who purchased it fromtwo local breeders within 10 km of the vil-lage. The milk was immediately trans-ported to the dairy and transformed intocheese. The first step was to warm up themilk to 35 °C in a water bath, then it wasfiltered through folded white wool blan-kets into a clean churn, to remove most ofthe dirt. Salt (ca. 900 g/50 L milk) wasadded to the milk followed by the coagu-lant prepared just before use, as describedabove. The milk was left to coagulate for1 h while the temperature dropped to ca.31 °C. Both the amounts of salt and car-doon flower were empirically calculated atthe time by the experienced cheesemaker,according to the quantity of milk and theseason of the year.

Figure 1. Production of Serpa cheese at dairy B (traditional), showing partially drained curd (left)and partially-matured cheeses (right).


The coagulum was stirred vigorously inthe churn with the help of a wooden stick,until it broke down completely into a smoothgrainy mass. This was transferred in smallportions into a large mould on the workingtable. The mould consisted of a perforatedflexible wooden strip, that could be formedand tied into a ring, folded and closed at theextremes, so that it could be made smallerwhile the curd was getting dryer by thedraining of the whey. The whey was col-lected in a vessel at the narrowed end of theworking table and could be used later forthe production of “Requeijão”, a traditionalwhey cheese that is highly appreciated byitself or used in the local pastry industry,thus also being of great economic benefitfor the region.

The curd was worked in this big moulduntil it became sufficiently dry to be distrib-uted into small round open-ended stainless-steel moulds (Fig. 1, left). The working andhand pressing was continued in thesemoulds for a while and then the curd wasleft to rest. After ca. 1 h, the moulds wereturned over and left for another hour. Themoulds were turned over again and placedin the maturation room, with a mean tem-perature of 13 °C and mean relativehumidity (RH) of 90%.

Cheeses were demoulded the next dayand turned every day until they were 8–10 dold. At this stage, cheeses were washed withwarm diluted whey to control the growthof the surface microflora that naturallyforms during this stage of ripening and ischaracteristic of these cheeses. A whitecotton bandage was put around the side toavoid breakage of the fine rind that hadstarted to form on the soft flattening paste.The bound cheeses were then transferred toa second maturation room, which was lesshumid and warmer, for the second stage ofmaturation (Fig. 1, right).

The maturation was carried out in anambient environment, and consequently, atvariable values for temperature and humid-ity depending on the weather conditions(temperatures ranging from 9–17 °C and

RH ranging from 67–100% during the min-imum 30 d maturation period). The cheese-maker used his experience in controllingthese temperature/RH parameters, by closingor opening screened windows at the dairy.During the experimental period, the rangesof temperature and relative humidity wererecorded at the two stages of maturation bya Hygrolog® apparatus that was set to reg-ister temperature (ºC) and RH (%) at hourlyintervals. The data were stored and unloadedinto proprietary software for further analy-sis. These cheeses have a seasonal produc-tion period that usually goes from lateOctober until the end of May.

2.1.2. Semi-industrial manufacture

2.1.2.1. Preparation of the coagulant

The semi-industrial dairy used a similarprocedure to prepare the coagulant. Approx-imately 30–80 g of flowers were used per100 L of milk, depending on a visual assess-ment of their quality. The weighed flowerswere first washed with tap water to takeaway the gross impurities, then squeezedto remove surplus water. The squeezed wetflowers were placed in an electric blenderand comminuted into a brown paste. Thispaste was added to 250 mL cold water andwell mixed. The flowers were squeezedagain and the brownish aqueous extractwas filtered through a cotton cloth andadded to the milk. This extract was moreconcentrated than that obtained manually.

2.1.2.2. Cheesemaking process

The milk was obtained by machine-milking from a herd of more than 1800 ewes,and was kept refrigerated in tanks at 4 °C,usually until the next day. From there itwas transferred to reception tanks thenpassed through a heat exchanger where themilk was warmed to 30 °C and directed tothe 1000 L mechanised water-jacketed vat.At discharge to the vat, the milk was fil-tered through four white wool blankets thatretained the gross impurities. This stagenormally took 1.5–2 h while the bulk of the


milk was maintained at 30 °C under gentlestirring. The aqueous extract of the car-doon flowers, prepared while the milk wasbeing filtered, was then added. The coagu-lation took ca. 1.5–1.75 h. The coagulumwas cut with vertical and horizontal knivesthat circulated around the vat until thesmall curd grains were ca. 5 mm in size.The curd was then left to settle for 10 minand the whey was drained and pumped intoa different vat, where it was boiled to makethe whey cheese “Requeijão”.

After whey drainage, the curd mass waspressed in the vat. All these steps weremechanised but with the assistance of twocheesemakers. The curd was then manu-ally cut into blocks and salted (ca. 5.7 kgsalt per 1000 L of milk). Two blades werethen used to cut the curd and simultane-ously mix the salt thoroughly.

Portions of curd were then taken to aworking table by the six cheesemakers,who pressed it in white cotton cloths todrain most of the remaining whey. The curd,still inside the cloths, was put into whiteplastic perforated moulds with a plasticfollower. The filled moulds were placed ina hydraulic press at an air feed pressure of3 kg·cm–2 (0.3 MPa) for ca. 3.5–4 h, depend-ing on the amount of whey in the cheeses.Approximately 15 cheeses were put intoeach press. During this period, the cheeseswere turned over once and the cloths werewithdrawn.

The cheeses were demoulded after pressingand transferred to a maturation chamber,with temperature and humidity controlled atmean values of 10 °C and 88% RH, respec-tively, for 17 d. After that period, the cheeseswere transferred to a second chamber, wherethe temperature was higher and the humiditylower (mean values of 15 °C and 82% RH,respectively), and stayed there for 23–28 d.A Hygrolog® apparatus, (Rotronic Instru-ment Corp., Huntington, New York, USA)was also installed in the two maturationchambers to register the range of tempera-ture and relative humidity during theexperimental period. Between these two

stages of maturation, the cheeses were washedwith water and a white cotton band wasplaced around the side to avoid deforma-tion of the rind. During the maturationperiod, cheeses were turned every twodays. Cheeses were washed again beforegoing onto the market.

This dairy produces an average of 250cheeses per day, and although the high sea-son of production is from January to May,it normally produces Serpa cheese all theyear round.

2.2. Sample collection and analysis

2.2.1. Sample collection

Milk, whey and fresh curd were collectedat the two dairies on the day of manufacture.During the maturation period, three cheeseswere collected after 30, 45 and 60 d, respec-tively, from dairies B and C, and wereweighed and measured before preparationfor analysis. This procedure was repeatedfor each cheesemaking trial.

2.2.2. Chemical analysis

Chemical analyses were performed inall milk, whey, fresh curd and cheese sam-ples collected from the two dairies, in orderto characterise these products. The methodsof analysis used were mainly according toPortuguese standards (NP) or internal methodsbased on Portuguese and/or IDF standards.Duplicate analyses for each determinationwere performed.

pH was determined for all samples bydirect measurement using a Metrohm 713 pHMeter (Metrohm Ltd., Herisau, Switzerland).Titrable acidity [11], fat [10], total nitrogen[16], dry matter [12], salt [2] and ash [13]content were also determined for all samples.For nitrogen determination in cheese, a sam-ple was prepared according to the methoddescribed in [26]. Ash content in cheesewas determined by adapting the standardmilk method for the cheese sample prepa-ration.


Milk and cheese samples were charac-terised for their main mineral content (Ca,K, Mg and Na) according to a draft IDFstandard [17].

2.2.3. Microbiological analysis

A survey was done of the microbiologi-cal quality of the milk and the cheese pro-duced by each dairy. Total viable counts(TVC) were done at 30 °C [14] and lacticacid bacteria (LAB) were enumerated byplate counts [21]. Yeasts and moulds werealso enumerated [19]. Presumptive colif-orms were enumerated by incubation at30 °C [20] with confirmation of Escheris-chia coli by incubation at 44 °C [1]. Coag-ulase positive Staphylococcus aureus wereenumerated [18] while Listeria monocy-togenes was determined by an enzymelinked fluorescent assay [29].

2.3. Statistical analysis and data treatment

Descriptive statistics of the results(namely, average and standard deviation)and analysis of variance (ANOVA) wereperformed using StatView® 5.0.1 from theSAS Institute Inc. The effect of milk fromthe dairies B and C was evaluated for themicrobiological quality and chemical compo-sition of the milk and also for the cheese-making process throughout the maturationtime of the respective cheeses, using thefactorial model analysis of variance based ona completely randomised block experimen-tal design. The interaction effect “dairy ×maturation of cheeses” for each determina-tion was also determined by evaluating thedifferences between means at the 95%level calculated from the residual meansquare using Fisher’s test.

3. RESULTS

B and C cheeses had a flat cylindricalshape with a soft texture, presenting thedimensions for weight, diameter and height

at 60-day maturation time of, respectively,1502 ± 81 g, 19 ± 0.7 cm and 5 ± 0.4 cmfor B cheese and 1261 ± 41 g, 16 ± 0.4 cmand 6 ± 0.8 cm for C cheese.

3.1. Proximate analysis

3.1.1. Milk results

Table I shows the mean results for thecomposition for 23 samples of raw ovinemilk collected in the artisanal dairy (B), and17 samples collected in the semi-industrialdairy (C) during the experimental work. Inmost of the analytical parameters (i.e.except Ca, K and Na) the milks from dairy Bwere significantly different from those ofdairy C, the most significant differencesbeing for acidity (P < 0.001), protein(P < 0.001) and ash (P < 0.001), all ofthem higher for milk B.

The average values (cfu·mL–1) for themicrobiological quality of 13 samples of rawovine milk collected from dairy B and 11samples collected from dairy C during theexperimental work revealed, respectively,for dairies B and C, the values of 8.6 × 106

Table I. Composition of raw ovine milk fromB and C dairies1.

B CpH (direct) 6.75a ± 0.05 6.77b ± 0.10Acidity(cm3 NaOH N·dm–3)

22.1a ± 1.7 19.9b ± 2.2

Composition (g·kg–1)Fat 75a ± 16 68b ± 8Dry Matter 191a ± 21 176b ± 18Protein 61.7a ± 6 56.9b ± 4Ash 9.9a ± 1.0 9.2b ± 0.4Ca 1.83 ± 0.26 1.81 ± 0.16K 0.99 ± 0.19 0.94 ± 0.14Mg 0.19a ± 0.20 0.17b ± 0.30Na 0.60 ± 0.18 0.57 ± 0.181 Means from 23 samples of milk B and 17 sam-ples of milk C in duplicate. For each determina-tion, means in the same row followed by differentletters differ significantly (P < 0.05).


Tabl

e II

. Mea

n an

d st

anda

rd d

evia

tion

valu

es f

or th

e ch

eese

com

posi

tion

from

B a

nd C

dai

ries

dur

ing

mat

urat

ion1

.

0 d

30 d

45 d

65 d

BC

BC

BC

BC

pH (

dire

ct)

6.64

a ±

0.1

36.

46b

± 0

.08

5.59

d ±

0.0

65.

23 e

± 0

.16

5.63

d ±

0.1

85.

26 e

± 0

.19

5.74

c ±

0.1

75.

28 e

± 0

.21

Aci

dity

(g

·kg–

1 la

ctic

aci

d)1.

1d ±

0.1

1.1d

± 0

.47.

5 c

± 0

.69.

8 b

± 2

.18.

1 c

± 1

.110

.2 b

± 2

.77.

5 c

± 1

.611

.0 a

± 3

.4

Com

posi

tion

(g·

kg–1

)

Fat (

DM

) 48

1d ±

48

538b

± 3

250

8 c

± 2

557

6 a

± 2

450

7 c

± 2

457

0 a

± 1

752

1 bc

± 1

356

8 a

± 2

3

Moi

stur

e (F

FB

) 80

2a ±

17

725b

± 2

070

8 c

± 1

068

7 d

± 3

169

5 d

± 1

066

6 e

± 1

869

2 d

± 1

064

7 f ±

14

Pro

tein

(D

M)

353

b ±

20

357

b ±

25

408a

± 5

236

0b ±

17

406a

± 2

835

7b ±

25

402a

± 1

636

1b ±

13

Sal

t in

Moi

stur

e (S

/M)

(g·k

g–1

NaC

l)26

.5 f

± 2

.835

.3 e

± 3

.136

.1 e

± 3

.244

.3 c

± 6

.040

.3 d

± 3

.853

.5 b

± 6

.042

.9 c

d ±

4.4

60.5

a ±

7.8

Ash

(D

M)

97.5

a ±

7.0

86.1

b ±

2.6

86.3

b ±

2.5

80.9

d ±

2.5

86.0

b ±

3.4

85.8

b ±

2.9

83.6

c ±

3.8

87.5

b ±

3.3

1 M

eans

fro

m 1

8 sa

mpl

es o

f ch

eese

B a

nd 2

4 sa

mpl

es o

f ch

eese

C in

dup

lica

te.

For

each

det

erm

inat

ion,

mea

ns in

the

sam

e ro

w f

ollo

wed

by

diff

eren

t let

ters

dif

fer

sign

ific

antl

y (P

< 0

.05)

.

Tabl

e II

I. M

ean

and

stan

dard

dev

iatio

n va

lues

for

the

prin

cipa

l min

eral

s in

che

ese

from

B a

nd C

dai

ries

dur

ing

mat

urat

ion1

.

Min

eral

s(g

·kg–

1 D

M)

30 d

45 d

60 d

BC

BC

BC

Ca

13.1

1a ±

0.6

412

.48a

b ±

1.2

212

.10b

± 2

.09

12.7

1ab

± 1

.09

12.2

7b ±

0.5

512

.71a

b ±

0.6

0

K

2.14

± 0

.35

1.94

± 0

.34

1.71

± 0

.28

1.83

± 0

.33

1.92

± 0

.44

2.72

± 0

.36

Mg

1.26

a ±

0.1

40.

69d

± 0

.03

1.19

b ±

0.0

50.

73 c

d ±

0.1

21.

22 a

b ±

0.1

00.

75 c

± 0

.06

Na

13.3

2 d

± 2

.02

12.5

8 d

± 2

.33

13.7

1 cd

± 3

.72

15.1

1 ab

c ±

3.7

815

.62

ab ±

2.0

416

.62

a ±

2.4

4

1 M

eans

fro

m 1

8 sa

mpl

es o

f ch

eese

B a

nd 2

4 sa

mpl

es o

f ch

eese

C in

dup

lica

te.

For

each

det

erm

inat

ion,

mea

ns in

the

sam

e ro

w f

ollo

wed

by

diff

eren

t let

ters

dif

fer

sign

ific

antl

y (P

< 0

.05)

.


and 1.4 × 105 for the total viable count at30 °C (TVC); 2.6 × 103 and 6.7 × 104 forthe lactic acid bacteria count (LAB);6.5 × 102 and 1.1 × 102 for the coliformscount; 56.6 and 1.4 for E. coli count. Listeriamonocytogenes was not detected in a 25 mLsample for all samples.

There was no significant difference inhygienic quality between the milks, despitemilk C being obtained by machine-milkingand milk B by hand-milking in the field. Asignificant difference was found in the nat-urally-occurring lactic acid bacteria count(P < 0.001), being higher in milk C.

Presumptive coliform counts were lowbut variable, without significant differencebetween the milk sources. A small propor-tion of these counts were confirmed asE. coli.

3.1.2. Cheese results

The composition of the cheese through-out maturation and the mineral content areshown in Tables II and III, respectively.

The results for temperature and relativehumidity were recorded by the Hygrolog®

in each of the dairies throughout the matu-ration period. These observations resultedin mean values for ripening temperature of13.5 ± 1.7 °C and 12.5 ± 3.5 °C; and mean val-ues for relative humidity of 89.9 ± 6.9% RHand 85.2 ± 5.2% RH, respectively, for dairiesB and C.

One cheesemaking trial was chosen ran-domly at each of the dairies to evaluate themicrobiological quality throughout the pro-cedure of cheesemaking. The results areshown in Table IV.

Though the transformation of milk tocurd in dairy B was accompanied by agreater rise in TVC, it would appear that thiswas due to the growth of the endogenousLAB since no starter addition is used forthis type of cheese. From 30 d maturationonwards there was practically no differencein the microbial counts between cheesesfrom the two dairies, except for yeasts.Moulds were not detected in either of thedairies’ products at any stage. Coliformsand E. coli were practically absent in milkfrom both dairies, but present in curd andcheese throughout maturation. The presence

Table IV. Microbiological results of raw ovine milk (cfu·mL–1) and cheese (cfu·g–1) throughoutthe cheesemaking processes in B and C dairies1.

Determination Dairy Milk 0 d 30 d 45 d 60 d

Total viable countat 30 °C

BC

9.5 × 103

5.0 × 1033.0 × 109

5.0 × 1067.8 × 108

5.6 × 1082.1 × 109

6.3 × 1088.6 × 108

4.6 × 108

Lactic acid bacteria

BC

8.5 × 102

9.3 × 1034.8 × 108

8.6 × 1051.5 × 108

2.5 × 1084.0 × 108

4.1 × 1085.6 × 108

1.6 × 108

Coliforms BC

115

3.8 × 102

5.5 × 1056.8 × 105

1.5 × 1055.1 × 106

7.9 × 1041.1 × 106

6.3 × 104

E. coli BC

11

1.2 × 102

0.9 × 1023.1 × 104

1.4 × 1038.7 × 104

1.8 × 1031.6 × 105

1.2 × 103

Staph. aureus (coagulase positive)

BC

-< 10 4

< 10< 10 4

-< 10 4

-< 10 3

-< 10 3

Listeria monocytogenes(25 mL or 25 g)

BC

--

--

--

--

--

Yeasts BC

2.1 × 102

8.9 × 1034.4 × 105

1.1 × 1041.3 × 105

2.3 × 1037.2 × 102

1.1 × 1041.1 × 103

2.9 × 102

1 Mean results for duplicate determinations.


of S. aureus in dairy C was considerablyhigh, in contrast with dairy B where S.aureus were practically absent.

4. DISCUSSION

The specifications for Serpa cheese andthe corresponding results obtained for Band C cheeses are shown in Table V. Thebreed of sheep is not specified but mayhave an effect. It can be seen from thistable that values of cheeses B and C forparameters such as maturation tempera-ture, relative humidity and the index ofproteolysis previously determined [26] donot agree with the specifications describedin the Serpa cheese decree [6].

Despite the differences between cheesesB and C, both are within the dimensionlimits of normal Serpa cheese. These usedto be bigger, but the recent market tendencyis towards a cheese of ca. 1 kg, which cer-tainly affects the maturation and typicity ofthe cheese.

4.1. Milk results

The differences between milk B andmilk C were probably due to specificgenetic characteristics of the two breeds,

milking and feeding systems. The Merinobreed produces low quantities of milkwhich is richer in its composition [23] thanthat from other, more productive, breedssuch as Lacaune. Additionally, free-grazinganimals in the fields all day tend to producea richer milk, in terms of fat and protein,than animals that spend part of the dayindoors and eat concentrated feed. Skilledshepherds also know which herbs andplants give off-flavours to the milk andconsequently to cheese, and avoid leavingtheir animals in fields where these herbsgrow. It also appears that hand-milkingextracts more milk, especially towards theend of milking, while machine-milkingtends to leave a certain amount of residualmilk in the udder, which normally is moreconcentrated [3].

In a similar investigation of raw ovinemilk used in the manufacture of raw ovinemilk cheeses, Gomez et al. [15] foundcounts (cfu·mL–1) of 7.1 × 105 for TVC and2.0 × 104 for coliforms. These figures werevery similar for TVC but higher for coliformswhen compared with the ones for Serpamilk. Thus, these Portuguese raw ovinemilks can be regarded as being of a superiormicrobiological quality. The immediaterefrigeration of milk C after milking and

Table V. Specifications for Serpa cheese and corresponding results for B and C cheeses.

Decree No. 39/87 [6] B C

Milk Pure raw ovine milk Yes Yes

Coagulant Aqueous extract from C. cardunculus L. Yes Yes

Whey drainage Slow overnight in press (3.5–4 h)

Maturation time 30 d (minimum) Yes 45 d (minimum)

Maturation temperature (°C) 6–12 13.5 ± 1.7 12.5 ± 3.5

Maturation relative humidity (%) 85–90 89.9 ± 6.9 85.2 ± 5.2

Moisture (FFB) (%) 61–69 ‡ 70.8 ± 1.0 ‡ 66.6 ± 1.8

Fat (DM) (%) 45– < 60 ‡ 50.8 ± 2.5 ‡ 57.0 ± 1.7

Maturation index 45 (minimum)(method not specified)

‡* 34 (pH4.4-SN/TN) ‡* 29 (pH4.4–SN/TN)

‡ At time of sale from each dairy. * Roseiro et al. [26].


the minimal delay between milking andcheesemaking for milk B may be regardedas contributory factors.

4.2. Cheese results

The highly significant differences (P<0.001) observed for certain parameters (fat,dry matter and moisture in fat-free basis,salt-in-moisture and ash) between curds fromthe two dairies were naturally related todifferences in both the milks and cheese-making practices in the dairies. Wheydrainage was aided by manual manipula-tion of curd in dairy B, while in dairy Cboth the cutting of the curd and wheydrainage were mechanised. This resulted ina higher loss of fat to whey (data notshown), leaving a lower level of fat in drymatter (FDM) in curd from dairy B in com-parison with dairy C, despite the higher fatlevel in the milk. However, losses of proteinand/or fat into the whey are not considereda major cheese yield problem. All the wheyobtained as a by-product of the cheesemak-ing is traditionally used for the productionof “Requeijão”, a whey cheese made bysimply boiling the whey and scooping theheat-precipitated protein (together with fatand the fines from curd) into a cotton cloth,which is hung and left to drain.

Differences in pressing techniques alsoled to dry matter (DM) being considerablyhigher in curd C. Protein in dry matter,however, showed no significant differencebetween the two curds. Salt-in-moisture(S/M) and ash were also significantly higherin curd C (P < 0.001), this difference beingrelated to the amount of salt used and thesalting procedures in the dairies (dairy Badded salt to the milk before cheesemak-ing, while dairy C added salt directly to thecurd after whey drainage).

Overall, there was a highly significantdifference (P < 0.001) between cheesesfrom the two dairies. The metabolism ofresidual lactose in the curd reduced the pHof the cheeses to minima at 30 d, the slightupward drift thereafter being attributableto proteolysis and to lactate metabolism by

the yeasts. A similar pattern was observedfor titrable acidity, where differences inprotein also contributed to variation betweencheeses. Differences in storage conditionswill also have an effect; Carmona et al. [4]observed that similar ewe’s milk cheesesmatured in a controlled chamber exhibitedhigher lactic acid levels than those storedunder ambient conditions. Moisture, fatand FDM were also significantly different(P < 0.001) between B and C cheeses. S/Mvalues were significantly higher (P < 0.001)in C cheeses throughout maturation. Mostof the salt added to the milk in B was lostin whey, while at dairy C the salt was addedto the curd after whey drainage. The higherinitial moisture combined with lower S/Mfor cheeses from dairy B contributed to ahigher percentage of moisture loss duringmaturation, 23% compared with 19.5% forcheeses from dairy C.

B and C cheeses were also within thelimits for fat-in-dry matter set by the regu-lations for Serpa cheese [6], though someother parameters do not agree with thesespecifications. Some differences betweenthe cheeses may be attributable to the dif-ferent ewe’s milks used, coming from bothdifferent sources and different breeds.

The ash content in the cheese was influ-enced by the salt added and those mineralcomponents retained from the milk. Thecalcium, sodium and potassium levels indry matter showed no significant differ-ence between cheeses B and C throughoutmaturation, but magnesium in dry matterwas significantly higher in B cheeses (P <0.001). Sanjuán et al. [28] determined thecontent of minerals in Los Pedroches cheese,a Spanish Merino ewe’s milk cheese alsocoagulated with Cynara cardunculus L.They obtained results for Ca, K and Na indry matter for cheeses at 30, 43 and 60 dripening similar to those presented inTable III; the results for Mg in dry matterwere closer to those obtained for C cheeses.The higher results obtained for Mg inB cheeses might be due to genetic charac-teristics of the milk or to the composition


of the feed, since the Mg level was also sig-nificantly higher in the milk (Tab. I).

Dairy C showed a considerable presenceof S. aureus in milk and throughout cheesematuration in comparison with dairy B.This may be an indirect result of machine-milking, which is associated with a higherincidence of mastitis. The presence ofS. aureus in cheese is not desirable becausehigh levels of coagulase-positive entero-toxin–producing strains may lead to foodpoisoning. Listeria monocytogenes was notdetected in a 25 g sample for all samplesfrom both dairies.

Consideration must also be given tothe possible contamination of the cardoonextracts used as coagulants. Gomez et al.[15] obtained similar results for TVC andcoliforms in curd and cheese from rawovine milk using Cynara L. as coagulant at30 and 60 d maturation. However, whenthese results were compared with similarcheeses made with animal rennet, theyconcluded that considerable contaminationhad originated from highly contaminatedcardoon extracts.

A highly significant difference (P <0.001) was also observed for ripening tem-perature and relative humidity conditionsbetween the two dairies. These differenceswould contribute to the development of thedifferent microflora and to differences inmoisture losses between the two dairies.The specified values for temperature andrelative humidity were difficult to attain,especially for the B cheeses, which werematured in ambient conditions.

It should also be noted that the decreedoes not specify the method for measuringthe maturation index. Assuming that thematuration index was based on the percent-age of nitrogen soluble in water, then thisminimum value for the maturation indexwas not achieved by the percentage ofnitrogen soluble at pH 4.4 (pH4.4-SN/TN)at the maturation time of 30 d, as could beobserved from the results obtained for theevaluation of proteolysis for these cheesesamples previously published [26]. In the

experiments described here, the minimum“45” value established was achieved forB cheese at 60 d maturation time, while theC cheese only achieved a mean proteolysisindex of 31. This value is very muchdependent on the cheesemaking procedureand maturation conditions, which haveundergone variations for this type of cheesefor decades, namely with the introductionof maturation chambers. These considera-tions lead to the suggestion that Serpacheese should have a minimum maturationtime of 60 d, in order to achieve the idealstate of maturity. This would imply not onlygreater safety, bearing in mind that it is araw milk cheese, but also an improvementin the organoleptical characteristics. Accord-ing to those results, there should be a moredetailed study of the maturation index basedon the percentage of nitrogen soluble atpH 4.4 in order to establish a more accu-rate minimum value, consistent with goodcheesemaking practice and satisfactory sen-sory properties for the consumer.

Additionally, the results obtained fromthe two dairies studied also suggested thata revision of the specifications in the regu-lations for Serpa cheese should be consid-ered, since a certain degree of heterogeneityis also characteristic of Serpa cheese. Thisvariability has also been observed for othersimilar cheeses, e.g. La Serena cheese, pro-duced with Cynara cardunculus L. ascoagulant [7, 8]. The heterogeneity of thevegetable coagulant, differences in thecheesemaking procedures between dairies,and use of raw milk with high total countsand no added starter culture, lead to somevariations between cheeses. These are, likeother similar cheeses, traditional products,linked to a geographic origin and local know-how which cannot be reproduced exactlyin another place, thus contributing to thepreservation of a natural diversity of fla-vours and to the variety of cheeses.

This investigation has highlighted theneed for a more detailed study of the mat-uration of Serpa cheese. Such a studyshould be complemented by an evaluation


and definition of the sensory and rheologi-cal properties of the cheese. The study ofthe specific cheese flora during ripeningcan also be an important factor [22]. Suchwork would be consistent with develop-ment of the concept of PDO products,which is a significant factor in maintainingboth variety and quality in food productionwithin Europe.

It is also considered important that otherparameters, which have a relevance to meas-urement of proteolysis, should be includedin the cheese specifications, namely moistureand salt-in-moisture content.

Aknowledgements: The authors are indebtedto the cheesemakers at dairies B and C in theDemarcated Region of Serpa cheese, for theirinvaluable collaboration and support.

Special thanks are due to F. Ravasco,S. Pinto and C. Marrocos for their collaborationin sample preparation and chemical analysesand to H. Cordeiro (UMA), M.J. Borges (UBAI)and C. Neves (UQA) for, respectively, per-forming microbiological, mineral, salt and ashanalyses, and to their respective coordinators.

The authors are grateful to the “Fundaçãopara a Ciência e Tecnologia, Ministério daCiência e do Ensino Superior” (FCT/MCES),for their sponsorship of this project (POCTI35257/2000) and the Ph.D. fellowship forL.B. Roseiro. Part of the work was also spon-sored by a British Council grant.

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