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An overview of applied CoRFiLaC research
to support traditional cheeses.
S. Carpino, CoRFiLaC, Ragusa, Italy
1° WwTCa Conference
28 January, 2012Consorzio
Ricerca
Filiera
Lattiero-
Casearia
Mission
• Satisfying the actual needs of producers and consumers and exploring future needs.
• Offering opportunity of job and specialization to many technicians and researchers.
• Developing research of the historic Sicilian cheeses and their production, in order to evidence the importance of tradition, biodiversity and respect of nature in a civilized society.
Tools
• Extension Service
• Center for Elaboration of Data, “CED”
• PDO Certification
• Laboratories
• Cheese Pilot Plant and Aging Center
• Mediterranean Institute of Culinary Art of Sicily, “Micas”
• Cacioteca Regionale
• Marketing
uikoTools• Extension service
• It gathers producer’s needs and understands their future requirements.
• The Extension service involves in its experimental protocols over 300 dairy farms, producing more than 60% of Sicilian milk for productive and reproductive data analysis.
• The data, processed in real time in collaboration with CED, are communicated to farmers offering them scientific and technical support for management decisions.
Tools• CED
It receives and elaborates data from:
• productive and reproductive events of dairy cows,
• analytical data from the laboratories,
• and the results from the experimental protocols.
Tools• Laboratories
• Laboratory for cattle feed analysis
• Laboratories for Milk and Dairy Products
• Laboratory for Antioxidants Analysis
• Flavour Laboratory
• Sensory Analysis Laboratory
• Biochemistry of Protein Laboratory
• Microbiology Laboratory
Tools• Laboratories
• Laboratory for cattle feed analysis
created in order to provideinformation on Sicilian andsouthern feeds and foragethat grows in the Mediterranean
area.
Tools• Laboratories
• Laboratory for milk and dairy products
– monitores milk and cheese quality, animal metabolic diseases and reproduction efficiency through milk urea, ketons, progesterone tests, plus all those tests necessary to certify PDO cheeses.
Tools• P.D.O. Certification
• The scientific knowledge acquired in the 1990’s on typical Sicilian dairy productions allowed us to evaluate the key-factors of producing and aging of typical Sicilian cheeses, especially Ragusano and Pecorino Siciliano.
• P.D.O. recognition in1996.
• CoRFiLaC was awarded from “MIPAF” the authorization to certify Ragusano and Pecorino Siciliano with the P. D.O. label in2000.
Tools• Laboratories
• Laboratory for Antioxidants Analysis
– Literature has reported that several milk components have anti-carcinogenic properties. Conjugated linoleic acid “CLA”, has been demonstrated to have anti-carcinogenic effects (in vivo and in vitro) anti-atherogenic effects, effects on the alteration of the distribution of the nutrients, on lipid metabolism, on the treatment of type II diabetes by reducing hyperglycemia, on the immune response and on the mineralization of the bone.
– This lab studies potential healthy qualities of dairy products.
Tools
• Laboratories
• Flavour Laboratory
Gas Chromatography Olfactometry (GCO)
GC Mass
Retronasal Aroma Simulator (RAS)
Smart nose
Tools• Laboratories
• Sensory Analysis Laboratory
– panelists have been highly trained .
– Experimental cheeses as well those aiming for the PDO recognition are tested and a sensory profile is traced.
Tools• Laboratories
• Protein Biochemistry Laboratory
– analyzes caseins and whey proteins factors influencing cheese quality and
characteristics
– studies typical molecules of traditional Sicilian cheeses deriving from proteolytic processes during cheese ripening.
Tools• Laboratories• Microbiology Laboratory
– studies the microflora of historic Sicilian cheeses, and the importance of wooden
equipment for cheese production,
– supports the milk quality program of the Extension Service,
– supports the cheese pilot plant’s activity with microbiological tests ,
– uses the Scanning Electronic
Microscope, “SEM”.
Tools
CoRFiLaC’s cheese makers learned to produce quality cheeses
that were similar or equal to the quality
of the local cheeses.
This gives creditto research results,
and facilitates
transmitting
these results to
the real world.
• Cheese pilot plant and aging center
Tools• Mediterranean Institute of Culinary Art
of Sicily, “Micas”
short specializedcooking courses
famous chefs, researchers and experts organize
for opinion leaders, gourmets,
journalists, and lovers of the
world of gastronomy.
to witness the wine and gastronomic heritage of the
Mediterranean basin, searching for tradition and using it as a carrier of history, culture and customs.
CoRFiLaC, a link between consumers and producers
CoRFiLaC
PR
OD
UC
TIO
N
MarketingMICAS
Extension servicePDOLaboratories
CED
Cheese plant/Aging center
CO
NS
UM
PT
ION
Consumer directed
Producerdirected
RESEARCH
Market Research
Request for Consulting and Service/
Information aboutProduction and
Management
Service, Consulting,
Information, Education
Information,Education,Promotion
Quality Guarantee
Consumer
Producer
Producer directed Research-Measurement of feed particles by image analysis
Usually forage particle size analysis is done by sequential sieving
Having the proper particle size distribution of forages is important for dairy nutrition
Development of a method to measure single feed particles by “image analysis” using Matlab®
Consumerdirected Research
The real challenge is to demonstrate scientificallythat traditionally produced cheeses are “different”.
Biodiversity factors- Healthy properties
- Food safety and traditional production systems- Aromatic and sensorial profile
of traditional cheeses
Consumerdirected Research –
Lactic acid bacteria from a natural biofilm of Tina, a wooden vat, potential contributors to Ragusano
cheese fermentation
-To determine the difference of the biofilm among stainless steel, plastic, and wooden tina.
Objectives:
-To verify the release of bacterial cells
-from the biofilm into the milk .
-To characterize the natural biofilm
-of the wooden tina.
-Sterile swabs inside the tina-Filled with sterile (UHT) milk
-@ 34°C (50L each)-Milk left into the tina until 20’
Analysis of milkand wooden tina
0
1
2
3
4
5
6
7
8
Bact
eria
Cou
nt
(log10)
Stainless
Steel
Plastic Wooden
Tina
Consumerdirected Research –
Lactic acid bacteria from a natural biofilm of Tina, a wooden vat, potential contributors to Ragusano
cheese fermentationLcc. lactis subsp. Lactis
Ec. durans
Str. macedonicus
Ec. Faecium
Stc. thermophilus
The porous structure of the tina allows the settlement of a niche of bacterial
communities which are released into milk during the Ragusano cheese making process.
and goes through the
surface inside the external
wood vessels
CLSM confirms that bacteria also colonize the internal
wood layers
Consumerdirected Research –
Microstructure of traditional Sicilian
Cheeses by scanning electron
microscope (SEM)Cheese microstructure:
Globular casein micelles that join together
into clusters and chains
Relative proportions ofcasein, calcium,moisture and fat.
To examine the internal microstructure of the main traditional Sicilian cheeses, by scanning electron microscope (SEM)
-9 (5 pressed and 4 pasta filata)
typical Sicilian cheeses were analysed
Consumerdirected Research –
Microstructure of traditional Sicilian
Cheeses, and quantitative analysis by
scanning SEM imagesFibrous microstructure
Pasta Filata cheesesSpounge-like
microstructure Pressed cheesesRagusano DOP 9 Months
Moisture: 34,63%
Porosity: 0,2377
Pores: 828
Fiore Sicano 1 Month
Moisture: 40,92 %
Porosity: 0,3525
Pores: 1004
Consumerdirected Research –
Measurement of Gas Holes and Mechanical
Openness in Cheese by Image Analysis
Gas production in cheeses:-Typical characteristic-defect
Development of an image analysis method
using Matlab®to measure gas hole areas
in cheese slices
Calculation of the black pixel area as a percentage of the
total surface area
Measurement of gas productionand quality control incheese manufactoring
Consumerdirected Research –
Microbiological safety of Ragusano PDO cheese through traditional farmhouse
manufacturing
Factors affecting the microbiological
safety
Raw milk pathogens
Natural antibacterial activities of raw milk
-Fast acidification of the curd-High temperatures and
-exposure time of the curd- Salt content
Cheese manufacturingconditions
Geographic discrimination between Pecorino Siciliano cheeses by electronic nose and sensory analyses
J. Horne, S. Mallia, S. Carpino*, G. Licitra & J.O. Bosset
CoRFiLaC, Regione Siciliana - Italy
Results
Sensory PCA
Cheeses separated by
geographic area mainly
on PC1, with SE and
NE cheeses separating
on PC2
W
C
SE
NE
Important attributes in
NE and SE cheeses
were overall aroma and
butyric aroma
intensities, saltiness,
spiciness and hardness
Important attributes in
W cheeses were
moistness, mushroom,
floral and fruity aromas
C. Pediliggieri1, S. Carpino1* and G. Licitra1,2
1CoRFiLaC, Regione Siciliana, 97100 Ragusa, Italy; 2D.A.C.P.A., University of Catania, Catania, Italy.
INTRODUCTIONPecorino Siciliano is an artisanal pressed cheese made using raw ewe’s milk and traditional equipments. The specificity of Protected Denomination of Origin assigned to Pecorino Siciliano (CEE Decree n. 1107, 12/06/1996) implied that its unique sensorial characteristics are related to the production areas, the race and the nutrition of the ewes and the traditional cheese manufacturing practices (Gazzetta Ufficiale, N° 1263, Luglio 1996).
OBJECTIVESThe aim of the present study was to evaluate the influence of different production areas in Sicily, on the dynamics of the microbial communities of artisanal cheese by Temporal Temperature Gel Electrophoresis (TTGE).
MATERIAL AND METHODSSeven farms were selected, by microbiological and chemicalcomposition of cheese samples, between 21 farms allocated in sevendifferent areas of Sicily (Fig. 1): Iblean (I), Etnea (E), South Center(SC), North Centre (NC), Western (W), Western Centre (WC) andPeloritan (P); for each farm, four cheese-making were carried out. Milkand curd at 0 days were sampled just for first and third trial whilecheese ripened for two, four and eight months were obtained fromeach cheese-making. All cheese samples were analysed in two layers:internal and rind.PCR-TTGE was performed on all experimental samples: the DNAextraction from different samples and the amplification of V3 region ofthe 16S rRNA gene were carried out as described by Licitra et al.(2007).
RESULTS AND DISCUSSIONTTGE profiles derived from PCR amplicons of cheese samplesmanufactured at farms of different areas were showed in figure 2. It wasselected one cheese-making for each farm and the list of mainmicroorganism used as reference was illustrated in Table 1.The predominance of Lc. lactis subsp. cremoris, Lb. delbruekii subsp. lactis
and S. termophilus was observed. The presence of a bande relative to S.
gallolyticus subsp. macedonicus was detected too and Staphilococcus
equorum was observed to be peculiar in the rind layers of cheese samples.An high GC bacteria as propionibacteria and corinebacteria were identifiedtoo.
Corresponding author: [email protected]
CONCLUSIONSThis study showed the dominance of Lc. lactis, St..termophilus, Lb. delbruekii and St. equorum in the traditionalartisanal Pecorino Siciliano cheese. Some differences in the bacterial ecosystem, among all farms, were found asthe dominance of Lb. fermentum in the Centre area, E. faecium in the West area and high variability ofPropionibacteria in the East area. The bacterial distribution in milk samples based on farm location, wasapproximately maintained in cheese samples profile. This relationship between microorganism detected in milksamples and that one detected in cheese samples suggested that the quality of starter raw milk, probably affectedby geographic area, was thought to be directly related to variability development in bacterial ecosystem of cheese.
P
E
I
SC
NCWCW
Fig.1.Geographic distribution of seven farms selected.The samples were obteined from the follow areas: Iblean (I), Etnea (E),
South Center (SC), North Centre (NC), Western (W), Western Centre (WC) and Peloritana (P).
E
L M C0 2P1 2P4 4P1
4P4 8P1 8P4
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j
d d d
a a
rq
r r
v
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a a
xxy y y zzx
e
h
P
L M C0 2P1 2P4 4P1 4P4
8P1 8P4
vv v
o
k
l
j
x x xx
n nm
def
L M C0 2P1 2P4 4P1
4P4 8P1 8P4
ddd
l
m
k
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e ef
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I
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SC NC
L M C0 2P1 2P4 4P1
4P4 8P1 8P4
L M C0 2P1 2P4 4P1
4P4 8P1 8P4a a a ab bb b be be d d d
bd d dc
h hf f f
ll
kjj
us s r
v
q
u
q
WC W
L M C0 2P1 2P4 4P1
4P4 8P1 8P4
L M C0 2P1 2P4 4P1
4P4 8P1 8P4
l
be
h h
e ec c c cd d d
jk
v
q q q q
m m m
l
kj
he
y y y
d d d d
Fig. 2. TTGE profiles of milk samples (M), curds (C0) and cheese samples withdrawn at
2 , 4
And 8 month analysed in two different layers: rind (P1) and internal (P4). Lane L:
identification ladder. The samples were obteined from seven differents farms
rapresentative of each area: Iblean (I), Etnea (E), South Center (SC), North Centre (NC),
Western (W), Western Centre (WC) and Peloritana (P).
For TTGE analysis, a DCode universal mutation detection system was used to separate the V3 region PCR product.Migration was performed at 41 V for 16h with a temperature gradient of 63°C to 70°C (rate of 0.4°C 1h-1) for bacterialspecies with low Guanine an Citosine content (GC). After staining with ethidium bromide the gels were photographed ona UV transillumination table and analyzed using Gel Compare software (Bionumerics -Applied Maths, Version 4.6).The bands were identified by comparison with a TTGE species database developed by Parayre et al. (2007). PCR-TTGE is a rapid molecular method based on direct analysis of DNA in the environment and on the separation of DNAmolecules that differ by singles based allowing to analyse diversity within bacterial communities (Mayers et al. 1987).
a. Lb. plantarum
b. Lb. fermentum
c. Leuconostoc cremoris/Ln. mesenteroides
d. Staphylococcus equorum subsp. linens
e. E. faecium
f. Lb. helveticus/Lb. acidophilus/Lb. crispatus
g. Lb. delbrueckii subsp. bulgaricus
h. Lb. delbrueckii subsp. lactis
i. E. faecalis
j. S. gallolyticus subsp. macedonicus
k. Lc. lactis subsp. cremoris
l. S. thermophilus
m. C. variabile
n. C. flavescens
o. M. gubbeenense/Lb. reuteri
p. Bifidobacterium longum
q. Lb. paracasei/Lb. casei/Lb. rhamnosus/Lb.
zeae/B. infantis
r Corynebacterium casei
s Arthrobacterium nicotianae/A. globiformis/B.
tyrophermentas
t Propionibacterium microaerophilum
u B. alimentarium/B. linens
v P. acidipropionici
w B. casei
x P. jensenii/P. thoenii
y P. freuderechii
z P. cyclohexanicum
T
a
b
l
e
1
.
Main microrganism used for
reference set
Tab. 1. List which provides decoding informations for the main microrganism detected
by comparing with database set.
Characterization of bacterial ecosystem in Pecorino Siciliano Cheese produced in different areas of Sicily # M 62
Only in the Centre areas (farms NC e SC) acostant presence of Lb. fermentum was detected;Lc. lactis resulted to be dominant in East areafarms (P, I, and E) and in less extent in the farmW, while it decreased in the Centre area farms.Lb. delbruekii subsp. lactis was strongly detectedin West area farms (WC and W) and in Centrearea farms (SC and NC) while in East area farmswas observed to be weakly represented by twobands in the internal layers of cheese samples at2 and 4 months of the Etnea area (farm E).
Some bands, with low intensity, correspondent to Lb.
plantarum and Ln. cremoris were identified too; Lb.
plantarum was detected only in some samples of farm I,E and SC while Ln. cremoris was observed to bepeculiar of the internal layers of cheese samples infarm WC.As regards the high GC microorganisms, a consistentvariability of propionibacteria was observed in theprofiles of East area samples, mainly in farm P andfarm E, due to detection of bands relative toPropionibacterium acidipropionici, P. jensenii, P.
freuderechii and P. cyclohexanicum of cheese samples,as showed from comparison of TTGE profile of cheesesrind (Fig. 4). The high GC region profiles of other areaswere instead characterized by the presence of Lb.
paracasei, Arthrobacter nicotianae andCorynebacterium variabile.
Lc lactis
Farm W MIII
Farm W MI
Farm WC MIII
Farm WC MI
Farm NC MIII
Farm NC MI
Farm SC MIII
Farm SC MI
Farm P MI
Farm E MIII
Farm E MI
Farm I MIII
Farm I MI
Farm P MIII
E. faecium
Propionibacteria
Lb.fermentum
Fig. 3. Comparison of TTGE profiles of milk samples (M) withdrawn during first (I) and third (III) cheesemaking from each from
seven differents farms rapresentative of each area: Iblean (I), Etnea (E), South Center (SC), North Centre (NC), Western (W),
Western Centre (WC) and Peloritana (P).
Lb delbruekii lactis
St. termophilus
St. termophilus
PropionibacteriaSt. equorum
Lc lactis
Lb. fermentum
Fig. 4. Comparison of TTGE profiles of rind (P1) of cheese samples withdrawn at 2, 4, and 8 months of ripening from seven
differents farms rapresentative of each area: Iblean (I), Etnea (E), South Center (SC), North Centre (NC), Western (W), Western
Centre (WC) and Peloritana (P).
Some differences due to farmslocation were detected, as displayedeven by comparison of milk samplesTTGE profiles (Fig.3).The band relative to St. equorum, inthe milk samples withdrawn fromPeloritan area farms (farm P), wasobserved to be predominant in allcheese samples too; E. faecium,predominant in the West areasamples (Farm W) and with lessextent in the others areas, wasobserved to be absent in the NorthCentre farm.
REFERENCESLicitra et al. 2007. Applied and Environ. Microbiol. 73 : 6980-6987.Mayers et al. 1987. Methods Enzymol. 155: 501-527.Parayre et al. 2007. J. Microbiol. Methods 69 : 431-441.Gazzetta Ufficiale. Regolamento (CE) no.1263/96. Ser. L163, 02/07/1996: p.0019-0021.
D.A.C.P.A., University of Catania,
Catania, Italy
Consumerdirected Research –
Investigation of the potential role of the tina biofilm to
generate aroma compounds when inoculated into milk
Pasteurized
milk
Farm A
Farm B
Farm C
All milk samples inoculated with biofilm
showed a clear separation from the
pasteurized milk indicating a potential
difference in aroma compounds caused by the
growth of the biofilm.
International Dairy Products
Molecular characterization of Algerian Cheese Bouhezza by PCR-TTGE # M61
INTRODUCTION
For all the population of the world, the interest of identification and characterization of traditional products became more and more important. Characterization of Bouhezza traditional cheese, which manufactured in the East of the
Algeria, has started on some aspects since a few years (Aissaoui Zitoun et Zidoune, 2006). Bouhezza can be made from goat, ewe or cow’s raw and/or fermented milk “lben” and its production was limited at rural area and only for a
self consumption. It is obtained by spontaneous coagulation and utilizes a goat skin container for draining and ripening during more than three weeks. The aim of this work is to approach the microbiology of the cheese and it evolution
during cheese-making and ripening specialty with the PCR-Temporal Temperature Gel Electrophoresis method.
REFERENCES
Aissaoui Zitoun, O., and M.N., Zidoune. 2006. Communication. Séminaire d’Animation
Régional «Technologies douces et procédés de séparation» INSAT-AUF-GP3A, Tunis.
Sommaires, 118-124.
Licitra, G. et al. 2007. Applied and Environ. Microbiol. 73:6980-6987.
Parayre et al. 2007. J. Microbiol. Methods 69:431-441.
Mannu et al, 2000. J. Applied Microbiol. 2000, 89, 191-197.
Coeuret et al. Lait 83 (2003) 269–306.
MATERIALS AND METHODS
RESULTS and DISCUSSION
Traditional Bouhezza cheese is manufactured and characterized.
1) The microflora is essentially formed by the indigenous lactic acid bacteria: lactobacillus and lactococcus (106 to 107fcu/g).
2) Lc. lactis was the predomina nt and the most stable species in the cheese. It is the most common bacterium in cheese ecosystem (Mannu et al, 2000; Coeuret et al., 2003). Lb. plantarum is found in almost
samples.
3) Comparing Bouhezza cheese TTGE profiles to Ragusano cheese profiles, S. thermophilus was showed to be not the main microorganism of bacterial ecosystem in Bouhezza Algerian cheese; otherwise in
Ragusano (Licitra et al., 2007) cheese it has a predominant role.
4) The presence of lactobacilli in Bouhezza cheese could be due to their tolerance of low pH, high salt content and the lack of fermentable carbohydrate.
5) No difference in PCR-TTGE profiles due to pepper adding and now difference detected regarding the two cheese-making.
Evolution of microbiological characterization of Bouhezza cheese (Figure 2)
Total aerobic flora was grown in PCA (30°C/48h); lactobacilli on MRS (30°C/48 h); lactococci sur M17 (30°C/72h); yeast and
mould on OGYE (21°C/7j). Identification of the bacterial ecosystem of Bouhezza by PCR-TTGE: the DNA extraction from
different samples and the amplification of V3 region of the 16S rRNA gene were carried out as described by Licitra et al. (2007).
The bands were identified by comparison to a TTGE specie database developed by Parayre et al. (2007). In order to specifically
identify the main microorganism detected by TTGE profiles, a species-specific PCR assay was performed using specific primers.
The Lactococcus lactis specific assay was carried out using 1RL and LacreR primers on cheese samples and on single colony
isolated from M17 agar of Bouhezza cheese at 7 days.
o Total aerobic flora is between 108 to 109fcu/g; its essentially formed by the
indigenous lactic acid bacteria: lactobacillus and lactococcus (106 to 107 fcu/g).
They are the major flora witch contribute to the maturation of cheese.
o Yeasts and moulds are present at all stage of cheese-making/ripening (105 to
106 fcu/g).
TTGE profiles of Bouhezza cheese, made from two cheese-making C1 and C2 at different ripening days, were
compared (Figure 3) using database bacterial species as reference (line M). The use of universal primers, PCR
TTGE revealed:
• The predominance in all samples of Lc. lactis, which presence was confirmed by specific PCR tests.
• Also Lb. plantarum is detected in almost samples.
• The presence of Lb. haelveticus and Lb. delbruekii was detected too. They are used as starter in differents
cheeses.
• The presence of some bacterial species sounds depending of ripening days. For example: the band
corresponding to Leuconostoc cremoris prevails in cheese samples at few ripening days (until 21 d), while Lb.
helveticus was detected at about 21 days of ripening; S. gallolyticus subsp. macedonicus was observed to be
slightly present till 41 days to disappear later while the Lb. delbruekii subsp. lactis was evident after 41 days.
• The bands corresponding to S. equorum, Lb. delbruekii and S. thermophilus were detected in cheeses after 41
days probably due to addition of whole raw milk.
• The presence of a few high-GC-content species, like coryneform bacteria was observed.
• Other species shows sporadic presence along the 70 days of ripening (Lb. paracasei, B. cereus,
Corynebacterium variabile, Corynebacterium flavescens, Lb. buchneri and Bifidobacterium longum). The
evolution of these species must be confirmed.
1.2.Adopted Diagram for experimental cheesemaking
(1) Starting the manufacture in «chekoua» by salted lben (25 g/L);
(2) Continuous addition (all 3 days) of salted lben for six weeks;
(3) Finishing and correcting additions by whole raw milk, for acid and salted tastes, during the last
weeks. Once the cheese is elaborated, it is spied on with red hot pepper.
O. Aissaoui Zitoun1, C. Pediliggieri2, S. Lortal3, S. Carpino*2, G. Licitra2,4, and M.N. Zidoune1;
Laboratoire de Nutrition et de Technologie Alimentaire (LNTA)- INATAA. Université Mentouri de Constantine Route de Ain El Bey, Algeria1; CoRFiLaC, Regione Siciliana, 97100 Ragusa, Italy2; INRA, Agrocampus
Rennes, UMR1253 Science et Technologie du Lait et de l’Oeuf, 65 Rue de Saint-Brieuc, F-35000 Rennes, France3; D.A.C.P.A. University of Catania, Italy4.
Corresponding author: [email protected]
1. Cheese-making based on Traditional Diagram
1.1. Crude Matter and Materials
•Lben = Spontaneous fermented raw milk, churned and partiely skimmed.
•Cow’s raw milk.
•Chekoua = permeable container made with goat skin treated in a mixture of salt + juniper and
then washed with soapy water and rinsed.
2. Analysis:
Lben, raw cow’s milk and Bouhezza cheese are studied. Microbiological evolutions are studied at 7, 15, 21, 41, 56, and 70 days on two experimental cheese-making
(C1 and C2) in similar conditions. Like in traditional consumption, red pepper is added to a separated sample of the final cheese (70 days) for a rapid comparison
(figure 1).
The indigenous LAB of lben are the main flora occurring in the refining of the cheese. The raw milk brings
from its part a complement of other species which can growth in the conditions of the cheese and participate
too in the refining.
CONCLUSION
ACKNOWLEDGEMENTS
The authors thank CoRFiLaC staff for support in the lab analysis.
This research was funded with APQ Algeria Program by the "Assessorato Agricoltura e Foreste della
Regione Siciliana", Palermo, Italy.
Microbiological Identification with PCR-TTGE (Figure 3 and 4)
a
i
ca a a a
d d d dc c
jh h/jhjj j
mm
n n
l l l l
p
r rs s
w
s
xx
Φ Φ
aa a a a
d d dc c
ji
jj j
n n
l l l l
p
rss
w
s
x
zΦ
n
a
bi i
j
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i gg g
Φ
7d 15d 21d 41d 56d 70d
NP
70d
WP7d 15d 21d 41d 56d 70d
NP
70d
WPM M M
Cheese-making C1 Cheese-making C2
Fig.3. TTGE profiles of Bouhezza cheese made from two cheese-making (C1 and C2) at different
ripening time: 7d (days), 15d, 21d, 41d, 56d and 70d without pepper (np) and with pepper (wp).
Lane M: identification ladder.
a: Lactobacillus plantarum/gasseri, c: Leuconostoc cremoris/mesenteroides, d, Staphilococcus
equorum subsp. linens, h: S. xylosus, i: Lb brevis or xylosus, j: Lb helveticus/
acidophilus/crispatus, m: E. faecalis/S. succinus, n: S. gallolyticus subsp. macedonicus, p: Lc.
lactis subsp. cremoris, r: Lb. buchneri, s: B. cereus/B. licheniformis, w: C. variabile, x: C.
flavescens, Φ: Lb. paracasei/Lb. casei/Lb. rhamnosus/Lb. zeae/B. infantis, ?: Unknown band.
238 bp
7d 15d 21d 41d 56d70d
NP
70d
WP
Cheese-making C1
7d 15d 21d 41d 56d70d
NP
70d
WP
Cheese-making C2
M B C+ C-
Fig. 4. Species-specific PCR products from Bouhezza cheese samples of two cheese-
making C1 and C2 at different ripening time: 7 days, 15d, 21d , 41d, 56d, 70d without
pepper (70d NP) and with pepper (70d WP). Lactococcus lactis and Lactobacillus
plantarum strains were used as positive (C+) and negative (C-) control respectively. Lane
M: DNA molecular weights marker 100 bp.
Chekoua (skin goat bag)
Fig. 1 Bouhezza cheese at 70 days
4D.A.C.P.A., University of Catania,
Catania, Italy
Aroma profile characterization of traditional Algerian Bouhezza cheese # M60
REFERENCES
Acree T.E., J., Barnard.1994. Gas chromatography-olfactometry using Charm analysisH. Maars 35, 211–220.
Agresti A. 1996. An introduction to categorical data analysis. New York & Chichester: John Wiley and Sons INC.
Marin A.B., T.E., Acree, J., Barnard. 1988. Variation in odor detection thresholds determined by Charm analysis. Chemical Senses. 13, 435–444.
MATERIALS AND METHODS
RESULTS and DISCUSSION
The high variability of Bouhezza cheese was clearly detected by Smart Nose because the high
resolution power of the instrument in detecting the single aromatic ion mass, these results clearly
indicated the peculiarity of the single farm producing cheese. On the other side GCO analyses
showed different profiles with some odor active compounds in common of the Bouhezza samples
analyzed. Both instruments, at different levels and with different statistical results, confirmed that
the farm traditional cheese-making process, the milk and the different spices used during
manufacturing contribute to the uniqueness of this product. More farms and cheese samples should
be investigated to prove the linkage of the Bouhezza cheese to the territory of production in order to
demonstrate the uniqueness of the product.
CONCLUSION
S. Carpino*1, T. Rapisarda1, G. Belvedere1, O. Aissaoui Zitoun2, M.N. Zidoune2, and G. Licitra1,3;
CoRFiLaC, Regione Siciliana, Ragusa, Italy1; Laboratoire de Nutrition et de Technologie Alimentaire (LNTA)- INATAA. Université Mentouri de Constantine Route de Ain El Bey Algeria2; D.A.C.P.A. University of
Catania, Italy3.
Corresponding author: [email protected]
INTRODUCTION
For all the population of the world, the interest of identification and characterization of traditional products became more and more important. Characterization of Bouhezza traditional cheese, which manufactured in the East of the
Algeria, has started on some aspects since a few years (Aissaoui Zitoun et Zidoune, 2006). This cheese can be produced by goat, sheep or cow milk. It can be also made from mixed milks in different proportions. Bouhezza cheese
manufacturing requires the preparation of both a natural container "chekoua" and a spontaneous fermented milk, rather than skimmed and little acid "lben". The chekoua is a bag obtained from goat or sheep skin, treated with salt
and juniper berry. It has also an important role as separator of serum and solid phase (ultrafilter). Manufacturing process is curried out by adding "lben" and raw milk in order to correct both acidity and saltiness. On the average,
cheese making process is completed after 70 days when pepper id added. The aim of this preliminary study was to characterize the volatile organic compounds (VOCs) of Bouhezza cheese produced in different farms.
Five traditional Bouhezza samples obtained from five different farms (F2, F3, F4,F5, F6) were analyzed by:
SMart Nose (SN) system which allowed the direct analysis by MS of volatile organic compounds (VOCs). The ion fragments
obtained by SN were then processed by a multivariate statistical PCA.
Dynamic headspace purge and trap apparatus (Tekmar 8900) in combination with a gas chromatograph olfactometry/mass
selective detector (GC/MS/O) (modified Hewlett Packard 6890 gas chromatograph by Datu Inc., Geneva, NY) to analyze odour
active compounds. Statistical software was used to elaborate GCO results (JMP® 8.0.1).
PCA applied to SN results showed a good separation (PC1 77,93%;
PC2 13,05%) among Bouhezza cheeses indicating the uniqueness of
the homemade products (figure 1).
Olfactometry results (Marin et al., 1988; Acree & Barnard 1994)
confirmed a high variability for the aroma profiles (table 1). F2
sample had the richest profile (23 VOCs) among all farms, followed
by F5 (19 VOCs), F3 (18 VOCs), F4 (15 VOCs) and F6 (13 VOCs).
All samples were mainly characterized both by aldehyde and ester
compounds. Among aldehydes there were octanal, nonanal, (E,E)-
2,4-decadienal, (E)-2-nonenal that gave, respectively, orange, apple,
hay, and green notes. There were identified the following free fatty
esters: ethyl octanoate, ethyl hexanoate, ethyl butanoate and propyl-
1-methyl-acetate. These released respectively, wine, floral, and fruity
notes on cheese aroma profiles.
Statistical analysis were performed using a nominal logistic model
on the presence of VOCs compounds. Data showed that probability
of VOCs presence is marginally dependent from farms, thus farm
traditional method of production did not significantly affected
cheese VOCs presence (Wald Statistic χ2df=4=7.99; p=0.09) with the
exception of F2 presenting a higher significative number of VOCs
compared to the others farms (χ2df=4=4.55; p<0.05).
ACKNOWLEDGEMENTS
The authors thank Catia Pasta, from CoRFiLaC, for her help in the data analysis.
This research was funded with APQ Algeria Program by the "Assessorato Agricoltura e Foreste della Regione Siciliana", Palermo, Italy.
Figure 1 – Score Plot Traditional Bohuezza cheeses
4D.A.C.P.A., University of Catania,
Catania, Italy
Table 1 - Volatile compounds in Bouhezza cheese by P&T-GC/MS/O.
Compounds Chemical Class Descriptor LRI a Identb F2 F3 F4 F5 F6
NI _ potato 1068 _
NI _ ranci 1148 _ *
NI _ fruity 1154 _
butyric acid acid sweaty 818 PI *
acetic acid acid sour/garlic 815 MS,PI *
nonadienol alcohol green 1167 PI * * * * *
1-butanol, 3-methyl alcohol rancid,garlic 838 MS,PI * *
octanal aldehyde orange,lemon 1030 MS,PI * * * *
nonanal aldehyde milk 1121 MS,PI * * * * *
heptanal aldehyde unpleasant 946 MS,PI * * *
4-heptenal aldehyde cheese,rancid 917 PI *
2-hexenal aldehyde apple 852 PI *
(Z)-2-nonenal aldehyde hay 1181 PI * * * * *
(E,E)-2,4-decadienal aldehyde fried oil 1236 PI * * * *
(E)-2-nonenal aldehyde green 1175 PI * * * * *
octanoic acid ethyl ester ester wine 1214 MS,PI * * * * *
methyl geranate ester floral 1294 PI *
hexanoic acid ethyl ester ester apple,orange 1028 MS,PI * * * * *
ethyl valerate ester apple 911 PI * *
butanoic acid ethyl ester ester apple up 877 MS,PI * * * * *
acetic acid 1-methyl propyl ester ester apple 850 MS * *
1-butanol, 3-methyl acetate ester apple,apricot 929 MS *
3,5-octadienone ketone mushroom 1101 PI * * * * *
1-octen-3-one ketone mushroom up 1008 PI * * * * *
diethyl methyl pyrazine pyrazine burnt 1118 PI * * * * *
azine pyridine pungent 830 PI *
thiophane sulfur rancid,garlic 799 PI * * * * *
methional sulfur cooked potato 954 PI * *
isodihydro carveol terpene red pepper 1202 PI *
geraniol terpene wine,floral 1278 PI * * *
(+)-cis-rose-oxide terpene floral,orange 1126 PI * *
TOTAL 23 18 15 19 13a LRI, Linear Retention Index, HP-5 capillary column.
b Identification: MS (Wiley library); PI (Internet Data Base: flavornet)
NI: not identified
Figure 5. PCR-TTGE profiles of following samples : 1. raw milk, 2. Lben before contact, 3. Fresh goat
skin biofilm (FS1), 4. Lben after 10’ in FS1, 5. Lben after 24h in FS1, 6. Fresh goat skin biofilm (FS2), 7.
Lben after 10’ in FS2, 8. Lben after 24h in FS2. Lanes M: Genomic DNA marker (Lb. plantarum, Lb.
fermentum, Ec. faecium, Lb. helveticus, E. faecalis, Lc. lactis, Str. thermophilus, C. moorparkense, Lb.
paracasei, Arthr. nicotianae, and B. casei).
Figure 6. Cluster analysis of PCR-TTGE profiles of following
samples: raw milk (RM), Lben before contact (LbC), Fresh
goat skin biofilm (FS1, FS2), Lben after 10’ in FS1 (L10FS1),
Lben after 16 h in FS1 (L16FS1), Lben after 10’ in FS2
(L10FS2) and Lben after 16 h in FS2 (L16FS2).
PCR-TTGE profiles showed in Figure 5 revealed thepredominance of L. lactis subsp cremoris and St. gallolyticus
subsp macedonicus; these bands, as well as Lb. delbruekii
subsp. bulgaricus and Lb. plantarum, were observed to be mostlypresent in milk profiles. The bands corresponding to Lb.
fermentum, E. faecalis and St. thermophilus were insteaddetected only in skin biofilm profiles. Lb. brevis and S. equorum
subsp. linens were detected only in milk after contact with goatskin and in fresh skin samples. Lb. helveticus was observed to beslightly present in the molecular profiles of milk before contactwith goat skin, but its band was very intense in one of the twofresh skins, to disapper in Lben at once after contact with skin.
As displayed from comparison of all molecular profiles in cluster analysis (Fig. 6), all milksamples (before and after contact) resulted similar between them and revealed somedifferences with skin profiles, above all in the high-GC region. Indeed the presence of a fewbands corresponding to Bif longum, Arth nicotianae, P. microaerophilum, B. brevis, Lb.
paracasei and C. flavescens were found in fresh skin biolfilm. A feeble band corresponding toP. cycloexanicum was also detected, even if its presence was observed only in Lben beforecontact and in milk samples after 10’ in goat skin.
Microbiological analisys showed no pathogens and an increasing of mesophilic LAB and yeast in Lben after contact.
SEM micrographs showed fresh “Chekoua” colonized by bacilli anddiplococci. The skins examined after Lben adjunct were mainly populated bybacilli; diplococci were few and appeared isolated or joined in short chains(Fig. 3A). CLSM picked out mainly alive bacteria (Fig. 3B).
Yeasts were absent in FS1, FS2, whereas they were founded in FSL1,FSL2, appearing isolated or joined to form small colonies (Fig. 4).
Goat skins surface resulted lacking in bacteria. Samplesobserved before Lben addition (FS1, FS2) were less populated(Fig. 2A, 1B) than the skins examined after Lben adjunct (FSL1,FSFL2) (Fig. 2C, 1D).
Just isolated bacteria entrapped in the network of the epidermictissue were detected in FS1, FS2 (Fig. 2B).
10
0
80
60
40
20
Pearson correlation [0.0%-100.0%]
V3 bas GC V3 bas GC
FS1
FS2
LbC
RM
L10FS1
L10FS2
L16FS2
L16FS1
a. Lb. plantarum/Lb. johnsonni/Lb. gasseri
b. Lb. fermentum
c. Leuconostoc cremoris/Ln. mesenteroides
d. Staphylococcus equorum subsp. linens
e. E. faecium
f. E. faecium/Lb.brevis
g. Lb. brevis
h. S. xylosus
i. Lb. brevis/S. xylosus
j. Lb. helveticus/Lb. acidophilus/Lb. crispatus
k. Lb. delbrueckii subsp. bulgaricus
l. Lb. delbrueckii subsp. lactis
m. E. faecalis/S. succinus
n. S. gallolyticus subsp. macedonicus
o. S. carnosus
p. Lc. lactis subsp. cremoris
q. S. carnosus subsp. carnosus/Lc. lactis subsp. cremoris
r. Lb. buchneri
s. B. cereus/B. licheniformis
t. S. thermophilus
u. E. coli
v. B. breve/Hafnia alvei
w. C. variabile
x. C. flavescens/ B. pumillus
y. M. gubbeenense/Lb. reuteri
z. Bifidobacterium longum
Φ Lb. paracasei/Lb. casei/Lb. rhamnosus/Lb. zeae/B. infantis
Ω Corynebacterium casei
α Arthrobacterium nicotianae/A. globiformis/B. tyrophermentas
β Propionibacterium microaerophilum
γ B. alimentarium/B. linens
δ P. acidipropionici
ε B. casei
η P. jensenii/P. thoenii
θ P. freuderechii
µ P. cyclohexanicum
? Unknown band
Band Species or subspecies
Table 1. Main microrganisms used for the reference set.
a. Lb. plantarum/Lb. johnsonni/Lb. gasseri
b. Lb. fermentum
c. Leuconostoc cremoris/Ln. mesenteroides
d. Staphylococcus equorum subsp. linens
e. E. faecium
f. E. faecium/Lb.brevis
g. Lb. brevis
h. S. xylosus
i. Lb. brevis/S. xylosus
j. Lb. helveticus/Lb. acidophilus/Lb. crispatus
k. Lb. delbrueckii subsp. bulgaricus
l. Lb. delbrueckii subsp. lactis
m. E. faecalis/S. succinus
n. S. gallolyticus subsp. macedonicus
o. S. carnosus
p. Lc. lactis subsp. cremoris
q. S. carnosus subsp. carnosus/Lc. lactis subsp. cremoris
r. Lb. buchneri
s. B. cereus/B. licheniformis
t. S. thermophilus
u. E. coli
v. B. breve/Hafnia alvei
w. C. variabile
x. C. flavescens/ B. pumillus
y. M. gubbeenense/Lb. reuteri
z. Bifidobacterium longum
Φ Lb. paracasei/Lb. casei/Lb. rhamnosus/Lb. zeae/B. infantis
Ω Corynebacterium casei
α Arthrobacterium nicotianae/A. globiformis/B. tyrophermentas
β Propionibacterium microaerophilum
γ B. alimentarium/B. linens
δ P. acidipropionici
ε B. casei
η P. jensenii/P. thoenii
θ P. freuderechii
µ P. cyclohexanicum
? Unknown band
Band Species or subspecies
Table 1. Main microrganisms used for the reference set.
Results
Figure 2.
A) CLSM micrograph of
goatskin before Lben
addition.
B) SEM micrograph of
goatskin before Lben
addition.
C) CLSM micrograph of
goatskin after Lben addition.
D) SEM micrograph of
goatskin before Lben
addition.
A C
B D
Figure 4. Isolated colonies of yeasts in goatskin after Lben addition.
Figure 3. A) Bacilli and diplococci in goat skin samples. B) Microflora
population of goat skin. Alive and dead microorganisms are coded in green
and red, rispectively.
Main microorganisms used for the reference
set
AcknowledgementsThe authors thank CoRFiLaC staff for support in the lab analysis.This research was funded with APQ Algeria Program by the"Assessorato Agricoltura e Foreste della Regione Siciliana",Palermo, Italy.
Ecosystem characterization of “goatskin” biofilm
ObjectiveThe aim of this work was to study the Chekoua bacterial microflora by Confocal Laser Scanning
Microscopy, Scanning Electron Microscopy and by PCR-temporal temperature gel electrophoresis.
O. Aissaoui Zitoun1, N. Fucà2, C. Pediliggieri2, L.Tuminello2, M. N. Zidoune1, G. Licitra2,3, S. Carpino2*
1 Lab. LNTA, INATAA, University of Mentouri, Constantine, Algeria; 2 CoRFiLaC, Regione Siciliana, Ragusa, Italy; 3 DISPA, Catania University, Catania, Italy.
ConclusionsThe biofilm ecosystem of the final Chekoua was composed by bacteria and yeasts. The coexistence of those groups
might be the result of their commensalistic interaction, which is fundamental for the cheese safety. The combination
of such conditions inhibits many spoilage bacteria and filamentous fungi, thereby substantially increasing shelf life
and safety of Bouhezza cheese.
TTGEThe DNA extraction from different samples and the amplification of V3 region of the 16S rRNA gene by the use ofuniversal primers, were carried out as described by Licitra et al. (2007).
A DCode universal mutation detection system was used to separate the V3 region PCR product. Migration wasperformed at 41V for 16h with a temperature gradient of 63°C to 70°C (rate of 0.4°C h) for bacteria of low GCcontent. After staining with ethidium bromide the gels were photographed on a UV transillumination table andanalyzed using Gel Compare software. The bands were identified by comparison to a TTGE specie databasedeveloped by Parayre et al. (2007).
MicrobiologyDetection of pathogens was performed on biofilm samples freshly collected, by using BAX® System (DuPontQualicon, UK). Enrichment for Salmonella was carried out using modified tryptone soya broth (Oxoid) at 36°C for24 h; 10 µL were then transferred in 500 µL BHI (Oxoid) for a further incubation for 3h at 37°C. Enrichment forListeria was done in Listeria selective enrichment broth base (Oxoid), at 30°C for 24 h. A volume of 0.1 mL ofenriched broth was transferred in 9.9 mL of MoPS-BLEB (Oxoid), and incubated at 37°C for 18 h. Enrichment forEscherichia coli O157:H7 was done with E.C. broth (Reduced Bile Salt) added with novobiocin (Oxoid), at 36°C for24h. Lysis was performed following the BAX® System instructions.
BackgroundChekoua is the goatskin bag used for draining and ripening of the traditional Bouhezza cheese. Before use, thegoatskin is firstly treated with salt and juniper berry, tied and rinsed. The obtained container is filled up with Lben,
stored overnight and then rinsed. After cheese making, the Chekoua can be rinsed and dried for another cheesemaking.
A B
ReferencesLicitra et al. 2007. Applied and Environmental Microbiology 73: 6980-6987. Lortal et al. 2009. Int J Food Microbiol. 132: 1-8Parayre et al. 2007. J. Microbiol. Methods 69 : 431-441.
Material and MethodsSamplingTwo replicates of fresh skin (FS) were used, prepared,sampled and analyzed before (FS1, FS2) and after contactwith Lben (FSL1, FSL2).
SEMThe samples were dehydrated dehydrated (Lortal etal., 2009) in a graded ethanol series (75, 85, 95 and100%) and dried by the critical point method in CO2.The dried samples were mounted on SEMaluminium stubs and gold-palladium coated. Thesamples were analyzed by the scanning electronmicroscope Jeol JSM 5900LV (Jeol, Tokyo, Japan),operating at an accelerating voltage of 15 kV, aworking distance of 12 - 18 mm and a magnificationof 5000 - 10000X.
CLSMThe specimens were directly fluorescently stained onmicroscope slides. An aliquot of 5 µL of LIVE/DEAD®BacLight™ Bacterial Viability Kit stock solution wasadded for microorganisms detection and 10 µl ofConcanavalin A linked with Alexa Fluor 633 1% in 0.1M NaHCO3 were used as polysaccharides stain. After15 minutes of incubation in the darkness, the imageswere collected using a 60x objective lens.
Figure 1.Prepared Chekoua
characteristics (a) and biofilm
sampling (b). Surface in the blue area, 10 cm x 10
cm.
TRACHANAS CHEESES
S. Carpino, T. Rapisarda, G. Belvedere, P. Papademas, M. Neocleous, I.Schadt, C. Pasta, and G. Licitra. 2010. Effect of dehydration by sun or byoven on volatiles and aroma compounds of Trachanas. J. Dairy Sci. and Tech.90: 715–727.
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