Small Ruminant Research 101 (2011) 1732

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Small Ruminant Research

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Microbiological characteristics of Greek traditional cheeses

Evanthia Litopoulou-Tzanetaki , Nikolaos Tzanetakis
Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

a r t i c l e

i n f o

a b s t r a c t
Cheesemaking in Greece has a centuries-old tradition and ancient Greeks considered the cheese as a divine invention and gift. Numerous traditional cheeses are made throughout Greece today and twenty of them were recognized as PDO. As a total, Greek traditional cheeses are grouped, according to their technology of manufacture, as: cheeses in brine, soft cheeses, semi-hard cheeses, hard cheeses and whey cheeses. Their predominant microora throughout ripening is consisted of NSLAB that proliferate and contribute by their biochemical activities to cheese ripening and the development of avor characteristics. Microorganisms indicative of their hygienic quality, Enterobacteriaceae and coliforms, decline during ripening and are usually found at negligible levels in the mature products. 2011 Elsevier B.V. All rights reserved.

Article history: Available online 2 October 2011 Keywords: Greek traditional cheeses Microora Biochemical activities NSLAB

1. Introduction Cheese making in Greece has a centuries old tradition. Homer in 8th century BC in his Odyssey describes well the shepherd and cheese maker Polyphemus of the 12th century BC, as well as his cheeses. Diodorus (Diodorus Siculus, 1st century B.C), the Greek historian from Sicily, wrote that Aristeus, son of Apollo and grandson of Zeus, who had learned the art of cheese making from his nannies, the nymphs, was sent by the Gods at Olympus mountain to teach the Greeks how to make cheese. Given the value of cheese as a food, it is not surprising that ancient Greeks considered the cheese as a divine invention and gift. The cheese that Homer described seems possible to be the ancestor of Feta and it is the main cheese manufactured in Greece from the ancient times till today. Meanwhile, various cheese types evolved through the centuries, so that nowadays each area, almost every island has its individual unique tradition in cheese manufacture. Lately, the EU supports the local products and the traditional technologies. Typical foods are considered as safer

and healthier by the consumer and typicality has been established as a recent way to understand the quality of a product (Rubino et al., 1999). There is also the Council Regulation (EEC) no. 2087/92 (1992), which attempts to unify different concepts of typicity and lays down rules on the protection of agricultural products intended for human consumption. The PDO/PGI labels guarantee specic sensory properties and imply that particular characteristics are present in the product. Numerous traditional cheeses are made throughout Greece today. Some of them are in fact types of the same cheese variety, have somewhat different steps in technology or possibly the same technology, but known with different local names. Twenty of them were recognized as PDO (Protected Designation of Origin) cheeses and there are others, waiting for their recognition. As a total, Greek traditional cheeses could be grouped, according to their technology of manufacture, as: cheeses in brine; soft cheeses; semi-hard cheeses; hard cheeses; whey cheeses. 2. Cheeses in brine (Pickled cheeses) 2.1. Batzos cheese, PDO Batzos is a low-fat, semi-hard, white brined cheese with a sourish and lightly piquant taste and a large number of

This paper is part of the special issue entitled Products from Small Ruminants, Guest Edited by A. Govaris and G. Moatsou. Corresponding author. Tel.: +30 2310991727; fax: +30 2310991674. E-mail address: ganet@agro.auth.gr (E. Litopoulou-Tzanetaki). 0921-4488/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2011.09.022

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E. Litopoulou-Tzanetaki, N. Tzanetakis / Small Ruminant Research 101 (2011) 1732 Table 1 Species of lactic acid bacteria isolated from ripened curd of Batzos cheese from raw ovine milk, made in spring and summer. Isolates from two batches of cheese in each season. % of isolates from cheese made in Species Lactococci Lc. lactis subsp. lactis Lactobacilli Lb. paracasei subsp. paracasei Lb. paraplantarum Lb. pentosus Enterococci E. faecalis E. faecium E. durans E. pseudoavium Pediococcus spp. Total Spring 22.22 14.82 7.41 7.41 40.74 3.70 3.70 100.00 Summer 32.98 30.85 13.83 21.28 1.06 100.00

holes in the body. It is made from either caprine milk, or ovine milk or mixtures of both milks. The cheese is made at home by the traditional method as well as at creameries of Western Macedonia and the nearby Thessaly, using modications of the traditional method and technological innovations. The milk is not heat-treated and coagulation takes place within 50 min at 2830 C. When the milk just begin to gel, the manufacturer hits the milk with a thick wooden stick about 150200 times; the milk is then left to curdle for 3540 min before being hit again for 300350 times. During these hitting stages, a large proportion of fat is transferred to the whey. The curd is then left to settle, but some cheese makers would apply mild heat before placing the curd, cut in appropriate pieces, in cheese cloths and hanging them to drain and ripen for 24 h. The next day, the ripened curd is cut into slices, salted with coarse salt, placed in tins and covered with brine (Anifantakis, 1991; Zygouris, 1956). Cheese is either consumed fresh or stored in cool rooms. The microbial counts in the curds of Batzos cheese from ovine milk in summer (Fig. 2) were higher than in curds of spring cheeses (Fig. 1) for all microbial groups except staphylococci (Nikolaou et al., 2002). Lactic acid bacteria (LAB), Enterobacteriaceae and coliforms are the major components of the microora during ripening; LAB grown on M17 agar was the most abundant microbial group in the curd and cheese during storage for one month at 4 C. Enterobacteriaceae and coliforms, declined faster in cheeses made in the summer. This was possibly related to the lower pH values of cheeses made in the summer and the higher number of LAB present in curds of summer cheeses. Among LAB lactobacilli predominated in cheeses made in summer; in spring enterococci were the most abundant
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bacteria (Table 1). Lactobacillus paracasei subsp. paracasei and Lb. paraplantarum were isolated frequently from the ripened curds of Batzos cheese in summer, while Enterococcus faecium was the most frequently isolated LAB species in spring. Lactococcus lactis subsp. lactis was found only in the curds of spring cheeses. Hydrolysis of both s - and -casein (CN) proceeded more rapidly in spring than in summer; s -CN is also degraded faster than -CN. Lipolysis products increased gradually with storage and accumulated at higher levels in cheese made in the summer. For the cheese from caprine milk no signicant differences were observed in the counts related to the season, but signicant changes in the microbial counts throughout
Curd (after draining) Ripened curd

10

8 log10 cfu/g

0
TC Coliforms

Enterobacteriaceae

Staphylococci

Yeasts

Lactobacilli

Lactococci

Enterococci

Fig. 1. Log counts (x SD) of microbial groups in the curd of Batzos cheese from raw ovine milk, made in summer. Mean of two cheesemaking trials.

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12

Curd (after draining) Ripened curd

10

8 log10 cfu/g

TC

Coliforms

Enterobacteriaceae

Staphylococci

Yeasts

Lactobacilli

Lactococci

Enterococci

Fig. 2. Log counts (x SD) of microbial groups in the curd of Batzos cheese from raw ovine milk, made in spring. Mean of two cheesemaking trials.

ripening and storage were recorded (Psoni et al., 2003). As also observed for cheese from ovine milk, LAB, Enterobacteriaceae and coliforms are the principal microbial groups in cheese from caprine milk. LAB, are the most abundant microbial group throughout storage. Ripening and storage had a signicant inuence on the number of Enterobacteriaceae and coliforms (Fig. 3). The lactation season has a clear effect on the predominance of certain LAB in each season. Thus, in cheese made in winter lactococci and enterococci predominated. Lactococci were less frequently isolated from cheeses made in spring and summer, whereas enterococci were rarely found among the predominant LAB. However, the frequency of isolation of lactobacilli was increased in spring and summer cheeses. The main lactococci species were Lc. lactis subsp lactis, found more frequently in winter cheeses. In spring and summer cheeses facultatively heterofermentative lactobacilli predominated. Lactobacillus plantarum and Lb. paraplantarum were the principal species of lactobacilli isolated. Enterococcus durans was the predominant Enterococcus in cheese made in winter. In an attempt to improve the microbiological quality of the cheese from raw milk, selected Lc. lactis subsp lactis strains were used as starter to make cheese from raw (RS), pasteurized (PS) and pasteurized standardized (PSS) milk. RS and PS cheese were made by the traditional method of excluding fat from the milk. PSS cheese was made from partially skimed milk (Psoni et al., 2006). Surprisingly, the microbial loads of lactococci in cheeses made from raw milk (R) and raw milk with starter and the traditional method were similar and the same was observed with their pH. Only when standardized milk (25 g/L fat) was used for

cheese production without hitting and stirring during coagulation, higher levels of LAB and lower pH than the other cheese types were recorded. A higher rate of decrease of undesirable microorganisms was recorded in this cheese. Degradation of s -CN was in R > RS and in PS > PSS, while a small reduction of -CN during ripening and storage was recorded. As expected, degradations of s -CN occurred at a faster rate than -CN and proteolysis and lipolysis products increased throughout ripening and storage. Thus, conclusively, cheese milk pasteurization and use of a native starter from the traditional cheese has resulted in better microbiological quality Batzos cheese. However, standardized milk with low fat content must be used and not the traditional method of excluding fat into the whey. 2.2. Feta cheese, PDO Feta is a semi-soft, white brined cheese, with mildly rancid and slightly acid taste, rm and smooth texture. No gas holes should be present, but irregular small mechanical openings are desirable. It is made from ovine or from mixtures of it with caprine milk (up to 30%). The cheese is produced all-over the country. However, its production is not common in the islands. The coagulation of the milk is traditionally made by rennet from abomasa of lambs and kids, within 50 min to 1 h. After curdling, the curd is cut and transferred into either cheese cloths or perforated moulds for draining without pressing. The drained curd is subsequently cut into blocks and salted by coarse salt on cheese-tables, until it contains about 33.5% salt. The cheese remains on the cheese-table for about 15 days. During this period a surface microora is developed which is considered to contribute to cheese

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E. Litopoulou-Tzanetaki, N. Tzanetakis / Small Ruminant Research 101 (2011) 1732

Fig. 3. Log counts (x SD) of Enterobacteriaceae and coliforms throughout ripening of goat milk cheese made in spring and summer.

ripening and avor. The cheese is afterwards transferred into barrels or tins with brine (78%) to continue ripening in cold rooms. The modern technology follows the traditional steps and standardized conditions. The milk is usually standardized to a fat content of 5.86.0%; it is then pasteurized (72 C, 15 s) and immediately cooled at 3234 C for renneting. Lactic starters (for example yoghurt, Lc. lactis + Lb. bulgaricus) are added 20 min prior to renneting. According to Greek standards (Greek Food Code, 1998) the cheese ripens for at least two months. The predominant surface microora of fresh cheese (at 4d, when the microbial slime starts appearing) is composed of lactic acid bacteria (LAB), yeasts and salt tolerant microbes. Leuconostoc, Lc. lactis and Lb. plantarum constitute the predominant LAB microora developed at day 4, while at the end of ripening in the warm room the LAB microora is composed of lactobacilli, with Lb. plantarum being the predominant species. Aminopeptidase activities are largely due to lactobacilli, while lipolytic activities are either not detected or weak in these organisms (Tzanetakis and Hatzikamari, 1994). The predominant yeast species at 4 days are Saccharomyces cerevisiae and Debaryomyces hansenii, while at the end of ripening in the warm room S. cerevisiae is the predominant yeast. Yeasts also contain leu- and val-aminopeptidase that is higher than the cysaminopeptidase activity. In addition, their esterase/lipase activity is higher than the esterase activity, and for some strains only lipase activity is found (Tzanetakis et al., 1996).

The halotolerant surface microora is composed of staphylococci, micrococci, enterococci and coryneform bacteria. Staphylococcus saprophyticus predominates the halotolerant bacteria content on the surface of cheese at day 4, while coryneforms are commonly found in the 20-dayold cheese. Staphylococci isolates from Feta cheese surface do not exhibit any aminopeptidase activity; however, staphylococci show quite strong, in general, esterase and esterase/lipase enzyme activity (Tzanetakis et al., 1995a,b). Thus, lactobacilli of the cheese surface may affect proteolysis, yeasts participate to both, proteolysis and lipolysis, and staphylococci contribute to cheese lipolysis. The non starter lactic acid bacteria (NSLAB) proliferate during Feta cheese from pasteurized milk ageing and may reach numbers as high as 107 108 cfu/g at the end of ripening in the warm room (Tzanetakis and LitopoulouTzanetaki, 1992; Manolopoulou et al., 2003). Mesophilic lactococci increase signicantly their numbers during this period and then decrease; enterococci also showed a similar behavior (Manolopoulou et al., 2003). On the other hand, micrococci, yeasts and coliforms seem to reach their maximum levels at day 4, for most cheeses. Facultatively heterofermentative lactobacilli are an important group throughout cheese ripening. Lb. plantarum is the dominant species constituting the 50% of the NSLAB population, and Lb. paracasei subsp. paracasei and Lb. brevis are also often found. In addition, enterococci and pediococci are among the predominant NSLAB of Feta cheese (Tzanetakis and Litopoulou-Tzanetaki, 1992). Other species, such as

E. Litopoulou-Tzanetaki, N. Tzanetakis / Small Ruminant Research 101 (2011) 1732 Table 2 Log counts (x SD) of microbial groups during ripening of Feta cheese from raw ewes milk. Days at 16 C 1 TAC Enterobacteriaceae Coliforms Staphylococci LAB on MRS agar LAB on M17 agar Lactobacilli Enterococci Yeasts Gram ve 9.94 7.25 7.89 6.50 8.87 9.01 7.94 5.98 6.37 7.04 1.6 0.3 1.0 0.1 0.3 0.3 0.4 05 1.6 0.9 4 8.95 6.24 6.43 5.91 8.80 8.65 8.04 6.83 5.86 6.25 0.3 0.8 0.8 1.0 0.2 0.3 0.0 0.2 2.2 0.7 Days at 4 C 30 9.18 3.04 3.08 3.54 8.48 7.68 8.36 5.68 4.43 4.92 0.8 2.6 2.7 3.1 0.6 0.2 0.3 0.7 0.8 0.7 60 8.37 2.49 2.65 4.88 8.29 6.80 8.18 5.53 4.77 2.60 0.2 2.2 2.3 0.2 0.1 0.1 0.1 0.2 0.3 2.3

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Lb. paraplantarum and Lb. pentosus may also be found frequently (Manolopoulou et al., 2003). In the cheese made from raw milk, all microbial groups decrease their counts from one to four days, except for lactobacilli and enterococci (Table 2). After one month of ripening, Enterobacteriaceae, coliforms, yeasts and Gram negative bacteria decreased their numbers rapidly. Enterococci also decline, and lactobacilli increase slightly (Vassiliadis et al., 2009). Microorganisms indicative of the hygienic quality of the product, Enterobacteriaceae and coliforms, were not detected in the 60-day-old cheese. The NSLAB microora of raw milk Feta cheese is complex (Table 3). Lactococci were the most abundant microbial group in the fresh cheese, with Lc. lactis subsp. lactis predominating. Lactobacilli were found more frequently than enterococci in the fresh cheese and Lb. paraplantarum prevailed over the other NSLAB species in the 30-day-old cheese. This species was also equally frequently found with Lb. paracasei subsp. paracasei in the cheese after 60 days. The traditional cheesemakers of Feta cheese either use raw milk and no starter at all or apply a milder-than-HTST heat treatment of milk and usually traditional yogurt as a starter. Proteolysis products increased with ageing and they were higher in cheese made from raw (R) than thermized (TS) milk throughout ripening. Amino acid levels accumulated in the cheese increased with ripening in both cheese types (Vassiliadis et al., 2009). Degradations of s CN occurred at a faster rate than -CN. At 60 days the amount of undegraded s -CN was similar in both cheese types. -Casein remained almost unattacked in R cheese, while in TS it underwent a low degree of degradation, increasing with ripening. The lipolysis products accumulated in the cheese after 1 month of ripening were 50% less in TS than R cheese. FFA content then decreased, possibly due to further degradation in R cheese, while in TS increased to higher than R levels (Table 4). Lb. plantarum isolates from Feta cheese exhibit lipolytic and peptidolytic activities (Xanthopoulos et al., 2000a,b). The amounts of amino acids accumulated in the milk are low and strain-dependent. Strains of slow and medium rate of acidication are also found. An array of peptidolytic enzymes can be detected in Lb. paracasei subsp paracasei isolates from Feta such as endocellular dipeptidase, aminopeptidase, endopeptidase, and carboxypeptidase enzyme activity (Bintsis et al., 2003). Their dipeptidyl

aminopeptidase activity is low, except for the diagnostic substrate Gly-Pro-pNA. This is an important feature for cheese ripening since caseins have high-proline content. In addition, Lb. paracasei subsp paracasei may degrade caseins (Bintsis et al., 2003). The amount of amino acids accumulated in the milk after 24 h is low and strain dependent (Mama et al., 2002). Moreover, the esterase activity increases with increasing carbon chain length (Bintsis et al., 2003). Less frequently isolated during ripening of Feta,

Table 3 Changes in numbers and kinds of non starter lactic acid bacteria (NSLAB) during ripening of Feta cheese from raw ewes milk. Ripening time (days) 1 Species Lc. lactis ssp. lactis Lc. lactis ssp. cremoris Lc. rafnolactis Ent. durans Ent. faecalis Ent. faecium Ent. saccharolyticus Lb. buchneri Lb. brevis W. confusa Ln. mesenteroides ssp. dextranicum Ln. mesenteroides ssp. mesenteroides Lc. lactis ssp. lactis Ent. faecalis Ent. avium Ent. saccharolyticus Ent. hirae Lb. paraplanarum Lb. paracasei ssp. paracasei Lb. plantarum Lb. rhamnosus Lb. buchneri Lb. brevis W. confusa Ln. mesenteroides ssp. mesenteroides Lb. paraplanarum Lb. paracasei ssp. paracasei Lb. paracasei ssp. tolerans Lb. plantarum Lb. buchneri Lb. brevis TOTAL No. of isolates 12 2 3 2 1 1 8 1 4 5 3 1 30 9 1 3 1 1 18 18 9 2 135 %

43.59

10.26

23.08

23.08

30

8.16

89.98 2.04

60

100

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Table 4 Values (x SD)a of pH, moisture %, NaCl-in-moisture %, proteolysis (o-PA)b , degradation of caseins and lipolysis (acid degree value ADV) during ripening of Feta cheese made from raw (R) and/or thermized (TS) ewes milk. Days at 16 C Cheese type pH Moisture% NaCl-in-moisture% Proteolysis (o-PA) % degradation of caseins s -CN -CN Lipolysis (ADV)
a

Days at 4 C 4 30 0.2 0.0 2.2 2.4 2.6 0.3 4.69c 4.83c 56.13c 58.63c 6.098d 6.724d 5929.77d 5241.73d 57.07c 39.4c 0.44c 1.73c 0.559c 0.244c 0.5 0.3 6.7 5.9 1.7 0.3 257.87 948.70 13.67 7.68 0.24 0.59 0.008 0.132 60 5.06c 5.06c 60.99c 62.0c 5.355cd 4.582cd 7383.21d 5087.02d 66.7c 65.2c 0.44c 10.17d 0.288d 0.349c 0.6 1.0 9.7 7.4 0.5 0.3 659.23 1125.64 19.01 10.38 0.24 2.66 0.094 0.08

1 4.96c 4.88c 55.03c 55.59c 0.1 0.1 1.4 0.7

R TS R TS R TS R TS R TS R TS R TS

4.81c 5.0c 51.1c 53.8c 1.956c 1.582c

2412.21c 245.93 1997.96c 272.72

Mean of three cheesemaking trials. In ppm of l-glycine equivalents. c,d Values of each raw of the same cheese differ (P < 0.05).
b

heterofermentative lactobacilli and leuconostocs, produce esterase, and degrade preferentially short-chain fatty acids (Vafopoulou-Mastrogiannaki et al., 1996). Leuconostoc strains may exhibit peptidase activities and hydrolyze the caseins (Vafopoulou-Mastrogiannaki and LitopoulouTzanetaki, 1996). Pediococcus pentosaceus isolates from Feta produce acid slowly and often form diacetyl and acetaldehyde when grown in milk (Litopoulou-Tzanetaki et al., 1989). These organisms also have dipeptidyl aminopeptidase and dipeptidase activity for peptides containing proline (Vafopoulou-Mastrogiannaki et al., 1994). P. pentosaceus strains also exhibit carboxypeptidase activities with signicant inter-strain differences. Their overall proteolytic activity on -casein is considerably higher than on s1 casein. Quite low esterase activities are found in P. pentosaceus compared to other organisms. Enterococci isolates from Feta are poor acidiers and some of them exhibit low proteolytic activity (Ambadoyiannis et al., 2005; Sarantinopoulos et al., 2001). Low peptidase activities are also exhibited by enterococci and their esterolytic system is rather complex; enterococci seem to preferentially degrade substrates with low molecular weight fatty acids (Sarantinopoulos et al., 2001). The main volatile compounds produced by enterococci are acetaldehyde, ethanol and acetoin. Enterococci isolates from cheese were able to withstand low pH (3.0) and high bile (1%) concentrations and exhibited antagonistic activities towards enterococci, LAB and Cl. sporogenes (Ambadoyiannis et al., 2005). Selected isolates had interesting probiotic and technological properties and could be used as adjuncts to make cheese. Isolates of yeasts from the surface of Feta cheese were also found to exhibit interesting probiotic properties (Psomas et al., 2001) and assimilated cholesterol into the cells (Psomas et al., 2003). It may be concluded, therefore that, the predominant LAB microora during ripening of Feta cheese may contribute to proteolysis and avor development of the cheese by virtue of an active proteolytic system, the ability to form

carbonyl compounds, their lipolytic activity and their acidifying abilities. The inuence of the various starter combinations on the microbiological, physicochemical and sensory characteristics of Feta cheese has been studied in several investigations. When thermophilic starter cultures, combinations of mesophilic LAB (Lc. lactis subsp lactis, Lactobacillus casei) and adjunct cultures (E. durans, Leuconostoc cremoris) were used to make Feta cheese, signicantly lower pH values were attained in the cheese with the mesophilic LAB and the adjuncts. At the same time, during the later stages of ripening the adjunct E. durans population accumulated higher concentrations of amino acids and small peptides, producing cheese with signicantly better avor (Litopoulou-Tzanetaki et al., 1993). Similar effects were also observed by the addition of E. faecium as an adjunct culture to make Feta cheese. The cheese avor was improved via enhanced degradation of s - and -CN and increasing concentrations of free amino acids (Sarantinopoulos et al., 2002). The main volatile compounds formed were ethanol, acetate, acetaldehyde, acetoin and diacetyl, with highest amounts determined for ethanol, followed by acetate. When P. pentosaceus was included in the starter culture for Feta cheese production, the cheese pH was reduced to lower levels and the amount of soluble nitrogen, even in the fresh cheese, was signicantly higher than in the control cheese (Vafopoulou-Mastrogiannaki et al., 1990). The levels of acetaldehyde in cheese made with P. pentosaceus were also signicantly higher and the avor was improved. P. pentosaceus accelerated cheese ripening and the maturation time of Feta cheese by 1 month. 2.3. Touloumissio cheese (Touloumotyri) Touloumissio is a cheese that ripens and is preserved in skin bags (Touloumia). It is in fact Feta cheese with some modications in technology. The cheese is manufactured from raw ovine or caprine milk or mixtures of them. The

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milk is curdled as for Feta. After salting and ripening, the cheese is cut in small pieces and put into skin bags. The production of this cheese has been dramatically reduced nowadays, mainly due to its preservation. The pH of the cheese is sometimes higher than in Feta (4.655.40), lactobacilli constitute the predominat microora (107 cfu/g) and enterococci and yeasts may also be found at high levels (range of log10 cfu/g 5.008.27 and 2.236.07, respectively) (Tzanetakis, 1990). It also seems, that the combined effect of low pH and high salt content (brine concentration 5.567.35%) regulate the survival of undesirable microorganisms. Thus, 35% of the retail samples were negative for coliforms. 2.4. Kalathaki of Limnos, PDO This cheese type is made at Limnos island with a technique similar to Feta, with the exception that the curd is drained into round baskets, where it is also surface salted. 2.5. Sfela (Feta of re), PDO Sfela is a semi-hard cheese produced in South Peloponnesus, particularly in Messinia province. The cheese is often called Feta of re because the curd is cooked at 3638 C. The cheese is made from raw ovine milk, mixtures of ovine and caprine milk, and it nowadays ripens in barrels, like Feta. The milk is curdled like Feta. The cooked curd is drained in cheese cloths and it is then put on tables, where it is inverted regularly, in order to obtain a round shape. The cheese is then cut in pieces, the size of Feta, and it is dry-salted (Anifantakis, 1991). 2.6. Teleme Teleme is a soft and pleasant in taste white-brined cheese, made from all milk types or mixtures of them. Teleme seems to have originated from northern Romania. It is manufactured all over the country, in the mainland, mainly. The technology of manufacture of Teleme cheese differs of that used for Feta in the procedure of draining and salting. The moulded curd of Teleme, is subjected to pressure to expel the whey. After draining the blocks of Teleme, they are immediately immersed into brine (usually 18% NaCl, 20 h) to allow penetration of salt (Anifantakis, 1991). LAB growth and evolution in Teleme cheese from ewe milk is similar to that in Feta, with Lb. plantarum becoming the predominant species with the progress of ripening. Lactococci are present in curd and cheese until the age of up to 75 d, while leuconostocs constitute an important microbial group until 30 d of ripening (Tzanetakis and LitopoulouTzanetaki, 1992). Isolates of P. pentosaceus, lactobacilli and leuconostocs from Teleme are also similar to isolates from Feta, in respect of their proteolytic and lipolytic activities (Mama et al., 2002; Tzanetakis and Litopoulou-Tzanetaki, 1989; Vafopoulou-Mastrogiannaki and LitopoulouTzanetaki, 1996; Vafopoulou-Mastrogiannaki et al., 1994, 1996). When mesophilic starter cultures (Lc. lactis subsp lactis and Lb. casei) were used concurrently with Ln. cremoris and E. durans to make Teleme the pH of the cheese

was lower than in cheese made with thermophilic starter cultures (Tzanetakis et al., 1995a,b). In addition, the amino acid levels in these cheeses were higher and the cheese avor was improved. P. pentosaceus is also benecial as an adjunct starter culture (Tzanetakis et al., 1991). In its presence, the growth of LAB was enhanced, the pH of the cheese was signicantly lower than in the control and the cheese proteolysis was accelerated. Thus, a concomitant acceleration of ripening by 1 month was noticed. The use of different starter cultures to make cheese from different types of milk was found to affect the rate of cheese proteolysis (Pappa et al., 2006). The thermophilic (yoghurt culture) and mesophilic (Lc. lactis subps lactis and subsp cremoris) starter resulted in cheeses with the highest and lowest degree of proteolysis, respectively. Proteolysis was of a descending order in cheeses made from sheeps, goats and cows milk on the basis of nitrogenous fractions. It was also concluded that the mixed (thermophilicmesophilic) or the thermophilic cultures might be more appropriate in the making of Teleme cheese from all milk types, as they resulted in cheeses of higher levels of proteolysis. 3. Soft cheeses 3.1. Anevato, PDO Anevato is a spread type cheese made from raw ovine or caprine milk or mixtures of both. The traditional cheese has a pleasant taste, especially the product made from goat milk. The cheese is produced at home as well as at creameries in the mountain region of Western Macedonia in north-west Greece and the nearby Thessaly. Traditionally, Anevato cheese was produced in Western Macedonia by shepherds with large ocks of goats and sheep. They renneted milk obtained in the morning just before taking the cattle out for feeding. During the day, the curd was raised and was ready to be drained on their return late in the afternoon. The shepherds used to visit their villages once a week to see their families and sell their cheese. The microbial counts of the traditional cheese from goat milk made throughout the whole lactation season were high (Hatzikamari et al., 1999). Lactic acid bacteria (Fig. 4) and Gram-negative organisms are the major components of the cheese microora throughout the entire lactation season. However, Enterobacteriaceae and coliforms numbers are decreased even during curd draining, due possibly to unfavourable conditions created by the increasing population of LAB and the decreasing pH. In addition, more acid conditions and higher levels of LAB in summer than spring and/or winter cheese coincided with lower levels of Enterobacteriaceae and coliforms in the respective cheeses. Both microbial groups also disappeared rapidly from cheese made in May and they underwent a more accelerated decline in cheese made in March than the cheese made in January. Lactococci dominate in the cheese until 15 days of storage at 4 C and lactobacilli become predominant in the 30and 60-day-old cheese. Leuconostoc spp. were present at low level initially, but occurred more frequently in cheese after salting. Lactococci were identied, mainly, as citrate

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E. Litopoulou-Tzanetaki, N. Tzanetakis / Small Ruminant Research 101 (2011) 1732

Fig. 4. Counts (log10 cfu/g; x SD) of lactic acid bacteria during manufacture and storage of Anevato cheese made throughout the whole lactation season.

fermenting. Lc. lactis subsp lactis, Ln. paramesenteroides and Lb. plantarum were the most frequently isolated species among Leuconostoc spp. and Lactobacillus spp., respectively. The isolates of lactococci differed considerably in respect of their acidifying, caseinolytic, aminopeptidase and esterase/lipase activities (unpublished data). Cheeses from heat-treated milk lacked the typical avor of raw milk cheese, despite the use of Lc. lactis subsp lactis strains as starter cultures, isolated from the traditional cheese (Xanthopoulos et al., 2000a,b).

3.2. Galotyri, PDO Galotyri is a soft cheese with sourish and brakish taste, made from ovine milk at the end of the lactation season. The cheese is made in all regions of the country by processes differing from region to region. The milk is boiled and put in a clay pot, and after 24 h it is salted (4% NaCl) and left for another two days. During this time the milk is mixed every 3 h. It is then transferred to a skin bag. This is repeated several times until the bag is lled. The cheese ripens for three months (Zygouris, 1956). Nowadays, the cheese usually ripens in barrels instead of skin bags. In this case, in order to avoid the growth of moulds, the cheese surface, is covered with melted fat. Retail cheeses have a low pH (3.744.0) and high moisture content (av. 76.4%). The predominat population of LAB is found at levels of 8.0 log10 cfu/g and undesirable bacteria (pseudomonads, enterobacteria and pathogenic staphylococci) are present at low levels (<2.0 log10 cfu/g). However, L. monocytogenes was found in some samples (Samelis and Kakouri, 2007). The main spoilage agent of the product is yeasts and L. monocytogenes is the main safety concern of this cheese.

Artisanal cheese manufactured by heating the milk to 8590 C and then adding natural starter, contained microbial populations ranging from 1.66 (enterobacteria) to 8.0 (lactococci) log10 cfu/g (Samelis and Kakouri, 2007). The NSLAB microora of the cheese samples was composed, primarily, of heterofermentative arginine-negative mesophilic lactobacilli and, secondary, of lactococci. Gas producing NSLAB of the Leuconostoc group made up a minor part of the NSLAB microora. Yeasts were counted at quite high levels (104 cfu/g). Although the population of NSLAB did not undergo major changes during storage, yeast counts grew signicantly to levels exceeding 7.0 log10 cfu/g after 14 days of storage at 4 C, due to favourable for their growth conditions in the cheese (pH 4.1 at 14 days). Yeast growth resulted in slimy yellowish colonization spots on the cheese surface after 14th day at 4 C, which made the cheese unt for human after 21 days. When a commercial starter containing Lc. lactis (80%) and S. thermophilus (20%) was used to make Galotyri, an amount of 28 volatile compounds were found, including alcohols, aldehydes, ketones, esters, sulphur compounds and others (Massouras et al., 2006).

3.3. Katiki, PDO Katiki is a soft cheese made at Domokos area and other parts of mountainous Greece. The milk is left at room temperature (2022 C) to sour and curdle with or without rennet. It is then drained in cheese cloths, salt is added, and the cheese is kept in cold stores until it is consumed. Pasteurized milk is nowadays used for cheese production. In retail samples the cheese pH was low (3.84.2) and may regulate the microbial growth. The predominant microora, LAB and yeasts, were counted at mean levels

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25

107 and 106 cfu/g, respectively. Enterococci were abundant (60.8% of the isolates), lactobacilli were found quite often (23.5%) and weisselae and leuconostocs were considerably less frequently (9.8% and 5.9%, respectively) isolated (Fourkiotis et al., 2004). Some samples were contaminated by coliform bacteria. It also seems that the low salt content (brine concentration 1.62.2%) may slightly affect the microbial growth. 3.4. Kopanisti, PDO Kopanisti is a cheese variety with a characteristic peppery taste made, mainly, from cows milk, produced in the islands of Aegean Sea, Cyclades. Its manufacture is not standardized but there are some common steps followed by the producers. Thus, cows milk is used, which is clotted with a small quantity of rennet over a period of a few hours. The curd is drained in a cheese cloth after which a quantity of older cheese (mana) is added and the curd is kneaded by hand and formed into balls, the size of a small orange. The cheese is then left in clay pots for several days until a green or blue green mould is formed on the surface. The cheese balls are then sprinkled with ne salt and kneaded again, so that the mould spreads evenly throughout the mass of the cheese. The cheese ripens for 12 months during which it is kneaded several times (Tzanetakis et al., 1987; Zygouris, 1956). In retail cheese samples, lactobacilli were >105 cfu/g in 86% of the cheeses, yeastsmoulds were >103 cfu/g in 78%, and coliforms were absent in 60% of the samples. The cheese pH (in 74% of the cheeses <5.0), moisture (in 72% of the samples >60%) and salt (in 52% of the cheeses <4.0%) ranged greatly. Lb. plantarum and Lb. casei subsp casei were the dominant lactobacilli and enterococci and P. pentosaceus were also isolated. Among yeasts, Pichia membranefasciens dominated while moulds were characterized as Penicillium commune (Tzanetakis et al., 1987). 3.5. Pichtogalo Chanion, PDO It is a soft spreadable cheese. The milk is left to sour for 24 h. The cheese is drained, salt is added at 1% and the product is ready for consumption. The product is made at Chania of Crete island. The gross composition of this cheese was found to be as follows (Papageorgiou et al., 1998): moisture content 61.63% 4.67; fat in dry matter 54.03% 7.73; protein content 14.23% 1.72; salt content 1.02% 0.38; aw 0.99 0.003; and pH 4.36 0.25. Salmonella and L. monocytogenes were not detected, coagulase positive staphylococci were present in 6.45% of the samples and B. cereus and sulte-reducing clostridia were isolated from 14.51% and 40.32% of the samples, respectively. The cheeses contained high populations of coliforms, enterococci, yeasts and moulds and psychrotrophic bacteria, while lactic acid bacteria constituted the predominant cheese microora. It was also shown that high quality of Pichtogalo Chanion cheese can be produced using a pasteurized mixture of ewes and goats milk and 4% of mesophilic starter culture.

4. Semi-hard cheeses 4.1. Kasseri, PDO Kasseri is a traditional Greek cheese of pasta lata type with a pleasant avor, manufactured from ovine milk or mixtures of ovine and caprine milk. The cheese was introduced to Greece from the neighboring Balkan countries at the end of the 19th century, initially to Thessaly, Central Greece, from where its manufacture was spread to other areas of the country. Plain ovine or mixtures of it with caprine or bovine (510%) milk are used for the cheese manufacture (Anifantakis, 1991; Zygouris, 1956). The milk is curdled at 32 C, the coagulum is cut and it is usually cooked at 38 C. The curd is cut in large pieces, and the baski is left to drain in a cheese cloth and ripen over a period of 824 h at 1820 C. The ripened baski is cut into small thin uniform slices, which are put into a basket (kanelo). The basket is dipped into water (at 70 C), where it is turned in a way, in order to facilitate the penetration of water between the slices. The cheese thus becomes soft, elastic and its uniformity is achieved by its thorough mixing with a wooden ladle. Then, the cheese is cut in pieces, formed to the shape of spheres and introduced into the moulds, where the cheese remains for 24 days. The cheese is salted every 23 days, until it accepts 1214 saltings. The ripening in the warm room (18 C) lasts for at least 70 days; it is then transferred to cold stores (02 C). The pH of the traditional fresh cheese is over 5.0 and it is increased slightly throughout ripening. Moisture content range between 42 and 46%, fat content in dry matter is around 45% and the brine concentration of the mature cheese is around 3.0%. LAB constitutes the predominant microora throughout cheese ripening (107 cfu/g) and enterococci are present at quite high levels (104 106 cfu/g), depending on the milk quality. Staphylococci, coliforms and yeasts are found at levels from negligible to quite low in the fresh cheese and decrease their numbers during ripening. In the fresh cheese (baski) pediococci and lactobacilli constitute the 25% of the predominant LAB microora and enterococci are the most abundant microbial group (50%). The proportion of pediococci is increased with the progress of ripening and at 120 days the composition of the LAB microora is: pediococci 50%, enterococci 30% and lactobacilli 20%. Selective isolates of pediococci and enterococci were found to produce small quantity of acid and this makes them appropriate as adjuncts. Pediococci were more proteolytic than enterococci and may contribute to the acceleration of cheese ripening and avor through their strong aminopeptidase and other proteolytic activities (unpublished data).

4.2. Krassotyri (Possias) Krassotyri or Possias has slightly sour taste and organoleptic properties very much affected by the properties of wine sediment, where the cheese is put after ripening (Anifantakis, 1991). The wine sediment is called Possia, from which the cheese was named. The cheese

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is made from ovine or caprine milk or mixtures of both, mainly in Kos island, Dodekanisos. The milk from the afternoon milking is heated until boiling and it is then transferred into swallow clay pots. The next morning, the milk fat risen on the milk surface is removed and the boiled milk is thoroughly mixed with fresh milk from the morning milking. The milk is coagulated with rennet (usually traditional), the curd is cut and then is transferred into baskets (tyrovolia) to drain for 24 h. After that, the baskets are removed the surface of the cheese is salted and then is put in vessels. The cheese is transferred in cool places to ripen for 2030 days and after that it is removed from the vessels and put on shelves to dry. The dry cheese is again put in the vessels and wine sediment is added to cover the cheese. In this environment the cheese ripens for one more week. At the end, the cheeses get a reddish colour of the sediment (Anifantakis, 1991). The NSLAB constitute the predominant microora of the cheese and are found at similar levels on the cheese surface and interior (Kalavrouzioti et al., 2004). The salttolerant and the coliform microoras were less on the cheese surface than the interior by 1 log10 cfu/g. This was possibly due to the effect of possia and not of the pH and/or the NaCl, which were similar for the cheese surface (4.38 and 2.38%, respectively) and interior (4.38 and 2.49%, respectively). Possia also affected the composition of the lactic microora. Thus, the surface LAB microora was composed of lactobacilli and enterococci (50% each), with E. faecium predominating. The LAB microora of the cheese interior consisted of lactobacilli, enterococci and lactococci, but lactobacilli predominated. Lb. plantarum and E. faecium were the most frequently isolated species. In addition, the species Lb. rhamnosus, Lb. casei and Lb. paracasei were not isolated from the cheese surface, while no isolates of Lb. buchneri, E. gallinarum and E. pseudoavium were found in the cheese interior. The mean moisture of the cheese surface and interior was 54.4 and 55.6%, respectively. 5. Hard cheeses 5.1. Graviera, PDO Graviera is the nest cheese among the Greek hardcheeses. It is distinguished for its pleasant aroma and

its ne taste. Sheeps milk is mainly used for cheese manufacture, but cows milk or mixtures of it with sheeps and goats milk are also used, the latter not exceeding 20%. The mature cheese has a smear rind and, usually, exhibits small or larger irregular openings and could be characterized as slits of holes rather than eyes. The Greek Graviera was initially manufactured in the second decade of the 20th century. Nowadays, many types of Graviera are traded and are distinguished from the name of the region where they are produced (Graviera of Crete, Naxos, Mytilini, Tinos, Lamia, Kerkyra, Larissa, Dodoni and others). The milk is renneted at 3336 C with a quantity of rennet able to curdle the milk within 2530 min. The use of starter is optional (Zygouris, 1956). The curd is cut in small pieces, the size of corn and cooked at 4850 C under continuous stirring. The curd is cut in pieces, each one enough to ll one mould. The cheese in the moulds is pressed for 24 h. After pressing, the cheese is put on shelves in the salting rooms (1416 C) where it is surface salted by 3540 or even 60 saltings depending on the cheese height. The cheese ripens for 34 months. Nowadays, the cheese milk is pasteurized and starter (mesophilic + thermophilic or fresh yoghurt) is used for cheese manufacture in modern dairies (Anifantakis, 1991). The traditional Graviera Kritis cheese (from raw ewes milk) contains Enterobacteriaceae and coliforms at high populations (Fig. 5). In addition, NSLAB constitute the most abundant microbial group during ripening. They increase their numbers in the cheese before salting and then gradually decrease till the end of storage (6 months). The NaCl-in-moisture of the cheese was found to range between 2.42 and 3.05% and moisture content of the mature cheese was 31.52%. At this time the 65.36% and 14.39% of s - and -CN, respectively, of the cheese was reduced (Table 5) by the native ora, mainly the lactic. At the various steps of cheese manufacture and ripening, different NSLAB species were found (Table 6). In the curd of the cheese lactococci predominated. The main Lactococcus species was Lc. lactis subsp. lactis. Its high frequency in the curd allows us to speculate that Lc. lactis population act as starter to drive the initial acidication of the curd. Leuconostocs in the curd constituted a signicant part of

Table 5 Values (x SD)a , of pH, moisture%, NaCl-in-moisture%, degradation of caseins, proteolysis (o-PA)b and lipolysis (acid degree value ADV) during ripening of Graviera Kritis cheese made from raw ewes milk. Days at 17 C Before cooking pH 6.27 0.23 Moisture% 67.1 4.15 NaCl-in-moisture% % degradation of caseins s -CN -CN Proteolysis (o-PA) Lipolysis (ADV)
a b

After cooking 6.48 0.15 53.69 9.48

Before salting (1 day) 5.3 0.049 45.51 3.01

After salting (4 days) 5.38 0.075 41.92 4.14 3.05 0.82

45 days 5.38 0.2 39.44 1.1 2.53 0.28 33.12 12.15 8013.23 0.3315 12.91 3.94 986.81 0.212

90 days 5.07 0.079 33.6 1.96 2.417 0.14 65.36 14.39 9016.8 0.5759 2.88 2.77 1902.03 0.045

0 0 7069.72 2774.35 0.227 0.03

Mean of three cheesemaking trials. In ppm of l-glycine equivalents.

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9 8 7 6 log10 cfu/g 5 4 3 2 1 0 Before cooking After cooking

Coliforms Enterobacteriaceae

45

90

Time of ripening (days)

Fig. 5. Populations (log10 cfu/g; x SD) of Enterocacteriaceae and coliforms throughout manufacture and ripening of Graviera Kritis cheese.

lactic microora. Leuconostoc mesenteroides subsp. dextranicum and Ln. pseudomesenteroides found in the curd, may be greatly responsible for the formation of small holes in the cheese body. Lb. paracasei subsp. paracasei, Lb. plantarum
Table 6 Changes in numbers and kinds of NSLAB isolates during ripening of Graviera Kritis cheese made from raw ewes milk (number from three cheesemaking trials). Ripening time Before cooking Species Lc. lactis ssp. lactis Lc. lactis ssp. cremoris Lc. rafnolactis Lb. paracasei ssp. paracasei Lb. brevis Leuc. mesenteroides ssp. dextranicum Leuc. pseudomesenteroides E. faecium Lb. paracasei ssp. paracasei Lb. paracasei ssp. tolerans Lb. plantarum Lb. paraplantarum Lb. curvatus Lb. brevis Lb. buchneri C. piscicola Leuc. pseudomesenteroides E. durans Lb. paracasei ssp. paracasei Lb. plantarum Lb. paraplantarum Lb. biffermentans Lb. coryneformis Lb. brevis Lb. parabuchneri Leuc. mesenteroides ssp. mesenteroides Leuc. mesenteroides ssp. dextranicum W. confusa E. faecalis E. malodoratus No. of isolates 4 5 2 1 1 3 6 1 8 2 7 1 1 5 1 1 1 1 24 6 3 1 1 1 1 1 1 1 3 1 %

47.83 8.7

39.13 4.34

45 days

89.29

3.57 3.57 3.57

90 days

84.09 4.55 2.27 9.09

and heterofermentative lactobacilli, found in the curd in low proportions may play a minor role to the open body formation by gas production from citrate or lactose, respectively. The proportion of lactococci isolation was decreased with ripening and these organisms were not present in the cheese at 45 days. On the contrary, the facultatively heterofermentative lactobacilli increased their frequency with ripening. Therefore, their contribution to cheese ripening seems possible. Lb. paracasei subsp. paracasei predominated over the NSLAB in the mature cheese. Enterococci participated at a quite low percentage in the predominant lactic microora throughout cheese ripening. Leuconostocs constituted a low proportion of the cheese microora throughout ripening and weissellae and carnobacteria emerged at some ripening stages of the cheese (unpublished data). Graviera cheese made from cows milk had a high pH (>5.3) and brine concentration ranged from 0.43 to 4.10 during ripening for 180 days. The WS-N increased rapidly until the cheese was taken out of the warm room, due to the activity of the increasing population of proteolytic microorganisms. Proline formation was mainly due to the starter (L. bulgaricus, propionibacteria) activity and peak formation of diacetyl at the end of ripening in the warm room was related to the rapid increase of almost all the desirable microbial groups. The amounts of C2 to C6 fatty-acids were increased throughout ripening for 180 days. Highest quantities of fatty acids from C10 to C16 were recorded at the age of 3 months with palmitic acid being in highest amounts followed by myristic acid (Zerridis et al., 1984). Addition of combination of thermophilic and/or mesophilic and thermophilic starters along with Propionibacterium freundereichii subsp. shermanii did not affect the gross composition of the ripening cheeses. However, the pH and the enterococci counts in the cheeses made with starters were signicantly lower than the control and coliforms were not detected (Kandarakis et al., 1998).

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E. Litopoulou-Tzanetaki, N. Tzanetakis / Small Ruminant Research 101 (2011) 1732 Table 7 Log microbial counts, pH, moisture % and NaCl-in-moisture % (x SD) of Orinotyri cheese from raw sheeps milk. Fresh cheesea Enterobacteriaceae Coliforms Yeasts pH Moisture % NaCl-in-moisture%
a b

5.2. Kefalograviera, PDO Kefalograviera is a hard cheese, with a rm body and many holes or eyes, mild to medium piquant avor and colour from nearly white to slight yellow. A mixture of 60/40 cow/sheep milk is usually used, but goats milk may also be incorporated in a proportion not higher than 20%. Kefalograviera is manufactured, mainly, in mountains of Greece. As the name of the cheese is indicating its technology of manufacture has elements of both, Kefalotyri and Graviera cheese (Anifantakis, 1991). When full-fat and low-fat cheeses were manufactured with different starters, it was observed that the type of starter did not affect the composition of the low-fat cheese but the latter received signicantly higher avor scores that the low-fat control (Katsiari et al., 2002). In addition, the fat content affected the pattern of proteolysis, water soluble nitrogen was signicantly affected by the addition of adjunct cultures and the production of low molecular mass nitrogenous compounds was enhanced (Michaelidou et al., 2003). The experimental cheese had signicantly higher levels of acetone at 90 days and acetic acid, diacetyl and acetoin at 180 days than the control low-fat cheese, which had signicantly higher levels of butan-2-ol and butan-2one than the former cheeses at both sampling ages. The type of starter also affected the total levels of free fatty acids (Kondyli et al., 2003). 5.3. Kefalotyri Kefalotyri (Kefali + tyri = head + cheese) is characterized by high hardness, salty taste and strong avor. It is made from sheeps or goats milk or mixtures of them. Cows milk may also be used. The cheese technology was introduced from Italy to Greece centuries ago. It is manufactured in various parts of Greece with technologies that differ from area to area and it is traded under the name of the region where it is manufactured (Crete, Naxos, Thessaly, Kefalonia, Epirus, etc.). The milk is partially skimmed (5.86.0% fat content) and it is usually pasteurized. Starter cultures are added in the cheese milk. After coagulation, the coagulum is cut into small particles. The temperature is then raised to 4345 C under continuous stirring. The coagulum is cut into pieces, the size of one mould. The pieces are put into moulds and they are then put into press. The cheese is placed in the ripening room (1416 C) for 24 h and then in brine (1820 B) for 12 days. It is transferred again in the ripening room where it is given 2025 dry saltings. The cheese is nally washed with brine and transferred into a cold store (24 C) to continue ripening. The pH of the cheeses sold in retail shops was found to be >5.0, the percent moisture in the majority of the cheeses was 38% and the brine concentration 6.8%. The lactic microora present in the fresh cheese from raw cows milk was composed of streptococci (S. thermophilus), lactococci (Lc. lactis), enterococci (E. faecalis, faecium and durans), lactobacilli (obligately as well as facultatively heterofermentative) and leuconostocs (predominantly Ln. lactis). Streptococci and lactococci were not isolated after the 15th

Three-months-old cheeseb 4.93 0.91 4.65 1.08 ND2 5.79 0.28 36.4 3.05 6.64 1.85

7.94 7.41 1.99 6.31 49.16 4.13

0.19 0.29 0.77 0.19 5.82 1.83

Means of ve cheesemaking trials. Not detected.

day of ripening; leuconostocs were also present among the predominant LAB of the 2-month-old cheese (LitopoulouTzanetaki, 1990). In Orinotyri (Orinos + tyri = mountainous + cheese), a local name for Kefalotyri, made from raw ewes milk, the following were observed (Prodromou et al., 2001). Enterobacteriaceae and coliform counts in the tenday-old cheese, were high; their average counts in the three month-old-cheese were smaller by 3.02 and 7.76 log10 cfu/g, respectively, than counts in the fresh cheese (Table 7). At the same time, the pH value of the fresh cheese was high (>6.31 pH units) and it was reduced by 0.52 pH units in the mature cheese. Yeasts were counted at low levels in the fresh cheese (200 cfu/g) and they were not detected in the mature product. S. aureus was not detected in either fresh or mature cheese. The levels of NSLAB were decreased signicantly with ripening. Enterococci predominated in the fresh Oritotyri. Lactococci, lactobacilli and pediococci were also less frequently found in the fresh cheese. The NSLAB microora was complex in both, 10-day and 3-month-old cheese (Table 8). Fifteen different species were characterized in the fresh cheese, with E. faecalis being predominant, followed by P. pentosaceus, Lc. lactis subsp. lactis and Lb. curvatus. In the 3-month-old cheese fourteen different species were found and the predominant at decreased frequencies were: E. faecalis, E. faecium, Lb. paracasei subsp. paracasei and Lb. coryniformis subsp. torquens. There were also species found in the fresh cheese (E. faecalis var. liquefaciens, E. malodoratus, Lc. garvieae, Lb. paracasei subsp. tolerans, Lb. curvatus) which were not detected in the mature cheese and others that emerged as ripening progressed and were found in the 3-month-old product (Lc. rafnolactis, Lb. coryneformis subsp. torquens, Lb. plantarum, Lb. paraplantarum, Lb. homohiochii).

5.4. Ladotyri, PDO Ladotyri is a hard cheese, produced mainly in the island of Mytilini. It is considered as a type of Kefalotyri of good quality. The cheese is made from ewes or mixtures of ewes and goats milk raw at home or pasteurized in dairies. Ladotyri is made by different methods but the overall technology is very similar to that used for Kefalotyri manufacture. Ladotyri is preserved in olive oil, as indicated by its name (ladi = olive oil; tyri = cheese).

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In cheese made from raw ewes milk microbial counts were measured at high levels (mean counts of coliforms 1.8 105 cfu/g; yeasts 5.8 104 cfu/g; halotolerant bacteria 1.5 105 cfu/g; psychrotrophs 1.3 106 cfu/g) and lactic acid bacteria constituted the predominant microora (mean counts 3.2 108 cfu/g and 4.1 108 cfu/g for cocci Gram positive and catalase negative and lactobacilli, respectively). Enterococci predominated (58%) over lactobacilli (36.2%), lactococci, S. thermophilus and leuconostocs, the cheese pH was over 5 and its moisture and NaCl% content were 31% and 3%, respectively (Fista et al., 1996). 5.5. Manoura Manoura is a hard traditional farmhouse Greek cheese variety manufactured by producers of Sifnos island in the Aegean sea (Cyclades complex) from raw ovine or a mixture (50:50) of raw ovine and caprine milk from local herds. The cheese is made as follows: the milk is not heattreated and it is not inoculated with a starter culture. Animal rennet deriving from the stomach of a young animal is used to curdle the milk. The coagulum is cut into pieces, the size of a nut, and after a thorough stirring, it is left to settle in the bottom of the vat. The curd is transferred to clean baskets (tyrovolia) to drain. The cheeses are salted for 23 days with coarse salt, on a different side each day, after removal from the basket and inversion. After that, the cheeses are removed from the baskets and put on beds of straw to dry for 34 months. The cheeses are then put into barrels with red wine to soften for about a week (510 days). They are then put into barrels and covered with wine
Table 8 Species of lactic acid bacteriaa isolated from fresh and three-month-old Orinotyri. % of the isolates Fresh Lactococcus lactis ssp. lactis Lc. garvieae Lc. rafnolactis Enterococcus faecalis E. faecalis var. liquefaciens E. faecium E. hirae E. saccharolyticus E. malodoratus Enterococcus spp. Weisella paramesenteroides Pediococcus pentosaceus Lactobacillus paracasei ssp. paracasei Lb. paracasei ssp. tolerans Lb. curvatus Lb. coryneformis ssp. coryniformis Lb. coryniformis ssp. torquens Lb. plantarum Lb. paraplantarum Lb. homohiochii TOTAL 16.5 1.6 24.7 1.6 6.6 1.6 1.6 1.6 1.6 1.6 23.0 1.6 1.6 11.6 3.2 100 Three-month-old 25.0 1.5 27.9 10.3 1.5 1.5 4.4 2.9 8.8 2.9 8.8 1.5 1.5 1.5 100

sediment for 1 day. The cheeses are afterwards moved to empty barrels, where they are kept until sold (Gerasi et al., 2003). Changes in the microora of Manura from raw ovine milk were studied during ripening (Gerasi et al., 2003). In general, the various microbial groups developed better on the cheese surface than in the interior. Red wine treatment had an inhibitory effect on their growth and microbial counts decreased more rapidly on the cheese surface than in the interior (Fig. 6). Enterobacteriaceae and coliforms were reduced sharply during ripening on a straw bed (3 months) and they were not detected in mature cheese. Lactic acid bacteria predominated over the other microbial groups throughout ripening. Ln. mesenteroides subsp. cremoris, P. pentosaceus and Lb. paracasei subsp. paracasei, frequently found in maturing cheese, could be used as starters to make this cheese. Moreover, the lactic acid bacteria predominating in mature cheese, such as Weissella paramesenteroides, Lb. bifermentans and Lb. brevis, may contribute to cheese ripening though their biochemical activities. 5.6. Xinotyri Xinotyri is a farm cheese variety, manufactured from raw goats milk from indigenous breeds in the island of Naxos (Cyclades complex). The milk is renneted and curdled in about 24 h at room temperature. Sometimes, a small amount of cheese whey from the previous day is used as starter. The curd is transferred in a cheese cloth to drain for 23 h and then dry salt at 1.5% is added and the curd is kneaded for the uniform dispersion of salt. The curd is transferred into plastic truncated conical moulds for 34 days and turned over daily. After that, the moulds are removed and the cheese is left to ripen on wooden shelves for 3045 days (Bontinis et al., 2008). In the traditional cheese the following microbiological changes were observed (Bontinis et al., 2008). Early in ripening (6-day), the cheeses contained signicantly higher populations of mesophilic LAB and lactococci than thermophilic LAB and streptococci. During the later stages of ripening, there were major declines in populations of NSLAB, signicantly higher for the mesophilic as compared to the thermophilic part of the NSLAB ora. Enterococci and yeasts were counted at quite high levels (105 and 107 cfu/g, respectively), early in ripening and declined to levels as low as 104 cfu/g for enterococci, and 103 cfu/g for yeasts. 5.7. Melichloro (melipasto) Melichloro is the traditional cheese of Limnos island. The cheese is made from raw ewes milk at the end of the lactation season. The milk, warm as it is after milking, is coagulated within 1 h. The curd is cut into very small pieces and transferred into baskets for draining on wooden shelves. During draining, the cheese in the baskets is reversed every 1 h for 34 times. Ten hours after curdling the cheese is dry salted several times on each side. Then, the baskets are put in a wooden construction like a cage, hung from a tree, under the sun to dry and the baskets are inversed every day. After

a Isolates from RA, M17 agar and CAA. All isolates from CAA were enterococci. Isolates from M17 agar were; fresh cheese: lactococci 11 and enterococci 11 strains; three-month old cheese: lactococci 18 and enterococci 10 strains. Isolates from RA were lactobacilli, P. pentosaceus and W. paramesenteroides.

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Table 9 Mean log microbial counts (log10 cfu/g; x SD) during storage of Manouri cheese in spring and summer (mean of three cheesemaking trials). Means in the same row with different superscripts were signicantly different (P < 0.05). Days at 4 C Microbial group Total aerobic count Sample from Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Surface Interior Season Spring Spring Summer Summer Spring Spring Summer Summer Spring Spring Summer Summer Spring Spring Summer Summer Spring Spring Summer Summer Spring Spring Summer Summer Spring Spring Summer Summer Spring Spring Summer Summer 0 (cheese after draining) 4.25a 4.73a 4.83a 4.91 2.67a 0.00a 3.16a 1.59a 2.28a 0.98a 3.06a 1.17a 5.07a 4.98 4.13a 5.17 3.97a 3.08a 4.16a 4.45a 2.90a 2.25a 2.50a 2.86ab 4.90 4.65 3.53a 2.41a 4.06a 3.92 3.64a 3.37 0.24 0.75 0.03 0.42 1.12 0.00 1.25 0.51 0.64 0.68 0.82 0.81 0.53 0.55 0.65 0.39 0.76 1.60 0.40 1.00 1.39 0.67 0.10 1.18 1.06 1.01 0.22 1.14 0.98 1.42 0.52 1.03 5 6.01b 5.10ab 6.45b 6.25 3.04a 2.93b 5.69b 2.94a 3.45ab 3.29b 5.12b 2.47a 5.52ab 5.10 5.01ab 4.76 5.52ab 4.55ab 4.73b 4.95a 4.97ab 2.65a 4.26b 2.54a 5.30 4.85 5.41b 4.65b 6.21b 3.89 5.49b 3.43 0.44 1.25 0.99 1.22 0.04 0.14 0.95 1.94 0.74 0.71 0.79 0.99 0.47 0.48 0.87 0.71 0.47 0.48 0.42 0.26 1.09 0.97 0.23 0.99 0.23 0.57 0.52 0.50 0.96 0.52 0.29 0.83 10 6.69bc 5.82abc 7.48bc 6.31 5.65b 5.14c 7.14bc 5.32b 4.39bc 2.41ab 6.59c 5.65b 5.76ab 5.05 5.71bc 5.23 5.78bc 5.09ab 6.11b 5.10a 5.15ab 3.30ab 5.30c 4.26bc 4.93 4.67 5.65b 5.22b 5.92b 4.15 5.63b 4.12 0.96 0.68 0.65 0.70 1.25 1.13 0.82 1.09 0.99 0.39 0.45 0.53 0.54 0.18 0.72 0.23 0.56 0.11 0.67 0.25 1.76 1.21 0.46 0.35 0.25 0.58 0.97 0.10 0.12 0.69 0.45 0.85 20 7.32c 6.87c 8.34c 6.33 7.26b 5.37c 8.32c 5.89b 5.34c 1.78ab 8.32c 5.75b 6.54b 5.17 7.05c 5.16 7.02c 5.36b 8.21c 6.40b 6.42b 4.66b 7.17d 4.75c 6.46 5.40 6.81b 5.34b 6.68b 5.12 6.64c 4.39 0.64 0.84 0.66 0.75 1.59 1.18 0.56 0.77 1.15 1.23 0.56 0.97 1.04 0.46 0.65 0.32 1.18 0.41 0.38 0.42 1.45 1.05 0.35 0.69 0.81 0.37 1.09 0.32 0.76 0.16 0.12 0.47

Enterobacteriaceae

Coliforms

Total LAB

Lactococci

Enterococci

Yeasts

Staphylococci

Fig. 6. Counts (log10 cfu/g; x SD) of different microbial groups at three main points of ripening of Manura cheese.

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45 days the baskets are removed and the cheese wheels are left in the cage for further drying. When they are hard enough, the cheeses are washed with sea water and dried with a cloth. The mean pH of ve cheese samples was 4.49 0.2, its moisture content 31.4% 1.5 and the mean brine concentration was measured at 5.8% 2.1. The log counts of various microbial groups ranged between 1.63 (coliforms) and 7.11 (lactococci). The NSLAB of the mature cheese consisted, mainly, of Lb. paraplantarum (56% of the isolates) and Lb. paracasei subsp. paracasei (34%) (unpublished data). 6. Whey cheeses 6.1. Manouri, PDO Manouri is a whey cheese that has many similarities with myzithra, but it is creamier and less salty, with a delicate texture and superior sensory quality (Anifantakis, 1991; Zygouris, 1956). Manouri is a traditional product of Western Macedonia, in northern Greece, and it has long been made from the whey obtained during the production of Batzos, a semihard cheese derived from caprine and ovine milk. Today, Manouri is also produced from the whey of cheeses made solely from ovine milk, and although a whey cheese, it is the main product of this cheesemaking process. The overall technology of Manouri manufacture has much in common with the process for making myzithra, but the nal moisture content (5060%) is lower than that of the latter cheese. Selected microbial groups were counted throughout storage for 20 days at 4 C in batches of Manouri cheese (Lioliou et al., 2001). The counts of all the microbial groups increased (P < 0.05) throughout storage and reached higher levels in cheeses made in summer than in those made in spring. Moreover, the micro-organisms developed better on the cheese surfaces than in the interiors, especially in the summer (Table 9). The pH (6.787.33) and salt-in-moisture content (2.533.72) of the cheeses did not seem to affect the growth of bacteria and yeasts. The isolates of Enterobacteriaceae were mainly Hafnia, while the isolates from Baird-Parker medium were mainly staphylococci. There was a great diversity of yeasts species, but Debaryomyces hansenii and Pichia membranefasciens predominated. The isolates of Enterobacteriaceae, staphylococci and-to a lesser extent- yeasts were proteolytic, but the free amino acid (NNH2) content of the cheese did not increase signicantly during storage (136.4225.2 mg/kg glycine equivalent). It also seems that milk fat was not degraded to any great extent (acid degree value 0.090.19) by the lipolytic activity of the strains. The main enzymes detected in selected isolates of Enterobacteriaceae were leucine aminopeptidase and phosphohydrolase. 6.2. Myzithra Myzithra is a traditional Greek whey cheese made by heating the whey at a temperature of 8892 C (Zygouris, 1956). The whey heated under continuous stirring to obtain a temperature of 8892 C within 4045 min. For a better quality of Myzithra, whole milk is added to the whey

(in a proportion of 35%) when it obtains a temperature of 6570 C. Salt (11.5%) may also be added to the whey while it is heated. 6.3. Xinomyzithra, PDO This type of Myzithra is made from the whey obtained during the production of Kefalotyri and Graviera from sheeps and goats milk. It is a traditional product of Crete island. For the production of Xinomyzithra, Myzithra is rst manufactured, which is cooked and drained more severely and it is then pressed for one week. Salt is added after that and the cheese is mixed. Then, the cheese is placed in barrels which are transferred in rooms of 510 C temperature to ripen for a period of two months. 6.4. Xinotyri (Klotsotyri, giza, artymi or prentza) Xinotyri (xyno = sour + tyri = cheese) is the cheese type that is made from the whey obtained during the production of butter with a pleasant sourish and salty pleasant taste. For its production, the whey is put into a copper vat, where it is heated until curdling. The cheese is left to drain in a cheese cloth for several hours. The drained cheese is salted and then put in either barrels or skin bags or tins for two or three months to ripen. The NSLAB are detected at low levels in this cheese. The cheese pH is <5.0, mean moisture % is 40 and mean NaCl% is 1.4. Mean % fat on dry matter is 14.8 (unpublished data). Conict of interest statement None of the authors (E. Litopoulou-Tzanetaki, N. Tzanetakis) has a nancial or personal relationship with other people or organisations that could inappropriately inuence or bias the paper entitled Microbiological characteristics of Greek traditional cheeses. References
Ambadoyiannis, G., Hatzikamari, M., Litopoulou-Tzanetaki, E., Tzanetakis, N., 2005. Probiotic and technological properties of enterococci isolates from infants and cheese. Food Biotechnol. 18, 307325. Anifantakis, E.M., 1991. Greek Cheeses: A Tradition of Centuries. National Dairy Committee of Greece, Athens. Bintsis, T., Vafopoulou-Mastrogiannaki, A., Litopoulou-Tzanetaki, E., Robinson, R.K., 2003. Protease, peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine. J. Appl. Microbiol. 95, 6877. Bontinis, T.G., Mallatou, H., Alichanidis, E., Kakouri, A., Samelis, J., 2008. Physicochemical, microbiological and sensory changes during ripening and storage of Xinotyri, a traditional Greek cheese from raw goats milk. Int. J. Dairy Technol. 61, 229236. Fista, G., Tzanetakis, N., Zerridis, G., 1996. Microbiological characteristics of Ladotyri of Mytilene a traditional Greek cheese. In: Fifth Symposium on Lactic Acid Bacteria: Genetics, Metabolism and Applications, Veldhoven, The Netherlands (Feredation of European Microbiological Societies, Netherlands Society for Microbiology, Foundation Antonie van Leeuwenhoek). Fourkiotis, A., Vaadou, E., Litopoulou-Tzanetaki, E., Tzanetakis, N., 2004. Microora of traditional cheese Katiki Domokou. In: 20th Medical Congress of Force Forces, Thessaloniki, Greece. Gerasi, E., Litopoulou-Tzanetaki, E., Tzanetakis, N., 2003. Microbiological study of Manura, a hard cheese made from raw ovine milk in the Greek island Sifnos. Int. J. Dairy Technol. 56, 117122.

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E. Litopoulou-Tzanetaki, N. Tzanetakis / Small Ruminant Research 101 (2011) 1732 Samelis, J., Kakouri, A., 2007. Microbial and safety qualities of PDO Galotyri cheese manufactured at the industrial or artisanal scale in Epirus, Greece. Int. J. Food Sci. XIX (1), 81. Sarantinopoulos, P., Andrighetto, C., Georgalaki, M.D., Rea, M.C., Lombardi, A., Cogan, T.M., Kalantzopoulos, G., Tsakalidou, E., 2001. Biochemical properties of enterococci relevant to their technological performance. Int. Dairy J. 11, 621647. Sarantinopoulos, P., Kalantzopoulos, G., Tsakalidou, E., 2002. Effect of Enterococcus faecium on microbiological, physicochemical and sensory characteristics of Greek Feta cheese. Int. J. Food Microbiol. 76, 93105. Tzanetakis, N., 1990. Microbiology and composition of Touloumisio and Xynotyri, two traditional Greek sheeps milk cheeses. MicrobiologieAliments-Nutrition 8, 281288. Tzanetakis, N., Hatzikamari, M., 1994. La ore lactique supercielle du fromage Feta. In: Colloque de Societe Francaise de Microbiologie: Gestions des Populations Microbiennes dans les Industries Agroalimentaires, Dijon, France. Tzanetakis, N., Hatzikamari, M., Litopoulou-Tzanetaki, E., 1996. Yeasts of the surface microora of Feta, a traditional Greek cheese. In: Symposium FIL-IDF: Yeasts in the Dairy Industry: Positive and negative aspects, Copenhagen, Denmark. Tzanetakis, N., Litopoulou-Tzanetaki, E., 1989. Biochemical activities of Pediococcus pentosaceus isolates of dairy origin. J. Dairy Sci. 72, 859863. Tzanetakis, N., Litopoulou-Tzanetaki, E., 1992. Changes in numbers and kinds of lactic acid bacteria in Feta and Teleme, two Greek cheeses from Ewes Milk. J. Dairy Sci. 75, 13891393. Tzanetakis, N., Litopoulou-Tzanetaki, E., Manolkidis, K., 1987. Microbiology of Kopanisti, a traditional Greek cheese. Food Microbiol. 4, 251256. Tzanetakis, N., Litopoulou-Tzanetaki, E., Vafopoulou-Mastrogiannaki, A., 1991. Effect of Pediococcus pentosaceus on microbiology and chemistry of Teleme cheese. Lebensm. Wis. Technol. 24, 173176. Tzanetakis, N., Litopoulou-Tzanetaki, E., Zerridis, G., 1995a. Etude de la ore supercielle du fromage Feta: les micrococcaces. In: 4me Congres de la SFM, Tours, France. Tzanetakis, N., Vafopoulou-Mastrojiannaki, A., Litopoulou-Tzanetaki, E., 1995b. The quality of white brined cheese from goat milk made with different starters. Food Microbiol. 12, 5563. Vafopoulou-Mastrogiannaki, A., Litopoulou-Tzanetaki, E., 1996. Protease and peptidase activity from whole cells and crude cell-free extracts of Leuconostoc mesenteroides and Leuconostoc paramesenteroides isolates from cheese. Microbiologie-Aliments-Nutrition 42, 167174. Vafopoulou-Mastrogiannaki, A., Litopoulou-Tzanetaki, E., Tzanetakis, N., 1990. Effect of Pediococcus pentosaceus on ripening changes of Feta cheese. Microbiologie-Aliments-Nutrition 8, 5362. Vafopoulou-Mastrogiannaki, A., Litopoulou-Tzanetaki, E., Tzanetakis, N., 1994. Proteinase, peptidase and esterase activity of crude cell-free extracts of Pediococcus pentosaceus isolated from cheese. Lebensm. Wis. Technol. 27, 342346. Vafopoulou-Mastrogiannaki, A., Litopoulou-Tzanetaki, E., Tzanetakis, N., 1996. Esterase activities of cell-free extracts from wild strains of leuconostocs and heterofermentative lactobacilli isolated from traditional Greek cheese. Lett. Appl. Microbiol. 23, 367370. Vassiliadis, A., Psoni, L., Nikolaou, Arvanitis, L., Tzanetakis, N., LitopoulouTzanetaki, E., 2009. Changes in microbial populations, kinds of lactic acid bacteria and biochemical characteristics of Greek traditional feta cheese during ripening. Int. J. Dairy Technol. 62, 3947. Xanthopoulos, V., Polychroniadou, A., Litopoulou-Tzanetaki, E., Tzanetakis, N., 2000a. Characteristics of Anevato cheese made from raw or heat-treated goat milk inoculated with a lactic starter. Lebensm. Wis. Technol. 33, 483488. Xanthopoulos, V., Hatzikamari, M., Adamidis, T., Tsakalidou, E., Tzanetakis, N., Litopoulou-Tzanetaki, E., 2000b. Heterogeneity of Lactobacillus plantarum isolates from Feta cheese throughout ripening. J. Appl. Microbiol. 88, 10561064. Zerridis, G.K., Vafopoulou-Mastrogiannaki, A., Litopoulou-Tzanetaki, E., 1984. Changes during ripening of commercial Gruyre cheese. J. Dairy Sci. 67, 13971405. Zygouris, N.P., 1956. Milk Industry. Ministry of Agriculture, Athens, Greece.

Hatzikamari, M., Litopoulou-Tzanetaki, E., Tzanetakis, N., 1999. Microbiological characteristics of Anevato: a traditional Greek cheese. J. Appl. Microbiol. 87, 595601. Kalavrouzioti, I., Hatzikamari, M., Litopoulou-Tzanetaki, E., Tzanetakis, N., 2004. Production of hard cheese from caprine milk by the use of two types of probiotic cultures as adjuncts. Int. J. Dairy Technol. 58, 3038. Kandarakis, G., Moschopoulou, E., Moatsou, G., Anifantakis, E.M., 1998. Effect of starters on gross and microbiological composition and organoleptic characteristics of Graviera Kritis cheese. Lait 78, 557568. Katsiari, M.C., Voutsinas, L.P., Kondyli, E., 2002. Improvement of sensory quality of low-fat Kefalograviera-type cheese with commercial adjunct cultures. Int. Dairy J. 12, 757764. Kondyli, E., Massouras, T., Katsiari, M.C., Voutsinas, L.P., 2003. Free fatty acids and volatile compounds in low-fat Kefalograviera-type cheese made with commercial adjunct cultures. Int. Dairy J. 13, 4754. Lioliou, K., Litopoulou-Tzanetaki, E., Tzanetakis, N., Robinson, R.K., 2001. Changes in the microora of manouri, a traditional Greek whey cheese, during storage. Int. J. Dairy Technol. 54, 100106. Litopoulou-Tzanetaki, E., 1990. Changes in numbers and kinds of lactic acid bacteria during ripening of Kefalotyri cheese. J. Food Sci. 55, 111113. Litopoulou-Tzanetaki, E., Vafopoulou-Mastrojiannaki, A., Tzanetakis, N., 1989. Biotechnologically important metabolic activities of Pediococcus isolates from milk and cheese. Microbiologie-Aliments-Nutrition 7, 113122. Litopoulou-Tzanetaki, E., Tzanetakis, N., Vafopoulou-Mastrogiannaki, A., 1993. Effect of the type of lactic starter on microbiological, chemical and sensory characteristics of Feta cheese. Food Microbiol. 10, 3141. Mama, V., Hatzikamari, M., Lombardi, A., Tzanetakis, N., LitopoulouTzanetaki, E., 2002. Lactobacillus paracasei subsp. paracasei heterogeneity: the diversity among strains isolates from traditional Greek cheeses. Int. J. Food Sci. 14, 351362. Manolopoulou, E., Sarantinopoulos, P., Zoidou, E., Aktypis, A., Moschopoulou, E., Kandarakis, I.G., Anifantakis, E.M., 2003. Evolution of microbial populations during traditional Feta cheese manufacture and ripening. Int. J. Food Microbiol. 82, 153161. Massouras, T., Pappa, E.C., Mallatou, H., 2006. Headspace analysis of volatile avor compounds of Teleme cheese made from sheep and goat milk. Int. J. Dairy Technol. 59, 250256. Michaelidou, A., Katsiari, M.C., Voutsinas, L.P., Kondyli, E., Alichanidis, E., 2003. Effect of commercial adjunct cultures on proteolysis in low-fat Kefalograviera-type cheese. Int. Dairy J. 13, 743753. Nikolaou, E., Tzanetakis, N., Litopoulou-Tzanetaki, E., Robinson, R.K., 2002. Changes in the microbiological and chemical characteristics of an artisanal low-fat cheese made from raw ovine milk during ripening. Int. J. Dairy Technol. 54, 1217. Papageorgiou, D.K., Abrahim, A., Bori, M., Doundounakis, S., 1998. Chemical and bacteriological characteristics of Pichtogalo Chanion cheese and mesophilic starter cultures for its production. J. Food Prot. 61, 688692. Pappa, E.C., Kandarakis, I.G., Zerridis, G.K., Anifantakis, E.M., Sotirakoglou, K., 2006. Inuence of starter cultures on the proteolysis of Teleme cheese made from different types of milk. Lait 86, 273290. Prodromou, K., Thasitou, P., Haritonidou, E., Tzanetakis, N., LitopoulouTzanetaki, E., 2001. Microbiology of Orinotyri, a ewes milk cheese from the Greek mountains. Food Microbiol. 18, 319328. Psomas, E., Andrighetto, C., Litopoulou-Tzanetaki, E., Lombardi, A., Tzanetakis, N., 2001. Some probiotic properties of yeast isolates from infant faeces and Feta cheese. Int. J. Food Microbiol. 69, 125133. Psomas, E., Fletouris, D.J., Litopoulou-Tzanetaki, E., Tzanetakis, N., 2003. Assimilation of cholesterol by yeast strains isolated from infant feces and Feta cheese. J. Dairy Sci. 86, 34163422. Psoni, L., Tzanetakis, N., Litopoulou-Tzanetaki, E., 2003. Microbiological characteristics of Batzos, a traditional Greek cheese from raw goats milk. Food Microbiol. 20, 575582. Psoni, L., Litopoulou-Tzanetaki, E., Tzanetakis, N., 2006. Characteristics of Batzos cheese made from raw, pasteurized and/or pasteurized standardized goat milk and a native culture. Food Control 17, 533539. Rubino, R., Morand-Fehr, P., Renieri, C., Peraza, C., Sarti, F.M., 1999. Typical products of the small ruminant sectoer and the factors affecting their quality. Small Ruminant Res. 34, 289302.