International Journal of Food Properties

This article was downloaded by:[University of Newcastle] On: 24 April 2008 Access Details: [subscription number 772827708] Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

International Journal of Food Properties Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713597259

Lactic Microflora Present in Liqvan Ewes' Milk Cheese Javad Barouei a; Ahmad Karbassi a; Hamid B. Ghoddusi b; Ali Mortazavi b a Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran b Department of Food Science and Technology, School of Agriculture, Ferdowsi University, Mashad, Iran

Online Publication Date: 01 April 2008 To cite this Article: Barouei, Javad, Karbassi, Ahmad, Ghoddusi, Hamid B. and Mortazavi, Ali (2008) 'Lactic Microflora Present in Liqvan Ewes' Milk Cheese', International Journal of Food Properties, 11:2, 407 - 414 To link to this article: DOI: 10.1080/10942910701436883 URL: http://dx.doi.org/10.1080/10942910701436883

PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

Downloaded By: [University of Newcastle] At: 03:54 24 April 2008

International Journal of Food Properties, 11: 407–414, 2008 Copyright © Taylor & Francis Group, LLC ISSN: 1094-2912 print / 1532-2386 online DOI: 10.1080/10942910701436883

LACTIC MICROFLORA PRESENT IN LIQVAN EWES’ MILK CHEESE Javad Barouei1, Ahmad Karbassi1, Hamid B. Ghoddusi2, and Ali Mortazavi2 1

Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran 2 Department of Food Science and Technology, School of Agriculture, Ferdowsi University, Mashad, Iran This study aimed to isolate and characterize Lactic Acid Bacteria (LAB) in Liqvan Ewes’ milk cheese. A total of 117 Lactic Acid Bacteria were isolated and identified phenotypically. They belonged to 4 genera and 17 species. The dominant LAB found in Liqvan cheese were from the genus Lactobacillus (75.21%) consisted of 70.08% facultatively heterofermentative and 5.12% obligately heterofermentative lactobacillus species. Other isolates were classified as Pediococci (5.12%), Enterococci (5.98%), and Leuconostocs (13.67%). Lb. paracasei subsp. paracasei was the predominant species accounted for 36.75%. Likewise, predominant species of each genus were Lb. paracasei subsp. paracasei, P. pentosaceus, E. faecalis, and Leu. lactis. The preponderance of isolates (86.32%) was referred to be as members of Non Starter Lactic Acid Bacteria (NSLAB). Keywords: Liqvan cheese, Ewes’ milk cheese, Lactic acid bacteria.

INTRODUCTION Liqvan cheese exemplifies a traditional Iranian white-brined cheese made from raw ewes’ milk. It is known as the best cheese in the country for its typical piquant flavor.[1–3] It is classified as a semi soft cheese with a desired sour taste, cream color, high fat content and crumbly texture.[1] The name comes from Liqvan valley near Tabriz city. Its production is limited to a part of the northwestern mountainous area of Iran.[3] Conventionally, coagulation is achieved by natural lamb rennet, and starter cultures are not used. The final coagulum is cut and drained using cheesecloth and pressed. Curing the produced cheeses in brine is carried out in underground rooms dug with access through narrow galleries in width enough for passing just one man.[3] Similarly, the ripening process takes place in these rooms for up to 6 months. The rooms have been used for centuries in this way, and the individual responsible for the creation of these mysterious rooms, and the exact time at which this occurred, remains unknown. The cheese quality entirely depends on different ripening conditions, variations in composition of milk and indigenous microflora. Received 13 November 2006; accepted 8 May 2007. Address correspondence to Javad Barouei, Microbiology Lab., Life Science Building, School of Environmental and Life Sciences, The University of Newcastle, Callaghan 2308, NSW, Australia. E-mail: Javad.Barouei@ newcastle.edu.au 407


408

BAROUEI ET AL.

The resultant cheese is well known for its typical sensory characteristics. There have been no previous studies examining the diversity of Lactic Acid Bacteria in liqvan ewes’ milk cheese, and understanding what organisms are involved may lead to a better understanding of the development of its desirable organoleptic qualities. MATERIALS AND METHODS Cheese Samples Eight premium quality Liqvan cheese samples were obtained from traditional cheese manufacturers in Liqvan village. The samples were kept refrigerated (4°C) until examination. Isolation and Identification An amount of 25 g of each cheese sample was suspended in 225 ml of 0.5% bactopeptone (Difco, Detroit, USA), homogenized by Stomacher® 400 Lab Blender (Seward Medical, London, UK) and serial decimal dilutions were prepared. Diluted samples were plated on MRS agar,[4] and incubated anaerobically at 30±1°C for 48 hrs. Selected bacterial colonies successively transferred 3 times on MRS agar for purification, and checked for catalase and Gram’s stain reactions.[5] Gram positive and catalase negative isolates were propagated in MRS broth and stored at −25°C in a 2:1:1 glycerol/MRS broth/reconstituted 20% skim milk powder mixture. When needed, recovery of isolates was undertaken by two consecutive subcultures in MRS broth prior to testing. Each isolate was tested for oxidase, gas production from glucose, growth at different temperatures (10, 15, 40, and 45°C), growth in presence of different NaCl concentrations (2, 4, and 6.5%), Voges-Proskauer, nitrate reduction, polysaccharide formation, haemolysis and ammonia production from arginine.[5] D- and L-isomers of lactate produced by isolates were measured using a previously described colorimetric method.[6] Carbohydrate fermentation patterns of isolates were determined using an API 50 CH kits (bioMerieux sa, Marcy-I'Etoile, France) according to manufacturer's instruction. Finally the isolates were identified using morphological, cultural and biochemical tests according to criteria of Holt;[7] Schleifer and Kilpper-Bälz;[8] Schleifer et al.;[9] Mundt;[10] Devries et al.;[11] Leblanc;[12] Holzapfel et al.;[13] Garvie;[14–15] Björkroth and Holzapfel;[16] Kandler and Weiss,[17] and Hammes and Hertel.[18] RESULTS AND DISCUSSION Bacterial isolates were identified based on phenotypic characteristics. Number and percentage of each genus/strain among the sum of isolated LAB has been summarized in Table 1. The Gram positive and catalase negative organisms were divided into two morphological groups. These were coccal or ovoid forms and rods. Each group was then subdivided into homofermentative and heterofermentative by examining their fermentation products. Heterofermentative activity was determined by the ability of strains to produce carbon dioxide as a fermentation product from glucose. The majority of LAB isolated from Liqvan cheese belonged to Lactobacilli (75.21%). Facultatively heterofermentative (FHL) and obligately heterofermentative lactobacillus species comprised 70.08% and 5.12% of the isolates respectively.[17–19]


LACTIC MICROFLORA IN MILK CHEESE

409

Table 1 LAB isolated from Liqvan cheese. Genus/Species

na

%b

Facultatively Heterofermentative Lactobacilli Lb. plantarum Lb. paracasei subsp. paracasei Lb. casei Lb. rhamnosus Lb. coryniformis subsp. coryniformis Lb. curvatus subsp. curvatus Lb. bifermentan Lb. pentosus Obligately Heterofermentative Lactobacilli Lb. brevis Lb. fermentum Pediococci P. penctosaceus P. acidilactici P. parvulus Enterococci E. faecum E. faecalis E. durans Leuconostoc Leu. lactis Total

82

70.08

14 43 12 2 1 4 3 3 6

11.96 36.75 10.25 1.70 0.85 3.41 2.56 2.56 5.12

5 1 6 3 2 1 7 4 2 1 16 16 117

4.27 0.85 5.12 2.56 1.70 0.85 5.98 3.41 1.70 0.85 13.67 13.67 100

a : Number of isolates; and b: percentage of genus/strains among the sum of isolated LAB.

Of the isolates, eighty two were FHL (70.08% of total isolates). Eight species were characterized in this group. The strains that fermented ribose, mannitol and melezitose, but not rhamnose, grew at 15°C and formed L-Lactic acid, were identified as L. paracasei subsp. paracasei (43 strains). Strains that fermented ribose and gluconate, but not glycerol and xylose, grew at 15°C, but not at 45°C, formed DL-Lactic acid and catabolised α-methyl-D-mannoside, were identified as Lb. plantarum (14 strains). The strains that did not ferment ribose and mannitol, grew at 15°C and 45°C and formed L-Lactic acid, were identified as L. casei (12 strains). Four strains that did not produce ammonia from arginine, fermented ribose, but not mannitol, xylose and melibiose, grew at 15°C, but not at 45°C and formed DL-Lactic acid, were identified as L. curvatus. The strains that fermented ribose, mannitol, but not melezitose, grew at 15°C, but not at 45°C and formed L-Lactic acid, were identified as Lb. bifermentan (3 strains). And finally, the strains that fermented ribose, gluconate, glycerol and xylose, grew at 15°C, but not at 45°C and formed DL-Lactic acid, were identified as Lb. pentosus (3 strains). The strains that fermented ribose and rhamnose grew at 15°C and 45°C and formed L-Lactic acid seemed to be Lb. rhamnosus (2 strains). The strain that did not ferment ribose, but mannitol, grew at 15°C, but not at 45°C, formed DL-Lactic acid (more D isomer than L isomer), were identified as Lb. coryniformis subsp. coryniformis (one strain).[18] Of Lactobacilli, six belonged to obligately heterofermentative group (5.12% of total isolates). The species


410

BAROUEI ET AL.

identified consisted of 5 Lb. brevis, and one Lb. fermentum. The strains that fermented ribose, galactose and melibiose, but not mannose, melezitose and gluconate, grew at 15°C, but not at 45°C were identified as Lb. brevis (5 strains). The strain that did not ferment gluconate or grow at 15°C but did so at 45°C was identified as Lb. fermentum (one strain).[18] FHL form a significant portion of the NSLAB community in most cheese varieties during ripening.[20] In the case of raw milk cheeses, FHL are mainly derived from the cheese milk.[20] They usually increase from a low number in fresh curd to dominate the microflora of ripened cheese.[21] Non starter Lactobacilli can easily tolerate the hostile environment of cheese typically characterized by low pH, low moisture content, high salt concentration, and nutrient deficiency.[22] Non starter lactobacilli have a wide range of proteolytic and lipolytic activities. Proteolytic and lipolytic products contribute to and/or act as precursors for flavour development in cheese.[23] On a negative note, some species of obligately heterofermentative lactobacilli can produce some end products contributing to texture and off-flavour defects, if present at adequate levels.[23–24] Lactobacilli usually grow as a heterogeneous community in the cheese matrix.[25] The most frequently strains of facultatively and obligately heterofermentative lactobacilli found in raw ewes’ milk cheeses include; Lactobacillus paracasei subsp. paracasei, Lb. plantarum, Lb. curvatus, Lb. rhamnosus, Lb. coryniformis, Lactobacillus casei subsp. casei and Lb. brevis.[25–33] Of the coccal/ovoid isolates, 16 were Leuconostoc. Heterofermentative activity and failure to produce ammonia from arginine were characteristics that allowed Leuconostoc to be distinguished from the other coccal isolates. They were also differentiated from morphologically similar heterofermentative lactobacilli in that they produced no ammonia from arginine and formed D-, but not DL-lactic acid from glucose. All 16 Leuconostoc were identified as Leu. Lactis. They gave a positive Voges-Proskauer test and did not hydrolyze esculine; however they fermented lactose and sucrose. These isolates also did not ferment ribose, cellobiose, trehalose, mannitol and xylose.[14,16,34] Technologically, Leuconostocs are not main producers of lactic acid in milk and dairy products. Particular strains of Leuconostocs (Leu. mesenteroides subsp. cremoris and Leu. lactis) play a significant role in developing flavour and aroma in certain varieties of cheese.[34– 35] They typically contribute to the production of flavour compound diacetyl through carbohydrate heterofermentation in associative growth with some other LAB.[34] Cogan et al.[35] studied 4379 lactic acid bacteria from European artisanal dairy products including some cheeses; they identified about 10% of the strains as Leuconostoc spp. Previously Leu. lactis was isolated as the predominant Leuconostoc from some ewes’ milk cheeses.[25,33] Of the isolates of homofermentative cocci, seven were characterized as Enterococcus by their ability to grow at 10°C and 45°C, as well as their ability to grow in the presence of 6.5% NaCl and at pH 9.6 without any tetrad formation becoming evident. In addition, these homofermentative coccal isolates produced ammonia from arginine, but not carbon dioxide from glucose. There were four E. faecalis, two E. faecium and one E. durans identified. The E. faecalis was differentiated from E. faecium and E. durans by its ability to both reduce triphenyl tetrazolium chloride (TTC) and produce acid from sorbitol. In addition, E. faecalis produced acid from D-arabinose and mannitol, which is not observed for E. faecium. The strain identified as E. durans was unpigmented and did not reduce TTC. Furthermore, it didn’t metabolize melibiose, saccharose, mannitol and raffinose.[7–8,10–12] Enterococci especially E. faecalis and E. faecium are frequently associated with the intestines of human and animals, and when found in food or water suggests faecal contamination.[36] These species are also the most common enterococcal species found in



411

cheese. Enterococci occur as NSLAB in variety of cheeses, especially raw ewes’ milk cheeses manufactured in Mediterranean and Middle Eastern countries.[25,37–44] Enterococci can survive and grow in the harsh environment of cheeses with high salt content and low pH.[45] They are likely to originate from the raw milk, and are considered to play an important role in cheese ripening and aroma development due to their proteolytic and lipolytic activities and diacetyl production.[46] It has been reported that the presence of the enterococci in cheese positively affected the growth of other NSLAB, increased the proteolytic indices and positively affected organoleptic properties of the cheese.[47] Furthermore, Enterococci produce bacteriocins that inhibit foodborne pathogens in cheese.[46,48] Pediococci were differentiated from other isolated homofermentative cocci on the basis that tetrads are formed during cell division. Pediococci constituted 6 isolates that were classified into four species. Two strains did not produce carbon dioxide from gluconate, fermented rhamnose and D-arabinose but not maltose and grew at 48°C, were identified as P. acidilactici. Whereas, P. penctosaceus (3 strains) fermented maltose and did not grow at 48°C. One isolate was identified as P. parvulus. This isolate was characterized by its ability to ferment rhamnose, D-arabinose and galactose, but not ribose and lactose and to grow at pH 7.0 and 35°C.[7,13,15] The role of pediococci in dairy products is not as important as that of Lactobacilli, Lactococci, Streptococcus thermophilus or certain Leuconostocs.[13] Pediococci have been occasionally involved in ripening and flavor production of some cheeses.[49] They have been isolated as NSLAB from raw ewes’ milk cheeses such as Kurdish,[25] Batoz,[28] and Manura,[27] where they may facilitate the cheese making process via their metabolic activities, especially proteolytic activity.[50–51] P. acidilactici and P. pentosaceus are the most frequent species and important members of NSLAB group and are involved in the maturation and ripening of cheeses.[20,52–54] Moreover, P. acidilactici and P. pentosaceus produce a wide range of pediocins as biopreservatives that can inhibit some harmful bacteria in cheese.[13] With the exception of the Leuconostoc, the LAB isolated in this study (86.32%) are members of Non Starter Lactic Acid Bacteria (NSLAB). NSLAB contribute to flavour development.[20,55] Furthermore, among isolates, there were some potential probiotic strains including Leu. lactis, Lb. plantarum, Lb. casei, Lb. rhamnosus, Lb. fermentum, E. faecalis, E. faecium, and P. acidilactici, however they need to fulfill requirements in this way.[56] CONCLUSIONS Liqvan cheese is considered to be a supreme product for its flavour and other sensory characteristics. Its qualities are linked to the type of milk (ewes’ milk), absence of heat treatment and most importantly, the desired metabolic activities of indigenous microflora of milk during cheese ripening. A wide diversity of LAB mainly Lactobacilli was found to be present in the cheese. Up to 86.32% of all isolates belonged to NSLAB. This diversity implies that LAB may give various attributes to cheese. In addition, among the isolates, there were potential probiotic strains that may contribute to consumer health. Further testing of these naturally occurring bacteria may provide new strains for dairy fermentations. ACKNOWLEDGMENT The authors wish to thank Mrs. Rosalind Smith for her language assistance in this paper.


412

BAROUEI ET AL.

REFERENCES 1. Khamsan, A.; Mirhadi, A.; Mahdavi-Adeli, H.R. Study on Flavor Compounds in Liqvan Cheese by Comparison with Golpayegan White Brined Cheese. Research Report No. 00583019; Iranian Animal Science Research Institute (ASRI): Tehran, Iran, 1999. 2. Khazen, S. Study on Tabriz Cheeses in Comparison to Imported Cheeses, Phar. D. Thesis, No. 337880; Medical University of Tabriz: Tabriz, Iran, 1997. 3. Phelan, J.A.; Renaud, J.; Fox, P.F. Some Non-European Cheese Varieties. In Cheese; Chemistry, Physics and Microbiology; Fox, P.F., Ed.; Chapman and Hall: London, 1993; 421–465. 4. De Man, J.C.; Rogosa, M.; Sharp, M.E. A Medium for the Cultivation of Lactobacilli. J. Appl. Microbiol. 1960, 23, 130–135. 5. Harrigan, W.F. Laboratory Methods in Food Microbiology; Academic Press: London, 1998; 100–118, 343–348. 6. Swearingen, P.A.; O’Sullivan, D.J.; Warthesen, J.J. Isolation, Characterization and Influence of Native Nonstarter Lactic Acid Bacteria on Cheddar Cheese Quality. J. Dairy Sci. 2001, 84, 50–59. 7. Holt, J.G. Bergey’s Manual of Determinative Bacteriology, 9th ed.; Williams and Wilkins: Baltimore, 1994; 528–67. 8. Schleifer, K.H.; Kilpper-Bälz, R. Molecular and Chemotaxonomic Approach to the Classification of Streptococci, Enterococci and Lactococci: A Review. System. Appl. Microbiol. 1988, 10, 1–19. 9. Schleifer, K.H., Kraus, J., Dvorak, C., Kilpper-Bälz, R., Collins, M.D.; Fischer, W. Transfer of Streptococcus lactis and Related Streptococci to the Genus Lactococcus gen. nov. System. Appl. Microbiol. 1985, 6, 183–195. 10. Mundt, J.O. Enterococci. In Bergey’s Manual of Systematic Bacteriology, 8th ed., Vol. 2; Sneath, P.H.A., Mair, N.S., Sharp, M.E., Holt J.G., Eds.; Williams and Wilkins: Baltimore, 1986; 1063–1065. 11. Devries, L.A.; Pot, B.; Collins, M.D. A Review, Phenotypic Identification of Genus Enterococcus and Differentiation of Phylogenetically Distinct Enterococcal Species and Species Groups. J. Appl. Bacteriol. 1993, 75, 399–408. 12. Leblanc, D.J. Enterococcus. In Prokaryotes, Vol. 4; Dworkin, M., Ed.; Springer-Verlag: New York, 2006; 175–204. 13. Holzapfel, W.H.; Franz, C.M.A.P.; Ludwig, W.; Back, W.; Dicks, L.M.T. The Genera Pediococcus and Tetragenococcus. In Prokaryotes, Vol. 4; Dworkin, M., Ed.; Springer-Verlag: New York, 2006; 229–266. 14. Garvie, E.I. Genus Leuconostoc. In Bergey’s Manual of Systematic Bacteriology, 8th ed., Vol. 2; Sneath, P.H.A., Mair, N.S., Sharp, M.E., Holt, J.G., Eds.; Williams and Wilkins: Baltimore, 1986; 1071–1075. 15. Garvie, E.I. Genus Pediococcus. In Bergey’s Manual of Systematic Bacteriology, 8th ed. Vol. 2; Sneath, P.H.A., Mair, N.S., Sharp, M.E., Holt, J.G., Eds.; Williams and Wilkins: Baltimore, 1986; 1075–1079. 16. Björkroth, J.; Holzapfel, W. Genera Leuconostoc, Oenococcus and Weissella. In Prokaryotes, Vol. 4; Dworkin, M., Ed.; Springer-Verlag: New York, 2006; 267–319. 17. Kandler, O.; Weiss, N. Genus Lactobacillus. In Bergey’s Manual of Systematic Bacteriology, 8th ed., Vol. 2. Sneath, P.H.A., Mair, N.S., Sharp, M.E., Holt, J.G., Eds.; Williams and Wilkins: Baltimore, 1986; 1209–1234. 18. Hammes, W.P.; Hertel, C. Lactobacillus and Carnobacterium. In Prokaryotes, Vol. 4; Dworkin, M., Ed.; Springer-Verlag: New York, 2006; 320–403. 19. Hammes, W.P.; Vogel, R.F. The Genus Lactobacillus. In Lactic Acid Bacteria, Vol. 2; Wood, B.J.B., Holzapfel, W.H., Eds.; Blackie Academic & Professional: London, 1995; 19–54. 20. Cogan, T.M.; Beresford, T.P. Microbiology of Hard Cheese. In Dairy Microbiology Handbook; Robinson, R. K., Ed.; Wiley Interscience: New York, 2002; 515–560.



413

21. Peterson, S. D.; Marshall, R. T. Non-Starter Lactobacilli in Cheddar Cheese: A Review. J. Dairy Sci. 1990, 73, 1393–1410. 22. Turner, K.W.; Lawrence, R.C.; Levriere, J. A Microbiological Specification for Milk for Aseptic Cheese Making. N. Z. J. Dairy Sci. Technol. 1986, 21, 249–254. 23. Khalid, N.M.; Marth, E.H. Lactobacilli-Their Enzymes and Role in Ripening and Spoilage of Cheese: A Review. J. Dairy Sci. 1990, 73, 2669–2684. 24. Crow, V.; Curry, B.; Christison, M.; Hellier, K.; Holland, R.; Liu, S.Q. Raw Milk Flora and NSLAB as Adjuncts. Aust. J. Dairy Technol. 2002, 57, 99–105. 25. Mortazavi, A.; Ghandi, A.; Barouei, J.; Moussavi, M. Diversity of Lactic Acid Bacteria Isolated from Kurdish Ewes’ Milk Cheese. Aust. J. Dairy Technol. 2007, 62 (3), 185–190. 26. Mannu, L.; Riu, G.; Comunian, R.; Fozzi, M.C.; Scintu, M.F.A. Preliminary Study of Lactic Aacid Bacteria in Whey Starter Culture and Industrial Pecorino Sardo Ewes’ Milk Cheese: PCR-Identification and Evolution During Ripening. Int. Dairy J. 2002, 12 (1), 17–26 27. Gerasi, E.; Litopoulou-Tzanetaki, E.; Tzanetakis, N. Microbiological Study of Manura, A Hard Cheese Made from Raw Ovine Milk in the Greek Island Sifnos. Int. J. Dairy Technol. 2003, 56 (2), 117–122. 28. Nikolaou, E.; Tzanetakis, N.; Litopoulou-Tzanetaki, E.; Robinson, R.K. Changes in the Microbiological and Chemical Characteristics of An Artisanal, Low-Fat Cheese Made from Raw Ovine Milk During Ripening. Int. J. Dairy Technol. 2002, 55 (1), 12–17. 29. Durlu-Ozkaya, F.; Xanthopoulos, V.; Tunail, N.; Litopoulou-Tzanetaki, E. Technologically Important Properties of Lactic Acid Bacteria Isolates from Beyaz Cheese Made from Raw Ewes’ Milk. J. Appl. Microbiol. 2001, 91, 861–870. 30. Fortina, M.G.; Ricci, G.; Acquati, A.; Zeppa, G.; Gandini, A.; Manachini, P.L. Genetic Characterization of Some Lactic Acid Bacteria Occurring in An Artisanal Protected Denomination Origin (PDO) Italian Cheese, the Toma piemontese. Food Microbiol. 2003, 20 (4), 397–404. 31. Prodromou, K.; Thasitou, P.; Haritonidou, E.; Tzanetakis, N.; Litopoulou-Tzanetaki, E. Microbiology of ‘Orinotyri’, A Ewes’ Milk Cheese from the Greek Mountains. Food Microbiol. 2001, 18 (3), 319–328. 32. Angelis, M. de.; Corsetti, A.; Tosti, N.; Rossi, J.; Corbo, M.R.; Gobbetti, M. Characterization of Non-Starter Lactic Acid Bacteria from Italian Ewe Cheeses Based on Phenotypic, Genotypic, and Cell Wall Protein Analyses. Appl. Environ. Microbiol. 2001, 67 (5), 2011–2020. 33. Perez-Elortondo, F.J.; Echobaria, P.A.; Albisu, M.; Barcina, Y. Indigenous Lactic Acid Bacteria in Idiazabal Ewes’ Milk Cheese. Int. Dairy J. 1998, 8, 725–732. 34. Garvie, E.I. Separation of Species of the Genus Leuconostoc and Differentiation of the Leuconostocs from Other Lactic Acid Bacteria. In Methods in Microbiology, Vol. 16; Bergan, T., Ed.; Academic Press: London, 1984; 147–178. 35. Cogan, T.M.; Barbosa, M.; Beuvier, E.; Bianchi-Salvadori, B.; Cocconcelli, P.S.; Fernandez, I.; Gomez, J.; Gomez, R.; Kalantzopoulos, G.; Ledda, A.; Medina, M.; Rea, M.C.; Rodriguez, E. Characterization of the Lactic Acid Bacteria in Artisanal Dairy Products. J. Dairy Res. 1997, 64, 409–421. 36. Murray, B.E. The Life and Times of the Enterococcus. Clin. Microbiol. Rev. 1990, 3, 46–65. 37. Ordoñez, J.A.; Barneto, R.; Ramos, M.; Studies on Manchego Cheese Ripened in Olive Oil. Milchwissenschaft. 1978, 33, 609–612. 38. Trovatelli, L.D.; Schiesser, A. Identification and Significance of Enterococci in Hard Cheese Made from Raw Cow and Sheep Milk. Milchwissenschaft. 1987, 42, 717–719. 39. Del Pozo, B.F.; Gaya, P.; Medina, M.; Rodr´guez-Mar´n, M.A.; Nun∼ez, M. Changes in the Microflora of La Serena Ewes’milk Cheese During Ripening. J. Dairy Res. 1988, 55, 449–455. 40. Tzanetakis, N.; Litopoulou-Tzanetaki, E. Changes in Numbers and Kinds of Lactic Acid Bacteria in Feta and Teleme, Two Greek Cheeses from Ewes’ Milk. J. Dairy Sci. 1992, 75, 1389–1393. 41. Macedo, A.C.; Malcata, F.X.; Hogg, T.A. Microbiological Profile in Serra Ewes’ cheese during ripening. J. Appl. Bacteriol. 1995, 79, 1–11.


414

BAROUEI ET AL.

42. Franz, C.M.A.P.; Holzapfel, W.H.; Stiles, M.E. Enterococci at the Crossroads of Food Safety? Int. J. Food Microbiol. 1999, 47, 1–24. 43. Durlu-Ozkaya, F.; Xanthopoulos, V.; Tunail, N.; Litopoulou-Tzanetaki, E. Technologically Important Properties of Lactic Acid Bacteria Isolates from Beyaz Cheese Made from Raw Ewes’ Milk. J. Appl. Microbiol. 2001, 91, 861–870. 44. Giraffa, G. Review, Functionality of Enterococci in Dairy Products. Int. J. Food Microbiol. 2003, 88, 215–222. 45. Wessels, D.; Jooste, P.J.; Mostert, J.F. Technologically Important Characteristics of Enterococcus Isolates from Milk and Dairy Products. Int. J. Food Microbiol. 1990, 10, 349–352. 46. Franz, C.M.A.P.; Stiles, M.E.; Schleifer, K.H.; Holzapfel, W.H. Enterococci in Food - A Conundrum for Food Safety. Int. J. Food Microbiol. 2003, 88, 105–122. 47. Sarantinopoulous, P.; Kalantzopoulous, G.; Tsakalidou, E. Effect of Enterococcus faecium on Microbiological, Physicochemical and Sensory characteristics Characteristics of Greek Feta Cheese. Int. J. Food Microbiol. 2002, 76, 93–105. 48. Ennahar, S.; Aoude-Werner, D.; Assobhei, O.; Hasselmann, C.; Antilisterial Activity of Enterocin 81, A Bacteriocin Produced by Enterococcus faecium WHE 81 Isolated from Cheese. J. Appl. Microbiol. 1998, 85, 521–526. 49. Ray, B. Fundamental Food Microbiology; CRC Press: Boca Raton, 2001; 113 pp. 50. El-Soda, M.; Ezzat, N.; El-Howby, T.; Zeiday, I. The Peptide Hydrolase System of Pediococci: Cell-Bound Enzymatic Activities. Microbiol. Aliments Nutr. 1994, 12, 145–154. 51. Vafopoulou-Mastrojiannaki, A.; Litopoulou-Tzanetaki, E.; Tzanitakis, N. Proteinase, Peptidase and Eesterase Activity of Crude Cell-Free Extracts of Pediococcus pentosaceus Isolated from Cheese. Lebensmittel Wissenschaft und Technologie. 1994, 27 (4), 342–346. 52. Fox, P.F.; Lucey, J.A.; Cogan, T.M. Glycolysis and Related Reactions During Cheese Manufacture and Ripening. Crit. Rev. Food. Sci. Nutr. 1990, 29, 237–253. 53. Olson, N.F. The Impact of Lactic Acid Bacteria on Cheese Flavor. FEMS Microbiol. Rev. 1990, 87, 131–147. 54. Callon, C.; Millet, C.; Montel, M. C. Diversity of Lactic Acid Bacteria Isolated from AOC Salers Cheese. J. Dairy Res. 2004, 71, 231–244. 55. Stanley, G. Microbiology of Fermented Milk Products. In The Technology of Dairy Products; Earley, R., Ed.; Blakie Academic & Professional: London, 1998; 50–80. 56. Gardiner, G.E.; Ross, R.P.; Kelly, P.M.; Stanton, C.; Collins, J.K.; Fitzgerald, G. Microbiology of Therapeutic Milks. In Dairy Microbiology Handbook; Robinson, R. K., Ed.; Wiley Interscience: New York, 2002; 431–478.