MICROBIAL CHARACTERIZATION OF VAN HERBY CHEESE USING MALDI-TOF MS AND EVALUATION OF STARTER CULTURE POTENTIAL OF SELECTED LACTIC ACID BACTERIA

Abstract

This study examined the microbial and biochemical properties of Van Herby Cheeses, produced using traditional dry salting and brining methods. Biochemical analyses showed that dry salted cheeses contained 54.41±4.80% dry matter, 0.74±0.09 water activity, pH 5.11±0.76, 30.4±5.32% fat, and 5.81±1.20% salt, while brined cheeses had 62.60±3.20%, 0.81±0.03, 4.68±0.57, 29.0±8.22%, and 7.58±0.98%, respectively. Microbial identification using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) revealed a predominance of Enterococcus, Lactobacillus, Leuconostoc, and Bacillus species, as well as yeasts such as Debaryomyces hansenii and Candida zeylanoides. A total of 17 LAB isolates were assessed for acidification, proteolytic activity, diacetyl production, antibacterial effects, and antibiotic resistance. Among these, E. faecium S431 and L. brevis B122 exhibited strong acidifying capacity and notable antimicrobial properties, highlighting their promising potential as functional starter cultures for traditional cheese production.

Keywords

• Van herby cheese
• MALDI-TOF MS
• Microbiota
• Lactic Acid Bacteria
• Starter Culture

Supporting Institution

Bolu Abant İzzet Baysal University Scientific Research Projects

Project Number

2022-10-01-1569

Thanks

All authors would like to thank Scientific Industrial and Technological Application and Research Center (SITARC).

VAN OTLU PEYNİRİNİN MALDI-TOF MS YÖNTEMİ İLE MİKROBİYAL KARAKTERİZASYONU VE SEÇİLİ LAKTİK ASİT BAKTERİLERİNİN STARTER KÜLTÜR POTANSİYELİNİN DEĞERLENDİRİLMESİ

Abstract

Bu çalışmada, geleneksel yöntemle basma ve salamura olarak üretilen Van Otlu Peynirlerinin mikrobiyal ve biyokimyasal özelliklerinin tespit edilmesi ve izole edilen bazı laktik asit bakterilerinin (LAB) starter kültür olma potansiyelleri değerlendirilmiştir. Çalışmada basma ve salamura peynirlerin kuru madde, su aktivitesi, pH, yağ ve tuz içerikleri sırasıyla %54.41±4.80-%62.60±3.20, %0.74±0.09-%0.81±0.03, %5.11±0,76-%4.68±0.57, %30.4±5.32-%29.0±8.22 ve %5.81±1.20-%7.58±0.98 olarak tespit edilmiştir. İzole edilen mikroorganizmalar MALDI-TOF MS yöntemi ile tür düzeyinde tanımlanmıştır. Örneklerde ağırlıklı olarak Enterococcus, Lactobacillus, Leuconostoc ve Bacillus cinslerine ait bakteri türleri belirlenmiş ve Debaryomyces hansenii ve Candida zeylanoides gibi maya türlerine de rastlanmıştır. Seçilen 17 LAB suşunun asidifikasyon yetenekleri, proteinaz ve diasetil üretim kapasiteleri ile antibakteriyel aktiviteleri ve antibiyotik duyarlılıkları değerlendirilmiştir. Özellikle E. faecium S431 ve L. brevis B122 suşları yüksek asidifikasyon ve antimikrobiyal aktiviteleriyle öne çıkmıştır. Çalışma sonuçları, Van Otlu Peynirinin çeşitlilik arz eden mikrobiyotasını ortaya koymakta ve izole edilen suşların starter kültür olarak kullanılabileceğini göstermektedir.

Keywords

• Van otlu peynir
• MALDI-TOF MS
• Mikrobiyota
• Laktik Asit Bakterileri

Project Number

2022-10-01-1569

References

1. Akkoç, Z., İncil, G. K., İlhak, O. İ. (2018). Otlu peynirlerde histamin, bazı mikrobiyolojik ve kimyasal özelliklerin araştırılması. Fırat Üniversitesi Sağlık Bilimleri Dergisi, 32(2), 87–92.
2. Al Bayatee, M. A., Alsammak, E. G. (2018). Phenetic and phylogenetic analysis of Kocuria palustris and Kocuria rhizophila strains isolated from healthy and thalassemia persons. Sci J Med Res, 2, 135-46.
3. Albayrak, Ç. B., Duran, M. (2021). Isolation and characterization of aroma producing lactic acid bacteria from artisanal white cheese for multifunctional properties. LWT, 150, 112053.
4. Alehosseini, E., McSweeney, P. L., Miao, S. (2025). Recent updates on plant protein-based dairy cheese alternatives: outlook and challenges. Critical Reviews in Food Science and Nutrition, 1-15.
5. Aydın, F. (2017). Yöresel bazı peynirlerdeki Enterococcus cinsi bakterilerin MALDI-TOF-TOF-MS yöntemi ile tanımlanması (Master's thesis, Aksaray Üniversitesi Fen Bilimleri Enstitüsü).
6. Azzouz, S., Ahadaf, S., Zantar, S., El Galiou, O., Arakrak, A., Bakkali, M., Laglaoui, A. (2024). Microbial Communities of Raw Milk Cheeses, A Review. Journal of Food Science and Technology (2008-8787), 21(150).
7. Barzegar, H., Alizadeh Behbahani, B., Falah, F. (2021). Safety, probiotic properties, antimicrobial activity, and technological performance of Lactobacillus strains isolated from Iranian raw milk cheeses. Food Science and Nutrition, 9(8), 4094-4107.
8. Bozoudi, D., Kotzamanidis, C., Hatzikamari, M., Tzanetakis, N., Menexes, G., Litopoulou-Tzanetaki, E. (2015). A comparison for acid production, proteolysis, autolysis and inhibitory properties of lactic acid bacteria from fresh and mature Feta PDO Greek cheese, made at three different mountainous areas. International Journal of Food Microbiology, 200, 87-96.

9. Case, R. A., Bradley, R. L., Williams, R. R. (1985). Chemical and physical methods. In G. H. Richardson (Ed.), Standard methods for the examination of dairy products (15th ed., pp. 327–404). American Public Health Association.
10. Champiri, I. D., Bamzadeh, Z., Rahimi, E., Rouhi, L. (2022). Isolation and Identification of Lactobacillus brevis from Local Cheese of Bazoft and Evaluation of Antimicrobial Activity Against Some Pathogenic Microorganisms. Iranian Journal of Medical Microbiology, 16(1).
11. Charteris, W. P., Kelly, P. M., Morelli, L., Collins, J. K. (1998). Antibiotic susceptibility of potentially probiotic Lactobacillus species. Journal of food protection, 61(12), 1636-1643.
12. Coelho, M. C., Malcata, F. X., Silva, C. C. (2022). Lactic acid bacteria in raw-milk cheeses: From starter cultures to probiotic functions. Foods, 11(15), 2276.
13. Coşkun, H. (2024). Otlu Peynir, 2. Baskı. Astana yayınları. Çankaya/Ankara. ISBN:9786256501966. Çelik, S. E., Özyürek, M., Altun, M., Bektaşoğlu, B., Güçlü, K., Berker, K. I., Apak’, R. (2008). Antioxidant capacities of herbal plants used in the manufacture of Van Herby Cheese: ‘Otlu peynir’. International Journal of Food Properties, 11(4), 747-761.
14. Domingos-Lopes, M. F. P., Stanton, C., Ross, P. R., Dapkevicius, M. L. E., Silva, C. C. G. (2017). Genetic diversity, safety and technological characterization of lactic acid bacteria isolated from artisanal Pico cheese. Food microbiology, 63, 178-190.
15. Ekici, K., Okut, H., Isleyici, O., Sancak, Y. C., Tuncay, R. M. (2019). The determination of some microbiological and chemical features in herby cheese. Foods, 8(1), 23.
16. Emirmustafaoğlu, A., Coşkun, H. (2012). Keçi sütü, inek sütü ve bu sütlerin karışımından yapılan otlu peynirlerde olgunlaşma boyunca meydana gelen değişmeler. Gıda, 37(4), 211-218.
17. Encu, Ş. B., Soykut, E. A., Çakır, İ. (2022). Geleneksel yoğurtlardan izole edilen laktik asit bakterilerinin MALDI TOF MS Biotyper sistemi ile tanımlanması ve bazı starter kültür özelliklerinin belirlenmesi. Gıda, 47(6), 1059-1082.
18. Ertürkmen, P., Öner, Z. (2015) Beyaz Peynir örneklerinden izole edilen laktik asit bakterilerinin başlatıcı (starter) kültür özelliklerinin biyokimyasal yöntemlerle belirlenmesi, Süleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 19(3): 9-16. sdufbed.25545.
19. Ertürkmen, P. (2025). Microbiota composition of buffalo colostrum and characterization of potential probiotic bacteria with high exopolysaccharide production and cholesterol assimilation capacity. Journal of Food Quality, 2025(1).
20. Farahani, Z., Rasooli, I., Owlia, P. (2017). Isolation, identification and characterization of indigenous lactic acid bacteria for flavour improvement. International Food Research Journal, 24(1).
21. Fox, P.F. (1989). Proteolysis during cheese manufacture and ripening, Journal of Dairy Sciences. 72: 1379–1400. https://doi.org/10.3168/ jds.S0022-0302(89)79246-8.
22. Gad, G. F. M., Abdel-Hamid, A. M., Farag, Z. S. H. (2014). Antibiotic resistance in lactic acid bacteria isolated from some pharmaceutical and dairy products. Brazilian journal of Microbiology, 45, 25-33.
23. Gardener, B. B. M. (2004). Ecology of Bacillus and Paenibacillus spp. in agricultural systems. Phytopathology, 94(11), 1252-1258.
24. Grady, E. N., MacDonald, J., Liu, L., Richman, A., Yuan, Z. C. (2016). Current knowledge and perspectives of Paenibacillus: a review. Microbial cell factories, 15, 1-18.
25. Gueimonde, M., Sánchez, B., G. de los Reyes-Gavilán, C., Margolles, A. (2013). Antibiotic resistance in probiotic bacteria. Frontiers in microbiology, 4, 202.
26. Gülmez, M., Üner, S., Bayhan, K. Y. (2024). Experimental Studies on the Siirt Herby Tulum Cheese: I. Development of Industrial Process Model. Dicle Üniversitesi Veteriner Fakültesi Dergisi, 17(1), 65-77.
27. Gvozdyak, O. R., Nogina, T. M., Schumann, P. (1992). Taxonomic study of the genus Brachybacterium: Brachybacterium nesterenkovii sp. nov. International Journal of Systematic and Evolutionary Microbiology, 42(1), 74-78.
28. Hajikhani, R., Onal Darilmaz, D., Yuksekdag, Z. N., Beyatli, Y. (2021). Assessment of some metabolic activities and potential probiotic properties of eight Enterococcus bacteria isolated from white cheese microbiota. Antonie Van Leeuwenhoek, 114(8), 1259-1274.
29. Halkman, A. K. (2019). Gıda mikrobiyolojisi. Başak Matb. ve Tanıtım Hiz. Ltd, Ankara. PP, 187. Harris, L. J., Daeschel, M. A., Stiles, M. E., Klaenhammer, T. R. (1989). Antimicrobial activity of lactic acid bacteria against Listeria monocytogenes. Journal of food protection, 52(6), 384-387.
30. İșleyici, Ö., Akyüz, N. (2009). Detection of lactic acid bacteria and microbiota of Herby cheese sold in Van City. Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Dergisi, 20 (2), 59-64.
31. İşleroğlu, H., Yıldırım, Z., Yıldırım M. (2008). Yöresel peynirden antimikrobiyal aktiviteye sahip laktik asit bakterisinin izolasyonu ve tanısı. Gaziosmanpaşa Üniversitesi Ziraat Fakültesi Dergisi, 2008(1), 1-6.
32. Kamber, U. (2007). The traditional cheeses of Turkey: cheeses common to all regions. Food reviews international, 24(1), 1-38.
33. Karasu-Yalcin, S., Söylemez-Milli, N., Eren, O., Eryasar-Orer, K. (2021). Reducing time in detection of Listeria monocytogenes from food by MALDI-TOF mass spectrometry. Journal of Food Science and Technology, 58, 4102-4109.
34. Khushboo, Karnwal, A., Malik, T. (2023). Characterization and selection of probiotic lactic acid bacteria from different dietary sources for development of functional foods. Frontiers in microbiology, 14, 1170725.
35. Kırmacı, H. A., Kivik, İ. (2024). Artizanal Peynirlerin gastronomi turizmi kapsamında değerlendirilmesi: Van İli örneği. Turizmde Güncel Gelişmeler ve Araştırmalar, 53. Çizgi Kitabevi, ISBN: 978-625-396-432-0.
36. Kırmacı, H.A. (2010). Geleneksel Urfa peynirinde yer alan laktik asit bakterilerinin izolasyonu, moleküler karakterizasyonu ve starter kültür olarak kullanım olanakları. Harran Üniversitesi Fen Bilimleri Enstitüsü Gıda Mühendisliği Anabilim Dalı, Doktora Tezi, Şanlıurfa, Türkiye, 152s.
37. Kieliszek, M., Pobiega, K., Piwowarek, K., Kot, A. M. (2021). Characteristics of the proteolytic enzymes produced by lactic acid bacteria. Molecules, 26(7), 1858.
38. Kim, Y., Yoon, S., Shin, H., Jo, M., Lee, S., Kim, S. H. (2021). Isolation of Lactococcus lactis ssp. cremoris LRCC5306 and optimization of diacetyl production conditions for manufacturing sour cream. Food Science of Animal Resources, 41(3), 373.
39. Köse, Ş., Ceylan, M. M., Altun, İ., Erim Köse, Y., (2022). Determination of some basic properties of traditional Malatya cheese. Food Science and Technology (Brazil), vol.42.
40. Li, J., Huang, Q., Zheng, X., Ge, Z., Lin, K., Zhang, D., Shi, X. (2020). Investigation of the lactic acid bacteria in Kazak cheese and their contributions to cheese fermentation. Frontiers in Microbiology, 11, 228.
41. Lim, Y. H., Foo, H. L., Loh, T. C., Mohamad, R., Abdullah, N. (2019). Comparative studies of versatile extracellular proteolytic activities of lactic acid bacteria and their potential for extracellular amino acid productions as feed supplements. Journal of Animal Science and Biotechnology, 10, 1-13.
42. Mercha, I., Lakram, N., Kabbour, M. R., Bouksaim, M., Zkhiri, F., El Maadoudi, E. H. (2020). Probiotic and technological features of Enterococcus and Weissella isolates from camel milk characterised by an Argane feeding regimen. Archives of Microbiology, 202, 2207-2219.
43. Mortensen, H. D., Jacobsen, T., Koch, A. G., Arneborg, N. (2008). Intracellular pH homeostasis plays a role in the tolerance of Debaryomyces hansenii and Candida zeylanoides to acidified nitrite. Applied and environmental microbiology, 74(15), 4835-4840.
44. Nakagawa, H., Oyama, T. (2019) Molecular Basis of Water Activity in Glycerol–Water Mixtures. Front. Chem. 7:731. doi: 10.3389/fchem.2019.00731.
45. Nalepa, B., Markiewicz, L. H. (2022). Microbiological biodiversity of regional cow, goat and ewe milk cheeses produced in Poland and antibiotic resistance of lactic acid bacteria isolated from them. Animals, 13(1), 168.
46. Nicosia, F. D., Pino, A., Maciel, G. L. R., Sanfilippo, R. R., Caggia, C., de Carvalho, A. F., Randazzo, C. L. (2023). Technological characterization of lactic acid bacteria strains for potential use in cheese manufacture. Foods, 12(6), 1154.
47. Nielsen, S. S. (2003). Mohr titration food analysis laboratory manual (3rd ed.). New York: Kluwer Academic/Plenum Publishers.http://dx.doi.org/10.1007/978-1-4757-5250-2
48. Ocak, E., Javidipour, I., Tuncturk, Y. (2015a). Volatile compounds of Van Herby Cheeses produced with raw and pasteurized milks from different species. Journal of Food Science and Technology, 52, 4315-4323.
49. Ocak, E., Tunçtürk, Y., Javidipour, I., Köse, Ş. (2015b). Farklı tür sütlerinden üretilen Van Otlu Peynirlerinde olgunlaşma boyunca meydana gelen değişiklikler: II. mikrobiyolojik değişiklikler, lipoliz ve serbest yağ asitleri. Yuzuncu Yıl University Journal of Agricultural Sciences, 25(2), 164-173.
50. Oğuz, Ş., Andiç, S. (2024). Isolation identification and characterization of thermophilic lactic acid bacteria isolated from whey of Kars Kashar cheeses. Antonie van Leeuwenhoek, 117(85), 1–18.
51. Rüstemoğlu, M., Erkan, M. E., Cengiz, G., Hajyzadeh, M. (2023). Bacterial metagenome profiling of hand-made herby cheese samples utilizing high-throughput sequencing to detect geographical indication and marketing potential. Heliyon, 9(2).
52. Serrano, S., Morais, S., Semedo-Lemsaddek, T. (2024). Tradition unveiled: a comprehensive review of microbiological studies on Portuguese traditional cheeses, merging conventional and OMICs analyses. Frontiers in Industrial Microbiology, 2, 1420042.
53. Silva, L. F., Sunakozawa, T. N., Monteiro, D. A., Casella, T., Conti, A. C., Todorov, S. D., Barretto Penna, A. L. (2023). Potential of cheese-associated lactic acid bacteria to metabolize citrate and produce organic acids and acetoin Metabolites, 13(11), 1134.
54. Söylemez Milli, N., Ertürkmen, P., Alp Baltakesmez, D. (2025). The resistance abilities of some Bacillus species to gastrointestinal tract conditions: Whole Genome Sequencing of the novel candidate probiotic strains Bacillus clausii BA8 and Bacillus subtilis BA11. Food Science and Nutrition, 13(2), e70018.
55. Tunçtürk, Y., Ocak, E., Köse, Ş. (2014). Farklı süt türlerinden üretilen Van otlu peynirlerinin fiziksel ve kimyasal özellikleri ile proteoliz profillerinde olgunlaşma sürecinde meydana gelen değişimler. Gıda, 39(3), 163-170.
56. Türk Patent Kurumu, (2025). https://www.turkpatent.gov.tr/cografi-isaret, Erişim tarihi: 13.03.2025.
57. Vandera, E., Kakouri, A., Koukkou, A. I., Samelis, J. (2019). Major ecological shifts within the dominant nonstarter lactic acid bacteria in mature Greek Graviera cheese as affected by the starter culture type. International Journal of Food Microbiology, 290, 15-26.