SHANKLİSH PEYNİRİNDEN İZOLE EDİLEN ENDOJEN MAYALARIN MOLEKÜLER TANIMLANMASI VE ENZİMATİK KARAKTERİZASYONU

Abstract

Bu çalışmada, Ortadoğu’da uzun yıllardan beri tüketilen ve son yıllarda Türkiye’de de üretilmeye başlanan Shanklish peynirlerinden mayaların izolasyonu, Start Codon Targeted (SCoT) markör yöntemi kullanılarak identifikasyonu ve enzimatik aktivitelerinin belirlenmesi amaçlanmıştır. Olgunlaştırılmış peynirlerden 24 adet maya izole edilmiş, SCoT markör yöntemiyle DNA parmak izleri elde edilerek gruplandırılmış ve her gruptan temsili izolatlar sekanslanarak identifikasyon sonuçları elde edilmiştir. Bu sonuçlara göre, 19 adet Kluyveromyces lactis, 2 adet Pichia kudriavzevii, 1 adet Pichia fermentans, 1 adet Pichia membranifaciens ve 1 adet Clavispora lusitaniae suşu tanımlanmış ve API-ZYM enzim test kiti yardımıyla enzimatik karakterizasyonları belirlenmiştir. Bu suşlar arasından K. lactis ANO17 suşu yüksek esteraz lipaz, lösin arilamidaz, valin arilamidaz, sistin arilamidaz, asit fostataz, Naftol-as-bi-fosfohidroliz, α-glukosidaz ve β-glukosidaz aktivitesi gösterirken orta seviyede esteraz, β-galaktosidaz ve düşük seviyede alkalin fostataz aktivitesi göstermiş ve bu suş enzimatik aktivite yönünden en umut verici suş olarak tespit edilmiştir. Çalışma sonuçlarına göre, K. lactis ANO17 suşunun olası starter/destek kültür kombinasyonlarında laktik asit bakterileriyle birlikte kullanımının teknolojik yönden üstün peynir elde edilmesinde faydalı olacağı düşünülmektedir.

Keywords

• Shanklish
• maya
• enzimatik aktivite

MOLECULAR IDENTIFICATION AND ENZYMATIC CHARACTERIZATION OF ENDOGENOUS YEAST ISOLATED FROM SHANKLISH CHEESE

Abstract

In this study, the isolation of yeasts originated from Shanklish cheeses produced and consumed in Turkey, their identification using the SCoT marker method, and the determination of their enzymatic activities were aimed. Twenty-four yeasts were isolated from these ripened Shanklish cheeses and they are grouped by obtaining DNA fingerprints using the SCoT marker method and then representative isolates from each group were sequenced for identification. Based on the identification results, 19 Kluyveromyces lactis, 2 Pichia kudriavzevii, 1 Pichia fermentans, 1 Pichia membranifaciens and 1 Clavispora lusitaniae strains were identified and their enzymatic characterizations were determined using the API-ZYM enzyme test kit. Among these strains, K. lactis ANO17 showed high esterase lipase, leucine arylamidase, valine arylamidase, cysteine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-glucosidase, and β-glucosidase activities, while showing moderate esterase, β-galactosidase, and low-level alkaline phosphatase activities and so this strain was identified as the most promising strain in terms of its enzymatic activity. According to these study results, it is considered that the use of K. lactis ANO17 strain in potential starter/adjunct culture combinations with lactic acid bacteria can be utilized to obtain technologically superior cheese.

Keywords

• Shanklish
• yeast
• enzymatic characterization

References

1. Abou Younes, A., Salma A., Al-Eiq A. (2018). Identification lactobacilli according to the API and PCR techniques isolated from rustic Shanklish. Damascus University Journal of Agricultural Sciences, 34 (2).
2. Addas, M. (2013). Syrian Shanklish and its quality. Mediterranean Agronomic Institute of Chania. Master Thesis. https://drive.google.com/file/d/ 1T4BhJCSBssI8Db4UteOK4rkammQ3 -d6T/view.
3. Akuzawa, R., Fox, P. F. (2004). Acid phosphatase in cheese. Animal Science Journal, 75(5), 385-391, https://doi.org/10.1111/j.1740-0929.2004.00202.x
4. Álvarez-Martín, P., Flórez, A. B., López-Díaz, T. M., Mayo, B. (2007). Phenotypic and molecular identification of yeast species associated with Spanish blue-veined Cabrales cheese. International Dairy Journal, 17(8), 961-967, https://doi.org/ 10.1016/j.idairyj.2006.11.005
5. Amom, T., Tikendra, L., Apana, N., Goutam, M., Sonia, P., Koijam, A. S., ... Nongdam, P. (2020). Efficiency of RAPD, ISSR, iPBS, SCoT and phytochemical markers in the genetic relationship study of five native and economical important bamboos of North-East India. Phytochemistry, 174, 112330. https://doi.org/10.1016/ j.phytochem.2020.112330
6. Andrade, R. P., Melo, C. N., Genisheva, Z., Schwan, R. F., Duarte, W. F. (2017). Yeasts from Canastra cheese production process: Isolation and evaluation of their potential for cheese whey fermentation. Food Research International, 91, 72-79, https://doi.org/10.1016/j.foodres. 2016.11.032
7. Aponte, M., Pepe, O., Blaiotta, G. (2010). Identification and technological characterization of yeast strains isolated from samples of water buffalo Mozzarella cheese. Journal of Dairy Science, 93(6), 2358-2361, https://doi.org/10.3168/ jds.2009-2948
8. Atanassova, M. R., Fernández-Otero, C., Rodríguez-Alonso, P., Fernández-No, I. C., Garabal, J. I., Centeno, J. A. (2016). Characterization of yeasts isolated from artisanal short-ripened cows' cheeses produced in Galicia (NW Spain). Food Microbiology, 53, 172-181, https://doi.org/10.1016/j.fm.2015.09.012

9. Aydın, F., Özer, G., Alkan, M., Çakır, İ. (2022). Start Codon Targeted (SCoT) markers for the assessment of genetic diversity in yeast isolated from Turkish sourdough. Food Microbiology, 107, 104081, https://doi.org/10.1016/ j.fm.2022.104081
10. Berikten, D., Hoşgün, E. Z., Gökdal Otuzbiroğlu, A., Bozan, B., Kıvanç, M. (2021). Lipid production from crude glycerol by newly isolated oleaginous yeasts: strain selection, molecular identification and fatty acid analysis. Waste and Biomass Valorization, 1-10, https://doi.org/ 10.1007/s12649-021-01405-1
11. Biagiotti, C., Ciani, M., Canonico, L., Comitini, F. (2018). Occurrence and involvement of yeast biota in ripening of Italian Fossa cheese. European Food Research and Technology. 244 (11): 1921-1931, doi: 10.1007/s00217-018-3104-6
12. Ceugniez, A., Coucheney, F., Jacques, P., Daube, G., Delcenserie, V., Drider, D. (2017). Anti-Salmonella activity and probiotic trends of Kluyveromyces marxianus S-2-05 and Kluyveromyces lactis S-3-05 isolated from a French cheese, Tomme d'Orchies. Research in Microbiology, 168(6), 575-582, https://doi.org/10.1016/ j.resmic.2017.03.004
13. Chu, Y., Li, M., Jin, J., Dong, X., Xu, K., Jin, L., ... Ji, H. (2023). Advances in the application of the non-conventional yeast Pichia kudriavzevii in food and biotechnology industries. Journal of Fungi, 9(2), 170, https://doi.org/10.3390/jof9020170
14. Collard, B. C., Mackill, D. J. (2009). Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Molecular Biology Reporter, 27, 86-93, https://doi.org/10.1007/ s11105-008-0060-5
15. de Morais, S. B., Florentino, B. M., Marinsek, S. R. M., de Rezende Bastos, A. A., Quirino, B. F. (2023). The potential of β-glucosidases for aroma and flavor improvement in the food industry. The Microbe, 100004, https://doi.org/10.1016/ j.microb.2023.100004
16. Dodor, D. E., Tabatabai, M. A. (2007). Arylamidase activity as an index of nitrogen mineralization in soils. Communications in Soil Science and Plant Analysis, 38(15-16), 2197-2207, https://doi.org/10.1080/00103620701549132
17. Dos Santos, M.T.P.G., Benito, M. J., de Guía Córdoba, M., Alvarenga, N. And Herrera, S.R. M.S. (2017). Yeast community in traditional Portuguese Serpa cheese by culture-dependent and-independent DNA approaches. International Journal of Food Microbiology. 262: 63-70, doi: 10.1016/j.ijfoodmicro.2017.09.013
18. El Mayda, E. (2007). Manufacture of local cheese from raw milk in Syria. In Proceedings International Symposium on ‘Historical Cheeses of Countries around the Archipelago Mediterraneo’, Thessaloniki, 6-8 December, Greece (pp. 6-8).
19. Facchin, S., Barbosa, A. C., Carmo, L. S., Silva, M. C. C., Oliveira, A. L., Morais, P. B., Rosa, C. A. (2013). Yeasts and hygienic-sanitary microbial indicators in water buffalo mozzarella produced and commercialized in Minas Gerais, Brazil. Brazilian Journal of Microbiology, 44, 701-707, https://doi.org/10.1590/S1517-83822013000300006
20. Fadda, M. E., Mossa, V., Deplano, M., Pisano, M. B., Cosentino, S. (2017). In vitro screening of Kluyveromyces strains isolated from Fiore Sardo cheese for potential use as probiotics. LWT, 75, 100-106, https://doi.org/10.1016/ j.lwt.2016.08.020
21. Ferreira, A. D., Viljoen, B. C. (2003). Yeasts as adjunct starters in matured Cheddar cheese. International Journal of Food Microbiology, 86(1-2), 131-140, https://doi.org/10.1016/S0168-1605(03)00252-6
22. Gelen, S. U., Ceylan, Z. (2017). Isolation and identification of yeasts in white cheese. Journal of Advances in VetBio Science and Techniques, 6(2), 100-10, https://doi.org/10.31797/vetbio. 907007
23. Geronikou, A., Srimahaeak, T., Rantsiou, K., Triantafillidis, G., Larsen, N., Jespersen, L. (2020). Occurrence of yeasts in white-brined cheeses: Methodologies for identification, spoilage potential and good manufacturing practices. Frontiers in Microbiology, 11, 582778, https://doi.org/10.3389/fmicb.2020.582778
24. Gogoi, B., Wann, S. B., Saikia, S. P. (2020). Comparative assessment of ISSR, RAPD, and SCoT markers for genetic diversity in Clerodendrum species of North East India. Molecular Biology Reports, 47, 7365-7377. https://doi.org/10.1007/s11033-020-05792-x
25. Güneş, E., Aydin, F., Çakır, İ. (2021). Enzymatic characterization of yeast isolated from naturally fermented Herbs. Gıda, 46(5), 1081-1091, https://doi.org/10.15237/gida.GD21088
26. Gürkan, B. (2018). Çeşitli Kaynaklardan Probiyotik Mayaların İzolasyonu ve İdentifikasyonu. Yüksek Lisans Tezi, Fen Bilimleri Enstitüsü, Ege Üniversitesi.
27. Harju, S., Fedosyuk, H., Peterson, K. R. (2004). Rapid isolation of yeast genomic DNA: Bust n'Grab. BMC Biotechnology, 4(1), 1-6, https://doi.org/10.1186/1472-6750-4-8
28. Herreros, M. A., Fresno, J. M., Prieto, M. G., Tornadijo, M. E. (2003). Technological characterization of lactic acid bacteria isolated from Armada cheese (a Spanish goats’ milk cheese). International Dairy Journal, 13(6), 469-479, https://doi.org/10.1016/S0958-6946(03)00054-2
29. Karasu Yalçın, S., Ergül, Ş. Ş., Özbaş, Z. Y. (2011). Peynir mikroflorasındaki mayaların önemi. Gıda, 36(1), 55-62. Karasu-Yalcin, S., Senses-Ergul, S., Ozbas, Z. Y. (2019). Yeast strains with technological and probiotic traits isolated from Mihalic cheese. International Food Research Journal, 26(4), 1359-1370.
30. Karasu-Yalcin, S., Senses-Ergul, S., Ozbas, Z.Y. (2012). Identification and enzymatic characterization of the yeasts isolated from Erzincan tulum cheese. Mljekarstvo. 62(1): 53-61.
31. Kesenkaş, H., Akbulut, N. (2006). Mayaların peynir üretiminde destek starter kültür olarak kullanımı. Ege Üniversitesi Ziraat Fakültesi Dergisi, 43(2), 165-174.
32. Klein, N., Zourari, A., Lortal, S. (2002). Peptidase activity of four yeast species frequently encountered in dairy products—comparison with several dairy bacteria. International Dairy Journal, 12(10), 853-861, https://doi.org/10.1016/S0958-6946(02)00081-X
33. Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6), 1547, doi: 10.1093/molbev/msy096 Lenoir, J. (1984). The surface flora and its role in the ripening of cheese. International Dairy Federation Bulletin, 171, 3-20.
34. Magboul, A. A., McSweeney, P. L. (1999). Purification and characterization of an acid phosphatase from Lactobacillus plantarum DPC2739. Food Chemistry, 65(1), 15-22, https://doi.org/10.1016/S0308-8146(98)00255-6
35. Martin, N., Berger, C., Le Du, C., Spinnler, H. E. (2001). Aroma compound production in cheese curd by coculturing with selected yeast and bacteria. Journal of Dairy Science, 84(10), 2125-2135, https://doi.org/10.3168/jds.S0022-0302(01)74657-7
36. McSweeney, P. L. (2004). Biochemistry of cheese ripening. International Journal of Dairy Technology, 57(2‐3), 127-144. https://doi.org/10.1111/j.1471-0307.2004.00147.x
37. Merchán, A. V., Benito, M. J., Galván, A. I., de Herrera, S. R. M. S. (2020). Identification and selection of yeast with functional properties for future application in soft paste cheese. LWT, 124, 109173, https://doi.org/10.1016/j.lwt.2020.109173
38. Müjdeci, G. N. (2012). Gemlik Çeşidi Siyah Sofralık Zeytinlerin Doğal Fermantasyonlarında Yer Alan Mayaların Ve Teknolojik Özelliklerinin İncelenmesi, Doktora Tezi, Fen Bilimleri Enstitüsü, Hacettepe Üniversitesi.
39. Nehme, L., Salameh, C., Tabet, E., Nehme, M., Hosri, C. (2019). Innovative improvement of Shanklish cheese production in Lebanon. International Dairy Journal, 90, 23-27, https://doi.org/10.1016/j.idairyj.2018.10.005
40. Oliveira, D. R., Lopes, A. C. A., Pereira, R. A., Cardoso, P. G., Duarte, W. F. (2019). Selection of potentially probiotic Kluyveromyces lactis for the fermentation of cheese whey–based beverage. Annals of Microbiology, 69, 1361-1372, https://doi.org/10.1007/s13213-019-01518-y
41. Ozmen Togay, S., Capece, A., Siesto, G., Aksu, H., Sandikci Altunatmaz, S., Yilmaz Aksu, F., Romano, P., Karagul Yuceer, Y. (2020). Molecular characterization of yeasts isolated from traditional Turkish cheeses. Food Science and Technology, 40, 871-876, https://doi.org/10.1590/fst.24319
42. Patino, E. M., Giorgi, E. J., Mendez, F. Y. (1999). Shanklish cheese-an artisanal product from Corrientes Province, Argentina. Revista Argentina de Lactologia, 18(77), e81.
43. Psomas, E., Andrighetto, C., Litopoulou-Tzanetaki, E., Lombardi, A., Tzanetakis, N. (2001). Some probiotic properties of yeast isolates from infant faeces and Feta cheese. International Journal of Food Microbiology, 69(1-2), 125-133, https://doi.org/10.1016/S0168-1605(01)00580-3
44. Saqib, S., Akram, A., Halim, S. A., Tassaduq, R. (2017). Sources of β-galactosidase and its applications in food industry. 3 Biotech, 7, 1-7, https://doi.org/10.1007/s13205-017-0645-5
45. Spohner, S. C., Schaum, V., Quitmann, H., Czermak, P. (2016). Kluyveromyces lactis: an emerging tool in biotechnology. Journal of Biotechnology, 222, 104-116, https://doi.org/ 10.1016/j.jbiotec.2016.02.023
46. Suzzi, G., Lanorte, M. T., Galgano, F., Andrighetto, C., Lombardi, A., Lanciotti, R., Guerzoni, M. E. (2001). Proteolytic, lipolytic and molecular characterisation of Yarrowia lipolytica isolated from cheese. International Journal of Food Microbiology, 69(1-2), 69-77, https://doi.org/10.1016/S0168-1605(01)00574-8
47. Tempel van den, T., Jakobsen, M. (1998). Yeasts associated with Danablu. International Dairy Journal, 8(1), 25-31, https://doi.org/10.1016/S0958-6946(98)00013-2
48. Tikendra, L., Potshangbam, A. M., Dey, A., Devi, T. R., Sahoo, M. R., Nongdam, P. (2021). RAPD, ISSR, and SCoT markers based genetic stability assessment of micropropagated Dendrobium fimbriatum Lindl. var. oculatum Hk. f.-an important endangered orchid. Physiology and Molecular Biology of Plants, 27, 341-357. https://doi.org/10.1007/s12298-021-00939-x
49. Tokak, S., Kılıç, İ. H., Yalçın, H. T., Duran, T. (2019). Detection of extracellular lipases and genotypic identification from yeast causing spoilage of some dairy products produced in Gaziantep. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi, 22, 206-211, DOI:10.18016/ksutarimdoga.vi.555727
50. Toufeili, I., Shadarevian, S., Artinian, T., Tannous, R. (1995). Ripening changes and sensory properties of bovine, caprine and ovine shankleesh. International Dairy Journal, 5(2), 179-189, https://doi.org/10.1016/0958-6946(95)92209-M
51. White, T. J., Bruns, T., Lee, S. J. W. T., Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a guide to methods and applications, 18(1), 315-322.
52. Yildiz, M., Turgut, T., Cetin, B., Kesmen, Z. (2021). Microbiological characteristics and identification of yeast microbiota of traditional mouldy civil cheese. International Dairy Journal, 116, 104955, https://doi.org/10.1016/ j.idairyj.2020.104955
53. Zheng, X., Li, K., Shi, X., Ni, Y., Li, B., Zhuge, B. (2018). Potential characterization of yeasts isolated from Kazak artisanal cheese to produce flavoring compounds. Microbiology Open, 7(1), e00533, https://doi.org/10.1002/mbo3.533
54. Zheng, X., Shi, X., Wang, B. (2021). A review on the general cheese processing technology, flavor biochemical pathways and the influence of yeasts in cheese. Frontiers in Microbiology, 12, 703284, https://doi.org/10.3389/fmicb.2021.703284