Research Article
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DETERMINATION OF ENDOSPORE STRUCTURE AND VOLATILE COMPOUNDS OF CLOSTRIDIAL SPECIES DURING SPORULATION

Year 2023, Volume: 48 Issue: 3, 499 - 508, 16.06.2023

Pelin Ertürkmen Zübeyde Öner

https://doi.org/10.15237/gida.GD23028

Abstract

In this study, endospore structural and volatile compounds of clostridial species causing late blowing defect (LBD) in cheese were investigated during sporulation. The species were morphologically identified by Gram staining, catalase and oxidase tests, and anaerobic growth tests, and were confirmed by 16S rRNA gene sequence analysis. Strains with high gas production capacity were analyzed by Scanning Electron Microscopy (SEM) and gas chromatography-mass spectrometry (GC-MS) during sporulation. At the end of sporulation, alcohols such as butanol, pentanol, ethanol, hexanol, propanol, octanol, as well as sulfite, ester and acid group compounds were the prominent components. The cell wall residues and endospore structural components of Clostridium sporogenes strains with a total relative percentage area of >50% sulfite group compound were observed more intensively than Clostridium butyricum strains with high butanol production levels. The information obtained from this study will facilitate studies aimed at identifying endospore structural and volatile compounds of LBD clostridial species.

Keywords

Clostridium spp. GC-MS SEM late blowing 16S rRNA

Supporting Institution

SÜLEYMAN DEMİREL ÜNİVERSİTESİ

Project Number

FDK-2019-6943

Thanks

This study was part of a doctoral dissertation (Pelin Ertürkmen, FDK-2019-6943) supported by the Scientific Research Projects Unit of Suleyman Demirel University, Isparta, Türkiye.

References

  • Bakhtiary, F., Sayevand, H.R., Remely, M., Hippe, B., Indra, A., Hosseini, H., Haslberger, AG. (2018). Identification of Clostridium spp. derived from a sheep and cattle slaughterhouse by Matrix-Assisted Laser Desorption and Ionizationtime of Flight Mass Spectrometry (MALDI-TOF MS) and 16S rDNA sequencing. International Journal of Food Science Technology, 55(8):3232-3240.
  • Bassi, D., Cappa, F., Cocconcelli, P.S. (2009). A combination of SEM technique and X-ray microanalysis to study spore germination process of Clostridium tyrobutyricum. Research in Microbiology, 160:322-329.
  • Bassi, D., Fontana, C., Zucchelli, S., Gazzola, S., Cocconcelli, P.S. (2013). Taqman Real Time-Quantitative PCR targeting the phosphotransacetylase gene for Clostridium tyrobutyricum quantification in animal feed, faeces, milk and cheese. International Dairy Journal, 33:75-82.
  • Brändle, J., Domig, K.J., Kneifel, W. (2016). Relevance and analysis of butyric acid producing Clostridia in milk and cheese. Food Control, 67:96–113.
  • Brunt, J., Cross, L.K., Peck, M.W. (2015). Apertures in the Clostridium sporogenes spore coat and exosporium align to facilitate emergence of the vegetative cell. Food Microbiology, 51:45-50.
  • Cabrera-Martinez, R.M., Tovar-Rojo, F., Vepachedu, V.R., Setlow, P. (2003). Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis. Journal of Bacteriology, 185(8):2457-2464.
  • Chean, R., Kotsanas, D., Francis, M.J. (2014). Comparing the identification of Clostridium spp. by two matrix-Assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry platforms to 16S rRNA PCR sequencing as a reference standard: A detailed analysis of age of culture and sample preparation. Anaerobe, 30(12):85–89.
  • Cremonesi, P., Vanoni, L., Silvetti, T., Morandi, S., Brasca, M. (2012). Identification of Clostridium beijerinckii, C. butyricum, C. sporogenes, C. tyrobutyricum isolated from silage, raw milk and hard cheese by a Multiplex PCR assay. Journal of Dairy Research, 79:318–323.
  • D'Incecco, P., Pellegrino, L., Hogenboom, J.A., Cocconcelli, P.S., Bassi, D. (2018). The late blowing defect of hard cheeses: behaviour of cells and spores of Clostridium tyrobutyricum throughout the cheese manufacturing and ripening. LWT, 87:134-141.
  • Doyle, C.J., Gleesonc, D., Jordan, K., Beresford, T.P., Ross, R.P., Fitzgerald, G.F. (2015). Anaerobic sporeformers and their significance with respect to milk and dairy products. International Journal of Food Microbiology, 197:77–87.
  • Driehuis, F., Hoolwerf, J., and Rademaker, J.L.W. (2016). Concurrence of spores of Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus polymyxa in silage, dairy cow faeces and raw milk. International Dairy Journal, 63:70–77.
  • Ertürkmen P., Öner Z. (2023). Challenging the problematic detection of clostridial isolates causing late-blowing defect with MALDI–TOF MS. Czech Journal Food Science, 41(1):36-43.
  • Faille, C., Tauveron, G., Le Gentil-Lelievre, C., Slomianny, C. (2007). Occurrence of Bacillus cereus spores with a damaged exosporium: Consequences on the spore adhesion on surfaces of food processing lines. Journal of Food Protection, 70, 2346-2353.
  • Garde, S., Arias, R., Gaya, P., Nuñez, M. (2011). Occurrence of Clostridium spp. in ovine milk and Manchego cheese with late blowing defect: Identification and characterization of isolates. International Dairy Journal, 21:272–278.
  • Gómez-Torres, N., Ávila, M., Gaya, P., Garde, S. (2014). Prevention of late blowing defect by reuterin produced in cheese by a Lactobacillus reuteri adjunct. Food Microbiology, 42:82–88.
  • Jakob, E. (2011). Analytik Rund Um Die ButtersäAuregäArung. In Alp Forum (Vol. 85, Pp.1-23. Jones, D.T., Woods, D.R. (1986). Acetone-butanol fermentation revisited. Microbiological Reviews, 50:484-524. Julien, M.C., Dion, P., Lafrenie`re, C., Antoun, H., Drouin, P. (2008). Sources of clostridia in raw milk on farms. Applied and Environmental Microbiology, 74:6348–6357.
  • Keto-Timonen, R., Heikinheimo, A., Eerola, E., Korkeala, H. (2006). Identification of Clostridium species and DNA fingerprinting of Clostridium perfringens by amplified fragment length polymorphism analysis. Journal of Clinical Microbiology, 44:4057–65.
  • Lane, D.J. (1991). 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, Editors. Nucleic Acid Techniques in Bacterial Systematics. New York: Wiley. Pp. 115–175.
  • Le Bourhis, A.G., Dore, J., Carlier, J.P., Chamba, J.F., Popoff, M.R., Tholozan, J.L. (2007). Contribution of C. beijerinckii and C. sporogenes in association with C. tyrobutyricum to the butyric fermentation in emmental type cheese. International Journal of Food Microbiology, 113:154–63.
  • Lee, S.Y., Park, J.H., Jang, S.H., Nielsen, L.K., Kim, J., Jung, K.S. (2008). Fermentative butanol production by clostridia. Biotechnology and Bioengineering, 101(2):209-228.
  • Lequette, Y., Garenaux, E., Tauveron, G., Dumez, S., Perchat, S., Slomianny, C., Lereclus, D., Guerardel, Y., Faille, C. (2011). Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion to stainless steel. Applied and Environmental Microbiology, 77:4905-4911.
  • Lo´pez-Enr´ıquez, L., Rodr´ıguez-La´zaro, D., Herna´ndez, M. (2007). Quantitative detection of Clostridium tyrobutyricum in milk by Real-time PCR. Applied and Environmental Microbiology, 73(37), 47–51.
  • Matijasic, B.B., Rajsp, M.K., Perko, B., Rogelj, I. (2007). Inhibition of Clostridium tyrobutyricum in cheese by Lactobacillus gasseri. International Dairy Journal, 17:157–166.
  • Mc Sweeney, P.L.H. (2007). What is late gas blowing and how may this defect be avoided. In Mcsweeeny (Ed.) Cheese Problems Solved Boca Raton, USA:CRC Press, 198–199.
  • Oliveira, R.B.A., Lopes, L.S., Baptista, R.C., Chincha, A.A.I.A., Portela, J.B., Nascimento, J.S. (2018). Occurrence, populations, diversity, and growth potential of spore-forming bacteria in “Requeijão Cremoso”. LWT-Food Science and Technology, 89, 24–31.
  • Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J., Leonard, F.C. (2002). Veterinary Microbiology and Microbial Disease. Oxford, UK: Blackwell Science.
  • Qureshi, N., Blaschek, H.P. (1999). Production of Acetone-Butanol-Ethanol (ABE) by a hyper-butanol producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation. Biotechnology Progress, 15:594–602.
  • Qureshi, N., Ezeji, T.C. (2008). Butanol, “A Superior Biofuel” production from agricultural residues (Renewable Biomass): Recent Progress in technology. Biofuels, Bioproducts and Biorefinery, 2:319-330.
  • Rainey, F. A., Hollen, B. J., Small, A. (2009). Genus I. Clostridium prazmowski, 1880, 23al. In P. Vos, G. Garrity, D. Jones NR. Krieg, W. Ludwig, F. A. Rainey (Eds.), Bergey’s Manual of Systematic Bacteriology. The Firmicutes New York: Springer, 3:736-827.
  • Rees, C. A., Shen, A., Hill, J.E. (2016). Characterization of the Clostridium difficile volatile metabolome using comprehensive two-dimensional gas chromatography time-of-flight Mass Spectrometry. Journal of Chromatography, 1039:8-16.
  • Silvetti, T., Morandi, S., Brasca, M. (2018). Growth factors affecting gas production and reduction potential of vegetative cell and spore inocula of dairy-related Clostridium species. LWT, 92:32-39.
  • Turchi, B., Pero, S., Torracca, B., Fratini, F., Mancini, S., Galiero, A. (2016). Occurrence of Clostridium spp. in Ewe’s milk: Enumeration and identification of isolates. Dairy Science and Technology, 96(5):693-701.

GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ

Year 2023, Volume: 48 Issue: 3, 499 - 508, 16.06.2023

Pelin Ertürkmen Zübeyde Öner

https://doi.org/10.15237/gida.GD23028

Abstract

u çalışmada, çeşitli örneklerden izole edilen ve peynirde geç şişme kusuruna neden olan clostridial türlerin sporulasyon süresince endospor yapısal ve uçucu bileşikleri araştırılmıştır. Gram boyama, katalaz ve oksidaz testi, anaerobik gelişim testleri ile morfolojik olarak tanımlanan türler 16S rRNA gen sekans analizi ile doğrulanmıştır. Sporulasyon süresince, yüksek gaz üretim kapasitesine sahip suşlar Taramalı Elektron Mikroskobu (SEM) ve gaz kromatografisi-kütle spektrometresi (GC-MS) ile analiz edilmiştir. Sporulasyon sonunda bütanol, pentanol, ethanol, hexanol, proponol, oktanol gibi alkol bileşikleri; sülfit, ester ve asit grubu bileşikler öne çıkan bileşenlerdendir. Toplam bağıl yüzde alanı >%50 sülfit grubu bileşiğe sahip olan Clostridium sporogenes suşlarının hücre duvarı kalıntıları ve endospor yapısal bileşenleri; bütanol üretim düzeyi yüksek olan Clostridium butyricum suşlarına göre daha yoğun şekilde gözlemlenmiştir. Bu çalışmadan elde edilen bilgiler geç şişme etmeni clostridial türlerin endospor yapısal ve uçucu bileşiklerinin tespitine yönelik çalışmalara kolaylık sağlayacaktır.

Keywords

Clostridium spp. GC-MS SEM geç şişme 16S rRNA

Project Number

FDK-2019-6943

References

  • Bakhtiary, F., Sayevand, H.R., Remely, M., Hippe, B., Indra, A., Hosseini, H., Haslberger, AG. (2018). Identification of Clostridium spp. derived from a sheep and cattle slaughterhouse by Matrix-Assisted Laser Desorption and Ionizationtime of Flight Mass Spectrometry (MALDI-TOF MS) and 16S rDNA sequencing. International Journal of Food Science Technology, 55(8):3232-3240.
  • Bassi, D., Cappa, F., Cocconcelli, P.S. (2009). A combination of SEM technique and X-ray microanalysis to study spore germination process of Clostridium tyrobutyricum. Research in Microbiology, 160:322-329.
  • Bassi, D., Fontana, C., Zucchelli, S., Gazzola, S., Cocconcelli, P.S. (2013). Taqman Real Time-Quantitative PCR targeting the phosphotransacetylase gene for Clostridium tyrobutyricum quantification in animal feed, faeces, milk and cheese. International Dairy Journal, 33:75-82.
  • Brändle, J., Domig, K.J., Kneifel, W. (2016). Relevance and analysis of butyric acid producing Clostridia in milk and cheese. Food Control, 67:96–113.
  • Brunt, J., Cross, L.K., Peck, M.W. (2015). Apertures in the Clostridium sporogenes spore coat and exosporium align to facilitate emergence of the vegetative cell. Food Microbiology, 51:45-50.
  • Cabrera-Martinez, R.M., Tovar-Rojo, F., Vepachedu, V.R., Setlow, P. (2003). Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis. Journal of Bacteriology, 185(8):2457-2464.
  • Chean, R., Kotsanas, D., Francis, M.J. (2014). Comparing the identification of Clostridium spp. by two matrix-Assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry platforms to 16S rRNA PCR sequencing as a reference standard: A detailed analysis of age of culture and sample preparation. Anaerobe, 30(12):85–89.
  • Cremonesi, P., Vanoni, L., Silvetti, T., Morandi, S., Brasca, M. (2012). Identification of Clostridium beijerinckii, C. butyricum, C. sporogenes, C. tyrobutyricum isolated from silage, raw milk and hard cheese by a Multiplex PCR assay. Journal of Dairy Research, 79:318–323.
  • D'Incecco, P., Pellegrino, L., Hogenboom, J.A., Cocconcelli, P.S., Bassi, D. (2018). The late blowing defect of hard cheeses: behaviour of cells and spores of Clostridium tyrobutyricum throughout the cheese manufacturing and ripening. LWT, 87:134-141.
  • Doyle, C.J., Gleesonc, D., Jordan, K., Beresford, T.P., Ross, R.P., Fitzgerald, G.F. (2015). Anaerobic sporeformers and their significance with respect to milk and dairy products. International Journal of Food Microbiology, 197:77–87.
  • Driehuis, F., Hoolwerf, J., and Rademaker, J.L.W. (2016). Concurrence of spores of Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus polymyxa in silage, dairy cow faeces and raw milk. International Dairy Journal, 63:70–77.
  • Ertürkmen P., Öner Z. (2023). Challenging the problematic detection of clostridial isolates causing late-blowing defect with MALDI–TOF MS. Czech Journal Food Science, 41(1):36-43.
  • Faille, C., Tauveron, G., Le Gentil-Lelievre, C., Slomianny, C. (2007). Occurrence of Bacillus cereus spores with a damaged exosporium: Consequences on the spore adhesion on surfaces of food processing lines. Journal of Food Protection, 70, 2346-2353.
  • Garde, S., Arias, R., Gaya, P., Nuñez, M. (2011). Occurrence of Clostridium spp. in ovine milk and Manchego cheese with late blowing defect: Identification and characterization of isolates. International Dairy Journal, 21:272–278.
  • Gómez-Torres, N., Ávila, M., Gaya, P., Garde, S. (2014). Prevention of late blowing defect by reuterin produced in cheese by a Lactobacillus reuteri adjunct. Food Microbiology, 42:82–88.
  • Jakob, E. (2011). Analytik Rund Um Die ButtersäAuregäArung. In Alp Forum (Vol. 85, Pp.1-23. Jones, D.T., Woods, D.R. (1986). Acetone-butanol fermentation revisited. Microbiological Reviews, 50:484-524. Julien, M.C., Dion, P., Lafrenie`re, C., Antoun, H., Drouin, P. (2008). Sources of clostridia in raw milk on farms. Applied and Environmental Microbiology, 74:6348–6357.
  • Keto-Timonen, R., Heikinheimo, A., Eerola, E., Korkeala, H. (2006). Identification of Clostridium species and DNA fingerprinting of Clostridium perfringens by amplified fragment length polymorphism analysis. Journal of Clinical Microbiology, 44:4057–65.
  • Lane, D.J. (1991). 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, Editors. Nucleic Acid Techniques in Bacterial Systematics. New York: Wiley. Pp. 115–175.
  • Le Bourhis, A.G., Dore, J., Carlier, J.P., Chamba, J.F., Popoff, M.R., Tholozan, J.L. (2007). Contribution of C. beijerinckii and C. sporogenes in association with C. tyrobutyricum to the butyric fermentation in emmental type cheese. International Journal of Food Microbiology, 113:154–63.
  • Lee, S.Y., Park, J.H., Jang, S.H., Nielsen, L.K., Kim, J., Jung, K.S. (2008). Fermentative butanol production by clostridia. Biotechnology and Bioengineering, 101(2):209-228.
  • Lequette, Y., Garenaux, E., Tauveron, G., Dumez, S., Perchat, S., Slomianny, C., Lereclus, D., Guerardel, Y., Faille, C. (2011). Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion to stainless steel. Applied and Environmental Microbiology, 77:4905-4911.
  • Lo´pez-Enr´ıquez, L., Rodr´ıguez-La´zaro, D., Herna´ndez, M. (2007). Quantitative detection of Clostridium tyrobutyricum in milk by Real-time PCR. Applied and Environmental Microbiology, 73(37), 47–51.
  • Matijasic, B.B., Rajsp, M.K., Perko, B., Rogelj, I. (2007). Inhibition of Clostridium tyrobutyricum in cheese by Lactobacillus gasseri. International Dairy Journal, 17:157–166.
  • Mc Sweeney, P.L.H. (2007). What is late gas blowing and how may this defect be avoided. In Mcsweeeny (Ed.) Cheese Problems Solved Boca Raton, USA:CRC Press, 198–199.
  • Oliveira, R.B.A., Lopes, L.S., Baptista, R.C., Chincha, A.A.I.A., Portela, J.B., Nascimento, J.S. (2018). Occurrence, populations, diversity, and growth potential of spore-forming bacteria in “Requeijão Cremoso”. LWT-Food Science and Technology, 89, 24–31.
  • Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J., Leonard, F.C. (2002). Veterinary Microbiology and Microbial Disease. Oxford, UK: Blackwell Science.
  • Qureshi, N., Blaschek, H.P. (1999). Production of Acetone-Butanol-Ethanol (ABE) by a hyper-butanol producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation. Biotechnology Progress, 15:594–602.
  • Qureshi, N., Ezeji, T.C. (2008). Butanol, “A Superior Biofuel” production from agricultural residues (Renewable Biomass): Recent Progress in technology. Biofuels, Bioproducts and Biorefinery, 2:319-330.
  • Rainey, F. A., Hollen, B. J., Small, A. (2009). Genus I. Clostridium prazmowski, 1880, 23al. In P. Vos, G. Garrity, D. Jones NR. Krieg, W. Ludwig, F. A. Rainey (Eds.), Bergey’s Manual of Systematic Bacteriology. The Firmicutes New York: Springer, 3:736-827.
  • Rees, C. A., Shen, A., Hill, J.E. (2016). Characterization of the Clostridium difficile volatile metabolome using comprehensive two-dimensional gas chromatography time-of-flight Mass Spectrometry. Journal of Chromatography, 1039:8-16.
  • Silvetti, T., Morandi, S., Brasca, M. (2018). Growth factors affecting gas production and reduction potential of vegetative cell and spore inocula of dairy-related Clostridium species. LWT, 92:32-39.
  • Turchi, B., Pero, S., Torracca, B., Fratini, F., Mancini, S., Galiero, A. (2016). Occurrence of Clostridium spp. in Ewe’s milk: Enumeration and identification of isolates. Dairy Science and Technology, 96(5):693-701.
There are 32 citations in total.

Details

Primary Language Turkish
Subjects Food Engineering
Journal Section Articles
Authors

Pelin Ertürkmen 0000-0003-4321-7886

Zübeyde Öner 0000-0003-2557-0731

Project Number FDK-2019-6943
Early Pub Date April 4, 2023
Publication Date June 16, 2023
Published in Issue Year 2023 Volume: 48 Issue: 3

Cite

APA Ertürkmen, P., & Öner, Z. (2023). GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ. Gıda, 48(3), 499-508. https://doi.org/10.15237/gida.GD23028
AMA Ertürkmen P, Öner Z. GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ. The Journal of Food. June 2023;48(3):499-508. doi:10.15237/gida.GD23028
Chicago Ertürkmen, Pelin, and Zübeyde Öner. “GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ”. Gıda 48, no. 3 (June 2023): 499-508. https://doi.org/10.15237/gida.GD23028.
EndNote Ertürkmen P, Öner Z (June 1, 2023) GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ. Gıda 48 3 499–508.
IEEE P. Ertürkmen and Z. Öner, “GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ”, The Journal of Food, vol. 48, no. 3, pp. 499–508, 2023, doi: 10.15237/gida.GD23028.
ISNAD Ertürkmen, Pelin - Öner, Zübeyde. “GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ”. Gıda 48/3 (June 2023), 499-508. https://doi.org/10.15237/gida.GD23028.
JAMA Ertürkmen P, Öner Z. GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ. The Journal of Food. 2023;48:499–508.
MLA Ertürkmen, Pelin and Zübeyde Öner. “GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ”. Gıda, vol. 48, no. 3, 2023, pp. 499-08, doi:10.15237/gida.GD23028.
Vancouver Ertürkmen P, Öner Z. GEÇ ŞİŞME ETMENİ CLOSTRİDİAL TÜRLERİN SPORULASYON SÜRESİNCE ENDOSPOR YAPISAL VE UÇUCU BİLEŞİKLERİNİN BELİRLENMESİ. The Journal of Food. 2023;48(3):499-508.
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