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ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY

Year 2024, , 312 - 325, 15.04.2024
https://doi.org/10.15237/gida.GD23139

Abstract

The present study was conducted to identify acid-resistant lactic acid bacteria from fermented Turkish sucuk, detect phylogenetic affinities, and probiotic/biotechnological profiles. Samples were collected from popular fermented meat retail stores in Kayseri (n:20). The selected lactobacilli were exposed to different pH. Isolates resistant to pH2 were identified by sequencing following the 16s rRNA gene amplification and recorded in GenBank. The pH2 is distinctive for lactobacilli, as most (61.9%) of lactobacilli were inhibited (P <0.05). Isolates surviving at pH2 were determined to be Limosilactobacillus fermentum. The survival rates in bile salt, simulated gastrointestinal juices (between 97.13-106.60%), and autoaggregation, hydrophobicity, and coaggregation of isolates were statistically significant (P <0.05). L. fermentum S19 was the only isolate capable of producing exopolysaccharide; S19 had a high autoaggregation and hydrophobicity over 70%. Traditional Turkish fermented sucuk is a product with enormous potential, containing the newly isolated wild-type L. fermentum, which stands out for biotechnological/probiotic properties.

Project Number

Proje ile desteklenmemiştir.

References

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  • Asan-Ozusaglam, M., Gunyakti, A. (2019). Lactobacillus fermentum strains from human breast milk with probiotic properties and cholesterol-lowering effects. Food science and biotechnology, 28, 501-509.
  • Beganović, J., Kos, B., Pavunc, A. L., Uroić, K., Džidara, P., Šušković, J. (2013). Proteolytic activity of probiotic strain Lactobacillus helveticus M92. Anaerobe, 20, 58-64.
  • Bis-Souza, C. V., Barba, F. J., Lorenzo, J. M., Penna, A. B., Barretto, A. C. S. (2019). New strategies for the development of innovative fermented meat products: a review regarding the incorporation of probiotics and dietary fibers. Food Reviews International, 35(5), 467-484.
  • Bozdemir, M. (2021). Bozadan izole edilmiş laktik asit bakterilerinin bazı teknolojik ve fonksiyonel özellikleri (Master's thesis, Tekirdağ Namık Kemal Üniversitesi).
  • Bozdemir, M., Gümüş, T., Altan Kamer, D. D. (2022). Technological and beneficial features of lactic acid bacteria isolated from Boza A cereal-based fermented beverage. Food Biotechnology, 36 (3), 209-233.
  • Campedelli, I., Mathur, H., Salvetti, E., Clarke, S., Rea, M. C., Torriani, S., Ross, P. R., Hill, C., O’Toole, P. W. (2019). Genus-wide assessment of antibiotic resistance in Lactobacillus spp. Applied and environmental microbiology, 85(1), e01738-18.
  • Candogan, K. Acton, J. C. (2004). Proteolytic activity of bacterial starter cultures for meat fermentation. Journal of Muscle Foods, 15(1), 23-34.
  • Cevher, C. (2023). Socioeconomic Factors Affecting Sustainable Management of Improved Rangelands in Kayseri, Turkey. Rangeland Ecology & Management, 87, 44-54.
  • Clinical and Laboratory Standards Institute (CLSI) (2015) Performance standards for antimicrobial susceptibility testing: twenty-second informational supplement. In: CLSI document M100-S22. Clinical Laboratory Standard Institute, Wayne
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  • Han, Q., Kong, B., Chen, Q., Sun, F., Zhang, H. (2017). In vitro comparison of probiotic properties of lactic acid bacteria isolated from Harbin dry sausages and selected probiotics. Journal of Functional Foods, 32, 391-400.
  • Kaban, G. (2010). Volatile compounds of traditional Turkish dry fermented sausage (sucuk). International Journal of Food Properties, 13(3), 525-534.
  • Krausova, G., Hyrslova, I., Hynstova, I. (2019). In vitro evaluation of adhesion capacity, hydrophobicity, and auto-aggregation of newly isolated potential probiotic strains. Fermentation, 5 (4), 100.
  • Lambert, J., Hull, R. (1996). Upper gastrointestinal tract disease and probiotics. Asia Pacific Journal of Clinical Nutrition, 5, 31-35.
  • Li, T., Teng, D., Mao, R., Hao, Y., Wang, X., Wang, J. (2020). A critical review of antibiotic resistance in probiotic bacteria. Food Research International, 136, 109571.
  • Liu, C., Xue, W. J., Ding, H., An, C., Ma, S. J., Liu, Y. (2022). Probiotic potential of Lactobacillus strains isolated from fermented vegetables in shaanxi, China. Frontiers in Microbiology, 12, 774903.
  • Lorenzo, J. M., Munekata, P., Domínguez, R. (2017). Role of autochthonous starter cultures in the reduction of biogenic amines in traditional meat products. Current Opinion in Food Science, 14, 61-65.
  • Masco, L., Crockaert, C., Van Hoorde, K., Swings, J., Huys, G. (2007). In vitro assessment of the gastrointestinal transit tolerance of taxonomic reference strains from human origin and probiotic product isolates of Bifidobacterium. Journal of dairy science, 90(8), 3572-3578.
  • Montoro, B. P., Benomar, N., Gómez, N. C., Ennahar, S., Horvatovich, P., Knapp, C. W., Galvez, A., Abriouel, H. (2018). Proteomic analysis of Lactobacillus pentosus for the identification of potential markers involved in acid resistance and their influence on other probiotic features. Food microbiology, 72, 31-38.
  • Negrete-Romero, B., Valencia-Olivares, C., Baños-Dossetti, G. A., Pérez-Armendáriz, B., Cardoso-Ugarte, G. A. (2021). Nutritional contributions and health associations of traditional fermented foods. Fermentation, 7(4), 289.
  • Owusu-Kwarteng, J., Tano-Debrah, K., Akabanda, F., Jespersen, L. (2015). Technological properties and probiotic potential of Lactobacillus fermentum strains isolated from West African fermented millet dough. BMC microbiology, 15(1), 1-10.
  • Paulino do Nascimento, L. C., Lacerda, D. C., Ferreira, D. J. S., de Souza, E. L., de Brito Alves, J. L. (2022). Limosilactobacillus fermentum, current evidence on the antioxidant properties and opportunities to be exploited as a probiotic microorganism. Probiotics and Antimicrobial Proteins, 14(5), 960-979.
  • Phujumpa, P., Muangham, S., Jatuponwiphat, T., Koffas, M., Nakphaichit, M., Vongsangnak, W. (2022). Comparative genomics-based probiotic relevance of Limosilactobacillus fermentum KUB-D18. Gene, 840, 146747.
  • Potočnjak, M., Pušić, P., Frece, J., Abram, M., Janković, T., Gobin, I. (2017). Three new Lactobacillus plantarum strains in the probiotic toolbox against gut pathogen Salmonella enterica serotype Typhimurium. Food Technology and Biotechnology, 55(1), 48.
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  • Raveschot, C., Cudennec, B., Deracinois, B., Frémont, M., Vaeremans, M., Dugersuren, J., Demberel, S., Drider, D., Dhulster, P., Coutte, F., Flahaut, C. (2020). Proteolytic activity of Lactobacillus strains isolated from Mongolian traditional dairy products: A multiparametric analysis. Food chemistry, 304, 125415.
  • Rodríguez-Sojo, M. J., Ruiz-Malagón, A. J., Rodríguez-Cabezas, M. E., Gálvez, J., Rodríguez-Nogales, A. (2021). Limosilactobacillus fermentum CECT5716: mechanisms and therapeutic insights. Nutrients, 13(3), 1016.
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FERMENTE TÜRK SUCUKLARINDAN ELDE EDİLEN ASİDE DİRENÇLİ LİMOSİLACTOBACİLLUS FERMENTUM: PROBİYOTİK KARAKTERİZASYONU VE FİLOGENİNİN TARANMASI

Year 2024, , 312 - 325, 15.04.2024
https://doi.org/10.15237/gida.GD23139

Abstract

Bu çalışmada, geleneksel fermente Türk sucuğundaki aside dirençli laktik asit bakterilerini identifiye etmek, filogenetik afinitelerini ve probiyotik/biyoteknolojik profillerini tespit etmek amaçlanmaktadır. Örnekler (n:20) Kayseri'deki popüler fermente et perakende satış yerlerinden toplanmıştır. Seçilen laktobasiller farklı pH ortamlarına maruz bırakılmıştır. pH2'ye dirençli izolatlar, 16s rRNA gen amplifikasyonunu takiben dizileme yapılarak tanımlanmış ve GenBank’a kaydedilmiştir. Laktobasillerin çoğu (%61.9) inhibe edildiği için pH2, laktobasiller için ayırt edici bir ortamdır (P <0.05). pH2'de hayatta kalan izolatların Limosilactobacillus fermentum olduğu belirlenmiştir. L. fermentum izolatları arasında safra tuzu, simüle edilmiş gastrointestinal ortamlarında hayatta kalma oranları (%97.13-106.60) ve otoagregasyon, hidrofobiklik ve koagregasyon düzeyleri istatistiksel olarak anlamlı bulunmuştur (P <0.05). L. fermentum S19, ekzopolisakkarit üretme yeteneğine sahip tek izolat olduğu belirlenmiştir Ayrıca, S19 yüksek otoagregasyona sahip olup %70'in üzerinde hidrofobisite göstermiştir. Geleneksel Türk fermente sucuğu, biyoteknolojik/probiyotik özellikleriyle öne çıkan, yeni izole edilen yabani tip L. fermentum'u içeren, önemli potansiyele sahip bir üründür.

Project Number

Proje ile desteklenmemiştir.

References

  • Ammor, M. S., Flórez, A. B., Mayo, B. (2007). Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria. Food microbiology, 24(6), 559-570.
  • An, Y. H., Dickinson, R. B., Doyle, R. J. (2000). Mechanisms of bacterial adhesion and pathogenesis of implant and tissue infections. In Handbook of bacterial adhesion: principles, methods, and applications (pp. 1-27). Totowa, NJ: Humana Press.
  • Asan-Ozusaglam, M., Gunyakti, A. (2019). Lactobacillus fermentum strains from human breast milk with probiotic properties and cholesterol-lowering effects. Food science and biotechnology, 28, 501-509.
  • Beganović, J., Kos, B., Pavunc, A. L., Uroić, K., Džidara, P., Šušković, J. (2013). Proteolytic activity of probiotic strain Lactobacillus helveticus M92. Anaerobe, 20, 58-64.
  • Bis-Souza, C. V., Barba, F. J., Lorenzo, J. M., Penna, A. B., Barretto, A. C. S. (2019). New strategies for the development of innovative fermented meat products: a review regarding the incorporation of probiotics and dietary fibers. Food Reviews International, 35(5), 467-484.
  • Bozdemir, M. (2021). Bozadan izole edilmiş laktik asit bakterilerinin bazı teknolojik ve fonksiyonel özellikleri (Master's thesis, Tekirdağ Namık Kemal Üniversitesi).
  • Bozdemir, M., Gümüş, T., Altan Kamer, D. D. (2022). Technological and beneficial features of lactic acid bacteria isolated from Boza A cereal-based fermented beverage. Food Biotechnology, 36 (3), 209-233.
  • Campedelli, I., Mathur, H., Salvetti, E., Clarke, S., Rea, M. C., Torriani, S., Ross, P. R., Hill, C., O’Toole, P. W. (2019). Genus-wide assessment of antibiotic resistance in Lactobacillus spp. Applied and environmental microbiology, 85(1), e01738-18.
  • Candogan, K. Acton, J. C. (2004). Proteolytic activity of bacterial starter cultures for meat fermentation. Journal of Muscle Foods, 15(1), 23-34.
  • Cevher, C. (2023). Socioeconomic Factors Affecting Sustainable Management of Improved Rangelands in Kayseri, Turkey. Rangeland Ecology & Management, 87, 44-54.
  • Clinical and Laboratory Standards Institute (CLSI) (2015) Performance standards for antimicrobial susceptibility testing: twenty-second informational supplement. In: CLSI document M100-S22. Clinical Laboratory Standard Institute, Wayne
  • Collado, M. C., Sanz, Y. (2007). Induction of acid resistance in Bifidobacterium: a mechanism for improving desirable traits of potentially probiotic strains. Journal of applied microbiology, 103(4), 1147-1157.
  • D'ambrosio, S., Ventrone, M., Fusco, A., Casillo, A., Dabous, A., Cammarota, M., Corsaro, M. M., Donnarumma, G., Schiraldi, C., Cimini, D. (2022). Limosilactobacillus fermentum from buffalo milk is suitable for potential biotechnological process development and inhibits Helicobacter pylori in a gastric epithelial cell model. Biotechnology Reports, 34, e00732.
  • De Angelis, M., Gobbetti, M. (2004). Environmental stress responses in Lactobacillus: a review. Proteomics, 4(1), 106-122.
  • de Melo Pereira, G. V., de Oliveira Coelho, B., Júnior, A. I. M., Thomaz-Soccol, V., Soccol, C. R. (2018). How to select a probiotic? A review and update of methods and criteria. Biotechnology advances, 36(8), 2060-2076.
  • de Oliveira Coelho, B., Fiorda-Mello, F., de Melo Pereira, G. V., Thomaz-Soccol, V., Rakshit, S. K., de Carvalho, J. C., Soccol, C. R. (2019). In vitro probiotic properties and DNA protection activity of yeast and lactic acid bacteria isolated from a honey-based kefir beverage. Foods, 8(10), 485.
  • Dempsey, E., Corr, S. C. (2022). Lactobacillus spp. for gastrointestinal health: Current and future perspectives. Frontiers in immunology, 13, 840245.
  • Dincer, E., Kivanc, M. (2012). Characterization of lactic acid bacteria from Turkish Pastirma. Annals of microbiology, 62, 1155-1163.
  • Dincer, E., Kivanc, M. (2018). Lipolytic activity of lactic acid bacteria isolated from Turkish pastırma. Anadolu University Journal of Science and Technology C-Life Sciences and Biotechnology, 7(1), 12-19.
  • Dishan, A., Gönülalan, Z. (2024). Lacticaseibacillus paracasei AD22 Stress Response in Brined White Cheese Matrix: In Vitro Probiotic Profiles and Molecular Characterization. Probiotics and Antimicrobial Proteins, 1-14.
  • Food and Drug Administration. Microorganisms & Microbial-Derived Ingredients Used in Food (Partial List). Available online: https://www.fda.gov/food/generally-recognized-safe-gras/microorganisms-microbial-derived-ingredients-used-food-partial-list
  • Gueimonde, M., Sánchez, B., G. de los Reyes-Gavilán, C., Margolles, A. (2013). Antibiotic resistance in probiotic bacteria. Frontiers in microbiology, 4, 202.
  • Han, Q., Kong, B., Chen, Q., Sun, F., Zhang, H. (2017). In vitro comparison of probiotic properties of lactic acid bacteria isolated from Harbin dry sausages and selected probiotics. Journal of Functional Foods, 32, 391-400.
  • Kaban, G. (2010). Volatile compounds of traditional Turkish dry fermented sausage (sucuk). International Journal of Food Properties, 13(3), 525-534.
  • Krausova, G., Hyrslova, I., Hynstova, I. (2019). In vitro evaluation of adhesion capacity, hydrophobicity, and auto-aggregation of newly isolated potential probiotic strains. Fermentation, 5 (4), 100.
  • Lambert, J., Hull, R. (1996). Upper gastrointestinal tract disease and probiotics. Asia Pacific Journal of Clinical Nutrition, 5, 31-35.
  • Li, T., Teng, D., Mao, R., Hao, Y., Wang, X., Wang, J. (2020). A critical review of antibiotic resistance in probiotic bacteria. Food Research International, 136, 109571.
  • Liu, C., Xue, W. J., Ding, H., An, C., Ma, S. J., Liu, Y. (2022). Probiotic potential of Lactobacillus strains isolated from fermented vegetables in shaanxi, China. Frontiers in Microbiology, 12, 774903.
  • Lorenzo, J. M., Munekata, P., Domínguez, R. (2017). Role of autochthonous starter cultures in the reduction of biogenic amines in traditional meat products. Current Opinion in Food Science, 14, 61-65.
  • Masco, L., Crockaert, C., Van Hoorde, K., Swings, J., Huys, G. (2007). In vitro assessment of the gastrointestinal transit tolerance of taxonomic reference strains from human origin and probiotic product isolates of Bifidobacterium. Journal of dairy science, 90(8), 3572-3578.
  • Montoro, B. P., Benomar, N., Gómez, N. C., Ennahar, S., Horvatovich, P., Knapp, C. W., Galvez, A., Abriouel, H. (2018). Proteomic analysis of Lactobacillus pentosus for the identification of potential markers involved in acid resistance and their influence on other probiotic features. Food microbiology, 72, 31-38.
  • Negrete-Romero, B., Valencia-Olivares, C., Baños-Dossetti, G. A., Pérez-Armendáriz, B., Cardoso-Ugarte, G. A. (2021). Nutritional contributions and health associations of traditional fermented foods. Fermentation, 7(4), 289.
  • Owusu-Kwarteng, J., Tano-Debrah, K., Akabanda, F., Jespersen, L. (2015). Technological properties and probiotic potential of Lactobacillus fermentum strains isolated from West African fermented millet dough. BMC microbiology, 15(1), 1-10.
  • Paulino do Nascimento, L. C., Lacerda, D. C., Ferreira, D. J. S., de Souza, E. L., de Brito Alves, J. L. (2022). Limosilactobacillus fermentum, current evidence on the antioxidant properties and opportunities to be exploited as a probiotic microorganism. Probiotics and Antimicrobial Proteins, 14(5), 960-979.
  • Phujumpa, P., Muangham, S., Jatuponwiphat, T., Koffas, M., Nakphaichit, M., Vongsangnak, W. (2022). Comparative genomics-based probiotic relevance of Limosilactobacillus fermentum KUB-D18. Gene, 840, 146747.
  • Potočnjak, M., Pušić, P., Frece, J., Abram, M., Janković, T., Gobin, I. (2017). Three new Lactobacillus plantarum strains in the probiotic toolbox against gut pathogen Salmonella enterica serotype Typhimurium. Food Technology and Biotechnology, 55(1), 48.
  • QingWu, D., Ding, X. S., Zhao, B., An, Q., Guo, J. S. (2022). The essential role of hydrophobic interaction within extracellular polymeric substances in auto-aggregation of P. stutzeri strain XL-2. International Biodeterioration & Biodegradation, 171, 105404.
  • Raveschot, C., Cudennec, B., Deracinois, B., Frémont, M., Vaeremans, M., Dugersuren, J., Demberel, S., Drider, D., Dhulster, P., Coutte, F., Flahaut, C. (2020). Proteolytic activity of Lactobacillus strains isolated from Mongolian traditional dairy products: A multiparametric analysis. Food chemistry, 304, 125415.
  • Rodríguez-Sojo, M. J., Ruiz-Malagón, A. J., Rodríguez-Cabezas, M. E., Gálvez, J., Rodríguez-Nogales, A. (2021). Limosilactobacillus fermentum CECT5716: mechanisms and therapeutic insights. Nutrients, 13(3), 1016.
  • Sahadeva, R. P. K., Leong, S. F., Chua, K. H., Tan, C. H., Chan, H. Y., Tong, E. V., Wong, S.Y.W., Chan, H. K. (2011). Survival of commercial probiotic strains to pH and bile. International Food Research Journal, 18(4), 1515-1522.
  • Shehata, M. G., El Sohaimy, S. A., El-Sahn, M. A., Youssef, M. M. (2016). Screening of isolated potential probiotic lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity. Annals of Agricultural Sciences, 61(1), 65-75.
  • Silva, J. A., Marchesi, A., Wiese, B., Nader‐Macias, M. E. F. (2019). Technological characterization of vaginal probiotic lactobacilli: resistance to osmotic stress and strains compatibility. Journal of applied microbiology, 127(6), 1835-1847.
  • Soares, M. B., Martinez, R. C., Pereira, E. P., Balthazar, C. F., Cruz, A. G., Ranadheera, C. S., Sant'Ana, A. S. (2019). The resistance of Bacillus, Bifidobacterium, and Lactobacillus strains with claimed probiotic properties in different food matrices exposed to simulated gastrointestinal tract conditions. Food research international, 125, 108542.
  • Stingele, F., Neeser, J. R., Mollet, B. (1996). Identification and characterization of the eps (exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6. Journal of bacteriology, 178(6), 1680-1690.
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  • Suzuki, M. T., Giovannoni, S. J. (1996). Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Applied and environmental microbiology, 62(2), 625-630.
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There are 56 citations in total.

Details

Primary Language English
Subjects Meat Technology, Food Biotechnology, Food Microbiology
Journal Section Articles
Authors

Adalet Dışhan 0000-0001-8097-1648

Zafer Gonulalan 0000-0002-3935-6296

Project Number Proje ile desteklenmemiştir.
Publication Date April 15, 2024
Submission Date December 13, 2023
Acceptance Date March 18, 2024
Published in Issue Year 2024

Cite

APA Dışhan, A., & Gonulalan, Z. (2024). ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY. Gıda, 49(2), 312-325. https://doi.org/10.15237/gida.GD23139
AMA Dışhan A, Gonulalan Z. ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY. GIDA. April 2024;49(2):312-325. doi:10.15237/gida.GD23139
Chicago Dışhan, Adalet, and Zafer Gonulalan. “ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY”. Gıda 49, no. 2 (April 2024): 312-25. https://doi.org/10.15237/gida.GD23139.
EndNote Dışhan A, Gonulalan Z (April 1, 2024) ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY. Gıda 49 2 312–325.
IEEE A. Dışhan and Z. Gonulalan, “ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY”, GIDA, vol. 49, no. 2, pp. 312–325, 2024, doi: 10.15237/gida.GD23139.
ISNAD Dışhan, Adalet - Gonulalan, Zafer. “ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY”. Gıda 49/2 (April 2024), 312-325. https://doi.org/10.15237/gida.GD23139.
JAMA Dışhan A, Gonulalan Z. ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY. GIDA. 2024;49:312–325.
MLA Dışhan, Adalet and Zafer Gonulalan. “ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY”. Gıda, vol. 49, no. 2, 2024, pp. 312-25, doi:10.15237/gida.GD23139.
Vancouver Dışhan A, Gonulalan Z. ACID-RESISTANT LIMOSILACTOBACILLUS FERMENTUM ISOLATES RECOVERED FROM FERMENTED TURKISH SUCUK: SCREENING OF PROBIOTIC CHARACTERIZATION AND PHYLOGENY. GIDA. 2024;49(2):312-25.

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