Bacterial Dynamics of Hardaliye, a Fermented Grape Beverage, Determined by Highthroughput Sequencing

Abstract

Hardaliye is a traditional beverage produced by fermenting red grapes with mustard seeds and sour cherry leaves in the Thrace region of Turkey. There are few studies that have determined the microorganisms responsible for hardaliye fermentation, and these are limited to lactic acid bacteria (LAB) using culture-dependent techniques. The aim of this study was to determine the bacterial dynamics of hardaliye fermentation using a culture-independent approach, high-throughput sequencing of 16S rRNA amplicons. Hardaliye was produced using the traditional method, and samples were taken and analyzed on days 0, 2, 4, 6, and 10 of fermentation. During the fermentation period, pH decreased from 3.65 to 3.23. Amplicon sequencing showed that bacterial diversity was highest at 2 d, and lowest at 10 d, the final day. Although Enterobacteriaceae was the most dominant family at 0 and 2 d, Acetobacteriaceae, specifically Gluconobacter frateurii, became dominant with ~50% relative abundance at 4 d, and increased its abundance to >98% at 6 and 10 d. Although a slight increase in the relative abundance of ~1% (0 d) to ~5% (4 d) was observed in LAB, their presence was limited. This study showed that acetic acid bacteria should not be overlooked in hardaliye fermentation.

Keywords

• 16S rRNA targeted metagenomics
• acetic acid bacteria
• amplicon sequencing
• fermented foods
• Gluconobacter frateurii

References

1. Arici M & Coskun F (2001). Hardaliye: Fermented grape juice as a traditional Turkish beverage. Food Microbiology 18(4): 417–421 https://doi.org/10.1006/fmic. 2001.0413
2. Arici M, Coşkun F, Çeli̇kyurt G, Mi̇ri̇k M, Gülcü M & Tokatli N (2017). Some technological and functional properties of lactic acid bacteria isolated from hardaliye. Journal of Agricultural Sciences 23(4): 428–437 https://doi.org/ 10.15832/ankutbd.385870
3. Aydogdu H, Yildirim S, Halkman A K & Durgun T (2014). A study on production and quality criteria of hardaliye; a traditional drink from Thrace region of Turkey. Gida (The Journal of Food) 39(3): 193–145 http://doi.org/10.5505/gida.36844
4. Bartowsky E J & Henschke P A (2008). Acetic acid bacteria spoilage of bottled red wine—A review. International Journal of Food Microbiology 125(1): 60–70 https://doi.org/10.1016/j.ijfoodmicro.2007.10.016
5. Bayram M, Esi̇n Y, Kaya C, İlhan M, Akin G & Etdöğer R (2015). Geleneksel yöntemle müşküle üzümünden üretilen hardaliyenin bazı özelliklerinin belirlenmesi. Akademik Gıda (Academic Food Journal) 13(2): 119–126 (Article in Turkish).
6. Bokulich N A, Swadener M, Sakamoto K, Mills D A & Bisson L F (2015). Sulfur dioxide treatment alters wine microbial diversity and fermentation progression in a dose-dependent fashion. American Journal of Enology and Viticulture 66(1): 73 https://doi.org/10.5344/ajev.2014.14096
7. Caporaso J G, Kuczynski J, Stombaugh J, Bittinger K, Bushman F D, Costello E K, Fierer N, Peña A G, Goodrich, J K, Gordon, J I, Huttley G A, Kelley S T, Knights D, Koenig J E, Ley R E, Lozupone C A, McDonald D, Muegge B D, Pirrung M, … Knight R (2010). QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7(5): 335–336 https://doi.org/10.1038/ nmeth.f.303
8. Coskun F (2017). A traditional Turkish fermented non-alcoholic grape-based beverage, “hardaliye.” Beverages 3(2) https://doi.org/10.3390/beverages3010002

9. Coskun F & Arici M (2006). The effects of using different mustard seeds and starter cultures on some properties of hardaliye. Annals of Microbiology 56(4): 335 https://doi.org/10.1007/BF03175027
10. Çoşkun F & Arici M (2011). Hardaliyenin bazi ozellikleri üzerine farkli hardal tohumlari ve üzüm cesitleri kullaniminin etkisi. Akademik Gida (Academic Food Journal) 9: 6–11 (Article in Turkish)
11. Coşkun F, Arıcı M, Çelikyurt G & Gülcü M (2012). Farklı yöntemler kullanılarak üretilen hardaliyelerin bazı özelliklerinde depolama sonunda meydana gelen değişmeler. Tekirdağ Ziraat Fakültesi Dergisi 9(3): 62–67 (Article in Turkish)
12. De Roos J & De Vuyst L (2018). Acetic acid bacteria in fermented foods and beverages. Current Opinion in Biotechnology 49: 115–119 https://doi.org/10.1016/j.cop bio.2017.08.007
13. De Roos J, Verce M, Aerts M, Vandamme P, De Vuyst L & Schaffner Donald W (2018). Temporal and spatial distribution of the acetic acid bacterium communities throughout the wooden casks used for the fermentation and maturation of Lambic beer underlines their functional role. Applied and Environmental Microbiology 84(7): e02846-17 https://doi.org/10.1128/AEM.02846-17
14. De Vuyst L & Leroy F (2020). Functional role of yeasts, lactic acid bacteria and acetic acid bacteria in cocoa fermentation processes. FEMS Microbiology Reviews 44(4): 432–453 https://doi.org/10.1093/femsre/fuaa014
15. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M & Glöckner F O (2012). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research 41(1): e1–e1 https://doi.org/10.1093/nar/gks808
16. Li W, Fu L, Niu B, Wu S & Wooley J (2012). Ultrafast clustering algorithms for metagenomic sequence analysis. Briefings in Bioinformatics 13(6): 656–668 https://doi.org/10.1093/bib/bbs035
17. Magoč T & Salzberg S L (2011). FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27(21): 2957–2963 https://doi.org/10.1093 /bioinformatics/btr507
18. Martínez-Torres A, Gutiérrez-Ambrocio S, Heredia-del-Orbe P, Villa-Tanaca L & Hernández-Rodríguez C (2017). Inferring the role of microorganisms in water kefir fermentations. International Journal of Food Science & Technology 52(2): 559–571 https://doi.org/10.1111/ijfs.13312
19. Pehlivanoglu H, Gunduz H H, Ozulku G, Demirci M (2015) An investigation of antimicrobial activity of wheat grass juice,barley grass juice, hardaliye and boza. International Interdisciplinary Journal of Scientific Research 2(1): 8-14
20. Portillo M del C & Mas A (2016). Analysis of microbial diversity and dynamics during wine fermentation of Grenache grape variety by high-throughput barcoding sequencing. LWT - Food Science and Technology 72: 317–321 https://doi.org/ 10.1016/j.lwt.2016.05.009
21. Pothakos V, Illeghems K, Laureys D, Spitaels F, Vandamme P & De Vuyst L (2016). Acetic acid bacteria in fermented food and beverage ecosystems. In K. Matsushita H Toyama, N Tonouchi & A Okamoto-Kainuma (Eds.), Acetic Acid Bacteria: Ecology and Physiology (pp. 73–99) Springer Japan. https://doi.org/10.1007/ 978-4-431-55933-7_3
22. Schloss P D, Westcott S L, Ryabin T, Hall J R, Hartmann M, Hollister E B, Lesniewski R A, Oakley B B, Parks D H, Robinson C J, Sahl J W, Stres B, Thallinger G G, Van Horn D J & Weber C F (2009). Introducing mothur: Open-Source, platform-ındependent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology 75(23): 7537 https://doi.org/10.1128/AEM.01541-09
23. Villarreal-Soto S A, Bouajila J, Pace M, Leech J, Cotter P D, Souchard J P, Taillandier P & Beaufort S (2020). Metabolome-microbiome signatures in the fermented beverage, Kombucha. International Journal of Food Microbiology 333: 108778 https://doi.org/10.1016/j.ijfoodmicro.2020.108778