Starter Culture Modulates Microbial Diversity During Wine-Coffee Fermentation: A DGGE-Based Molecular Study
Main Article Content
Abstract
Wine-flavored coffee is a unique post-harvest product characterized by fruity, acidic, and winey notes, highly favored by Indonesian consumers and possessing strong potential in the global market. However, maintaining consistency in flavor and quality remains a challenge due to the variability of natural fermentation. This study aimed to evaluate the impact of yeast and bacterial starter culture inoculation on microbial community dynamics during wine-coffee fermentation, using a molecular approach based on PCR-DGGE. DGGE analysis revealed that natural fermentation without inoculation involved diverse populations of bacteria, yeasts, and filamentous fungi, with eukaryotic microbes dominating from the early stages. Sequencing identified prevalent yeast genera including Pichia, Torulaspora, Hanseniaspora, Saccharomyces, and Candida, as well as Aspergillus among filamentous fungi. Bacterial communities were dominated by lactic acid bacteria and members of Lactobacillus, Klebsiella, Enterobacter, Pantoea, and Bacillus. In contrast, controlled fermentation with inoculated starter cultures (Pichia kudriavsevii and Klebsiella sp.) showed a more stable microbial profile throughout the process. Shannon-Wiener diversity indices demonstrated a significant difference (p = 0.05) between natural and inoculated fermentations, with species dominance observed in the latter. Cluster analysis confirmed that starter culture inoculation significantly influenced microbial succession and community structure. These findings highlight the importance of controlled fermentation using selected microbial starters to ensure consistent microbial ecology, which in turn contributes to reproducible quality in wine-flavored coffee. The molecular profiling approach provides valuable insights for improving fermentation practices and developing reliable starter culture formulations tailored to enhance flavor consistency and product quality.
Downloads
Article Details
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
The copyright of the received article shall be assigned to the journal as the publisher of the journal. The intended copyright includes the right to publish the article in various forms (including reprints). The journal maintains the publishing rights to the published articles. Authors are allowed to use their articles for any legal purposes deemed necessary without written permission from the journal with an acknowledgment of initial publication to this journal.
The work under license Creative Commons Attribution-ShareAlike 4.0 International License.
References
Altschul, S., Gish, W., Miller, W., Myers, E., & Lipman, D. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.
Avallone, S., Guyot, B., Brillouet, J. M., Olguin, E., & Guiraud, J. P. (2001). Microbiological and biochemical study of coffee fermentation. Current Microbiology, 42, 252–257.
Avallone, S., Jean, M. B., Bernard, G., Eugenia, O., & Joseph, P. G. (2002). Involvement of pectolytic micro-organism in coffee fermentation. International Journal of Food Science and Technology, 37, 191–198.
Bassam, B. J., & Gresshoff, P. M. (2007). Silver staining DNA in polyacrylamide gels. Nature Protocols, 2, 26–49.
Coughlan, M. P., & Mayer, F. (1991). The cellulose-decomposing bacteria and their enzyme systems. In Balows, A., Trüper, H. G., Dworkin, M., Harder, W., & Schleifer, K. H. (Eds.), The Prokaryotes (Vol. 1, pp. 460–516). Berlin: Springer.
Daniel, H. M., Vrancken, G., Takrama, J. F., Camu, N., De Vos, P., & De Vuyst, L. (2009). Yeast diversity of Ghanaian cocoa bean heap fermentations. FEMS Yeast Research, 9, 774–783.
De Bruyn, F., Zhang, S. J., Pothakos, V., Torres, J., Lambot, C., Moroni, A. V., Callanan, M., Sybesma, W., Weckx, S., & De Vuyst, L. (2017). Exploring the impacts of postharvest processing on the microbiota and metabolite profiles during green coffee bean production. Applied and Environmental Microbiology, 83, e02398-16. https://doi.org/10.1128/AEM.02398-16
Ercolini, D. (2004). PCR-DGGE fingerprinting: Novel strategies for detection of microbes in food. Journal of Microbiological Methods, 56(3), 297–314. https://doi.org/10.1016/j.mimet.2003.11.006
Escobar-Zepeda, A., Sanchez-Flores, A., & Baruch, M. Q. (2016). Metagenomic analysis of a Mexican ripened cheese reveals a unique complex microbiota. Food Microbiology, 57, 116–127. https://doi.org/10.1016/j.fm.2016.02.004
Evangelista, S. R., Miguel, M. G. P. C., Cordeiro, C. S., Silva, C. F., Pinheiro, A. C. M., & Schwan, R. F. (2014). Inoculation of starter cultures in a semi-dry coffee (Coffea arabica) fermentation process. Food Microbiology, 44, 87–95.
FAO. (2025). Global coffee market and recent price developments (CD4706EN). Food and Agriculture Organization of the United Nations. https://www.fao.org/faostat/en/#data
Ferrocino, I., & Cocolin, L. (2017). Current perspectives in food-based studies exploiting multi-omics approaches. Current Opinion in Food Science, 13, 10–15. https://doi.org/10.1016/j.cofs.2017.01.002
Fromin, N., Hamelin, J., Tarnawski, S., Roesti, D., Jourdain-Miserez, K., Forestier, N., Teyssier-Cuvelle, S., Gillet, F., Aragno, M., & Rossi, P. (2002). Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns. Environmental Microbiology, 4, 634–643.
Hansen, E. B. (2002). Commercial bacterial starter cultures for fermented foods of the future. International Journal of Food Microbiology, 78, 119–131. https://doi.org/10.1016/S0168-1605(02)00238-6
International Coffee Organization (ICO). (2025, June). Coffee Market Report – June 2025. https://www.ico.org/documents/cy2024-25/cmr-0625-e.pdf
Istiadi, A. K. (2018). Optimasi perbandingan dan waktu fermentasi dalam pembuatan wine coffee [Tugas Akhir]. Institut Teknologi Bandung, Bandung.
Juanda, Muzaifa, M., Martunis, & Wahyuningsih, T. (2022). Analysis of Gayo wine-coffee processing facility development. IOP Conference Series: Earth and Environmental Science, 951, 012094.
Kim, D., Baik, K. S., Kim, S. M., Park, S. C., Kim, S. S., Rhee, M. S., Kwak, Y. S., & Seong, C. N. (2008). Acetobacter soli sp. nov., isolated from forest soil. Journal of Microbiology, 46, 396–401.
Krebs, C. J. (1985). Species diversity. In Ecology: The experimental analysis of distribution and abundance (pp. 507–534). New York: Harper and Row.
Lee, B.-H., Huang, C.-H., Liu, T.-Y., Liou, J.-S., Hou, C.-Y., & Hsu, W.-H. (2023). Microbial diversity of anaerobic-fermented coffee and potential for inhibiting Ochratoxin-produced Aspergillus niger. Foods, 12(15), 2967. https://doi.org/10.3390/foods12152967
Leesing, R. (2005). Identification and validation of specific markers for traceability of aquaculture fish for import/export [Disertasi Doktoral]. University of Montpellier II, France.
Lv, X.-C., Jiang, Y. J., Liu, J., Guo, W.-L., Liu, Z.-B., Zhang, W., Rao, P.-F., & Ni, L. (2017). Evaluation of different PCR primers for denaturing gradient gel electrophoresis (DGGE) analysis of fungal community structure in traditional fermentation starters used for Hong Qu glutinous rice wine. International Journal of Food Microbiology, 255, 58–65.
Madigan, M. T., & Martinko, J. M. (2014). Brock: Biology of microorganisms (14th ed.). San Francisco: Pearson Education.
Muyzer, G., De Waal, E. C., & Uitterlinden, A. G. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology, 59, 695–700.
Muzaifa, M., Abubakar, Y., Nilda, C., Andini, R., Olivia, B., & Putri, A. N. (2023). Physicochemical and sensory characteristics of three types of wine coffees from Bener Meriah Regency, Aceh Indonesia. IOP Conference Series: Earth and Environmental Science, 1183(1), 012061. https://doi.org/10.1088/1755-1315/1183/1/012061
Neu, A.-K., Pleissner, D., Mehlmann, K., Schneider, R., Puerta-Quintero, G. I., & Venus, J. (2016). Fermentative utilization of coffee mucilage using Bacillus coagulans and investigation of down-stream processing of fermentation broth for optically pure L (+)-lactic acid production. Bioresource Technology, 211, 398–405.
Odum, E. P. (1971). Fundamentals of ecology. Philadelphia: W. B. Saunders Company Ltd.
Ovreas, L., Forney, L., & Daae, F. L. (1997). Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Applied and Environmental Microbiology, 63, 3367–3373.
Payne, C., & Bruce, A. (2001). The yeast Debaryomyces hansenii as a short term biological control agent against fungal spoilage of sawn Pinus sylvestris timber. Biological Control, 22, 22–28.
Pereira, G. V. M., de Almeida, E. G., Ramos, C. L., Cardoso, P. G., Dias, E. S., & Schwan, R. F. (2010). Determination of dynamic characteristics of microbiota in a fermented beverage produced by Brazilian Amerindians using culture-dependent and culture-independent methods. International Journal of Food Microbiology, 140, 225–231.
Pereira, G. V. M., Miguel, M. G. C. P., Ramos, C. L., & Schwan, R. F. (2012). Microbiological and physicochemical characterization of small-scale cocoa fermentations and screening of yeast and bacterial strains to develop a defined starter culture. Applied and Environmental Microbiology, 78, 5395–5405.
Pereira, G. V. M., Neto, E., Soccol, V. T., Medeiros, A. B. P., Woiciechowski, A. L., & Soccol, C. R. (2015). Conducting starter culture-controlled fermentations of coffee beans during on-farm wet processing: Growth, metabolic analyses, and sensorial effects. Food Research International, 75, 348–356.
Pereira, G. V. M., Soccol, V. T., Brar, S. K., Neto, E., & Soccol, C. R. (2016). Microbial ecology and starter culture technology in coffee processing. Critical Reviews in Food Science and Nutrition, 57, 2775–2788.
Pereira, G. V. M., Soccol, V. T., Pandey, A., Medeiros, A. B. P., Andrade Lara, J. M. R., & Gollo, A. L. (2014). Isolation, selection and evaluation of yeasts for use in fermentation of coffee beans by the wet process. International Journal of Food Microbiology, 188, 60–66.
Ribeiro, L. S., Miguel, M. G., da Costa, C. P., Evangelista, S. R., Martins, P. M. M., van Mullem, J., Belizario, M. H., & Schwan, R. F. (2017). Behavior of yeast inoculated during semi-dry coffee fermentation and the effect on chemical and sensorial properties of the final beverage. Food Research International, 92, 26–32.
Silva, C. F. (2014). Microbial activity during coffee fermentation. In: Cocoa and coffee fermentations (pp. 397–430).
Silva, C. F., Batista, L. R., Abreu, L. M., Dias, E. S., & Schwan, R. F. (2008). Succession of bacterial and fungal communities during natural coffee (Coffea arabica) fermentation. Food Microbiology, 25, 951–957.
Silva, C. F., Vilela, D. M., Souza, C. C., Duarte, W. F., Dias, D. R., & Schwan, R. F. (2013). Evaluation of a potential starter culture for enhanced quality of coffee fermentation. World Journal of Microbiology and Biotechnology, 29, 235–247.
Silva, E. P., & Russo, C. A. M. (2000). Techniques and statistical data analysis in molecular population genetics. Hydrobiologia, 420, 119–135.
Tamang, J. P., Watanabe, K., & Holzapfel, W. H. (2016). Diversity of microorganisms in global fermented foods and beverages. Frontiers in Microbiology, 7, 377. https://doi.org/10.3389/fmicb.2016.00377
Tamura, K. (1992). Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Molecular Biology and Evolution, 9, 678–687.
Vale, A. d. S., Pereira, C. M. T., De Dea Lindner, J., Rodrigues, L. R. S., Kadri, N. K. E., Pagnoncelli, M. G. B., Kaur Brar, S., Soccol, C. R., & Pereira, G. V. d. M. (2024). Exploring microbial influence on flavor development during coffee processing in humid subtropical climate through metagenetic–metabolomics analysis. Foods, 13(12), 1871. https://doi.org/10.3390/foods13121871
Vilela, D. M., Pereira, G. V. M., Silva, C. F., Batista, L. R., & Schwan, R. F. (2010). Molecular ecology and polyphasic characterization of the microbiota associated with semi-dry processed coffee (Coffea arabica L.). Food Microbiology, 27, 1128–1135.