THE EFFECT OF STARTER CULTURE INOCULATION ON MICROBIAL COMMUNITY PROFILE DURING WINE COFFEE FERMENTATION: METAGENOMIC APPROACH

• Main Author: Handayani, Fitri
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/32563
• Institution: Institut Teknologi Bandung

Evaluation of microbial community involved during fermentation process of wineflavored coffee has been carried out. The taste of coffee that resembles grapes (winey) is in great demand by Indonesian society. Different from common coffee processing, in wine-flavored coffee production, fermentation was conducted using whole coffee cherry -including its skin. The unique taste sensation comes from the conversion of various compunds in coffee during processing. The coffee processing involves huge microbiota with species diversity that is very dependent on the processing method used. The microbiota that plays a role will contribute to the final quality of coffee brewing. The processing must be carried out in a controlled manner to ensure the growth of microbiota that promote high quality coffee drinks. Molecular approaches have been used extensively to easily evaluate the diversity and dynamics of microbial populations that play a role during coffee fermentation. The purpose of this study was to evaluate the community structure of microbiota in natural fermentation and controlled fermentation after inoculation of yeast and bacterial starter cultures with a ratio of 10: 1 (v / v) (10% (v / w)) in the processing of “wine” coffee using PCR-DGGE approach, and also to identify the prokaryotic and eukaryotic microbiota involved during natural fermentation of coffee wine. Metagenomic approach to independent-culture microbiota through polymerase chain reaction (PCR) denaturing electrophoresis gradient gel (DGGE) analysis was used to evaluate population dynamics that occured during processing. A universal primer set were used for targeting the V3 region of 16s rRNA gene and the ITS1 region of the Internal Transcribed Spacer (ITS) gene for amplification of prokaryotic and eukaryotic microbiota, respectively. DGGE profiles were analyzed using 2 different analysis methods. (1) Hierarchical cluster analysis of banding patterns was calculated and expressed as a dendogram. (2) Total diversity of microbiota was compared between three variations of fermentation by means of Shannon-Wiener and Simpson dominance index. DGGE community analysis on natural fermentation revealed 27 and 12 bands of eukaryotic and prokaryotic, respectively, obtained from all stages of “wine” coffee processing. Eukaryotic microbiota were abundant from the beginning of processing while the prokaryotics increased in the final stages of fermentation and drying. Based on DGGE profile, the microbial starter cultures NP and PB in the controlled fermentation tend to be stable compared to that of control. Based on diversity index, the dynamics of the microbiota after inoculation showed a significant difference (p = 0.05). Shannon-Wiener species diversity indices based on the number of DNA bands detected on DGGE profiles demonstrated that the total community of natural microbiota in coffee cherry decreased during fermentation and increased again in the drying process, while diversity after inoculation decreased and dominance indice increased during the coffee processing stage. These revealed that there is a species/genera dominance after inoculation of starter cultures in controlled coffee processing. Further dendogram analysis based on percent similarity of intensity and thickness of DGGE's band showed that diversity of microbes has a similarity level of 52% and classified the microbiota community into two main clusters based on the processing stages. Natural microbiota of fresh coffee cherry and those involved in first and second day of fermentation had similarities in diversity. On the other hand, microbiota on the third day of fermentation, drying and after hulling were in the same cluster. In controlled fermentation, the microbiota involved in each stage were in a cluster with similarity level (82%) higher than control (70%). This implies that the inoculation of starter culture can affect community dynamics during coffee processing. Sequencing results and phylogenetic analysis revealed various types of yeast from genera Pichia, Torulaspora, Hanseniaspora, Saccharomyces, and Candida and also filamentous fungi Aspergillus that played a dominant role during the fermentation and drying of natural process. In addition, lactic acid bacteria from genera Lactobacillus, Klebsiella, Enterobacter, Pantoea and Bacillus dominated bacterial community in this type of processing. In conclusion, molecular approach is useful to provide insight about the community structure of microbiota involved in post-harvest coffee processing, which can facilitate the improvement of starter culture for producing wine-flavored coffee. It also shows that inoculation of starter cultures Pichia kudriavsevii and Klebsiella sp. were able to change the microbial community structure by dominating the process and giving significant influence in creating a controlled processing of “wine” coffee.