Potensi Keragaman Khamir dari Madu Liar dan Buah-buahan serta pemanfaatannya pada Fermentasi etanol

Medhina Magdalena, Prof. Ir. Donny Widianto, Ph.D. ; Prof. Ir. Irfan D. Prijambada, Ph.D.

2025 | Disertasi | S3 Bioteknologi

Keragaman khamir telah dipelajari secara mendalam, namun masih banyak bioma, habitat, dan rentang geografis yang belum dieksplorasi. Studi berkelanjutan mengenai keragaman khamir perlu dilakukan dengan tujuan antara lain, memperbarui basis data keragaman khamir, memahami profil keragaman khamir di suatu habitat, serta menemukan spesies/strain khamir baru yang berpotensi memberi nilai tambah produk berbasis bioindustri. Penelitian ini dirancang dengan tujuan mengungkap keragaman khamir dari madu hutan dan buah-buahan, mendapatkan strain S. cerevisiae baru yang mampu menurunkan kadar gula pereduksi pada fermentasi etanol, dan menganalisis ekspresi gen Hxt1 dan Hxt3, serta keberadaan simporter Fsy1 pada S. cerevisiae selama fermentasi etanol. Tahapan penelitian dibagi menjadi tiga, yaitu isolasi khamir, karakterisasi isolat khamir hingga analisis keragamannya, dan fermentasi etanol menggunakan isolat S. cerevisiae serta analisis ekspresi gen transporter gula Hxt1, Hxt3 dan Fsy1 selama fermentasi.

Pada penelitian ini keragaman khamir dari buah-buahan lebih tinggi dibandingkan dari madu hutan. Isolat S. cerevisiae hanya didapatkan dari buah-buahan, yaitu Ag3 (anggur), NB1 (nanas), S1 (salak) serta BB3 (belimbing) dan keempatnya isolat digunakan untuk fermentasi etanol. Isolat NB1 dan Ag3 menunjukkan kadar gula pereduksi tersisa dengan jumlah paling sedikit pada perlakuan campuran glukosa-fruktosa, dan hasil ini berbeda nyata dengan perlakuan lain. Kadar etanol dari fermentasi fruktosa lebih kecil dibandingkan perlakuan gula lainnya, serta berbeda nyata dibandingkan dengan perlakuan glukosa dan campuran glukosa-fruktosa. Gen Hxt1 terekspresi tinggi pada fase logaritmik, namun menurun pada fase stasioner hingga fermentasi berakhir. Gen Hxt3 juga terekspresi tinggi di saat logaritmik, dan tetap lebih tinggi dibandingkan gen Hxt1 pada fase stasioner hingga akhir fermentasi. Ekspresi kedua gen tersebut terlihat hampir sama pada perlakuan gula dan isolat, namun terdapat perbedaan nyata antar isolat dan waktu. Ekspresi gen Hxt3 relatif rendah pada perlakuan glukosa saja dibandingkan hanya mengandung fruktosa saja dan berlaku pada semua strain, dengan eskpresi tertinggi ditunjukkan isolat NB1. Pada akhir fermentasi, ekspresi gen Hxt3 tertinggi ditunjukkan isolat NB1 pada perlakuan fruktosa saja, sedangkan pada perlakuan yang mengandung glukosa saja ekspresi tertinggi ditunjukkan oleh isolat Ag3. Keberadaan transporter Fsy1 tidak terdeteksi setelah amplifikasi gen tersebut, namun kenaikan pH sedikit teramati pada pengujian proton simpor setelah penambahan fruktosa, sehingga diduga perubahan pH disebabkan mekanisme lain. 

Yeast diversity has long been studied deeply, but many biomes, habitats, and geographical ranges remain unexplored. Continued research on yeast diversity is essential for updating diversity databases, characterizing yeast profiles within specific habitats, and identifying novel yeast species or strains with potential applications in bioindustry-related products. Therefore, this study was aimed at investigating the diversity of yeast species isolated from wild honey and a variety of fruits; to isolate and characterize new Saccharomyces cerevisiae strains with the capacity to reduce levels of reducing sugars during ethanol fermentation; and to analyze the expression profiles of the Hxt1 and Hxt3 genes, as well as to assess the presence of the Fsy1 symporter, in S. cerevisiae during fermentation. The study comprised of three primary stages, i.e., yeast isolation from honey and fruits; characterization and identification of isolates to assess their diversity, and conducting ethanol fermentation using S. cerevisiae new isolates while analyzing the expression of the sugar transporter genes Hxt1, Hxt3, and Fsy1 during the process.

            The results showed that yeasts from fruits showed higher diversity than those from wild honey. New S. cerevisiae strains were only obtained from fruits, namely Ag3 (grape), NB1 (pineapple), S1 (salak/dragonfruit), and BB3 (star fruit), which were subsequently employed for ethanol fermentation. Two isolates, NB1 and Ag3, showed the least amount of remaining reducing sugar in the glucose-fructose mixture, and this significantly differed from the other treatments. Fructose fermentation produced lower ethanol levels, significantly different from glucose and glucose-fructose mixture. The Hxt1 gene was highly expressed during the logarithmic phase but decreased throughout the stationary phase until the end of fermentation. Likewise, the Hxt3 gene exhibited high expression during the logarithmic phase, but maintained its expression higher than that of Hxt1 in the stationary phase until fermentation concluded. Both genes displayed similar expression levels across sugar and isolate treatments, although significant differences emerged between isolates and time. Expression of the Hxt3 gene was consistently lower in the glucose-only treatment compared to the fructose-only treatment across all isolates, with isolate NB1 exhibiting the highest expression levels overall. At the end of fermentation, isolate NB1 maintained the highest Hxt3 expression under fructose-only conditions, while isolate Ag3 showed the highest expression in the glucose-only treatment. Meanwhile, despite the fact that a slight increase in pH was observed in the proton symporter assay following fructose addition, the presence of the Fsy1 transporter gene was undetected after PCR amplification, indicating that the gene probably did not occur in S. cerevisiae, and other proton transport mechanisms may be involved. 

Kata Kunci : Khamir, keragaman, madu, etanol, transporter