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.
- Introduction
Hardaliye is a traditional grape-based beverage from the Thracian region of Turkey (Arici & Coskun 2001). It is produced by fermenting aromatic red grapes together with crushed raw mustard seeds, sour cherry leaves, sorbic and/or benzoic acid as a preservative for 7 to 10 d (Coşkun et al. 2012; Aydogdu et al. 2014). Yeast growth and ethanol fermentation in hardaliye are limited due to the protective effect of active compounds in mustard seeds together with the action of the preservative (Coşkun et al. 2012). After production, hardaliye is filtered and stored at 4 °C (Arici & Coskun 2001; Coskun 2017). Even if it is stored in the cold, there may be changes in its properties after one year of storage (Coşkun et al. 2012).
Fruits and fruit products are important components of a healthy diet due to their bioactive compounds, poor fat content, and low sodium and potassium (Rodríguez et al. 2021). Fermentation is an alternative preservation technique to extend the short shelf life of fruits; moreover, it improves the functional properties, including nutritional value and sensory attributes (Prado et al. 2008). Because the fermentation takes place with the seeds and skins of grapes, hardaliye is rich in antioxidant polyphenols, such as resveratrol, gallic acid, and flavonoids, such as quercetin and anthocyanidins (Amoutzopoulos et al. 2013). The 2013 study of Amoutzopoulos et al. (2013) showed that hardaliye consumption significantly decreased oxidative stress markers compared to the control group suggesting an antioxidative effect (Amoutzopoulos et al. 2013). Hardaliye is also shown to reduce the formation of the lipid oxidation product, malondialdehyde, in meat products during in vitro-digestion, indicating a potential health effect when consumed together with meat (Aksoy et al. 2022). Since it is non-alcoholic and plant-based, hardaliye is a beverage suitable for the consumption of a wide variety of consumer groups, including children and vegetarians (Prado et al. 2008).
Hardaliye has been described as a lactic acid fermented product (Arici & Coskun 2001; Bayram et al. 2015; Pehlivanoglu et al. 2015; Arici et al. 2017; Coskun 2017). Studies on the microbiology of hardaliye have predominantly focused on culture-dependent methods using plate counts, including total mesophilic aerobic bacteria, lactic acid bacteria (LAB), coliforms, and yeasts (Arici & Coskun 2001; Coşkun et al. 2012; Aydogdu et al. 2014; Bayram et al. 2015). In addition, two studies have described the isolation of LAB species from hardaliye (Arici & Coskun 2001; Arici et al. 2017).
Culture-independent microbial profiling techniques, which involve extracting DNA directly from samples, allow for the analysis of microorganisms without isolating and culturing them. Owing to the decrease in sequencing costs and the increasing availability of bioinformatics analysis tools, high-throughput sequencing (HTS) techniques have been ubiquitously used in fermented food community analysis for the past two decades (Chen et al. 2017; Ferrocino & Cocolin 2017; Rizo et al. 2020). In HTS, shotgun sequencing of whole genomes can be used for a comprehensive analysis involving identifying microbial communities and their functional potential (Rizo et al. 2020). Meanwhile, amplicon-based “targeted” approaches, which involve sequencing only an informative region of the genome, are more accessible and cheaper when the aim is to identify the community members and their relative abundance (De Filippis et al. 2017; Chen et al. 2017; Gołębiewski & Tretyn 2020). For the identification of bacteria, the most widely used taxonomically informative region is the 16S rRNA gene (Ferrocino & Cocolin 2017; Gołębiewski & Tretyn 2020). Amplicon sequencing is performed after amplification of 16S rRNA with universal primers. The resulting operational taxonomic unit (OTU) abundance is proportional to the number of reads, allowing the method to be quantitative (De Filippis et al. 2018).
Considering the scarcity of studies on hardaliye microbiology, this study’s aim is to determine the bacterial community of hardaliye during the 10-d fermentation period using a culture-independent method, namely, 16S rRNA-targeted amplicon sequencing, and to correlate this with the pH changes.
Bacterial Dynamics of Hardaliye, a Fermented Grape Beverage, Determined by Highthroughput Sequencing
Banu METİN Halime PEHLİVANOĞLU Esra YILDIRIM SERVİ Muhammet ARICI
Year 2023, Volume: 29 Issue: 3, 756–764
