Physicochemical, Functional and Microbiological Properties of Hardaliye Beverages Produced from Different Grapes and Collected from Different Households

Twenty-three hardaliye beverage samples were collected from different regions of Kirklareli city in Turkey and five hardaliye samples were produced under laboratory conditions according to traditional methods. Total phenolic compounds, monomeric anthocyanins (malvidin-3-glucoside), volatile acid, ethyl alcohol, total sugar contents, total acidity (lactic acid), pH, color values (L (brightness), a (red-green), b (yellow-blue)), total mesophilic aerobic bacteria, lactic acid bacteria, yeast and mold and coliform counts were determined. Acidity between 0.38 and 0.91%, pH from 3.54 and 4.33, color (L.a.b.) values from 7.57 to 13.74, from 0.71 to 7.68, from 0.73 to 4.50, respectively were determined. Total phenolics from 368.8 to 2647.5 mg L-1, ethanol between 0.4 and 6.0% in 7 out of 23 examples, TMAB number 3x1013.2x106 cfu mL-1, LAB 1x102-3x105 cfu mL-1 in 16 out of 23 samples, yeast and mold count 1x101-2,3x104 cfu mL-1 in 18 out of 23 samples were found. According to the results obtained, coliforms and Escherichia coli were not found in the analyzed samples. Statistical analysis results revealed that differences between samples were significant (P≤0.01). Given its bioactive characteristics, hardaliye beverage should be produced at large-scale and be recognized by large communities.


Introduction
Hardaliye is a lactic acid fermented beverage that is produced from red grape or grape juice with the addition of crushed mustard seeds and benzoic acid and it is widely consumed in the Thrace region of Turkey (Arici & Coskun 2001). As well known, grape is rich fruit interms of phenolic compounds (Shahidi & Naczk 1995). In vitro experimental systems also showed that flavonoids possess antiinflammatory, antiallergic, antiviral, and anticarcinogenic properties (Middleton 1998). Kanemaru & Miyamoto (1990) detected that 0.8% mustard extract showed bacteriostatic action against Staphylococcus aureus and Escherichia coli in culture medium. When essential oil of mustard was used, total mesophilic aerobic bacteria and lactic acid bacteria were significantly lower than the control after 2 days of storage (Lemay et al 2002). In a research conducted by Nielsen & Rios (2000), allyl isothiocyanate, a bioactive substance of mustard, was shown to have an inhibitory effect against Penicillium commune, P. roqueforti, Aspergillus flavus and Endomyces fibuliger. They also determined minimum inhibitory concentration to range between 1.8 and 3.5 mg mL -1 . Therefore, it is possible to say that the beneficial effects of hardaliye are not only related with grape which includes bioactive components but also with mustard which gained antibacterial and antifungal effects to hardaliye beverage.
The present study is composed of two parts. In the first part, 23 hardaliye beverage samples collected from local producers were analyzed in terms of their physicochemical, functional and microbiological properties. In the second part, fresh hardaliye beverage samples were produced from different grape types and their physicochemical and microbiological properties were investigated.

Material
In this research, twenty-three hardaliye samples were collected from different households of Kirklareli city in Turkey. In the second stage, hardaliye samples were produced in the laboratory using different types of grapes. These grape types were Dingil Kara, Kara Uzum, Carbernet Sauvignon, Yerli Izabella and Siyah Uzum and procured from Tekirdag Viticulture Research Institute.

Standard methods defined by FDA/Bacteriological
Analytical Manual for microbiological analysis were used for preparation of the samples for analysis. 25 mL hardaliye sample was added into Maximum Recovery Diluent (MRD) and it was homogenization. Other serial dilutions (10 -2 , 10 -3 ,10 -4 and 10 -5 ) were prepared using the initial dilution. These dilutions prepared were used for all microbiological analysis. Total bacteria were enumerated using plate count agar (PCA). For counting coliform bacteria, violet red bile agar (VRBA) and for counting E. coli, eosin methylene blue (EMB) agar were used. For counting yeast-mold, potato dextrose agar (PDA), was used. Numbers of total mesophilic aerobic bacteria, coliform bacteria, E. coli and yeast and mold were determined by providing the appropriate incubation temperature and duration (Anonymous 2001a;2001b;2001c;2002). De Man Rogosa Sharpe (MRS) agar was used for determination of lactic acid bacteria (Baumgart 1993).
In order to determine differences between samples, one-way ANOVA was performed using SPSS 18.0 statistical package. All statistical analyses were performed according to completely randomized design (SPSS 2009).

Results and Discussion
Some physicochemical properties of hardaliye samples collected from different households are shown in Table 1. Some chemical properties of hardaliye samples collected from different households are shown in Table 2. As can be seen from the tables, remarkable differences between the values of total phenolic, monomeric anthocyanins and total sugar contents were observed. Differences between the samples were found to be significant (P<0.01). Some microbiological properties of hardaliye samples collected from different households are shown in Table 3.
In the second part of the study, the fresh hardaliye samples were produced from different grape types according to the traditional production method. Their physicochemical and chemical properties are presented in Tables 4 and 5. Differences between the samples were found to be significant (P<0.01). Total bacteria, lactic acid bacteria and yeast-mold counts of the samples are shown in Table 6. Coliforms and E. coli were not observed.
In this study, pH values of hardaliye samples collected from producers are between 3.54 and 4.33. Arici & Coskun (2001) collected twentysix hardaliye samples from different spots of the Kirklareli city and the pH values determined in this study was similar to those determined in their study. pH values of samples produced in the laboratory are between 3.33 and 3.73. These values were lower than pH value (4.42) reported by Coşkun et al (2009). Total acidity values obtained in these two studies are similar. Volatile acid values of all samples were similar or slightly higher than value (0.25 g L -1 ) reported by Coşkun et al (2009).
Hardaliye was produced from papazkarası blueblack grapes by conventional method by Aşkın & Atik (2016). After having been exposed to lactic acid fermentation, it was bottled and then stored at +4 °C and 20 °C for 60 days. The analyses were carried out in prepared beverage within the 15, 30, 45 and 60 days of storage. The results of color parameters obtained show the highest proportion of red color in the samples at the beginning (dA%= 94.87). As expected, the brown color increased with storage time and the highest value was determined for 60 days depending on the storage temperature. Storage under 4 °C and 20 °C resulted in 60 and 78% losses in anthocyanin content, respectively.
In the present study, ethyl alcohol was found in some hardaliye samples collected from different households. The reason might be that preservatives such as benzoic acid and mustard seeds were not used at the sufficient amount. Moreover, improper storage conditions and prolonged storage time increases formation of alcohol.
In the study conducted by Coşkun et al (2009), hardaliye sample was produced using black grape from Malatya region's grape varieties by applying traditional. Total mesophilic aerobic bacteria count in the hardaliye sample was lower (4.7x10 4 ) than those in the samples produced in laboratory in this study. This may be caused by harvesting and fermentation conditions.
The results of this study were also in accordance with the study conducted by Arici & Coskun (2001) in which red colour (measured by Hunter Lab) of hardaliye samples ranged from 1.33 to 9.66 and total mesophilic aerobic bacteria, lactic acid bacteria and yeasts-molds counts ranged from 3.5x10 2 to 8x10 5 cfu mL -1 , 1.0x10 2 to 4.0x10 4 cfu mL -1 and 1.0x10 2 to 8.1x10 4 cfu mL -1 , respectively. Furthermore, coliforms and E. coli were not found in none of the samples.
Güven & Aksoy (2009) produced hardaliye using verigo variety of grapes. 2.5 g mustard seeds were used. Hardaliye was fermented for 21 day. On the 7 th and 21 st days of fermentation, pH and total acidity were detected to range from 4.17 to 3.94 and 3.39 to 10.40%, respectively. They found higher pH value than the present study, however, total acidity was comparable. They did not detect ethyl alcohol which was similar to the hardaliye samples produced by applying traditional method in the present study.

Conclusions
There are very few scientific studies about traditional hardaliye beverage although it has been produced traditionally for years. It brings the opportunity of consuming grapes in winter or spring seasons; thus, preventing the excess grapes from being wasted. Just like any other popular traditional food products, industrial-scale production of hardaliye should be realized and this traditional beverage should enjoy the large number of consumers' preference. In addition, more research is needed in order to its adaptation to industry.