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\title{Determination of Some Microbiological Properties of Kombucha Produced from Different Herbal Teas}
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             \author[1]{Fatma  Coskun}

             \author[2]{Serap  Kayisoglu}

             \affil[1]{  Tekirda? Nam?k Kemal University}

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\date{\small \em Received: 11 December 2019 Accepted: 4 January 2020 Published: 15 January 2020}

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\begin{abstract}
        


?Kombucha? is a beverage made by fermenting tea (generally black or sometimes green and oolong tea) and sugar, with a symbiotic culture of bacteria and yeast generally for 7- 10 days. The final product is a sour, slightly sparkling, acidic beverage. In this study, black tea, green tea, sage (Salvia fruticosa Mill.), peppermint (Mentha piperita L.), and linden (Tilia cordata) leaves were used in making kombucha. Some microbiological analyzes of the samples were carried out on the 3rd, 7th, 10th, and 14th days of fermentation, which lasted 14 days at 24oC. The number of Lactobacillus, Lactococcus, yeast, and acetic acid bacteria were average 5.73, 5.66, 5.63 and 5.55 log CFU/mL on the 3rd day of fermentation; average 5.77, 5.01, 5.70 and 3.55 log CFU/mL on the 14th day of fermentation, respectively. Generally, microorganisms increased until the 7th day of fermentation and then decreased. Different herbal teas can be used to produce kombucha enhanced functional properties

\end{abstract}


\keywords{kombucha, SCOBY, fermented black tea, fermented green tea, fermented linden tea, fermented sage tea, fermented mint tea}

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\let\tabcellsep& 	 	 		 
\section[{Introduction}]{Introduction}\par
ombucha is a type of refreshing beverage that is commercially produced by many companies and produced homemade. It is widely consumed in the world. It has a delicious taste, antioxidant properties of tea extracts, and beneficial effects of fermentative bacteria, which have antioxidant properties. Because of these, its consumption is increasing day by day. Phenolic substances found in the leaves of tea used in kombucha production also have antimicrobial effects on microorganisms such as Aeromonas, Bacillus, Clostridium botulinum, Clostridium perfringens, Enterobacter, Klebsiella, Proteus, Pseudomonas, Shigella, Staphylococcus aureus, Streptococcus \hyperref[b0]{[1,} {\ref 2]}.\par
Bacteria and yeasts lead a symbiotic life in the cellulosic film layer, where the microorganisms that play a role in the fermentation of kombucha are found. Gramnegative aerobic bacilli, yeasts, and lactic acid bacteria in the Acetobacteraceae family were identified in this film layer \hyperref[b1]{[3,} {\ref 4,} {\ref 5,}\hyperref[b5]{6]}. This cellulosic structure, which increases in thickness as fermentation progresses, provides the necessary oxygen for microorganisms \hyperref[b1]{[3,}\hyperref[b5]{6]}. These microorganisms create free phenolics using phenolic compounds in tea leaves, and they create useful fermentation products such as various organic acids, vitamins, and minerals for the health and also contribute to the sensory properties of the product. The metabolites formed depend on the variety of tea leaves, the variety of microorganisms in symbiotic culture and fermentation conditions \hyperref[b1]{[3,}\hyperref[b5]{6,}\hyperref[b6]{7,}\hyperref[b7]{8,}\hyperref[b8]{9,}\hyperref[b9]{10]}.\par
Kombucha is usually prepared from black tea and green tea. This research was carried out by considering it would be beneficial to prepare kombucha by sage, linden, and mint teas, which are highly valuable for health. In sage, flavones such as apigenin, luteolin, kaempferol, and quercetin were determined.\par
Carnosol and carnosic acids are also antioxidant phenolic compounds found in sage. Mint (Mentha piperita L.) is one of the commonly consumed herbal teas. The leaves of mint contain phenolic components rosmarinic acid and various flavonoids such as eriocitrin, luteolin, and hesperidin. Peppermint has significant antimicrobial and antiviral activities, strong antioxidant and antitumor activities, and some antiallergenic potential \hyperref[b11]{[11]}. Some of the beneficial effects of kombucha are anti-microbial, antioxidant, anti-carcinogenic, and anti-diabetic properties that have positive effects on stomach ulcers and high cholesterol. It is also recommended for use in the treatment of different diseases such as AIDS, arthritis, atherosclerosis, cancer, constipation, gallbladder diseases, hemorrhoids, hair growth, hypertension, and indigestion. It has also been shown to be effective in immune responses and liver detoxification \hyperref[b8]{[9]}. Kombucha regulates intestinal flora, strengthens cells, helps the body maintain acid-alkaline balance, and acts as a natural antibiotic \hyperref[b12]{[12]}. Great attention should be paid in home-made kombucha since pathogenic microorganisms are likely to contaminate the tea. Some people consuming homemade tea have dizziness, headache, nausea, and allergic reactions. For this reason, it can lead to contraindications in pregnant and lactating women and should be consumed with caution \hyperref[b7]{[8]}. However, as a result of tests carried out by the U.S. Year 2020 Food and Drug Administration, kombucha has been reported to be safe for human consumption \hyperref[b9]{[10]}.\par
In this study, black tea, green tea, sage, mint, and linden teas were used in making the kombucha. The fermentation period was kept for 14 days in making kombucha. 
\section[{II.}]{II.} 
\section[{Material and Methods}]{Material and Methods} 
\section[{a) Materials}]{a) Materials}\par
Black tea, green tea, mint, linden, sage, used for making kombucha in the research were obtained from local markets in Tekirdag. Kombucha mushrooms were purchased from the online shopping site via the internet. 
\section[{b) Preparation and Fermentation of Herbal Teas}]{b) Preparation and Fermentation of Herbal Teas}\par
In order to extract the teas, 10 g of each tea sample, which is used as a bacterial medium for fermentation, was left in 1 L of boiled water for 15 minutes  {\ref [13]}. After extraction, the suitable medium was prepared by adding 70 g/L of sucrose. The prepared tea samples were transferred to glass jars, which were sterilized at 121 o C for 20 minutes (Fig. \hyperref[fig_0]{1}). After we waited until these samples reached room temperature, all samples were inoculated with 7 g/L kombucha mushroom culture. After glass jars were covered with cheesecloth, left to fermentation at 24 ± 1 o C and in the dark for 14 days. Tea samples were taken from the incubator at 3-day intervals, and analyzes were performed. Serial dilutions of kombucha samples were prepared in aseptic conditions using sterile saline containing 0.89\% NaCl. MRS Agar (de Man Rogosa Sharpe, Merck) was used for counting lactobacilli, Petri plates were incubated for 72 hours at 30 o C \hyperref[b14]{[14]}. Lactococci were counted after 48 hours incubation at 30 o C in M17 Agar (Oxoid) \hyperref[b15]{[15]}. PDA (potato dextrose agar) was used for total yeast count, and Petri plates were incubated for three days at 25 o C \hyperref[b16]{[16]}. For total acetic acid bacteria count, a medium containing 1\% (Calcium Carbonate Agar) was prepared and sterilized, and then 3\% ethanol (95\%) (Merck 100983) was added. Then the Petri dishes were incubated for three days at 30 o C \hyperref[b17]{[17]}. Colonies were evaluated for all microorganisms at the end of the incubation, and the results were expressed as "log CFU/mL". 
\section[{d) Statistical analysis}]{d) Statistical analysis}\par
To test the differences between the parameters analyzed before and after fermentation in tea samples, two ways analysis of variance (ANOVA) was performed.\par
Significant differences between means were determined by Duncan's multiple range tests at P<0.05 significance level. 
\section[{III.}]{III.} 
\section[{Results and Discussion}]{Results and Discussion}\par
Kombucha is a fermented beverage. Ambient process conditions, and composition of the medium affect fermentation \hyperref[b18]{[18]}. As a result of this, the nutritional quality, the organoleptic properties, and other physicochemical properties of the product are affected. The different plant varieties, sugar concentrations, fermentation time, and the composition of the tea mushroom also affect fermentation and the properties of the fermented product \hyperref[b9]{[10,}\hyperref[b19]{19]}.\par
Kombucha is created with the help of a symbiotic culture. This symbiotic culture consists of acetic acid bacteria (Gluconobacter and Acetobacter sp., Komagataeibacter) \hyperref[b20]{[20]} lactic acid bacteria (Lactobacillus, Lactococcus) \hyperref[b18]{[18]}  
\section[{J Gl}]{J Gl}\par
Schizosaccharomyces pombe, Zygosaccharomyces bailii, Kloeckera apiculata, Torulaspora delbrueckii, Brettanomyces bruxellensis \hyperref[b21]{[21]}, Candida, Pichia, Lachancea, and Kluyveromyces \hyperref[b8]{[9,}\hyperref[b18]{18,}\hyperref[b21]{21]}.\par
Kombucha fermentation includes alcohol, lactic acid and acetic acid fermentation. In the fermentation of Kombucha, fermented tea and biofilm are formed as a result of the activity of different yeasts and bacteria species. First, in fermentation, sucrose breaks down into glucose and fructose as a result of yeast hydrolysis. Alcohol occurs as a result of fermentation of glucose and fructose. Then acetic acid bacteria convert alcohol into acetic acid. In addition to acetic acid, gluconic and glucuronic also occur \hyperref[b9]{[10]}.\par
Kombucha tea is usually made from black and green tea. In this study, different herbal teas known to be beneficial for health were also used. The aim is to make the benefit of the kombucha tea even more useful with these herbs. The number of Lactobacillus, Lactococcus, yeast, and acetic acid bacteria determined during fermentation were shown in Table  {\ref 1}.\par
Table  {\ref 1}: Changes of some microbiological properties of Kombucha samples during fermentation (log CFU/mL) * Capital letter refers to the difference between tea varieties. ** Lower case refers to the difference between fermentation days.\par
Lactobacillus number of black, green, and mint teas reached the highest value on the 7th day of fermentation. Lactobacillus number of linden, and sage teas reached the highest number on the 10th day of fermentation (Fig.  {\ref 2}). As a result of statistical analysis, the difference between the tea varieties in terms of lactobacilli numbers was found significant at the level of P<0.01 (F=7,716**). The difference between fermentation days was also found significant at P <0.01 (F=4,498**). Degirmencioglu et al. \hyperref[b5]{[6]} prepared the teas from white, green, black, oolong and pu'er tea leaves (100g / L sugar and 6g / L tea leaf, 100 mL / L fermented kombucha and SCOBY) at 30 o C after 21 days fermentation time. In their study, there was a rapid increase in lactic acid bacteria numbers until the 3rd day of fermentation, and this increase continued until the 12th day of fermentation. The highest value in LAB numbers was determined as 5.80 log CFU/mL in green tea. Their results are similar to those of this study. The Lactococcus number increased rapidly in all samples until the 3rd day of fermentation. Lactococcus numbers decreased in the kombucha samples prepared from black tea and mint tea after the 3rd day of fermentation and in the others after the 10th day of fermentation (Fig.  {\ref 3}). As a result of statistical analysis, the difference between the tea varieties in terms of Lactococcus numbers was found to be significant at the level of P <0.05 (F = 3.194*) and the difference between the fermentation days at the level of P <0.01 (F = 11.643 **). Due to lactic acid they produce, Lactic streptococci and lactobacilli can lower the pH to 4.5-4.3 and 3.5-3.2, respectively \hyperref[b22]{[22]}. At low pH, lactic acid is in toxic form for most bacteria and yeasts. Highly produced acetic acid by the lactic acid bacteria interacts with the cell membrane. It can affect intracellular acidity and cause protein denaturation. The acidity constant of acetic acid is higher than that of lactic acid (lactic acid 3.08, acetic acid 4.75). Lactic acid increases the effect of acetic acid by lowering the pH of the medium \hyperref[b23]{[23]}. K?l?ç \hyperref[b24]{[24]} stated that acetic acid is effective on Saccharomyces sp.  
\section[{J Gl}]{J Gl}\par
The number of yeasts increased rapidly in all samples until the 3rd day of fermentation. Yeast numbers decreased after the 3rd day of fermentation in the sample prepared from black tea, and after the 7th day of fermentation in other tea samples (Fig. \hyperref[fig_2]{4}). As a result of statistical analysis, the difference between tea varieties and days in terms of yeast numbers was found to be significant at the level of P<0.01. (F=9,715**; F=9,890**). In the study of Degirmencioglu et al. \hyperref[b5]{[6]}, the number of yeast in the green tea sample increased until the 12th day of fermentation, the number of yeast in the black tea sample was almost unchanged and decreased after the 12th day of fermentation. The yeast numbers in this study are similar to the yeast numbers in their study. The decrease in yeast numbers during fermentation may be due to a decrease in pH value. pH values decreased rapidly starting from the first days. These pH values are far below the pH value of yeasts that are effective in fermentation. However, it has shown that bacteria and yeasts in symbiotic culture can continue their activities. Y?km?? and Tu?güm \hyperref[b25]{[25]} produced kombucha from black tea leaves (10g / L tea leaves, 10\% sugar, and 10\% pre-prepared compost tea) after a fermentation that lasted ten days at 24 o C and stored at +4 o C. The yeast count of this sample was 2.51 log CFU/mL on the 1st day of storage. That value is considerably lower in our study than the number of yeasts on the 10th day of fermentation of kombucha produced from black tea leaves. Jayabalan et al. \hyperref[b26]{[26]} prepared kombucha from black tea leaves (1.2\% black tea leaves, 10\% sugar, 3\% SCOBY, 10\% fermented kombucha) at the end of fermentation (at 24 o C) for 21 days. While the number of yeast was around 45x10 7 in 7 days of fermentation, it reached 47.5x10 7 in the 14th day of fermentation. Yeast number is higher than determined in our study. The reason for this difference may be the microflora in the cultures used. The number of yeasts in the kombucha produced from mint leaf was lower than that of the others on all days of fermentation. The reason for this may be an inhibitory effect of peppermint against certain yeasts. Schelz et al. \hyperref[b27]{[27]} found that peppermint oil was very effective against S. cerevisiae 0425 52C and S. cerevisiae 0425 ? / 1 strains in their study.\par
Microbial interactions between Saccharomyces and non-Saccharomyces yeasts seem to be an advantageous option in mixed fermentation processing, having several benefits like avoiding the risks of a stuck fermentation, the addition of flavors and aromas allows the modification of undesired parameters, between others \hyperref[b29]{[28]}. The interaction of yeasts in Kombucha causes the formation of the desired properties in the fermented product \hyperref[b31]{[30]}.\par
The dominant bacteria of Kombucha tea culture are acetic acid bacteria \hyperref[b30]{[29]}. In this study, the number of acetic acid bacteria increased rapidly until the 3rd day of fermentation in all tea samples, and after the 3rd day of fermentation in samples produced from black, peppermint, linden, and sage tea showed a regular decrease. In the sample produced from green tea, it increased until the 10th day of fermentation and then decreased (Fig. \hyperref[fig_3]{5}). As a result of statistical analysis, the difference between the tea varieties in terms of the number of acetic acid bacteria was found to be significant at the level of P <0.01 (F = 214,793**). Differences among fermentation days were also significant at the level of P <0.01 (F = 146.318 **) In the study of Degirmencioglu et al. \hyperref[b5]{[6]}, the number of acetic acid bacteria increased rapidly until the 3rd day of fermentation, and the increase was slower until the 12th day. It was faster from the 12th to the 21st day of fermentation. In a study of Jayabalan et al. \hyperref[b26]{[26]}, while the number of acetic acid bacteria of the samples was around 41x10 4 on the 7th day of fermentation, it increased to around 45.5x10 4 on the 14th day of fermentation. In our study, the number of acetic acid bacteria decreased. Acetic acid bacteria show the best growth in the range of 25-30 °C. \hyperref[b31]{[30]}. The optimum pH range for the development of acetic acid bacteria is 5.5-6.5. They can develop even at low pH depending on the presence of oxygen in the environment. Acetophilic ones can develop at pH 3.5, acetotolerant ones can develop between pH 3.5-6.5 \hyperref[b5]{[6]}. In this study, since the pH fell below three after the 3rd day of fermentation, the number of acetic acid bacteria may decrease. The difference between our study and other researchers study may be the difference of the culture (SCOBY) used, the culture rate, the amount of sugar, the amount of tea leaf, the infusion time of the tea, and the fermentation conditions. Although other researchers added fermented kombucha in addition to SCOBY to the teas that will be produced, fermented kombucha was not used as a culture in this research.  The kombucha fermentation process leads to the formation of a floating biofilm on the surface of the growth medium due to the activity of certain strains of acetic acid bacteria \hyperref[b32]{[31]} (Acetobacter aceti, Acetobacter pasteurianus Acetobacter xylinoides, Bacterium gluconicum, and Gluconobacter oxydans) \hyperref[b7]{[8]}. It has been reported that the variety in the composition of yeast composition of Kombucha may be due to geographic, climatic and cultural conditions as well as local cross-contamination of yeasts and fungi or possible cross-contamination between cultures \hyperref[b6]{[7,}\hyperref[b33]{32]}.\par
In this study, the masses of biofilms formed in the kombuchas produced from black, green, mint, linden, and sage teas were determined as 43.85, 20.70, 7.44, 4.64, and 3.90g at the end of fermentation, respectively (Figure \hyperref[fig_5]{6,7}). The effect of peppermint, lobal Journal of Researches in Engineering ( ) Volume Xx X Issue II Ve rsion I linden, and sage on the growth and activity of some microorganisms may have affected biomass formation. The weight of the biomass formed in the kombuchas obtained from these plants is less than the weight of the biomass in the kombuchas obtained from black and green teas.   
\section[{Conclusion}]{Conclusion}\par
The number of Lactobacillus in the kombucha produced from green tea and linden was higher than that of other samples in all days of fermentation. While Lactococcus numbers in the kombucha samples produced from black tea and mint continuously decrease during fermentation, Lactococcus numbers in the other tea samples increased until the 10th day of fermentation and then decreased. While the yeast number of the kombucha produced from green tea, mint and linden was highest on the 7th day of fermentation, it was the highest on the 10th day of fermentation in the kombucha produced from sage. Acetic acid bacteria increased in only the green tea until the 10th day of fermentation and then decreased. Their number in other tea samples decreased from the 3rd to the 14th day of fermentation. Different herbal teas can be used to produce kombucha with enhanced functional properties. Researching the functional properties of these teas and publishing the results will enlighten the producers and consumers. \begin{figure}[htbp]
\noindent\textbf{1}\includegraphics[]{image-2.png}
\caption{\label{fig_0}Figure 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{23}\includegraphics[]{image-3.png}
\caption{\label{fig_1}Figure 2 :Figure 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{4}\includegraphics[]{image-4.png}
\caption{\label{fig_2}Figure 4 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{5}\includegraphics[]{image-5.png}
\caption{\label{fig_3}Figure 5 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{6}\includegraphics[]{image-6.png}
\caption{\label{fig_4}Figure 6 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{7}\includegraphics[]{image-7.png}
\caption{\label{fig_5}Figure 7 :}\end{figure}
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\end{bibitemlist}
 			 		 	 
\end{document}
