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Out of many Greek inventions, sport probably made the biggest career. Almost nobody practices philosophy anymore, almost nobody values science, but sport is on top. This last slogan probably best characterizes amateur sport, and already perfectly suits mass runs held in the mountains. Not only though. It seems that the entire contemporary culture is geared to crossing borders in various areas. In any case, many people like to describe their activity in this way. And it is hardly surprising since so much emotion is caused by such a prosaic activity as running. I also say this from personal experience. Every time I stand at the start of the competition I ask myself: what makes all these people make such a huge effort? What makes me follow them? I am talking now about ultra runs that are , or even more kilometers long and last 24 hours or even several days. So if to cross own limits, why not on Olympus, the highest mountain in Greece, which by definition is the border, between the world of gods and people of course. And best in July, when the chances of really extreme temperatures are greatest. There are at least 10 different competitions organize in the whole massif of the mountain of gods, I competed in several myself, but the Olympus Mythical Trail was a border-line experience, not comparable with anything else, neither before nor after. Olympus Mythical Trail is considered the most difficult mountain race in Greece. The route leads around the entire massif, partly along old, forgotten paths, brought back to life by the organizers. There are no villages or permanent human settlements along the way. The impression that you are on the edge of the inhabited world, and even beyond it, is very strong. The race starts on Friday at It is very warm, forecasts for that day promise 42 degrees C in the shade, but I want to believe that at an altitude of m will be a bit cooler. This beginning is beautiful. A high mixed forest that gives shade, a narrow but comfortable and wonderfully laid path. Trees everywhere, but many more species than in Poland. Every now and then the undergrowth is changing, once it is dense and full of bushes, again intensely green grass dominate. And everywhere a lot more colors, everything blooms as if it was the beginning of spring, not the high summer. Sometimes, in the open air, there are more sounds: cicadas and various other insects are playing. Litochoro can be seen somewhere below, the clear shape of the coastline besides, the blue of the sea and the Halkidiki peninsula on the other side of the bay. The OMT route is clearly divided into two sections. It runs quite gently up to meters above sea level to later run to The second, large loop is actually one great uphill to the main dome of Olympus and an equally great downhill. In detail, the route is much more diverse, and the downhills and ascents are intertwined much more often. Livadaki hut at a height of 2, m. On downhill the path is equally narrow and beautiful, but the way down doubles the pleasure. I accelerate, carried by the euphoria of the beginning, and deep down I explain to myself that I should run as many kilometers as possible in the light of day. In Enipeas Gorge gets warmer: stuffy, stagnant air, so thick that it can almost be cut with a knife. I run out of the ravine to return to it in a moment, in complete darkness, right next to the small pond Lambada. From here it is less than a kilometer to the food point in Litochoro. Too fast, of course, but who said madness is rational. I do not mind. It is good that it is night on this great 5-hour climb, when you overcome some 2, meters of elevation. The night brings great peace and quiet, although I am not sleepy at all. I run slowly so minimal light is enough for me. There is monotony and a slight exhaustion, but I try to celebrate them, they are such an important part of ultra run. Changes will inevitably come and I know that I am getting closer and closer to them with every step. In Mediterranean countries, the night is longer than in the north. Some time ago the forest ended. I run to the Muses Plateau and the sky begins to shine. Almost everything is still black and dark blue and densely set with stars, but in the east there is already a clearly brighter, reddish glow and the world comes alive. You can see the outlines of the peaks around and although the space still has only two dimensions, I have no doubt where I am. The plateau itself also starts to light up, or rather the Apostolidis Refuge which is located on the right and the headlights of other competitors who are approaching it. I know that the sun will rise soon and time will accelerate. At Olympus we are lucky that the downhill begins exactly with the sunrise. And the light is important, because the narrow path Zonaria, slightly descending and traverse the peaks of Stefani and Mytikas, encourages more madness. Right next to it is a cliff or very steep scree, so a bit of prudence is also useful. I run in ecstasy. The new day gives the runner new strength, almost does not feel that it is the 60th kilometer. At least until the next ascent. The large, sloping wall of Skolio, with meter elevation and loose stones escaping from under the feet. This time I deal with it much better than last year, although I still remember it as the most difficult part of the whole route. I still run alone, sometimes stop and take pictures but without much faith that the uniqueness of the moment can be preserved. Easy ascent and much more difficult descent. The path almost does not exist, there are only clumps of grass, fragile, moving stones and a tiny shelter, visible somewhere … far, far below. There is also no path for the next few kilometers of almost flat section, which is why the route markers painted on stones visible everywhere looks somewhat absurd. I am undoubtedly in a wild corner of the mountains, but gentle shapes and fresh shades of green grass at an altitude of over 2, m give a little heavenly impression. Imagine the path of your dreams. It was this hypsometry that fascinated me the most: each height painted in a different color…. The world of gentleness ends quite abruptly and decisively. Behind the pass between the Kakavrakas and Metamorfosi peaks everything changes. A huge wall, with the largest downhill on the route, with bends stretching almost indefinitely. Right to the south. The biggest hero of this competition enters the stage — the heat! The downhill takes the last of strength, and then there are two almost flat sections separated by an aid station, together no more than 24 km. The route leads through meadows and wide forest roads, near which trees grow rarely and give no shade at all. I do not know what is the objective temperature, but it seems to me that I have never felt so hot air on me. I feel the greatest need to cool a hot body. It helps only for a moment, but it works. The unique heat from heaven does not frighten me. I just miss water more and more radically. Not some big rain or sea, but those little springs and streams that flow here and there, and I am not surprised that they are considered sacred. I run into a food point, right on the road, in the middle of the forest. In front of it the stream flows out of the rock and a channel for watering animals and people. There is plenty of water. Out of people who started, only 86 reached the finish line in the extended time limit by one hour due to the heat. This is the smallest percentage of finishers in the entire history of the race. However, I would like to reassure running readers who would like to take part in these or other competitions in Greece: out of my 20 starts on the Aegean Sea, it has never and nowhere been so hot as during OMT The following report concerns the competition from It is worth mentioning that starting from , the start has been moved to Litochoro. The route is basically the same, the starting time has changed from 5. For me, this is definitely a more difficult configuration. Olympus Mythical Trail. Celebrating the Heat. Do you like my website? Peloponnese by Bike. Five weeks of cycling. This trip was not supposed to happen at all. A habit borrowed from northern countries … By bike in November, with a small tent? The person responsible for all of this is Lazaros Rigos on the left — a man whose contribution to the development of mountain running in Greece is hard to overestimate. An active and very good runner. Objectively very clearly, because there is a steep approach, but also proportionally, because I am overtaken by several runners. I save my strength for the rest, because the real run is just beginning. Again, I realize one of the basic features of ultra races — loneliness. At the forefront, the breaks between competitors are relatively large and I run alone to the very end. Even if someone overtakes me, he does it very quickly and soon disappears from my sight. Lost Trail. On Forgotten Paths on Olympus. This competition actually started on Friday, when it rained in Litochoro. Or maybe even a few days earlier, when on some weather map I saw that typhoon Janus was approaching Greece. But a typhoon tornado, etc. There is hardly any wind here. As if I was running completely effortlessly and had the impression that I could do it forever The Path of Your Dreams. From Chora Sfakion to Elafonisi Beach. Phaistos Disk. The messenger of the forgotten world. I saw the place where it was found. On the outskirts of the palace, right next to the northern, outer wall, in a completely inconspicuous place, neither now, nor probably 3, years ago. Yet it has become one of the most enigmatic objects ever found, a true hero of modern science and, to a greater extent, even popular culture. An object that evokes great emotions and prompts hundreds of people to create more or less meaningful theories, trying to explain it, understand it, and read it. I am getting closer and closer to the finish line, I am getting more and more tired and on the one hand I want to be in place, but on the other I do not want something so special to end. Warmth is life, dynamics and movement, and now I feel more than usual that I am part of it. On the last 12 km section from Pigadi to Agios Ioanis there are three small streams. I jump into each of them and immerse myself almost completely. I remember it as the most beautiful running section in my life. Euphoria drops down very slowly, in hot air I move as if slower, I have time to get used to the normal world. Only which is normal? Maybe real life is just on the run, and everything in between is just a necessary fulfillment? My 55 Peaks. Goumarostali Vertical Mile. From Prionia to Muses Plateau. My Life in Litochoro. Scroll to Top.

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Official websites use. Share sensitive information only on official, secure websites. This study implemented kombucha fermentation of Olympus Mountain tea Sideritis scardica sweetened with honey OMTWH in order to investigate the potential for producing a novel beverage with functional properties. The increase in the total count of bacteria and yeast suggests that the OMTWH acts as a viable substrate for supporting the proliferation of the microorganisms of the Kombucha symbiotic culture. The fermentation resulted in a reduction in pH and increased total titratable acidity. Total phenolics and antioxidant activity of the fermented beverage was significantly enhanced, as assessed by the method of Folin—Ciocalteu and ABTS assay, respectively. Hence, the process of kombucha fermentation can convert OMTWH into a novel beverage with enhanced functional properties. Keywords: kombucha, Sideritis scardica , thyme honey, antioxidant, bioactivities. The consumption of certain beverages for the prevention of chronic diseases and maintaining good health has now been recognized worldwide. The scientific community and consumers are keen to study and consume bioactive compound-rich beverages. Despite the availability of various beverages in the market with functional properties, there is a need to search for new combinations and ratios of raw materials to improve the bioactivity in the final products. Moreover, consumer lifestyles have changed in recent years and will be influenced by globalization, economic and social changes, economic growth, rapid advances in food science, food and lifestyle choices, and religious restrictions \[ 1 , 2 , 3 \]. Beverages are an excellent vector for the transfer of nutrients and bioactive compounds in the body, as well as facilitating bioavailability. Bioactive compounds, such as phytochemicals e. The presence of these compounds provides the prospect of using food as a valuable element in strategies to disease curing, particularly in the early stages of diseases \[ 4 , 5 , 6 \]. This is in line with the increasing trend in the consumption of functional drinks \[ 7 \] due to their nutritional content \[ 8 \]. Functional beverages can be classified according to the basic ingredients: milk, fruit and vegetables, fruit and legumes, cereals, coffee, and tea. Therefore, consuming these beverages can reduce the adverse effects of poor nutrition on health and the economy \[ 5 \]. Herbal infusions are well-known for their therapeutic properties. Recent research has shown advancements in the understanding of the health advantages associated with herbal infusions. The fermentation process of Kombucha resulted in an elevation in phenolic content, antioxidant activity, and a-amylase inhibition in five different types of teas \[ 10 \]. The decoction of Lemongrass Melissa officinalis L. Based on the citations provided in \[ 11 , 12 \], it was observed that there was an enhancement in the DPPH radical antioxidant activity. In a separate study, it was observed that the process of kombucha fermentation resulted in an enhancement of the taste of tea and the generation of many advantageous chemical compounds \[ 13 \]. Fermented beverages are considered to provide health benefits because of the presence of restructured polyphenols, gluconic, glucuronic, and lactic acids, vitamins, amino acids, and minerals resulting from the fermentation process \[ 11 \]. According to recent market research, Kombucha has emerged as the most rapidly expanding fermented beverage within the global functional drink industry \[ 14 \]. The conventional constituents used in the manufacture of Kombucha are black tea and sucrose. In recent times, many classifications of tea, such as white, green, oolong, and Pu-erh tea, have been used in the process of Kombucha fermentation \[ 15 \]. Furthermore, a number of scholars have used other sources of raw materials, including fruits \[ 16 \], vegetables, herb infusions \[ 17 \], and other substances \[ 18 \], to enhance the range of tastes and biological properties of kombucha. This is performed with the aim of catering to the varied preferences of customers. In Greece and the rest of the Mediterranean countries, the consumer public is familiar with the herbs provided by nature, and an increasing part of the population is incorporating them into their daily diet. One of the most popular is mountain tea Sideritis spp. Research suggests that Sideritis scardica has potential pharmacological properties similar to green tea Camellia sinensis \[ 19 \]. So far, it has been reported that Sideritis scardica has antioxidant \[ 20 , 21 \], anti-inflammatory \[ 22 \], and antibacterial effects \[ 21 \]. The numerous biological properties and the vast acceptance of mountain tea in Greece as a decoction make it a very interesting raw material for producing a functional beverage. In addition, mountain tea is more interesting when combined with honey as a sweetener instead of sugar. Honey is a highly complex natural liquid containing at least substances \[ 24 \]. Today, honey is one of the most nutritious food supplements and medicine, classifying it as functional food \[ 25 \]. The geomorphology of Greece, with its flora and the prevailing climatic conditions, form an excellent combination for producing many different kinds of honey with particular physicochemical and organoleptic characteristics. A particularly aromatic honey produced mainly in the island regions of the country is thyme honey, known since antiquity for its bright color and intense floral aroma. In this study, a new version of kombucha is produced with Greek raw materials. The tea Camellia sinensis is replaced with Olympus Mountain tea Sideritis scardica , and the sugar is replaced with thyme honey TH. This work aims to evaluate the effect of kombucha fermentation on the physicochemical and functional properties of this new version of kombucha with Olympus Mountain tea with honey OMTWH. Olympus Mountain tea OMT Bio Sideritis scardica-stems with leaves and flowers is a certified organic agricultural product of the harvest, from an agricultural holding in Litochoro, prefecture of Pieria, was purchased from the Evripidou store evripidou. All chemicals and enzymes were purchased from Sigma—Aldrich St. An amount of 3. The container was covered with a linen cloth to avoid contamination and infestation, mainly by the insect Drosophila melanogaster vinegar fly. The samples were placed in a shady place, unmoving for 4 days. On day 4, fermentation monitoring employing a taste test was started. The incubation period was terminated when optimal consuming acidity of 3. The solution was then filtered, filling the glass bottles, that were hermetically sealed. A sampling of the fermentation broth was performed daily. The pH value and titratable acidity were determined daily. The number of yeasts and acetic acid bacteria AAB was measured just after inoculation day 0 , day 2, and at the end of the fermentation. Analysis of color, total phenolic, and flavonoid content, a-amylase, and a-glucosidase inhibition, anticholinesterase, and antioxidant activities were performed in the broth before inoculation and at the end of the fermentation. The total color difference is defined by the expression:. The determination of titratable acidity was conducted using the methodology outlined in a previous study \[ 28 \]. In a concise manner, subsequent to the elimination of carbon dioxide from the fermentation solution, a 20 mL portion was extracted and subjected to titration using a 0. The populations of yeasts and acetic acid bacteria AAB in the fermentation broth were assessed using a plate pour technique. The tests were conducted in triplicate, maintaining consistent circumstances, with each amount being measured three times. The evaluation was carried out only in the broth since only the broth is typically consumed and not the pellicle. The ethanol concentration was determined using an enzymatic kit catalog no. The Ethanol Assay Kit, which is readily accessible for commercial purchase, includes all necessary components for conducting the examination. The determination of sugars, acids, and minerals was conducted using the Miura one, an enzymatic analyzer manufactured by TDI Barcelona, Spain , in accordance with the instructions provided by the provider. Vitamin C was evaluated with a Spectrophotometric method \[ 30 \]. An amount of 1. A standard curve of known vitamin C concentrations was used to quantify the vitamin C in samples under investigation. Briefly, 0. A signal was recorded from to nm, and chromatographs were registered at nm. Identification and quantification were performed with vitamin B-complex analytical standards. The Folin—Ciocalteu method was used to determine the total phenolics content \[ 32 \]. First, the reaction mixture was prepared by mixing Subsequently, the void was produced by the substitution of the specimen with distilled water. The quantification of total flavonoid content was conducted by the use of a colorimetric test \[ 33 \]. First, ethanol was mixed with After that Following a time interval of 5 min, a volume of Next, Finally, the blank was prepared by replacing the sample with distilled water. The absorbance at a wavelength of nm was measured immediately after the mixing process. The DPPH assay was conducted according to the procedure previously reported \[ 34 \]. The analytical protocol included the mixing of Following a 15 min incubation period in a light-restricted environment at ambient temperature, the reduction in absorbance at a wavelength of nm was quantified. The ABTS assay was carried out following the procedure as previously described \[ 35 \]. Amounts of After the mixtures were kept for 15 min in the dark at room temperature, the decrease in absorbance at nm was measured. The control group was comprised of samples that did not include any tea solutions. Briefly, Then, The sample solution After preincubation, the reaction was initiated by adding starch solution The reaction mixture was incubated for The reaction was stopped by adding HCl The Acetylcholinesterase AChE and Butyrylcholinesterase BChE activities were determined using a spectrophotometric method \[ 37 \] with some modifications. After that, Then the final blend was incubated for The blank was measured without extract. The findings were reported in terms of the mean value plus or minus the standard deviation SD based on three repeated measurements. In all solutions, an off-white cellulose membrane was formed on the first to the second day of fermentation, occupying the entire surface with a thickness that increased with the passage of days. A change in color towards lighter and turbidity towards clearer was also observed in the solutions with time, which was noted by other investigators \[ 39 \], where it was reported to be a result of the breakdown of polyphenols into smaller molecules due to enzymatic action by bacteria and yeasts in the acidic environment of kombucha. Also, the beverage started to smell fermented, and gas bubbles appeared from the carbonic acid produced during the fermentation. Finally, the mother culture sank to the bottom of the tea broth, which remained under the newly formed daughter culture, as previously described \[ 38 \]. The production of this mat occurs as a result of the development of a delicate cellulose layer that serves as a substrate for the attachment of bacterial and yeast cell masses \[ 38 \]. Cellulose is synthesized by the bacterial component of the microbial consortium. The primary agent involved in the formation of cellulose is Acetobacter xylinum. The presence of this cellulose network facilitates the interaction between the bacteria and the yeasts \[ 40 \]. The results of the chromatic parameter changes confirmed the visual observation and are presented in Table 1. Each average value was obtained from three observations. During fermentation, the pH values decreased almost the same for all three samples A, B, C. Changes in the pH values and titratable acidity at the time of inoculation day 0 and during fermentation days 0 through 4 of OMTWH. The small differences in the inoculation cultures can explain the slight differences. On the first day, there was a reduction in the pH value ranging from 0. Subsequently, on the subsequent days, the drop in pH was constrained to a range of 0. At the end of the process, on the fourth day, the pH value reached 3. These values are within the range considered safe for human consumption, which ranges from 2. Values below pH 2. TA increased from the beginning until the end of the fermentation process similarly for all samples Figure 2. The decrease in pH values Figure 2 follows the increase in acidity due to the Kombucha fermentation. These changes result from the metabolic activity of yeasts and AAB that produce mainly acetic acid. As it is evident, changes in titratable acidity towards the end of the fermentation are more significant than changes in pH, which the buffer capacity of the fermented broth could explain. Specifically, the process of fermentation results in the emission of carbon dioxide. This can cause more organic acid production but a smaller decrease in pH values, thus contributing to the buffer character of the system \[ 26 \]. Several other researchers have also noted similar pattern of pH and TA fluctuations \[ 42 , 43 \], which is often seen throughout the fermentation process of Kombucha. Based on the aforementioned information, the total acidity TA was used as a pivotal parameter in identifying the completion of kombucha fermentation, rather than relying on pH levels. In order to obtain a beverage with a desirable sourness, it is recommended to conclude the fermentation process when the total acidity TA reaches the optimal range of 4—4. This recommendation is supported by the experiences of long-term users of kombucha drinks \[ 26 \]. In our case, optimal consuming acidity was obtained in less than four days. In another study, the same acidity was received after nine days of fermentation of black tea and sugar \[ 44 \]. The drop in pH values and the increase in total acidity are fundamental indications that the fermentation of OMTWH from a symbiotic kombucha culture is progressing. Other parameters that suggest an active process of fermentation in OMTWH are mat formation, observed towards the end of the first day of fermentation, the color changes confirmed by the chromatic parameter changes before and after the fermentation, and residual sugars. Immediately after inoculation, there was a correlation between pH with TA in the samples. Sample B, with the highest total acidity, had the lowest pH value, and A, with the lowest total acidity, had the highest pH value. However, during fermentation, this linearity ceased to exist. The above is because we measure the acids without considering their strength by measuring total acidity. In contrast, with pH, we estimate the acidity by considering the power of each acid involved in forming its acidic taste. Figure 3 A,B show the average changes in microbiological parameters observed throughout the process of fermentation. Following the inoculation stage, significant variations in the quantities of yeasts and acetic acid bacteria AAB were observed across the samples. This discrepancy may be attributed to the use of three distinct symbiotic cultures of bacteria and yeast SCOBYs , each potentially containing varying quantities of viable cells. Nevertheless, by the end of the fermentation, acetic bacteria and yeasts were uniform among the three samples. A Acetic acid bacteria count at days 0, 2, and 4. B Yeasts count at days 0, 2, and 4. The total count of bacteria increased quickly daily to the maximum of 4. The initial number of yeasts was at 2. The increase was indicative of the ability of the OMTWH to act as a suitable substrate to sustain the proliferation of the microorganisms of the Kombucha symbiotic culture. The total count of yeasts and AAB by the end of fermentation is comparable with previous investigations. Another study showed counts of mesophilic bacteria 6. Similarly, Sreeramulu and others \[ 43 \] obtained 4. Some differences in chemical parameters, process duration, and cell counts may be expected because kombucha does not have a standardized microbiological and chemical composition \[ 45 \]. In another study \[ 47 \], during the kombucha fermentation of black tea sweetened with sugar, the viable counts of yeasts increased and followed the same trend as those of AAB. Also, in contrast with our study, cell concentrations of yeasts were generally higher than those of AAB, and the growth of yeasts was faster than that of the bacteria. The above concurs with the findings of \[ 48 , 49 \]. The above can be explained as follows: Yeast and bacteria in the Kombucha symbiotic culture use substrates in different and complementary ways. At the initial stage of fermentation, yeasts hydrolyzed sucrose to glucose and fructose, which were further utilized, with a preference for fructose, to produce ethanol and carbon dioxide \[ 40 , 50 \]. Then AAB used glucose to produce gluconic acid and ethanol to produce acetic acid \[ 38 \]. In our study, this is not the case, as the growth of acetic bacteria was faster than that of yeasts. The explanation lies in the chemical composition of the sugars in honey. Honey is composed mainly of fructose and glucose, while sugar is composed of sucrose. During the fermentation process, sucrose is first broken down into glucose and fructose by the yeasts through the enzyme invertase. In honey, the sugars are readily available, so the AAB can start their action without the prior action of the yeasts, mainly using the readily available glucose to produce gluconic acid and cellulose for the mat formation. Similarly, the yeasts hydrolyze some small amounts of sucrose that may be present in the honey and, at the same time, produce alcohol from glucose, preferably fructose, which is then used by the AAB for the production of acetic acid. The growth rate of the yeasts is lower than that of the acetic bacteria during the first two days, and then there is a reversal of this trend until the end of fermentation. AAB have the upper hand in the conditions prevailing at the beginning of fermentation, while yeasts have the upper hand after the second day. Yeasts, respectively, as facultative anaerobes, are favored. Liu and colleagues studied the relationship of the Kombucha organisms \[ 54 \]. Finally, it was noted that the simultaneous production of ethanol and acetic acid prevents the competition of other microorganisms. This relationship illustrates the defined level of symbiosis and compatibility between the organisms in the tea colony. The number of AAB decreased from 4. The results show that mountain tea with honey is a suitable substrate, respectively, with black tea and sugar, for microbial growth in symbiotic kombucha culture fermentation. Sugar in the fermentation medium is used as a carbon source for the growth of microorganism cells, in addition to the metabolic processes that produce cellulose and metabolites in the form of specific organic acids \[ 38 \]. On day zero, before inoculation, the total sugar content was Glucose and fructose contents were The results are in agreement with the literature. In almost all honey types, fructose is the primary sugar. Nevertheless, some types of honey, such as those derived from Brassica napus rape , Taraxacum officinale dandelion , and Trichostema lanceolatum blue curls , have higher levels of glucose concentration \[ 58 \]. These particular honeys deviate from the established norm. After fermentation, total sugars decreased by The drop in fructose value was almost double that of glucose for samples A and B. At the same time, it was similar for sample C. Thus, the fructose value after fermentation dropped to Glucose dropped to Generally, fructose content was lower than glucose during fermentation, suggesting that fructose was preferred as the carbon source by yeast cells. The results are consistent with previous studies \[ 38 , 63 \]. Yeast and bacteria in the Kombucha consortium use substrates in different and complementary ways. Yeast cells hydrolyze sucrose to glucose and fructose and then produce ethanol, with a preference for fructose as a substrate \[ 64 \]. In contrast, AAB utilize glucose to produce gluconic acid and cellulose and ethanol to produce acetic acid \[ 39 , 44 , 65 , 66 , 67 \]. More analytically, in the pentose phosphate pathway, the enzyme glucokinase converts glucose to glucosephosphate G6P , and then G6P is converted to glucosephosphate G1P with the help of phosphoglucomutase enzyme and converted back to uridine diphosphate UDP with pyrophosphorylase enzyme until it is converted to cellulose with the help of cellulose synthase enzymes \[ 68 \]. The result of cellulose formation will be seen as a pellicle on the surface of the fermentation medium. Besides producing cellulose, the metabolic process of Acetobacter xylinum will also produce primary metabolites in the form of acetic acid and other organic acids, including gluconic acid, glucuronic acid, malic acid, tartaric acid, citric acid, butyrate, and lactic acid \[ 38 , 69 \]. A slight differentiation is observed in sample C, with higher glucose and lower fructose consumption compared with samples A and B. From the results of the microorganism count, a higher number of acetic bacteria is recorded in sample C after the first day of fermentation and a lower number of yeasts until at least the third day compared with samples A and B. So, the higher number of acetic bacteria in C is responsible for the higher glucose consumption for conversion to gluconic acid and cellulose. In contrast, the reduced number of yeasts during the more extended fermentation period is reflected in the reduced consumption of fructose to ethanol compared with A, B. The sugar consumption and acid production observed suggest an active fermentation process in the OMTWH. The glucose value shows a decrease of Finally, the fructose value decreased by On day zero, before inoculation, gluconic, acetic, citric, tartaric, and lactic acids were found at concentrations of 0. Those concentrations corresponded to the honey added Honey contains organic acids in equilibrium with their corresponding lactones \[ 58 , 70 \]. They contribute to honey acidity and electrical conductivity. However, the vast majority of them are produced from nectar and honeydew sugars by the action of enzymes secreted by bees during ripeness and storage formic acid and others \[ 58 , 71 , 72 \]. Furthermore, in the process of storage, the osmophilic yeasts enzymatically convert honey sugars into alcohols and subsequently organic acids, facilitated by the action of glucose oxidase. This metabolic activity results in the production of various acids, including acetic acid, which might serve as potential markers of honey fermentation \[ 73 , 74 \]. It comes from glucose through the action of glucose oxidase. The presence of gluconic acid and gluconolactone in honey is known to exist in an equilibrium state \[ 72 , 73 \]. In addition to gluconic acid, a comprehensive analysis of honey has identified over 30 non-aromatic organic acids, including acetic, butyric, citric, formic, lactic, maleic, malic, oxalic, fumaric, pyroglutamic, succinic, pyruvic, and tartaric acids, among others \[ 74 \]. The presence of organic acids in honey serves as a distinguishing feature that reflects its botanical source \[ 76 , 77 , 78 \]. L-tartaric acid was present in all analyzed samples ranging from 0. The results were in accordance with \[ 79 \], where this acid ranged from 0. After fermentation, the dominant acid in the beverage for samples A and B was acetic acid with a concentration of 2. The second most abundant acid was gluconic acid, with concentrations of 1. All the other acids remained unchanged after fermentation. The gluconic to acetic acid ratio was 0. The most significant ratio is that of sample C, as expected by the equivalent consumption of sugars during fermentation. Also, the gluconic and acetic acid values correlate with the corresponding values of glucose and fructose, respectively. These conclusions are in agreement with the literature. As anticipated, acetic acid is the predominant organic acid present in kombucha beverages. All examined kombucha beverages yielded good results for the presence of this particular constituent, which is the most discernible byproduct derived from the fermentation process of kombucha. The manufacturing of AAB is derived from ethanol \[ 39 \]. In a study conducted by Sievers et al. The data were obtained using enzymatic analysis. Similarly, acetic acid was the primary acid produced in kombucha with black and green tea, with a concentration close to 3. Acetic acid was also the main organic acid found in tea fungus metabolites in a recent study \[ 27 \], and the concentrations of acetic acid in Zijuan tea-based kombucha increased with prolonged fermentation time. Additionally, gluconic acid was the other major organic acid found, and it reached a maximal concentration of 2. As reported, acetic and gluconic acids were the main organic acids in kombucha beverages \[ 81 \]. They found that both the time of fermentation and the type of substrate significantly affected the concentrations of acetic acid and gluconic acid in the kombucha and reached the maximum concentration of However, the presence of a high acetic acid negatively impacts the overall acceptability of kombucha due to its vinegar-like taste. On the contrary, gluconic acid was found to be beneficial with its mild, soft, and refreshing taste \[ 82 \]. It is essential to highlight that a higher content of gluconic acid in kombucha is related to a better sensory quality of the beverage \[ 83 \]. Sample C indicates that it is possible, with honey as a carbon substrate and a low count of yeast cells at the beginning of fermentation, that a more significant ratio of gluconic acid to acetic acid can be achieved with a positive impact on the overall acceptability of kombucha. The flavor and aroma profile of a batch of tea Kombucha is contingent upon the constituents included in the fermentation medium. The parameters included in this set of variables consist of the acetic acid to gluconic acid ratio, the carbon dioxide concentration, and the concentration of organic acids. The flavors generated by gluconic acid are characterized by their delicate nature, but those resulting from volatile acetic acid exhibit astringent and acidic qualities. Therefore, obtaining the desired quality of broth is possible by controlling the fermentation conditions. Organic acids present in kombucha also exhibit bioactivity. They play an essential role in biological processes through their involvement in metabolic pathways as intermediate or final products \[ 17 \]. A study has revealed the ability of acetic acid at a concentration of 1. Furthermore, the acidification of the cytoplasm and the accumulation of dissociated acid anions to potentially dangerous levels, induced by acetic acid and other organic acids, might have an impact on antibacterial efficacy \[ 12 \]. The gluconic to acetic acid ratio is 0. By reducing the contents of the acids to total acidity expressed as acetic acid, the value of sample C is lower than that of sample A and B. Finally, the decrease in total sugars correlates with the total acidity in the samples. Acetic acid has a concentration of 3. There is also a production of 0. Malic acid can be produced by yeasts \[ 85 \] and constitutes a byproduct of fermentation that helps detoxify the liver \[ 86 \]. On day zero, before inoculation, the ethanol content was zero. However, after fermentation, the ethanol content reached 5. The higher number of yeasts can explain the higher values in ethanol content in samples A and B during the more extended fermentation period that consumed more fructose to produce more ethanol than C. The values obtained were as per previous studies in traditional kombucha beverages. According to a recent study, it was shown that the concentration of ethanol in a kombucha beverage reached 6. According to Chen and Liu, ethanol concentration in kombucha increases with fermentation time, reaching an approximate maximum value of 5. Furthermore, Cardoso and colleagues found that the green and black tea kombucha had an alcoholic content of 7. According to a separate investigation, the ethanol concentration reached 0. The above trend is reflected in the total sugars decrease of This indicates an ongoing fermentation, and the insignificant change in the acetic and gluconic acid values in the samples, respectively, that can be explained by the anaerobic conditions in the sealed bottles. The results are in line with the study of Talebi and colleagues, where the ethanol concentration in all commercial kombucha products examined was 1. Moreover, it was noted that the ethanol concentration of commercial kombucha products changes with time. Moreover, previous studies have provided evidence indicating that the addition of sugar and the time of fermentation have a crucial role in determining the concentration of alcohol \[ 88 \]. The study of trace elements found calcium and potassium in concentrations of The essential minerals in honey are potassium, sodium, calcium, and magnesium \[ 89 \]. Vitamin C content increased from Vitamin C is the most common vitamin found in kombucha beverages Additionally, water-soluble vitamins in kombucha with 0. The values of vitamin C obtained in our study are higher than those obtained for traditional kombucha beverages after fermentation. The above can be explained by the different substrates used and their vitamin C content. Sugar in the traditional kombucha beverage does not contain vitamin C. On the other hand, the most important vitamin in honey is vitamin C \[ 94 \]. Vitamin C has been especially determined in honey because of its antioxidant effect, and it is often found in almost all kinds of honey \[ 95 , 96 , 97 \]. The vitamin C content of ninety kinds of honey has been previously reported \[ 98 \]. The results showed that vitamin C is present in all samples. In particular, multi-floral honey presented the highest vitamin C content 5. In unifloral honey, the content ranged between 2. These values were similar to those found in citrus and eucalyptus honey, while they were lower than those found in sulla honey by \[ 31 \]. Moreover, both leaves and flowers from Sideritis scardica have a high level of ascorbic acid \[ 99 \]. In kombucha fermentation, it is assumed that vitamin C is derived from glucose and synthesized by bacteria \[ \]. Biosynthesis of vitamin C in kombucha throughout the fermentation process is highly associated with fermentation temperature, duration of the fermentation process, symbiosis of the SCOBY culture, source of carbon atoms, and other conditions \[ \]. Previous studies have described that Gluconobacter species exhibit a preference for glucose and possess the ability to oxidize glucose via two distinct routes \[ , \]. The first process takes place intracellularly, wherein oxidation transpires through the pentose phosphate route. On the other hand, the subsequent process happens extracellularly and entails the production of gluconic and ketogluconic acid. Furthermore, gluconic acid serves as a metabolite of acetic acid bacteria AAB and has beneficial effects on human health. It acts as a precursor in the manufacture of naturally occurring vitamin C. Glucose is used as a substrate for the initiation of the reaction, specifically for the production of 2-oxo-L-gluonic acid, which serves as a precursor for vitamin C. In addition to glucose, other carbohydrates that may be considered include fructose, galactose, as well as arabinose, rhamnose, xylose, sorbose, or sucrose \[ \]. The precise mechanisms in the metabolic production of vitamin C in kombucha fermentation are to be determined \[ 86 \]. The stability in the vitamin C content can be attributed to the low temperatures and pH and the acetic acid content of the beverage. Natural mild organic acids, such as acetic acid, can maintain the stability of ascorbic acid in food \[ \]. Lower temperatures have been indicated as a factor to inhibit ascorbic acid degradation and thus limit the accumulation of various degradation products \[ \]. Low acidity is an adequate environment for storing vitamin C \[ \]. The results from vitamin B-complex quantification are presented in Table 2. Different letters in each column indicate significant differences. It has also been noted that vitamins of the B complex are synthesized during kombucha fermentation \[ 38 \]. Similar results in vitamin B complex were produced in the fermentation of soybeans to tempeh and the fermentation of lentils, black gram, and rice by Rhizopus species and Klebsiella pneumonia, respectively \[ 8 , \]. Also, increased levels of thiamine, pyridoxine, niacin, and pantothenic acid have been reported to be the result of lactic acid bacteria LAB fermentation in yogurt, buttermilk, cheese, and other fermented products \[ , \]. Moreover, the vitamin B2, B3, B6, B9, and B12 content has been shown to significantly increase during fermentation in cowpea and soybean tempeh \[ \]. The presence of vitamins in the Olympus Mountain tea with honey before fermentation sample A0 can be explained by the concentration of vitamin B-complex in the honey \[ 31 , , , , , \]. According to Behrendt and colleagues, a dietary supplement combining an herbal extract of Sideritis scardica and selected B vitamins can alleviate stress-induced impairment of executive functioning in regard to working memory, cognitive flexibility, and controlled behavioral inhibition after an intake period of six weeks \[ \]. A similar trend was observed in kombucha beverages after the fermentation by other investigators \[ 39 , 49 , 65 , , \]. The increment in the phenolic compounds was previously explained by Blanc \[ \]. The study showed that the enzymes phytases, in particular liberated by bacteria and yeast in the Kombucha consortium, and the increased acidic environment of the fermentation process, could release polyphenol compounds from the cellulosic backbone, resulting in an increase in polyphenols in the soluble fraction of the fermented beverage. It has also been identified that the enzymes liberated and low pH during the fermentation could also cause the bio-transformation or degradation of complex polyphenols to small molecules, resulting in increased phenolic compounds. This explanation has also been supported by other investigators \[ 39 , 49 , 65 , , \]. Only minor differences in content values were found between the individual samples. The same trend was observed in a recent study where total flavonoid content was lower in kombucha beverages with yarrow than in corresponding initial substrates and suggested that low pH in the fermented beverage caused the degradation of flavonoids \[ 17 \]. Furthermore, it has been referred that gut bacteria can cleave the C ring of flavonoids and release phenolic acid as 3- 4-hydroxyphenyl -propionic acid and 3-hydroxyphenyl acetic acid \[ \]. It was, therefore, possible that yeasts and bacteria of the kombucha consortium secreted enzymes capable of catalyzing the biodegradation of flavonoids and releasing phenolic acids. It has been shown that the content of total polyphenol compounds increased linearly with the fermentation time \[ 49 \]. That explains the correlation of TPC with TA, which change during fermentation more linearly than pH because of the buffer effect. On the other hand, the correlation between TF and pH is supported by a previous work, suggesting that low pH in the fermented kombucha beverage with yarrow caused the degradation of flavonoids \[ 17 \]. However, a slight increase was observed in sample C, which showed the lowest value before refrigeration. Now, the differences in the values of flavonoids and total phenolics between the three samples were almost annihilated. High total phenolic content enhances antioxidant activity \[ \]. Our study showed an increase in TPC during fermentation, explaining the enhancement of the antioxidant activity. Furthermore, it has been shown that the increase in antioxidant activity in kombucha samples mostly stems from the presence of low molecular weight polyphenols generated during the fermentation process. This observation has been made by scholars \[ 38 \]. The same trend was observed in a recent study, where despite the higher amount of phenolic compounds, the antioxidant activity was lower compared with the non-fermented blueberry beverage in relation to DPPH \[ \]. Thus, the lower pH of the fermented beverage may have influenced the ability to deactivate the free radicals of anthocyanins, leading to a lower antioxidant activity than the non-fermented beverage. Jayabalan and colleagues reported that the antioxidant capacity of kombucha had been significantly affected by the composition of SCOBY, fermentation period, temperature, and other metabolites produced during fermentation \[ \]. Moreover, properties of antioxidants such as polarity, ionization state, and steric hindrance also seemed to contribute to their antioxidant abilities, as mentioned by other studies \[ 28 , , , \]. When researching the impact that pH has on the antioxidant activity of a variety of substances, this information might be helpful. It is also helpful for measuring the antioxidant activity of samples extracted in acidic solvents. The essay is not a competitive reaction because DPPH is a radical probe and oxidant. Additionally, unrelated reactions can also contribute to the loss of color. The accessibility of steric factors plays a crucial role in determining the outcome of these reactions. Thus, this test shows that small molecules with better access to the radical site have higher apparent AOC. DPPH has a relatively small linear reaction range of only 2—3-fold. Because of steric inaccessibility, the reactivity of certain antioxidants toward peroxyl radicals may be hampered or rendered inactive when it comes to DPPH. The process of hydrogen atom abstraction from the neutral ArOH by DPPH becomes less favorable due to its sluggish kinetics in solvents with strong hydrogen-bond-accepting properties, such as methanol and ethanol. Furthermore, the researcher discovered that the presence of adventitious acids or bases in the solvent may significantly impact the ionization equilibrium of phenols, resulting in a decrease or increase, respectively, in the reported rate constants \[ \]. The use of the DPPH assay as a reliable method for measuring antiradical activity is compromised due to its chemical limitations. Under acidic circumstances, the reducing capacity of antioxidant compounds may be hindered as a result of protonation. Conversely, under basic conditions, the dissociation of protons from phenolic compounds would augment the reducing ability of a sample. Furthermore, a recent investigation revealed that the outcomes pertaining to the impact of flavonoids in the DPPH test were contingent upon the Bors criterion. Conversely, the association between the chemical structure and activity in the ABTS assay remained ambiguous. The differences between the three samples were negligible, justified by the respective elimination of differences in total phenolic and flavonoid values. These two enzymes are essential in the breakdown of carbohydrates, which are present in the small intestinal brush border. Therefore, the use of OMTWH beverage fermented with the Kombucha consortium as an anti-diabetic agent might be expected. Further in vivo and clinical studies are warranted to demonstrate their therapeutic potential to be relevant to human health. In a recent research, it was shown that the anti-acetylcholinesterase activity exhibited an increase when subjected to fermentation. This increase resulted in about an eight-fold higher value in both the ethyl acetate and aqueous extracts of B. Results suggested that kombucha fermentation may generate new active metabolites against AChE in the extracts. AD begins as a deficiency of acetylcholine ACh inhibitors. Studies indicated that various extracts of Sideritis species showed moderate cholinesterase inhibitory activity \[ , , , \]. Regarding BChE activity inhibition, the highest significant result was recorded for goldenrod honey That indicates an increase in the inhibition of AChE and BChE with the rise in the number of phenolic compounds in the samples. The above is in accordance with studies that suggested a high correlation between TPC and AChE inhibition activity \[ , \]. In a recent study, the high anti-ChE inhibitory activity of kinds of honey was linked with high polyphenolic contents \[ \], while a previous study showed that a high anti-AChE activity of studied kinds of honey was correlated with their high antioxidant activities, and consequently with the high TPC in the studied kinds of honey \[ \]. Furthermore, Zaidi et al. The honey samples were found to possess a considerable quantity of total phenolic content TPC. Furthermore, the investigators observed a statistically significant and strong correlation between the anti-acetylcholinesterase anti-AChE activities and total phenolic content TPC levels in the honey samples that were subjected to examination \[ \]. Similarly, a recent investigation \[ \] conducted on a total of 47 distinct samples of Polish honey has shown further evidence supporting a positive correlation between the activity of anticholinesterase enzymes anti-AChE and anti-BChE and the total phenolic content TPC. This study presents a novel beverage as well as its physicochemical analysis and an evaluation of its functional properties. The beverage was prepared with Olympus Mountain tea Sideritis scardica , sweetened with thyme honey, and fermented by kombucha culture. Four days of fermentation appeared sufficient to improve the nutritional and functional characteristics of the beverage. The total counts of bacteria and yeast increased daily to 4. The increase was indicative of the ability of the OMTWH to act as a suitable substrate to sustain the proliferation of the microorganisms of the kombucha symbiotic culture. Due to the kombucha fermentation, total sugars decreased, and ethanol and TA increased with acetic and gluconic as the dominant acids. The decrease in pH values followed the increase in acidity. In our case, optimal consuming acidity was obtained for less than four days of fermentation. The results showed that mountain tea with honey is a suitable substrate, respectively, with tea and sugar, for microbial growth in symbiotic kombucha culture fermentation. Moreover, total phenolic compounds, vitamin C, and vitamin B-complex content increased after fermentation. The IC 50 values of antioxidants for ABTS decreased after fermentation, which denotes increasing antioxidant activity. Therefore, kombucha fermentation can transform OMTWH into a novel beverage with enhanced functional properties. Conceptualization, I. All authors have read and agreed to the published version of the manuscript. The data used to support the findings of this study can be made available by the corresponding author upon request. The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Find articles by Ioannis Geraris Kartelias. Find articles by Haralabos Christos Karantonis. Find articles by Efstathios Giaouris. Find articles by Ioannis Panagiotakopoulos. Find articles by Constantina Nasopoulou. Ioannis Panagiotakopoulos : Investigation. Salam A Ibrahim : Academic Editor. Regina Kratzer : Academic Editor. Open in a new tab. Similar articles. Add to Collections. 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