GCMS elucidation of bioactive metabolites from fermented Kombucha tea

Introduction

Kombucha is a symbiotic growth of bacteria (such as Acetobacter xylinum, A. xylinoides, Bacterium gluconicum) and various yeast strains (including Schizosaccharomyces pombe, Saccharomycodes ludwigii, Saccharomyces cerevisiae) cultivated in sugared tea. The acetic acid bacteria and the yeast are in symbiotic association in which the yeasts by their invertases decompose sucrose and ferment its hexose units to ethanol and Carbondioxide. The Acetobacter genus oxidize ethanol through acetaldehyde to acetic acid. From glucose the Acetobacter synthesize gluconic acid and cellulose which occurs as a membrane on the surface of fermented liquid (Kersters, 2006) [9] . It is a popular beverage among many traditional fermented foods across the world. It was originated in North-East China (Manchuria) and later spread to Russia and rest of the world. It has sour, sweet and lightly carbonated taste. Kombucha tea or its extract show therapeutic properties, such as the ability to prevent cancers, decrease blood cholesterol level, reduce nephrotoxicity of pharmaceuticals, toxic metals and provide protection against the harmful effect of radiation. Several medical studies has revealed that kombucha has certain therapeutic value possessing antibiotic activity, positive effects on gastrointestinal activity, arthritis, gout, haemorrhoids, cholesterol value, arteriosclerosis and nervous system. Daily consumption of kombucha significantly reduces the risk of cancer. The synthesis of organic acids during fermentation protects the symbiotic colony from invasion by undesirable external microorganisms that are not associated with the tea fungus. In addition to tea and sugar components the beverage also contains acetic acid, gluconic acids, L-lactic acids, amino acids, biogenic amines, vitamins such as B-complex and vitamin C. One of the most important metabolite from therapeutical point of view is glucuronic acid, a carrier of detoxification activity of kombucha. Recent research on kombucha has proved that its antimicrobial activity against pathogenic microorganisms is largely attributable to acetic acid. Acetic acid is known to inhibit and destroy a number of Gram-positive and Gram negative microorganisms (Pinto et al., 2008) [23] . Dutta and Gachhui, 2007 [2] has indicated that kombucha is frequently called "tea fungus", although no fungus is actually involved in the fermentation process. Fermentation using kombucha colonies is composed of two portions: a floating cellulosic pellicle layer and sour liquid broth below the cellulosic layer formed by A. xylinum and yeasts. This fungus-like mixture of microorganisms and cellulose is likely the reason why kombucha is also called "tea fungus". Klemm et al., 2005 [10] has indicated that the cellulosic mat is an organic high dietary fiber rich food product, low in fat and calories and contains no cholesterol and further the cellulose is recognized by the FDA as edible and the Acetobacter is a non-pathogenic cellulose-producing food grade bacterium. Legaz et al., [13] has developed a non-edible biomaterial called Bioskin using Acetobacter xylinum. Cellulose formed from the culture medium was mechanically disrupted, filtered through a multi-layered cheese-cloth, pressed and dried. The final dry product was not dissolved in water or organic solvents, but when rewetted in distilled water absorbed about 250 per cent of its dry weight. Ultra structural studies by TEM reveal that Bioskin was an anastomous structure composed by fibers surrounding concrete bodies and was able to fix osmium tetroxide. The bioskin was lipidic or lipoproteic nature. SEM analysis shows that Bioskin is apparently formed by crossed fibers with very large interfibrillar spaces. Fibers showed a very regular structure without superficial adherences. Interfibrillar spaces were the basis of the water-absorbing property of bioskin. Some low molecular weight compounds, such as sucrose, dissolved in water, also enter the void spaces and they crystallize on the surface of fibers to which they remain adhered. Considering all the above, a research was carried to develop fermented organic extract from kombucha and the structural bioactive compounds was elucidated by GCMS.

Materials and Methods

Production of kombucha cultures

The Acetobacter xylinum strain (sju-1) used in this study was previously isolated and identified in our laboratory from fermenting sugarcane juice (Coimbatore, Tamil Nadu, India). The yeast namely Saccharomyces cerevisieae was obtained from MTCC 6507.The culture was resuscitated by incubation on YPM (25 g/L mannitol, 5 g/L yeast extract, 3 g/L peptone, and 15 g/L agar) at 30 °C for 2 days. Working cultures were routinely prepared on YPM and stored at 4 °C until use. The basic growth medium used for the Acetobacter strain was Hestrin and Schramm (HS) medium (20 g/L glucose, 5 g/L peptone, 5 g/L yeast extract, 2.7 g/L Na 2 HPO 4 , 1.15 g/L citric acid.H 2 O) (Hestrin et al., 1954) [6] . A traditional tea-based medium (80 g/L sucrose and 3 g/L tea) was also used to culture the organism. Inoculum was prepared by transferring a single colony from the YPM working culture plates into 100 ml of HS medium in 500 ml bottles and incubating the culture without agitation at 30 °C for 2 days. The broth was shaken vigorously to release the attached cells from the cellulose pellicle and then aseptically filtered through sterilized gauze. The resulting cell suspension was used for all subsequent experiments. The experiments were carried out by adding 10 ml of inoculum to a 500 ml bottle that contained 90 ml of medium. The bottle was then shaken at 30 °C for seven days either statically at 60 oscillations per minute.

Preparation of crude extract of kombucha culture

The fermentation media containing the cellulosic pellicle and the broth was homogenized well in a blender homogenizer and the cellulose was allowed to sediment and settle down. The crude extract of kombucha was obtained as per the procedures of Ibrahim et al., 2011 [7] . The supernatant was mixed with equal quantities of chloroform and methanol (2:1) and shaken well vigorously in a separating funnel and incubated overnight (Parliament, 2020) [22] .

Structural elucidation of bioactive compounds by GCMS

The Gas chromatography Mass Spectrometry (GCMS) analysis of the samples isolated from the chloroform and methanol (2:1) extract of fermented broth was performed using Thermo GC -trace ultra VER: 5.0,Thermo MS DSQ II equipped with DB 5 -MS capillary standard non-polar column of 30 Mts dimension, ID: 0.25 mm, film: 0.25 μm, for GC-MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium was used as carrier gas with a flow rate of 1.0 ml/min and oven temperature 70 °C raised to 250 °C at 6 °C/min. The chromatographic peak identification was carried out by comparing their mass spectra with those of the bibliography data of unknown compounds from the NIST library mass spectra database on the basis of the criterion similarity (SI)>800 (the highest value is 1000). The approximate quantification of volatile compounds was estimated using the Wanakhachornkrai and Lertsiri, 2003 [28] method, which involved using the Xcalibur software (Vienna, VA) to integrate the peaks on the total ion chromatogram. The peak area normalized (%) is used to display the results.

Results

GCMS Analysis of fermented kombucha extract

The result of GCMS analysis of kombucha extract is presented in table 1 and Figure 1. The result revealed the presence of seven volatiles compounds dominated by thiophene-2-carboxylic acid, santolin diacetylene (24.8%), 1,5-dihydroxyl-3,3-dimethyl-7-acetylaminomethyltricyclododecane (10.75%), 1-(2-hydroxyethylthio)-1phenyl-2-butanone (12.33%), Nonane, 4-hepatanol (6.53%) and 3-(4-nitrobenzoyl) cinnoline (5.8%).

Discussion

The relative abundance of alkane hydrocarbon or fatty acid molecules like dodecane, heptadecane, octadecane, hexadecane, octacosane, heneicosane, tricosane and nonane were higher in the methanolic extract of kombucha tea.

Haigh et al., 1973 [5] has illustrated that induction of bifringence (orientation of microfibrils) were due to the liberation of 1:1 mixture of saturated and monounsaturated tetra-hydroxy terpene. Previous publications reported that the compounds such as 1-octadecene, 1-heptadeceane found in algae, bacteria and plants show anticancer, antioxidant and antimicrobial activity (Lee et al., 2007; Mishra, 2007) [12, 19] . According to Santos et al., (2009) [25] , active fatty acids and lipids with antimicrobial properties are found in high concentrations in a variety of Gram-negative bacteria. These compounds kill microorganisms by rupturing the cellular membranes of bacteria, fungi, and yeasts. They accomplish this by penetrating the thick layer of peptidoglycan in the cell wall without leaving visible traces and reaching the bacterial membrane, which causes it to disintegrate. Kombucha was utilized to valorize tea wastes, and compounds such as long chain fatty acids and dienes, which were similar to the metabolites identified in the current findings, were found (Majumder et al., 2022) [16] . Majumder et al., (2020) [15] revealed the removal of caffeine in coffee kombucha and identified various alkaloids and

International Journal of Advanced Biochemistry Research https://www.biochemjournal.com undesirable metabolites, including amides, during fermentation. Their findings indicated that hexadecane had the most significant peak area. In a similar experiment conducted by Rinaldi, 2022 [24] about 65 different volatile compounds, including acids, alcohols, aromatic hydrocarbons, esters, phenols, terpenes, and others, were identified. The GCMS results revealed the presence of seven important volatile compounds essential for antibacterial activity in crude extract of kombucha. The typical flavour of the end product was due to the prevalence of thiophene-2-carboxylic acid, santolin diacetylene, acetic acid, neopentyl alcohol, lavanduyl acetate, 1-heptanol and 3-(4-nitrobenzoyl) cinnoline. Neopentyl-2-oxobutanoate is yet another flavour compounds present in crude extract. Benzaldehyde dioctyl acetal being an important ketone derivative is a prominant flavour compound that are normally present in most of the distilled beverages. Santolin diacetylene is an hepatoprotective agent and an important antioxidant compound. Compounds like acetamide and diacetamide are also present in the extract which are essential for stabilisation of cellulose derivatives which are formed as floating pellicle by the bacterium A. xylinum in the kombucha extract. Some other medically important compounds identified were piperazine, leucinol, crocusatin, sarracine, haliclorensin and quinoxaline. In earlier investigatons carried out by Czaja et al., 2006 [1] it has been indicated that bacterial cellulose obtained from A. xylinum can be used for making chronic and acute wound care products. Toda et al., 1997 [27] as reported that cellulose produced by acetic acid tolerant A. xylinum DA strain has an excellent moisture retaining capacity and continous production stability suited for industrial scale production. Because of the unique properties, resulting from the ultrafine reticulated structure several application such as skin grafts, face peeling, collagen, blood vessels and granulation have been proposed for this cellulosic layer by Lynd et al., 2002 [14] . From the present investigation, the pure cellulose obtained as a floating pellicle can better be applied for developing a wide array of rewettable supraadsorbent pads.

Conclusion

The relative abundance of antibacterial metabolites expressed in kombucha extracts might be due to the occurance of both hydrophobic and hydrophilic bioactive compounds. The various important metabolites identified serve as wholesome proof for the antibacterial propoerties of Kombucha tea. An improved knowledge of the composition, analysis and properties of A. xylinum with respect to antimicrobial compounds and bacterial cellulosic membrane would assist in efforts for the pharmaceutical application of this bacteria.

Acknowledgement

AcknowledgmentThe authors acknowledge Tamil Nadu Agricultural University, Coimbatore for granting the University Research Project fund to execute the work in a successful manner.

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