A Comparison of Kombucha SCOBY Bacterial Cellulose Purification
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Research Article A comparison of kombucha SCOBY bacterial cellulose purifcation methods Ananda S. Amarasekara1 · Deping Wang1 · Tony L. Grady1 Received: 2 October 2019 / Accepted: 6 January 2020 / Published online: 20 January 2020 © Springer Nature Switzerland AG 2020 Abstract Kombucha SCOBY pellicle formed as a waste product in the kombucha tea fermentation industry can be used as a poten- tial source for bacterial cellulose. We have compared four simple, scalable purifcation methods for the purifcation of this jelly like cellulosic matrix containing bacteria, yeast cells, proteins and polyphenols as impurities. The method using two successive 1.0 M NaOH washings at 90 °C, followed by the treatment with 1.5% (w/w) aq. NaOCl for 2 h, was the most efective method and gave the purifed cellulose sample with the highest leucometer whiteness value of 81.4 ± 4.8. The purifed cellulose samples were analyzed using FT-IR, TGA, SEM, EDX, X-ray and water absorption at room temperature. Keywords Kombucha · Scoby · Bacterial cellulose · Hydrogen peroxide · Sodium hypochlorite 1 Introduction vary, but generally include Acetobacter bacterial species, various Saccharomyces and a number other types of yeasts Kombucha is the common name given to a fermented [3]. Within this culture, anaerobic ethanol fermentation lightly alcoholic beverage prepared using sweetened black by yeast, organic acid fermentation and aerobic ethanol or green tea. Sometimes the beverage is called kombu- oxidation to acetate through bacteria can all take place cha tea as well to diferentiate from the bacteria and yeast concurrently along an oxygen gradient. In addition, Ace- culture used in the fermentation process. This centuries tobacter may well polymerize glucose residues forming a old beverage is believed to have been originated in north- bacterial cellulose mat supporting the microbial culture eastern china. The current broad attention to this classi- [3, 4]. cal beverage due to renewed interest in supposed health In recent years, with the surge of the kombucha bev- benefts has resulted a number commercial scale produc- erage industry, a number of research groups around the tions and a home brewing renaissance [1, 2]. Kombucha globe have studied the microbial culture, composition culture is composed of a symbiotic growth of acid produc- of the fermented liquid as well as the bacterial bioflm at ing bacteria and osmophilic yeast species in a zoogleal the water–air interface. The major components of the fer- mat. During the kombucha tea fermentation a gelatinous, mented media are gluconic, acetic, lactic acids and etha- cellulose-based bioflm or a pellicle is formed at the air–liq- nol; in addition, minor amounts of proteins, tea polyphe- uid interface. This bioflm is a Symbiotic Culture of Bacteria nols, minerals and vitamins (B1, 2, 6, 12 and C) are also and Yeast (SCOBY) and commonly known as a tea mush- reported [4, 5]. The pellicle formed at the air–liquid inter- room as well. The microbial populations in a SCOBY may face is mainly cellulose. Similar bacterial cellulose forms Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s4245 2-020-1982-2) contains supplementary material, which is available to authorized users. * Ananda S. Amarasekara, [email protected] | 1Department of Chemistry, Prairie View A&M University, Prairie View, TX 77446, USA. SN Applied Sciences (2020) 2:240 | https://doi.org/10.1007/s42452-020-1982-2 Vol.:(0123456789) Research Article SN Applied Sciences (2020) 2:240 | https://doi.org/10.1007/s42452-020-1982-2 are known for several decades and is traditionally isolated range using a scanning rate of 20 °C min−1. Cellulose flm from nata de coco, a gelatinous southeast Asian dessert thickness was measured using a Pittsburgh instruments made from coconut water [6]. In this preparation coconut digital micrometer model 895. Whiteness of purifed cellu- water serves as the carbon source for bacteria Acetobacter lose flms were measured using Landtek GM-6 Leucometer. xylinum and later converted to extracellular cellulose [6]. Scanning electron microscopy (SEM) and Energy Disper- In comparison with plant cellulose, microfbrils of bacte- sive X-ray Spectroscopy (EDX) analyses were performed rial cellulose are generally about 100 times smaller than in using a JEOL JSM-6010LA using InTouchScope software. plant cellulose and this fne structure leads to a number The backscattered electron images was collected using an of appealing properties of bacterial cellulose, such as high accelerating voltage of 10 kV and a load current ~ 90 µA water absorption capacity and strength [7, 8]. In addition, with a working distance of 9 mm. EDS spectra were col- the well arranged three dimensional nano-fber network lected at a magnifcation of 500× and the analyzed area in bacterial celluloses results the formation of hydrogel was 0.15 mm2 (110 µm × 135 µm). Qualitative and quan- sheets with high surface area, abundant surface hydroxyl tifcation elemental composition was performed with the groups, high porosity and good chemical-modifying characteristic x-ray using a silicon-drift detector. The semi- capacity [9, 10]. As a result bacterial cellulose has found in quantifcation is based on the theoretical correction of the numerous applications such as wound dressing [11, 12], ZAF efect. X-ray difraction experiments were performed raw material for food and as food packaging [13], biore- using a Bruker D8 Davinci spectrometer, wavelength actor, bio-processing [14], optoelectronics [15], polymer 1.54060 Å, with 40 kV, 15 mA/Cu source, and scan type: 2θ. electrolyte membranes [16], and many medical applica- tions such as: implants and scafolds for tissue engineering 2.2 Preparation of the kombucha pellicle of cartilage as well as carriers for drug delivery [17–19]. Our interest in processing cellulosic biomass for renew- Tea was brewed by using Lipton black tea (11.3 g, fve tea able fuels and chemical feedstock applications has led us bags) and 1.0 L of boiling water and allowed to stand for to study the kombucha pellicle formed as a waste product 20 min. The tea bags were then removed, sucrose (100 g, in the growing kombucha beverage industry as a poten- 0.292 mol) was dissolved in tea and allowed to cool to tial new sources of bacterial cellulose [20–22]. Under this 37 °C and kombucha SCOBY was added. The sweetened program, we have studied the probable industrially scal- tea with the starting SCOBY was then covered with a cot- able methods for the purifcation of kombucha pellicle to ton cloth and allowed to stand in a dark cupboard for produce bacterial cellulose. In this publication, we present 10 days. Then the new kombucha pellicle formed at the a comparison of four simple methods and identifcation air–water interface was carefully removed, washed with of the best conditions for the purifcation of bacterial cel- deionized water (2 × 500 mL) and pat dried with Kleenex lulose formed as a waste product in the kombucha tea tissues and used for the purifcation experiments. industry. 2.3 Purifcation of the bacterial cellulose in kombucha pellicle 2 Experimental The kombucha pellicle was cut into four approximately 2.1 Materials and instrumentation equal in size quarters and labeled as A, B, C and D and purifed using four diferent methods. The starting kombucha SCOBY was purchased from www. posey mom.com. Lipton black tea (100 tea bags, total 2.3.1 Purifcation of kombucha pellicle portion A 226 g) was purchased from local grocery store. Sodium hydroxide (> 99%), sucrose (> 99%), sodium hypochlorite Kombucha pellicle portion A was placed in 50 mL of 1.0 M solution (6%) and hydrogen peroxide (3%) were purchased aqueous sodium hydroxide solution and allowed to stand from Aldrich Chemical Co. Attenuated total reflection at room temperature (23 °C) for 1.0 h. Then removed from infrared (ATR-IR) spectra of cellulose flms were recorded in the sodium hydroxide solution and washed with deion- the 650–4000 cm−1 range on a Smiths IdentifyIR spectrom- ized water (6 × 500 mL), pat dried with Kleenex tissues and eter with a diamond ATR (Danbury, CT, USA). Thermogravi- weighed. The pellicle portion was then placed in a second metric analysis was carried out in air using a Perkin Elmer 50 mL of 1.0 M aqueous sodium hydroxide bath, allowed Diamond TG/DTA, High Temp 115. The mass of the thin to stand at room temperature (23 °C) for 1.0 h washed with flm piece used for a TG scan was approximately 10 mg, deionized water dried and weighed as in the earlier treat- and platinum crucibles were used in all experiments. The ment. Next the pellicle portion A was placed in 50 mL of TG curves were recorded in the 25–600 °C temperature 1.5% (w/w) aqueous hydrogen peroxide, allowed to stand Vol:.(1234567890) SN Applied Sciences (2020) 2:240 | https://doi.org/10.1007/s42452-020-1982-2 Research Article at room temperature (23 °C) for 2.0 h. Then removed from portion C was placed in 50 mL of 1.5% (w/w) aqueous the hydrogen peroxide bath and washed with deionized hydrogen peroxide, allowed to stand at room tempera- water (6 × 500 mL), pat dried with Kleenex tissues and ture (23 °C) for 2.0 h. Then removed from the hydrogen weighed. Finally, the pellicle portion A was transferred to peroxide bath and washed with deionized water (6 × 500 a watch glass and dried in an oven at 50 °C for 20 h and mL), pat dried with Kleenex tissues and weighed. Finally, weighed. The weights after each treatment are shown in the pellicle portion C was transferred to a watch glass and Table 1. dried in an oven at 50 °C for 20 h and weighed. The weights after each treatment is shown in Table 1.