Keywords

Introduction

Due to the growing demand for environmentally sustainable materials, biopolymers represent a viable replacement for synthetic polymers. Extensive research has aimed to obtain high-value-added products from natural polymers, including cellulose, starch, and chitin. Isolated primarily from plants, fungi, trees, and bacteria, cellulose is the most abundant biopolymer on Earth, with approximately 1.5 billion tons generated annually [1]. This material is of great value for producing food packaging, paper, textiles, nanofiber mats, pulp, construction, and fabric materials [2].

Moreover, kombucha is a traditional fermented Chinese beverage whose attributes were treasured by the Qin Dynasty as early as 220 BC. In 414 AD, Dr. Kombu brought kombucha tea from Korea to Japan. Over the following centuries, merchants popularized the drink in Russia, and during the nineteenth and twentieth centuries, it spread throughout Europe. During World War II, shortages of tea and sugar decreased kombucha consumption; however, after the war, it regained popularity in Germany, France, and Italy. Currently, kombucha is one of the most popular fermented drinks produced handcrafted on small scale, and commercial scales [3]; it was valued at 1.84 billion USD in 2019, and the kombucha market forecasts predict strong growth with projections of a 23.2% annual compound growth rate through 2027 [4]. The popularity of kombucha is attributed to its therapeutic effects, such as antimicrobial, antioxidant, anticancer, antidiabetic, detoxifying [5], antifungal, anti-inflammatory, antigenotoxic, and anti-stress properties [6]. It reduces cholesterol and blood pressure levels, promotes weight loss, improves glandular and gastric functions, reduces kidney calcification, combat acne, and inhibits cancer proliferation [7]. The preceding is because it contains different organic acids like gluconic and acetic; additionally, it comprises vitamins, lipids, proteins, polyphenols, minerals (manganese, copper, zinc, iron, cobalt, and cadmium), anions (chloride, fluoride, bromide, iodide, phosphate, and sulfate), etc. [8].

Kombucha is typically produced through the fermentation of sweetened green or black tea using a SCOBY (an acronym for “Symbiotic Culture of Bacteria and Yeast”) as a starter culture [9]. The SCOBY is a cellulosic byproduct that forms at the liquid–air interface during fermentation [10]. It consists of a gelatinous membrane composed of pure cellulose [11], generally biosynthesized by Gram-negative bacterial strains, including Acetobacter xylinum, Gluconacetobacter, Agrobacterium, Achromobacter, Sarcina, etc. [12], whose function is to protect the medium from factors such as ultraviolet light, some fungi, and spores [13]; in the medium there is also yeast such as Candida kefyr, Candida tropicalis, Dekkera anómala, to name some of them [14].

Bacterial cellulose (BC) was first reported in 1886 by R. M. Brown, who described it as “a sort of moist skin, swollen, gelatinous and slippery” [15], and has become a valuable biomaterial due to its range of favorable properties; these include exceptional water retention capacity, high degree of polymerization (2000–8000), high crystallinity, mechanical strength, high purity (free from lignin, hemicellulose, and pectin), non-allergenicity, moldability, as well as excellent biocompatibility and biodegradability [16,17,18] rendering it suitable for applications in tissue engineering, filtration, electronics, waste treatment, energy production [19], biomedicine, pharmaceuticals, fashion design (for the production of the so-called “vegan leather”), engineering, chemistry, environment; it was also categorized as GRAS (Generally Recognized As Safe) by the FDA (Food and Drug Administration) in 1992, being appropriate for food industry applications such as additive, stabilizer, and gelling in food, and packaging [20].

However, the characteristics, properties, and yield of BC are directly affected by numerous factors such as carbon source (sucrose, glucose, fructose, etc.) [21], incubation period (7–14 days), and temperature of fermentation (28–30 °C) [1]; oxygen pressure and, the amount supplied in the medium; pH (4–6), nitrogen source (yeast) [13]; agitated or static condition of the broth [22], even infusion times and geographical region in which kombucha is elaborated [3].

In this work, kombucha was prepared handcrafted employing a commercial starter inoculum. Then, KBC was characterized by SEM, TGA, and FT-IR to determine its thermal resistance, purity, and morphological characteristics. This is with the objective of obtaining a SCOBY for subsequent cultures, analyzing how these conditions influenced its properties, and determining KBC as a potential resource for its application in the preparation of biodegradable films in future works.

Materials and Methods

Kombucha culture (Vida Bebida), standard sugar, organic apple vinegar (Bragg), green tea, and black tea were purchased from a local market.

Kombucha Started Inoculum

The culture was prepared in a sterilized glass jar where 460 mL of organic vinegar, 235 mL of commercial unpasteurized kombucha, 1 L of water, 80 g of sugar, 2 mL of sugarcane alcohol, and one small homemade kombucha SCOBY were added, with an initial pH of 3. The glass jar was incubated statically at 30 °C for three days.

Preparation of the Infusion of Green Tea

20 g of green tea was boiled at 90 °C in 1 L of purified water and kept under infusion for 10 min, then sachets were removed and let the solution cool until room temperature (20 °C), following the best conditions described by Antolak et al., 2021 [3]. On the fourth day, green tea was added to the glass jar. It was kept incubated statically at 30 °C for 14 days.

Harvesting and Preparation of Kombucha BC

The KBC membrane formed at the liquid–air interface was carefully removed and washed with distilled water. A piece of the sample was placed on a Teflon sheet to dry at room temperature for three days and then cut into smaller pieces to be characterized.

Microbiological Analysis

Microbiological analysis of the kombucha and microscopic observations were conducted using Gram-staining fresh green tea preparation through a Carl Zeiss microscope.

Fourier Transform Infrared (FTIR) Spectrometry

The dried KBC film was characterized using a Perkin Elmer System 2000 Frontier FT-IR spectrophotometer over a wavenumber range of 400 to 4000 cm−1.

Scattering Electron Microscopy (SEM)

The morphology of the BC samples was obtained using a scanning electron microscope (Jeol-IT300) with a voltage of 30 keV.

Thermogravimetric Analysis (TGA)

Thermogravimetric analysis was conducted utilizing a Mettler Toledo TGA/SDTA851 thermogravimetric analyzer under the \({\text{N}}_{2}\) atmosphere. The sample was heated from 25 to 750 °C at a constant heating rate.

Results and Discussion

A sample of 6 mL of KBC was taken to perform Gram staining and visually analyze the colonies. As observed in Fig. 1a, rod-shaped, reddish homogeneous forms are distinguished, indicating Acetobacter xylinum, a Gram-negative bacterium that produces BC [23]. Likewise, a smear of the fresh KBC (Fig. 1a) was made, observing symbiosis between yeasts and bacteria (1.b.1), a yeast colony (1.b.2), and homogeneous bacteria (1.b.3) on the surface of the film.

Fig. 1
2 illustrations. A. An illustration of gram negative stain of K B C. Bacteria is highlighted in the stain. B. An illustrating of native visualization highlights symbiosis between yeasts and bacteria, a yeast colony, and a homogeneous bacteria.

a Gram-negative stain of KBC showing bacteria and b native visualization of bacteria and yeast in fresh KBC

Full size image

Fourier Transform Infrared (FTIR) Spectrometry

Figure 2, shows the FTIR spectrum where the intensity and position of the absorption peaks are indicated, giving details about the functional groups found in the KBC film, which are similar to the literature and validate the structure of bacterial cellulose.

Fig. 2
A line graph of transmittance percentage versus per centimeter for F T I R spectrum. A fluctuating line is illustrated.

FTIR spectrum of GTK SCOBY

Full size image

The band in 3286 \({\text{cm}}^{ - 1}\) indicates the –OH stretching vibrations [24]. The signal at 2920 \({\text{cm}}^{ - 1}\) is associated with the –CH stretching [24]. The band at 1645 \({\text{cm}}^{ - 1}\) corresponds to the –OH bending of the absorbed moisture of the film [25]. Additionally, the signals at 1417 \({\text{cm}}^{ - 1}\) and 1362 \({\text{cm}}^{ - 1}\) match with the \(- {\text{CH}}_{2}\) and CH bending, respectively [26, 27]; moreover, the band at 1034 \({\text{cm}}^{ - 1}\) is associated with either C–O–C and C-O–H stretching vibration of the sugar ring in cellulose [27, 28].

Scattering Electron Microscopy (SEM-E/EDX)

SEM was utilized to analyze the morphological characteristics of the KBC film. As Fig. 3a shows, the SCOBY surface is formed of bacteria in a fibrous configuration and disordered clusters. In Fig. 3b, the bacteria comprising the clusters can be identified, which is consistent with the microbiological observations using optical microscopy. It can be seen that the KBC film presents a high microbial population, as Villarreal et al., 2020 indicate, a substantial concentration of microbial cells can obstruct the interaction between fibrils in the network, leading to a decrease in the number of hydrogen bonds and consequently impacting the properties of the biofilm.

Fig. 3
2 S E M images. A. An illustration of G T K SC O B Y surface highlights a mashed surface. B. A S E M image of G T K S C O B Y of bacteria and yeast surface illustrates big noodle like structures, magnified up to 1500 times.

Scanning electron microscopy (SEM), a GTK SCOBY surface (350×), b GTK SCOBY surface (1500×)

Full size image

Thermogravimetric Analysis (TGA)

In Fig. 4, a TGA thermogram is displayed, the first endothermic peak from 25 to 100 °C approximately, corresponds to the evaporation of moisture from the sample resulting in a weight loss of 1.04%, the second peak from 193 to 230 °C consists of the thermal degradation of the KBC sample losing a 15.84% of the weight. From 359 to 700 °C it is observed the decomposition of the KBC corresponds to 20.4% of the weight loss.

Fig. 4
A line graph of weight percent versus temperature in degree Celsius illustrates a declining trend.

Thermogram of TGA, KBC film evaluated from 25 to 700 °C under N atmosphere

Full size image

In Fig. 4, a TGA thermogram is displayed. The first endothermic peak, occurring approximately between 25 and 100 °C, corresponds to the evaporation of moisture from the sample, resulting in a weight loss of 1.04%. The second peak, ranging from 193 to 230 °C, consists of the thermal degradation of the KBC sample, resulting in a 15.84% weight loss. Between 359 and 700 °C, the decomposition of the KBC is observed, corresponding to a 20.4% weight loss. According to Villarreal et al., 2020, the decline in thermal stability might be associated with a higher crystallinity index.

Conclusions

The green tea kombucha SCOBY film analyzed by SEM shows a high concentration of bacteria, consistent with the microbiological observations, where Gram-negative, rod-shaped, reddish homogeneous forms are observed. As the literature indicates, the properties of the film such as its thermal behavior are influenced by this excess of bacteria on the surface of the SCOBY. Therefore, it is necessary to improve the conditions of the culture and prove other substrates to nourish kombucha, for example, black tea, and carbon sources, apart from improvising other variables such as time of fermentation, temperature, pH, and methods for cleaning the films. Currently, these variables are under analysis and will be reported in the future; this initial experimentation phase allowed us to obtain the SCOBY, which was subsequently added to the new cultures.