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European Journal of Applied Sciences – Vol. 10, No. 4
Publication Date: August 25, 2022
DOI:10.14738/aivp.104.12869. Huerta, N. V., Iniesta, S. A., Cruz, B. S. S., Moran, P. Z., Diaz, A. O., Lozano, L. S. A., & Lopez, M. R. (2022). Kombucha Bacterial
Cellulose Synthesized by Liquid Fermentation on Black Tea (Camellia sinensis): Effect of the Sucrose. European Journal of Applied
Sciences, 10(4). 639-648.
Services for Science and Education – United Kingdom
Kombucha Bacterial Cellulose Synthesized by Liquid
Fermentation on Black Tea (Camellia sinensis): Effect of the
Sucrose
Natanael Victoriano Huerta
Centro de Investigación en Dispositivos Semiconductores
Instituto de Ciencias, Benemérita Universidad Autónoma
de Puebla, Puebla, México
Salvador Alcántara Iniesta
Centro de Investigación en Dispositivos Semiconductores
Instituto de Ciencias, Benemérita Universidad Autónoma
de Puebla, Puebla, México
Blanca Susana Soto Cruz
Centro de Investigación en Dispositivos Semiconductores
Instituto de Ciencias, Benemérita Universidad Autónoma
de Puebla, Puebla, México
Placido Zaca Morán
Instituto de Ciencias, Ecocampus Valsequillo
Benemérita Universidad Autónoma de Puebla, Puebla, México
Abdu Orduña Díaz
Instituto Politécnico Nacional, Centro de Investigación en
Biotecnología Aplicada, Tlaxcala, México
Leslie Susana Arcila Lozano
Instituto Politécnico Nacional, Centro de Investigación en
Biotecnología Aplicada, Tlaxcala, México
Marlon Rojas López
Instituto Politécnico Nacional, Centro de Investigación en
Biotecnología Aplicada, Tlaxcala, México
ABSTRACT
Bacterial cellulose membranes were synthesized by liquid fermentation of the
Kombucha strain into black tea (Camellia sinensis) at different concentrations of
sucrose. Structural properties of bacterial cellulose Kombucha, such as lateral
order index (LOI), total crystallinity index (TCI), hydrogen bond intensity (HBI),
fraction (fa), as well as their dependence on sucrose content were evaluated by
Fourier transform infrared spectroscopy, using different absorption bands of the
vibrational spectrum. It was thus observed that sucrose tends to crystallize
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European Journal of Applied Sciences (EJAS) Vol. 10, Issue 4, August-2022
Services for Science and Education – United Kingdom
bacterial cellulose, due to the increase in the total index of crystallinity and lateral
order, as well as the fraction (fa), while the index of hydrogen bonds decreased. The
addition of organic cocoa (Theobroma cacao) in the culture medium prior to
fermentation produced membranes with properties very similar to those prepared
only with black tea. Obtaining type I cellulose and crystallization controlled by this
process could contribute to obtaining high crystallinity membranes for biomedical
and bioelectronic applications.
Keywords: Bacterial Cellulose; Structural properties; Total Crystallinity Index; Lateral
Order Index; Hydrogen Bond Intensity; Fourier transform infrared spectroscopy.
INTRODUCTION
Kombucha bacterial cellulose (KBC) is a biodegradable thin membrane obtained from the
fermentation of black tea (Camellia sinensis), sucrose, and microbial culture known as SCOBY
(Symbiotic Culture of Bacteria and Yeasts), which has the consistency of a gel and is composed
of a consortium of bacteria of the genus Acetobacter, Gluconacetobacter, Leuconostoc,
Allobaculum, Ruminococcaceae, Enterococcus, Thermus[1–4] as well as yeasts such as
Zygossacharomyces, Brettanomyces, Saccharomyces [5,6]. Thus, this membrane has unique
characteristics such as high purity, biocompatibility, biodegradability [7,8], high water
absorption capacity and therefore greater biological adaptability [9], as well as high tensile
strength [10]. KBC microfibrils are 100 times smaller compared to plant cellulose [11], giving
it a unique three-dimensional micro/nanostructure, increasing its interest in biotechnology,
biomedicine, food, electronics, bioelectronics, nanocomposites and other applications including
tissue regeneration and scarring [12].
In this work, Fourier transform infrared spectroscopy (FTIR) was used to analyze the structural
properties of Kombucha bacterial cellulose, such as the lateral order index, the total
crystallinity index, the intensity of the hydrogen bonds, the
fraction and its dependence on
the concentration of sucrose and the composition of the medium.
MATERIALS AND METHODS
Reagents
Organic black tea brand Molienda Sagrada for infusion that will serve as a culture medium for
obtaining bacterial cellulose by SCOBY Kombucha. Reactive grade sucrose of the Meyer brand,
yeast powder extract of the Himedia brand. Peptone bacteriological grade brand Meyer. To
carry out the cleaning of the KBCs formed, Sodium Hydroxide (NaOH) was used in reactive
grade flakes, Meyer brand, and in all processes PiSA brand distilled water was used.
Preparation of Kombucha bacterial cellulose membranes
Two types of culture medium were made, both contain 1% black tea which is poured into 1L of
boiling water for 5 minutes allowing it to cool at room temperature, then four concentrations
of sucrose are used (5, 10, 15 and 20%). In addition, 1 g/L of peptone and 10 g/L of yeast extract
were added. Organic cocoa (Theobroma cacao) 10 g/L was added to the second medium. From
the medium obtained, 100 mL of the infusion is poured into cylindrical glass jars of 175 mL. The
samples (KBC-cocoa) were then sterilized at 121 °C for 15 min and the pH is measured.
Subsequently, an incubator was used to keep the cultures at a temperature of 26 ± 1 °C for 7
days. The obtained KBC membranes were washed for 30 minutes 3 times in a solution of NaOH
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Huerta, N. V., Iniesta, S. A., Cruz, B. S. S., Moran, P. Z., Diaz, A. O., Lozano, L. S. A., & Lopez, M. R. (2022). Kombucha Bacterial Cellulose Synthesized
by Liquid Fermentation on Black Tea (Camellia sinensis): Effect of the Sucrose. European Journal of Applied Sciences, 10(4). 639-648.
URL: http://dx.doi.org/10.14738/aivp.104.12869
1 M at 85 °C to remove cells and impurities. After washing, they were rinsed with distilled water
until a neutral pH was obtained. Finally, the KBC and KBC-cocoa membranes were filtered by
gravity [13] and dried at room temperature for 4 days. These membranes were prepared in
triplicate under the same conditions, varying only the concentration of sucrose.
Determination of the thickness and weight of the KBC membranes
KBC membrane thicknesses were measured using an Oumefar electronic micrometer with a
range of 0-12 mm and resolution of 0.01 mm. On the other hand, the weight of the membranes
was determined by an Amir digital balance with a resolution of 0.001 g, in a measuring range
of 0.05 to 500 g.
Scattered electronic microscopy
The morphology of the KBC membrane surface was examined using a scanning electron
microscope (Vega TS-5136SB, Tescan, Kohoutovice, Czech Republic) operating in low vacuum
mode. The samples were mounted on an aluminum base with carbon tape.
Fourier transform infrared spectroscopy
For the chemical and structural characterization of the bacterial cellulose samples, a FTIR
Bruker spectrometer was used, model VERTEX 70 in a wavenumber range of 4000 to 400 cm- 1, in total attenuated reflectance (ATR) sampling mode and a spectral resolution of 4 cm-1, with
120 scans per sample. Origin 6.0 software was used to analyze the absorption spectra.
Determination of crystallinity indices in KBC membranes
First, the FTIR spectra of the membranes were normalized at 1027 cm-1 (C-O extension
vibration), because this was the band of greater intensity in the samples analyzed. First, the
lateral order index (LOI) is defined as the ratio between the intensities between the bands at
1427 and 895 cm-1, I(1427)/I(895) [14,17]; while the total crystallinity index (TCI), is defined
as the ratio between the intensities between the bands at 1371 cm-1 and 2895 cm-1, I(1371)/
I(2895) [14,15]. The following structural property is hydrogen band intensity (HBI), calculated
as I(3340)/I(1337) [16] and finally, the
fraction of cellulose (fa) was determined using the
deconvoluted areas of the bands at 750 cm-1 and 710 cm-1 respectively, according to the
following equations [18–20]:
�a = 2.55 � �!" − 0.32 (1)
where
�!" = #!"#
#!"#$#!$#
(2)
RESULTS AND DISCUSSION
Physical appearance, thickness, and weight of the KBC membranes
Figure 1 (a, b) shows the physical appearance of a membrane of Kombucha bacterial cellulose
as obtained from the fermentation process into and out of the bottle after 7 days. Figure 2(a, b)
show the thickness and weight respectively of KBC membranes (wet and dry) as a function of
sucrose content observed after the fermentation process. In both cases an increase in both the
thickness and weight of the membrane with the sucrose content is observed. This can be
attributed to increased cellulose production by microorganisms of the Kombucha strain caused
by the increase in the amount of carbon source [21,22]. In the case of KBC membranes grown