Isolation of bacteria and yeast from Kombucha beverage:
Primary screening of BC production in HS basal medium was evaluated based on the ability to form a white gelatinous membrane on the surface of the medium under static conditions. A gram stain smear from the flasks which showed BC formation was examined by light microscope. As shown in Fig. 2 a, a symbiotic culture of bacteria and yeast (SCOBY) was illustrated. Separation and purification of bacterial and yeast isolates were carried out on HS and SDA media with antifungal and antibacterial agent, respectively, for further identification. The bacterial isolate was gram negative coccobacilli, while the isolated yeast appeared elongated with budding in some cells (Fig. 2 b, 2 c).
The efficiency of bacterial strain isolated from Kombucha beverage (KB) was examined by their ability to produce acid on GYC medium, where the colonies were surrounded by clear zone indicating the hydrolysis of CaCO3 as a result of acid production (Fig. 3 a). This result was confirmed by the changes in Carr medium color which containing bromocresol purple as indicator. The results showed that the color was changed from green into yellow after 24 h and then into green again (within 48 h) indicating that the isolate is belonging to Acetobacter genera (Fig. 3 b, 3 c). This result depended on a previous study which reported that the ability of the genera Acetobacter to oxidize acetate to CO2 and H2O was used to distinguish them from Gluconobacter members (Carr 1968).
Identification of bacterial and yeast isolates:
The identification of isolated culture by rapid biochemical tests using VITEK2 revealed that the bacterial and yeast isolates are: Acinetobacter lowffii and Candida krusei, respectively. Herellea was the first medium used for the isolation of Acinetobacter sp., where the colonies of Acinetobacter appeared as pale lavender on a yellow background (Jawad et al. 1994), as indicated in the current study (Fig. 4) and confirming VITEK results.
Production of BC in basal, alternative and modified media:
The current study uses BT as an available, simple, cheap and rich components alternative medium for BCNF production compared with three synthetic media (Fig. 5). The results indicated that BT medium gives the best BC production followed by HS then GEM (3.06, 1.8, and 1.2, subsequently), while, GACE record the lowest BC production (0.85 g/l). It was noticed that the BC membrane formed on the surface of HS medium was appeared as white, loose and thin compared with that formed on BT which was brownish, coherent and thick.
Different concentrations of Tea and sucrose in BT medium:
The effect of different concentrations of tea (0.0-1.0%) and sugar (0.0-10.0%) on BCNF production was illustrated (Table 2). BCNF membrane with dry weight (4.77, 4.65 and 4.09 g/l) were formed by using (0.2, 0.4 and 0.6%) tea concentration, respectively. While, (6.0%) sugar concentration resulted in the highest production (4.61 g/l). The media containing more concentration of sucrose (˃ 6.0%) may need an extra fermentation time for sugar consumption by microbial culture.
Table (2): Effect of different concentrations of tea and sugar on BC production (g/L)
Tea conc. (%)
|
BC dry weight (g/L)
|
Yield (%)
|
Productivity (%)
|
Sucrose conc. (%)
|
BC dry weight (g/l)
|
Yield (%)
|
Productivity (%)
|
0.0
|
0.787
|
1.31
|
7.87
|
0.0
|
0.04
|
-
|
0.40
|
0.2
|
4.77
|
7.95
|
47.7
|
2.0
|
3.15
|
15.75
|
31.5
|
0.4
|
4.65
|
7.75
|
46.5
|
4.0
|
4.19
|
10.47
|
40.9
|
0.6
|
4.09
|
6.81
|
40.9
|
6.0
|
4.61
|
7.68
|
46.1
|
0.8
|
3.88
|
6.46
|
38.8
|
8.0
|
4.39
|
5.50
|
43.7
|
1.0
|
1.81
|
3.01
|
18.1
|
10.0
|
4.41
|
4.41
|
44.1
|
* Sucrose 6.0 % (w/v) * Tea 0.4 % (w/v)
Modification of BT alternative medium:
A preliminary experiment aiming to optimize the production of BCNF in BT alternative medium was carried out by adding the components of HS medium; other than glucose; in addition to ethanol and acetic acid of GACE medium (Table 3). It was noticed that the addition of all HS components to BT medium resulted in turbidity, black precipitate and both microbial growth and BCNF production was negatively affected. This result can be attributed to the reaction of HS media components with one or more of tea constituents including; polyphenols, caffeine, adenine, catechins, gallic acids, tannins, amino acids, lipids, chloride, carotenoids, and volatile compounds (Aboulwafa et al. 2019; Dutta and Paul 2019)
Supplementation of BT medium with ethanol (1.0%), Na2HPO4 (0.27%), yeast extract (0.5%) and acetic acid (0.01%) individually were resulted in high BCNF production (7.85, 6.84, 5.73 and 5.05 g/l respectively). The results concluded that the addition of ethanol (1.0%) to BT medium not only increase the BCNF production, but also accelerates its synthesis in which the BC membrane started to formed after 2 days of incubation compared with the control (4-5 days).
Table (3): Effect of different supplements in BT on BCNF production (g/L)
Media
|
Supplements
|
Dry weight (g/l)
|
Yield (%)
|
Productivity (%)
|
Observation
|
*BT
|
-
|
4.77
|
7.95
|
47.7
|
-
|
BTHS
|
HS components
|
-
|
-
|
-
|
Turbidity and precipitation- inhibit BC formation
|
BT1
|
Yeast extract (0.5%)
|
5.73
|
9.55
|
57.3
|
Turbidity- increase BC formation
|
BT2
|
Peptone (0.5% (
|
4.33
|
7.21
|
43.3
|
Turbidity- slight increase BC formation
|
BT3
|
Na2HPO4 (0.27%)
|
6.84
|
11.4
|
68.4
|
Change the media to dark brown- enhance BC production
|
BT4
|
Citric acid (0.115%)
|
-
|
-
|
-
|
Change the media to light brown- inhibit BC formation
|
BT5
|
Acetic acid (0.01%)
|
5.05
|
8.41
|
50.5
|
No change in color- increase BC production
|
BT6
|
Ethanol (1.0%)
|
7.85
|
13.1
|
78.5
|
No change in color- enhance and accelerate BC production
|
* BT: Black tea medium (0.2% tea and 6.0% commercial sugar)
Production of BCNF using SCOBY without sterilization:
The present experiment was carried out to investigate the probability of BC production without sterilization of BT medium. The flasks showed the formation of BCNF membrane on the surface of the medium after 10 days of incubation, while the flask inoculated by ethanol showed more thick BC membrane. A smear from the flasks was prepared, stained and examined under light microscope to examine the presence of any contaminants. The slides showed no foreign microbial cells among the SCOBY except some residues from the media components.
Characterization of microbial isolates and BCNF using SEM:
The morphological characteristics of bacterial and yeast isolates; A. lowffii and C. krusei (P. kudriavzevii); were illustrated using SEM. The yeast cells appeared as elongated shape like the cells of P. kudriavzevii that mentioned in a previous study (Evy et al. 2013). In Fig. 6 a, the arrow pointed to the yeast budding, while the right arrow in Fig. 6 b, showed the autolysis of some yeast cells. The left arrow in Fig. 6 b pointed out to the bacterial cells which appeared as coccobacilli as described by (Koneman et al. 2006), who reported that Acinetobacter often has diploid arrangement.
On the other hand, Fig. 6 c and 6 d showed the micrographs of BCNF surrounding the microbial cells during fermentation and the diameter of the fibres was ranged between 68.36-97.88 nm. The surface of BCNF was distinguished before and after irradiation at 25 KGy as shown in Fig. 6 e and 6 f. The BCNF film without gamma irradiation was appeared as ultra- fine nano-fibrillary structure with irregular fibre arranged in a three-dimensional porous network as mentioned the previous studies (Jung et al 2010; Tsouko et al. 2015). While, the surface of BCNF film became dense and more compacted irradiation at 25 KGy compared to the control film.
Characterization of BCNF using FTIR:
Conformational characteristics of BCNF obtained from (HS), (SW) and (BT) media were analysed by FTIR spectrophotometer (Fig. 7). The infrared spectrum displayed an intense absorption peak at the wave numbers 3363 and 2987 cm-1 which attributed to stretching valence vibration of free hydroxyl –OH and C-H stretching groups, respectively. While, the peak appeared at 1660 cm-1 indicates C = O stretching vibration. The bands in the regions 1016, 1045 and 1055 cm-1 corresponded to the elongation of the C-C, C-OH, C- H ring. While C-O-C stretching at the β -(1-4) glycosidic bond in cellulose and O-H out of phase bending at the wave numbers 607,634 and 667 cm-1(Khan et al. 2020). There was an obvious similarity in the spectra of BCNF from the three investigated media. On the other side, FTIR spectra of BCNF after irradiation process at doses 5, 10, 15, 20 and 25 kGy showed the main characteristic absorption bands of BCNF with slight shifts of the peaks, implying the same chemical structure from different culture media.
Characterization of BCNF using XRD:
The crystalline structure as well as the change in crystallinity of the BCNF was evaluated using XRD analysis. As shown in Fig. (8), the peaks at 2θ angles of 14.54° and 22.84° for the BCNF control confirms the appearance of crystalline type -1cellulose. The previous study indicated that the peak at around 2θ = 22° is a main crystalline peak (Rosa et al. 2012). After irradiation by 5,10,15,20 and 25 kGy, XRD spectra of BCNF changed evidently and not only the intensity of major peaks increased, but also the specific peaks narrowed and sharped.