In our previous study, the optimum incubation time and protein concentration for production of BC was found to be 10 days and 2.75% (w/v), respectively (Bilgi et al. 2016). However, the wet weight of the facial mask (BC) sample produced under these conditions was over 200 g. As this weight was considered unsuitable as a facial mask, the incubation time was reduced from 10 days to 7 days to produce lighter and thinner BC. With the production by shortening the incubation period, products with a wet weight of 90 g (± 4.5 g) and a dry weight of 500 mg (± 13.5 mg) were obtained.
3.1. Endotoxicity tests
BC is widely used in the food industry as Nata de Cocco (Bernardo et al. 1998) and is generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) (Lin et al. 2013). Since the lipopolysaccharide found in the cell wall of Gram-negative bacteria is an endotoxin (Fang et al. 2009) and G. xylinus is a Gram-negative bacterium (Chiaoprakobkij et al. 2011), the potential endotoxin content should be considered. Although there are no restrictions on the use of this polymer, which is in the GRAS category, in sectors where the endotoxin threshold is very high, such as food, the effect of lipopolysaccharide residues on cells after washing should be evaluated, especially in biomedical, medical and cosmetic applications.
The results of the endotoxicity test with different dilutions of purified BC samples (1:1, 1:10, 1:100, 1:1000 and 1:10000) are given in Table 1. FDA states that biomedical devices that will come into direct contact with body fluids should not contain more endotoxin than 0.05 EU / ml of endotoxin. However, there is no clear limit for the skin. The reason is that skin protects our body against endotoxins and other different chemicals. Therefore, the endotoxin limit is high for the substances to be used on the skin surface as in the digestive system. When the test results of the samples produced in the study were examined, no gel formation was observed in 1:10 dilutions. The absence of gel formation indicates that the endotoxin content in that sample is below the limit that the test can detect. Accordingly, the samples intended to be used as facial masks contain endotoxin at a low concentration of 0,6 EU / ml or lower.
3.2. Bio-adhesion Tests
In previous studies Guilherme et al. used sensory tests (fragrance, skin adhesion and color, etc.) to qualitatively evaluate the performance of their BC masks (Pacheco et al. 2018). In another study using a BC based skin mask, Thanaporn Amnuaikit et. al. qualitatively measured user satisfaction and the decrease in sebum level. In their results after second application showed a decrease in sebum level (Amnuaikit et al. 2011). In our study, bio-adhesion test, which allows quantitative measurement of adhesion data, was preferred. How much distance the BC samples adheres to the apparatus after the applied force is shown in Table 2. During the experiment it appears that the highest strength was obtain with the products of CHb (0.102 kg sample) and HS (0.119 kg sample) media and contain neither Phenostat nor taurine. Taking into account the adhesion distances, the taurine and protective agent addition decrease the distance from 3.483 to 3.058 mm in products which are obtained in CHb medium, and from 2.950 to 2.133 mm in the products obtained in HS medium. In general, the addition of taurine and protective agent show less change in products of CHb (2.754-3.483) than in products of HS (2.133-3.208).
3.3. Cytotoxicity Results
Attachment tests were performed to determine the attachment time of HS2 cells to culture vessels, before cytotoxicity analysis. Attachment and growth kinetics of HS2 cell are given in Fig. 1. The results indicate that almost all cells were able to attach to the plates after 180 min (Fig 1. a) and the 12-day-old cells began to die as the culture medium did not replace during the experiment. (Fig 1. b).
To see if cell concentration under BC samples is decreased and/or morphology of cells changed, cells were dyed with Giemsa and investigated under light microscopy (Fig 2. and Fig 3). In the indirect cytotoxicity test, if the tested substance shows toxic effect, it is expected that the cells in the area under the part where the substance is placed are sparse or do not proliferate at all. When the images are examined no dilution is observed compared to the control. No difference is observed in the 72nd hour photographs taken at wide angle (4X) compared to the control (Fig 3.). BC did not show toxic effects on cells, these results are consistent with literature studies carried out in vitro or in vivo with BC or BC based products [(BC-alginat, BC-polyethylenglycol, BC-hydroxapatite, BC-gelatin, BC-Poly(3-hydroxybutyric acid-co-4 hydroxybutirate)) composites] (Bäckdahl et al. 2006; Fang et al. 2009; Cai and Kim 2010; Kim et al. 2010; Chiaoprakobkij et al. 2011). Indirect cytotoxicity tests were performed after cells reached to confluence. If the test is continued for a long time, the cells will soon die because they cannot find the surface area to attach. For this reason, the test was terminated after 72 hours to avoid false negative results. Long term in vivo biocompatibility studies on BC have also shown positive results (Klemm et al. 2001; Pértile et al. 2012). Based on these results, it was decided that BC could be used as a mask and samples were packaged by loading protective agent and taurine after characterization.
3.4. Microbial Stability
It is difficult to accurately predict the microbial stability and efficacy of a cosmetic product. Therefore, cosmetic products need to be tested against certain Gram-negative and Gram-positive bacterial strains that they may encounter during their shelf life (Russell 2003). In this way, the effectiveness of the protective agent can be tested. According to the European Union cosmetic laws, it is prohibited to subject cosmetic products to animal tests. Therefore, products must be tested without using animals and risking human health (Pauwels and Rogiers 2010). Although there are some difficulties, in vitro laboratory trials are proposed as an alternative to animal tests that was prohibited in 2013 (Adler et al. 2011).
These results show that 1% addition of preservative (Phenostat) allowed to be used in the range of 0.5-2% provides the desired effect. Protective stability and microbial stability tests were performed for products that were found to have no toxic effects on cells. After certain incubation periods it was determined that there was no microbial load on the samples tested (Table 3). These results shows that 1% addition of protective agent (Phenostat) allowed to be used in the range of 0.5-2% provides the desired protection effect. Since the contamination of S. aureus, P. aeruginosa and C. albicans was not present initially, their subsequent occurrence was not expected and these microorganisms were not sought after the initial time. It is seen that the effect of the 1% added protective agent is above the legal limit of 3 log to eliminate certain microorganisms (1x107-1x108 cfu/ml E. coli, S. aureus, P. aeruginosa, 1x106-1x107 cfu/ml C. albicans, 1x106 - 1x107A. niger) (Table 4). These results show that the product is effectively protected against microbial load for a long time (5 months), and microorganisms are effectively eliminated in case of contamination. According to the literature review none of the studies using BC as a facial mask or drug delivery system had long-term microbial stability analysis(Amnuaikit et al. 2011; Almeida et al. 2014; Pacheco et al. 2018).
3.5. DSC Analysis
BC is a highly crystalline and thermal stable polymer with inter and intramolecular hydrogen bonds. Although the thermal decomposition temperature of BC is 250 °C, in surface-modified BC samples or BC-polymer composites, this value may drop to 121 °C or even below 110 °C, the temperature used for steam sterilization (Badshah et al. 2018; Altun et al. 2019). The water holding capacity of BC is mostly provided by the hydrophilicity of the high amount of hydrogen bonds in its structure. In this study, since autoclave was used for the sterilization of BC samples, it was also investigated whether taurine loading had a negative effect on thermal stability. DSC, a widely used method for determining the thermal properties of biological molecules, (Spink 2008)allows the thermal capacity of the biological molecule in liquid solution to be obtained as a function of temperature (Lopez and Makhatadze 2002). The melting temperature of taurine is above 300 °C (White and Fishman 1936). At this temperature, disruption peaks are observed in biological polymers and studies have been conducted out in a range where we can observe possible glass transition temperatures. DSC plots of different BC samples are given in Fig 4. None of the formulations had a glass transition that was expected to be seen in the pure state of the products. This may be a sign that the material obtained is structurally different from the original material. It appears that addition of taurine and preservative shift degradation peaks in DSC plots. This also supports the conclusion that these additives contribute not only to microbiological and oxidative stress, but also to the thermal degradation as a result of dissolution in the material.
Structural difference can easily be seen in SEM images of BC samples with and without taurine loading (Fig 5). While the fibrous structure is preserved in freeze-dried BC samples without taurine, it is seen that the fibers are coated when taurine is added. EDS results of taurine (Table 5), dried BC and taurine on freeze dried BC indicate that taurine incorporation to BC samples were achieved successfully.
3.7. FT-IR analysis
FT-IR spectra of BC samples were given in Fig 6. in which, 2895, 1315, 1162, 1056, 665, 611 cm-1 and 3040, 2970, 1616, 1584, 1511, 1458, 1427, 1388, 1344, 1304, 1204, 1177, 1110, 1030, 962, 890, 848, 735, peaks were observed. In the FTIR spectrum, for pure cellulose, the peak at 3344 cm-1 and the peaks in the range of 3400-3500 cm-1 show OH stretching, strong absorption peak C-H binding at 2895 cm-1, peak at 1162 cm-1 shows C-O-C stretching, peaks at 1032 and 1056 cm-1 correspond to C-O bonds, the peaks at 1277 cm-1, 1335 cm-1, 1427 cm-1, indicating the presence of crystalline region and pure cellulose, correspond to C-H bending (C-H bending), O-H bending and CH2 stretch bonds, respectively which are correlated with literature (Wan et al. 2006; Castro et al. 2011; Liu et al. 2011; Halib et al. 2012). The peaks for BC-taurine (purple) around 3214, 3040, 2970, 1204 (S=O) and 962 (C-S-O) corresponds to taurine (Wang et al. 2015) which show that taurine loading to BC has been successfully completed.
3.8. Taurine Release Experiments
The formation and destruction of ROS and FR in the body occurs in a variety of ways lipid oxidation, the formation of ROS and FRs using oxygen which is between hypoxanthine and xanthine as a cofactor by xanthine oxidase (Quinlan et al. 1997; Finaud et al. 2006). Taurine has been shown to be effective in cell renewal and proliferation, to be a nutritive and supportive for cells, and helped to reduce or eliminate the harmful effects of ROS and FR (Devamanoharan et al. 1997; Chen et al. 1998; Guerin et al. 2001; Değim et al. 2002). Fig. 7 shows the taurine release characteristics which are profiled in different storage conditions namely 25 or 40 °C and 40 or 60% humidity.
The highest value was observed at the end of the 5th minute and the taurine concentration remained unchanged for the remaining 3 hours. A similar profile has been obtained in the following months Taurine concentrations stabilized at 2.5 and 3.0 μg/ml initially and gave the same result at the end of the following 1st and 6th months. Considering the limits that the LC-MS device can detect taurine linearly (Fig. 7a), BC samples were cut to 0.15 g/piece. BC samples immersed in 50 mM taurine solution were expected to contain 900-1000 μg taurine. During analysis the BC samples were kept in 20 ml PBS solution. Taurine concentration was predicted to be around 45-50 μg/ml. However, although the characteristic of release was consistent each month, the measured taurine concentration was found to be lower (2.5-3.5 μg / ml) than expected BC is known to have high hydrogen bonding capacity (Hakeem et al. 2016). When the molecular structure of taurine is examined, it is seen that the hydrogen bonding capacity of the hydrogen in the nitrogen and hydroxyl groups with unpaired electrons in each of the oxygen and nitrogen is present. Hydrogen bonds that may have occurred between taurine and BC may have caused the taurine release amount to be low. As a result, it is expected that a facial mask will release taurine after 5 minutes at 30 ± 3.5 mg (when it is thought that the final product produced is 90 ± 4.5 g). Studies in the literature of antioxidant activity of taurine indicate that taurine is capable of 11% reactive oxygen scavenging capacity at 10 μg/ml and 25% at 250 μg/ml concentration (Ripoll et al. 2012). It has also been shown with in vitro study that taurine protects the cells from toxic damage even at low concentrations such as 1-10 mM (Timbrell et al. 1995). The ability to achieve the highest release value of taurine in as little as five minutes indicates that the facial mask will have a short application period. This situation is considered to be good for user comfort.