Accepted: 12 September 2017

DOI: 10.1111/jocd.12441

ORIGINAL CONTRIBUTION

Bacterial cellulose skin masks—Properties and sensory tests

spoli de Mello MS2 |

Guilherme Pacheco MD1 | Carolina Ve

o PhD1,3 | Vera Lucia Borges Isaac PhD3 |
Bruna Galdorfini Chiari-Andre
Sidney Jose


Lima Ribeiro PhD2 | Edison Pecoraro PhD2 | Eliane Trovatti PhD1

1
Universidade de Araraquara, UNIARA,
Araraquara, Brazil Summary
2
UNESP - Instituto de Qu
ımica, Araraquara, Background: Bacterial cellulose (BC) is a versatile material produced by microorgan-
Brazil
isms in the form of a membranous hydrogel, totally biocompatible, and endowed
3
Faculdade de Cie^ncias Farmace^uticas, Univ
Estadual Paulista Julio de Mesquita Filho, with high mechanical strength. Its high water-holding capacity based on its highly
UNESP, Araraquara, Brazil porous nanofibrillar structure allows BC to incorporate and to release substances
Correspondence very fast, thus being suitable for the preparation of skincare masks.
Eliane Trovatti, Universidade de Araraquara, Aims: The preparation and characterization of cosmetic masks based on BC mem-
UNIARA, Araraquara, Brazil.
Email: [email protected] branes and active cosmetics.
Methods: The masks were prepared by the simple incorporation of the cosmetic
actives into BC membranes, used as a swelling matrix. The masks were character-
ized by Fourier transform infrared (FTIR), scanning electron microscopy (SEM), sen-
sory tests, and skin moisture tests on volunteers.
Results: The results of sensory tests revealed the good performance of BC, being
considered effective by the panel of volunteers, specially for adhesion to the skin
(7.7 at the score scale), and improvement of the skin moisture (the hydration effect
increased 76% in 75% of the volunteers that used vegetable extract mask formula-
tion [VEM]), or a decrease in skin hydration (80% of the volunteers showed 32.6%
decrease on skin hydration using propolis extract formulation [PEM] treatment),
indicating the BC nanofiber membranes can be used to skincare applications.
Conclusion: The results demonstrate the BC can be used as an alternative support
for cosmetic actives for skin treatment.

KEYWORDS
bacterial cellulose, cosmetic masks, sensory tests, volunteers

1 | INTRODUCTION substances, they also have to be biocompatible, nonallergenic, and

nontoxic.3 The facial masks are traditional presentations of cosmet-
The pursuit of beauty leads the cosmetics to a growing market and ics, which recently have undergone a makeover with new aspects,
the increasing variety of products. The substances responsible for ingredients, new vehicles and offer new perspectives for skincare.
1,2
improving the beauty, the cosmetics actives, are incorporated into Facial masks are used mainly to provide fast and deep moisturizing,
vehicles/excipients, which transport them to the skin, promoting skin restitution, and sebum control.4 They are promising cosmetic
penetration and/or permeation. The demand for innovative vehicles formulations because they are easy, effective, and the application is
grows at the same rate of cosmetics development. Creams, gels, gel- fast. The subcategories of facial masks include those that involve
creams, emulsions, lotions, among others are examples of conven- evaporation and those formulated into a substrate such as a cloth,
tional cosmetics presentations that, besides carrying the active patch, pad, or polymeric film.4 Natural polymers such as cellulose

J Cosmet Dermatol. 2017;1–8. wileyonlinelibrary.com/journal/jocd © 2017 Wiley Periodicals, Inc. | 1

2 | PACHECO ET AL.

pulp, hydrogels (silk, collagen), and cellulose nanofibers (biocellulose) Bacterial cellulose was already studied as a support for drug
and also the synthetic polymers poly(vinyl alcohol) and silicone have release, and the results showed its efficacy for fast release of
been used as the basic materials for the confection of innovative hydrophobic and hydrophilic drugs.17-20 Nevertheless, low number
4-6
facial masks as the support (or matrix) for the cosmetic actives. In of studies can be found in the literature with respect to the use of
general, hydrogels masks are mechanically weak and are supposed to BC for facial mask application and support for cosmetic actives.6 In
be used at the horizontal position, requiring the user to be laid down this study, we report the preparation of two cosmetic mask formula-
for its application. Cellulose masks have greater mechanical proper- tions for skincare, in which two well-known actives were incorpo-
ties when compared to hydrogel masks, which make them superior rated into BC membranes. The first was a skin moisturizing
in terms of handling and applications. However, pulp cellulose or association based on Hidrovitonâ and vegetable extracts (oat extract
nanofibers that come from pulp fibers tend to break when in contact and rosemary extract) and the second, propolis extract, an antiseptic
with wet environment. The effect is greater for ordinary pulp fiber and astringent active.21-23 The performance of the masks was stud-
because of the larger diameter of their fibers when compared to cel- ied by sensory analysis in human beings.
lulose nanofibers. However, cellulose is an interesting material for
facial mask because it is a natural high biocompatible polymer. An
2 | MATERIALS AND METHODS
alternative cellulosic substrate for facial mask is the bacterial cellu-
lose (BC), mainly because of its membrane-like shape endowed of
2.1 | Materials
high mechanical properties and other interesting properties for
actives loading and release. Hidrovitonâ (Symrise, Holzminden, Germany), oat extract, rosemary
Bacterial cellulose, as well as all the celluloses, is a polymer of extract, and calendula extract were purchased from Croda, and pro-
glucose, in which the pyranose rings are joint together by b-1,4 polis extract was purchased from DEG, Brazil. All the reagents were
bonds, giving rise to the macromolecules. It is a natural polymer of analytical grade and used with no modification. BC membranes
totally biocompatible and with great mechanical properties.7 Among were produced using the bacterial strain Gluconacetobacter xylinum
the celluloses, BC is a potential material for masks because of its (ATCC 23760) supplied by Fundaç ão André Tosello Pesquisa e
special properties such as 3-dimensional network of entangled Desenvolvimento, Brazil.
nanofibers with great mechanical properties, malleability, and easy
handling due to its membrane-like shape. The membrane-like shape
2.2 | Production of BC membranes
of BC is unique in nature, resulting from bacterial synthesis. The
network of BC membranes is the result of the complex process that Pre-inoculum preparation—the pre-inoculum was prepared inoculating
involves its synthesis at the bacterial cytoplasm, the extrusion, 100 mL of HS media with microorganism and incubated statically for
which takes place at the external membrane and the spatial 24 hour. Then, the flask was vigorously hand shaken, and the media
arrangement of the protofibrils, generating the nanofibrils at the were added to sterile media (10%, v/v).
external environment. In short, when synthesized, cellulose macro- Bacterial cellulose production—BC was produced by G. xylinum
molecules interact with each other by hydrogen bonds generating a (ATCC 23760) in Hestrin Schramm (HS)24,25 liquid media, which
supramolecular arrangement that gives rise to fibrilar structures was composed by 50 g/L glucose, 4 g/L of yeast extract, 0.73 g/L
called protofibrils.8-10 After extrusion by the pores of the microbial of MgSO4.7H2O, 2 g/L KH2PO4, 20 g/L ethanol, and distilled
membrane, the protofibrils fold each other and give rise to a fibrilar water, 1 L. The culture media were sterilized at 121°C/15 min, and
material that reaches the nanometric scale, the nanofibrils (3-20 nm ethanol was sterilized by filtration and added to the media at room
in diameter).11 The sequence of steps involved in BC synthesis hap- temperature. The cultivation process was carried out in aseptic
pens while the microorganism is moving (because of the movement conditions. The microorganisms were statically incubated in
of the liquid) within the culture media or at the surface of sub- polypropylene trays (25 9 36 9 12 cm), at 28°C for 48 hour. The
strates. Thus, the extruded nanofibers are woven in the form of trays were covered with a sheet paper to avoid contaminations
membranes in which the nanofibers are completely entangled and from the environment at the surface of membranes. The volume of
arranged in a 3-dimensional network that gives rise to its great culture media was 450 mL, and the inoculum volume was 50 mL
mechanical properties. BC membranes are composed of pure cellu- for each tray.
lose and water that represent about 99% wt of its total mass. BC
can be synthesized by several microorganisms, such as by bacteria
2.3 | BC purification
belonging to the genus Gluconacetobacter, Rhizobium, Agrobacterium,
Escherichia, Enterobacter, and Salmonella, among others.10 It is a Bacterial cellulose membranes were harvested and washed with tap
material with unique properties because of its high strength, its bio- water for 24 to remove all the residues from the culture media, and
compatibility, and specially its shape, which cannot be mimicked by then, they were treated with 0.1% solution of NaOH at 70°C for
artificial strategies.8,12,13 In fact, the enzymatic synthesis of cellulose 15 minutes in order to disrupt the bacterial cells. Then, the mem-
is well known,14 but the synthesis in large scale is in develop- branes were thoroughly washed with distilled water until neutral pH.
ment.15,16 The membranes were kept in distilled water at 8°C before use.
PACHECO ET AL. | 3

responded a questionnaire, in which the responses rated their

2.4 | Characterization of BC
degree of satisfaction with respect to buy the masks. The possible
Fourier transform infrared (FTIR) spectra were acquired using a Nico- responses were “I certainly would buy the product”; “I am not sure if
let Nexus Spectra equipped with a Golden Gate single reflection dia- I would buy the product” and “I certainly would not buy the
1
mond ATR accessory. Spectra were obtained at 4000-600 cm product.”
1
range, with a 4 cm resolution and 16 scans. Morphological analysis
were accessed by scanning electron microscopy (SEM) using a JEOL
2.7 | Skin moisture determination
JSM-7000F microscope.
Skin moisture at the region of the treatment was evaluated before
and at the end of the skin treatment with the masks using a cor-
2.5 | Preparation of the masks
€ ln, Germany CM 825), and it was
neometer (Courage Khazaka, Ko
Wet BC membranes were weighted, and 50% of its total mass was used as a parameter for evaluation of the BC matrix as a support for
removed by pressure.17 The membranes were immersed in a tray, release of the actives.
and the cosmetic actives (described below) were homogeneously dis-
tributed at their surface. The trays were shaken for 30 minutes. The
3 | RESULTS AND DISCUSSION
membranes were turned around every 5 minutes to provide a good
distribution of the actives. The membranes were cut in 4 9 4 cm
3.1 | BC synthesis, purification, and
pieces, packed in plastic hermetic bags, and sterilized by Gamma irra-
characterization
diation at 20 kGy.
Two cosmetic actives were used for the preparation of the The membranes were synthesized, purified, and dried for characteri-
masks. The first was a moisturizing association composed of oat zation. The morphology of BC and biomasks is a very important
extract (0.25 g), rosemary extract (0.25 g), calendula extract (0.25 g), parameter because it is closely related with their general perfor-
and hydroviton (0.25 g). One gram of the mixture was incorporated mance, including its physical aspect, touch and mechanical proper-
into 225 cm2 of BC membrane. The formulation was called veg- ties. SEM micrographs of the surface of the BC membranes are
etable extract mask formulation (VEM). The second formulation was shown in Figure 1. Figure 1A shows pure BC, Figure 1B shows BC
composed by 1 g of propolis extract at 30% (w/v, propolis/ with VEM, and Figure 1C shows BC with PEM. Pure BC shows its
polypropylene glycol). The formulation was called propolis extract typical 3-dimensional network of nanofibers, and no bacteria were
formulation (PEM). One gram of propolis extract was incorporated found into the purified samples. The diameter of the nanofibers was
2
into 225 cm of the BC membrane. The release of the actives was about 20-80 nm. SEM images of all the materials provided evidence
carried out in 20 mL of PBS solution. The samples were immersed that the incorporation of the active substances did not change the
within the Becker with PBS. The release was followed by FTIR. nanofiber morphology, keeping the BC nanofibrillar 3-dimensional
network. For BC with PEM (Figure 1B), it was possible to see
agglomerates into the fiber matrix; nevertheless, it is notable the
2.6 | Sensory tests
good dispersion within the fibers network. In addition, for BC with
Two methods were used for sensory tests, the descriptive analysis, VEM (Figure 1C), agglomerates were not observed, nor other struc-
and acceptance tests. For descriptive analysis, twenty volunteers ture that come from the actives, indicating its homogeneous disper-
(women in the range of 20-40 years old) evaluated the mask proper- sion. The images showed the process did not damage the fibers, an
ties and the improvement of skin characteristics. The parameters important feature for keeping their network and mechanical
evaluated were color, texture, skin adhesion, handling, smell, skin strength, avoiding damage of the product during application, also
improvement, and overall. The lowest and highest scores were 1 and avoiding the loss of cellulose residues when in contact with the face.
9, respectively. Two groups of ten volunteers received a pack with The structural characterization of BC was accessed by FTIR-ATR.
five masks of vegetable extract or propolis extract, which were ran- The spectra of BC membranes (Figure 2) and BC membranes loaded
domly distributed to guarantee they did not know the mask compo- with VEM and PEM showed the typical bands of the structure of
sition. The masks were used for 20 minutes for 5 consecutive days cellulosic substrates. These typical bands of cellulose are shown at
1
(5 days) at the forehead region. The volunteers did not use any pro- 1051 and 1154 cm corresponding to C–O symmetric stretching of
duct (makeup, sunscreen, etc.) at the forehead region during the primary alcohol and C–O–C antisymmetric bridge stretching, respec-
1
evaluation period. The study was previously approved by ethics tively, and at about 3323 cm (broad band) corresponding to OH
committee of Faculty of Pharmaceutical Sciences, S~ao Paulo State stretching. The band at 1315 cm 1
corresponds to the C–H defor-
University (UNESP), Araraquara, Brazil. All the volunteers signed the mation (CH3 or O-H in plane bending). Other bands related to O–H
1
consent form regarding to the possible skin irritation related to the bending of adsorbed water, at 1645 cm , CH stretching of CH2
1
use of the masks. All the volunteers were included in the study, and and CH3 groups at 2900 cm can also be seen. The release of the
no skin irritation or other adverse effect was detected while the actives from PEM and VEM was also followed by FTIR. The spectra
study was performed. For acceptance tests, all the volunteers of both samples, BC-PEM and BC-VEM, showed additional bands
4 | PACHECO ET AL.

FIGURE 2 FITR-ATR spectrum of bacterial cellulose (BC) and its

derivatives

3.2 | Preparation of BC masks

The incorporation of liquids into BC membranes remains a challenge,
even after so many years of research using it as support for drug
and cosmetic release. Previous work17 studied the kinetics of
absorption of liquids by BC membranes and showed that the mem-
branes should have a free volume for incorporation of the required
volume. In this sense, to guarantee the total absorption of the solu-
tions, the incorporation of the actives was performed after draining
50% of the membrane water content. Then, the membranes were in
contact with the active solutions for 40 minutes. The extracts used
for the masks preparation have hydrophilic nature, showing then,
high affinity for hydrophilic environments, such as the matrix BC
fibers. The incorporation of all the liquid material (cosmetic actives)
in which the membranes were immersed succeeded in 40 minutes,
with an evident swelling of the membranes. The active content of
the loaded BC membranes, prepared under such conditions, was
4.4 mg/cm2, and the thickness of the wet masks was about 0.7 mm.
The composition of both the actives include moisturizing substances
(Hydroviton26 in VEM masks and propylene glycol in PEM masks),
which can act as plasticizers, inferring flexibility to the mem-
branes,17,27,28 a desired feature for cosmetic masks, looking for
F I G U R E 1 scanning electron microscopy (SEM) image of pure
adhesion and adaption to the skin contour. As the sterilization pro-
bacterial cellulose (BC) membrane (A), BC-PEM (B), and BC-VEM (C).
PEM, propolis extract formulation; VEM, vegetable extract mask cess was performed at room temperature, the membranes kept their
formulation pristine properties and did not present loss of liquids.

1
3.3 | Evaluation of the performance of BC masks
that can be seen mainly at the region of 1500-1700 cm . After the
on volunteers
immersion in water, the spectrum of the samples (BC-PEM2 and BC-
VEM2) was quite similar to the spectrum of the pure BC, indicating Sensory tests are based on an appreciation of the product perfor-
the BC membranes are capable of releasing the actives in a short mance and give the information perceived or observed. The tests
period of time. were used to translate sensations into objective data, useful to study
PACHECO ET AL. | 5

the development of new products and to ensure that their perfor- respect to using the product. The acceptability of PEM was in high
mance meets the expectations of the consumers.29,30 With respect agreement with the descriptive sensory tests that reached lower rate
to the studied parameters for BC mask evaluation, the results of scores when compared to VEM. Besides the subjective nature of
showed only for the “handling” parameter the score was higher for the responses, the acceptance tests are predictive of marketplace
VEM (7.5) when compared to PEM (5.5) in the score scale ranging success, however, the tests should be performed in larger number of
from 0 to 9. Color, texture, smell, skin, and overall improvement panel lists.
reached 7.2, 6.8, 5.2, 6.3, and 6.2 score values, respectively, as
shown in Figure 3. For PEM, the results were 6.8, 6.3, 7.7, 5.5, 5.8,
3.4 | Skin moisture
6.3, and 6.7 for color, texture, skin adherence, handling, smell, skin,
and overall improvement. The parameter that reached the higher The skin moisture was strictly related to the efficiency of BC nanofi-
score, for VEM and PEM, was the adhesion to the skin. The result is ber masks as a support system to carrier and release the actives to
interesting considering the period of treatment with masks is nor- the skin, once the actives have known properties. The formulation
mally long, about 30 minutes, with no need of resting for the appli- based on vegetable extracts is a moisturizing composition, while the
cation. The adhesion properties of the mask are the result of the propolis extract formulation represents an antiseptic composi-
mechanical properties of BC, such as its high malleability, associated tion,30,31 for which the moisturizing properties are not properly stud-
with the composition of the formulation that include hydrophilic and ied and described in the literature. The increase in skin hydration
hydrophobic chemicals, providing a good interface between the skin effect was more pronounced for VEM treatment than for PEM treat-
and the mask. Pictures of the masks adhered to the face and hand ment. The increase in skin hydration effect was 76% (0.82) in 75%
skin are showed in Figure 4A and B, respectively. These images indi- of the volunteers that used VEM (at the end of the experiment,
cate the good adhesion of the mask to the skin. when compared to the moisture before treatment); for PEM treat-
The parameters analyzed in sensory tests correspond to the ment, only 20% of the volunteers showed an increase of the skin
association of intrinsic properties of BC with intrinsic properties hydration and that increase was low, 11.8% (0.20), as showed in
of the actives. For instance, the texture and the skin adhesion of Figure 5A. The decrease in hydration effect was more pronounced
the mask were highly improved by the actives, as they increased for PEM treatment than for VEM treatment. For PEM treatment,
17,27,28
the malleability of the BC and the adhesion between the 80% of the volunteers showed a decrease of 32.6% (0.55) in skin
masks and the skin. The smell was inferred to the masks by the hydration, and for VEM treatment, 25% of the volunteers showed a
extracts (propolis or vegetable extracts), as BC has no typical decrease of only 1.2% (0.03) in skin moisture, as showed in Fig-
smell. ure 5B. The results, in both cases, indicated that the use of the
The acceptability of the masks was evaluated by questionnaires masks for 30 minutes/d changed the skin moisture. The increase in
by all the volunteers after using the masks. All the volunteers that skin moisture for VEM was totally in agreement with the expected
used the VEM were in doubt if they would buy the masks and results based on the composition of the formulation. The lowering
66.67% of the volunteers that used PEM were also in doubt if they moisture effect for PEM is a result that can be associated to the
would buy the masks. For PEM, 33.33% responded they certainly propolis intrinsic properties and composition, which, to the best of
would buy the product. VEM reached higher scores in almost all the our knowledge, is not described in the literature. These results indi-
parameters analyzed in descriptive sensory tests, with some very cated the efficiency of BC membranes as a support for loading and
high scores, however, in acceptability tests, the responses did not fast release of the actives. In addition to the loading and release
correspond to the results, as all the volunteers were in doubt with capability of BC membranes, its occlusive effect leads to an

F I G U R E 3 Hedonic scale results of

descriptive sensory analysis of bacterial
cellulose (BC) masks
6 | PACHECO ET AL.

(A) (B)

FIGURE 4 Pictures of bacterial cellulose (BC) mask adhesion to forehead (A) and hand (B, indicated by arrow)

F I G U R E 5 Skin moisture results for vegetable extract mask formulation (VEM) and propolis extract formulation (PEM) on volunteers. A,
Increase in hydration and (B) decrease in hydration effect

increased permeation of the actives into the skin and protects the membranes removes the free water and keeps the molecules of the
skin against the loss of water.6 cosmetic actives trapped within the membranes; however, the chem-
ical interaction of the active cosmetic molecules and the cellulose
nanofibers is only possible at the surface of the nanofibers in which
3.5 | Relationship between BC nanofibers structure
their OH groups are free of bounded water. In addition, the size of
and morphology, sensory tests and moisture skin
the molecules (angstrom scale) is much lower than the nanofibers
tests
(nanometric scale); thus, the nanofibers can be seen as the frame-
BC morphology shows its nanofibrilar structure in which the nanofi- work for the active molecules. Therefore, the increased skin moistur-
bers diameter is about 20-100 nm. When wet, 99% of BC mem- izing effect in most of the volunteers that used VEM and low skin
branes mass correspond to water. The incorporation of drugs or moisturizing effect for most volunteers that used PEM, respectively,
other substances, such as the cosmetic actives used in this work, indicated the BC membranes behaved as good support for incorpo-
become soluble in the water hold within BC membranes. The water ration and release of the tested actives. The evaluation of the masks
molecules interact with hydroxyl groups (OH) at the surface of cellu- using descriptive sensory tests showed the material is appropriate
lose by hydrogen bonds. The other molecules (cosmetic actives, for for masks confection, handling, and to exert its role as a matrix for
instance) interact with such OH groups, only if their affinity for OH release of substances thanks to its unique network of entangled
is higher than the affinity of water to OH groups. The drying of the nanofibrils, capable of swelling, holding, and releasing liquids. In
PACHECO ET AL. | 7

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