Industrial Crops and Products 44 (2013) 378–382

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Industrial Crops and Products

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Production of cosmetic purpose collagen containing antimicrobial emulsion with

certain essential oils
Ali Yorgancioglu, Eser Eke Bayramoglu ∗
Department of Leather Engineering, Faculty of Engineering, Ege University, 35100 Izmir, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: In this study, reproducibility of the cosmetic purpose antimicrobial emulsion formulations containing
Received 25 May 2012 collagen hydrolysate by taking the advantage of the antimicrobial features of the industrial essential oil
Received in revised form 6 November 2012 plants including Thymus vulgaris, Origanum onites, Eucalyptus globulus and Mentha piperita was inves-
Accepted 9 November 2012
tigated. The formulations produced for this purpose were subjected to the microbiologic testings with
respect to the disc diffusion method. As the result of the studies, the formulations containing O. onites and
Keywords:
T. vulgaris were found to demonstrate the highest antimicrobial activity. In addition to this, the emulsions
Collagen hydrolysate
were subjected to the viscosity and particle size analysis and examined in organoleptic terms as well.
Emulsion
Essential oils © 2012 Elsevier B.V. All rights reserved.
Cosmetic
Antimicrobial activity

1. Introduction hydrolysate which is one of these materials has been known to be

Human beings always use cosmetic preparations in order to
blossom out. For the women have been identified with the con-
cept of ‘beauty’ in the history, they have been addressed as the
target audience from the side of the cosmetic industry. However, at
the present time, cosmetic products have particular importance for
both women and men separately. In this day and time, the people
are desirous of being well-groomed, feeling the indications of aging
later than expected, and behaving more consciously with respect
to protecting their health. Alterations in cultural and economic
conditions and changes in individuals’ outlook and expectations
on cosmetic products associated with the advancing science and
technology have projected the investigators to use novel active
substances and techniques in this field of research. Adaptation of
modern delivery systems to cosmetics and discovery of novel for-
mulations and active ingredients have provided new opportunities
for cosmetology.
Moisturizing and softening products have been gaining
increased importance as being used in maintenance of daily care
of normal skin and in the treatment of dry skin and as an ancillary
therapy in many other skin conditions (Loden, 2003). Particu-
larly in recent years, the use of natural bioactive materials have
become common in cosmetics (Bayramoglu et al., 2011). Collagen
∗ Corresponding author. Tel.: +90 232 311 2644; fax: +90 232 342 5376.
E-mail addresses: eserekebay@gmail.com, eser.eke@ege.edu.tr
(E.E. Bayramoglu).
used in cosmetic formulations for reasons such as protecting the
structure and the function of the skin, enhancing its appearance (Li
et al., 2005). Collagen is a natural biological material which is used
also in esthetic surgery, ophthalmology dentistry, pharmacy and
biotechnology besides cosmetic industry (Meena et al., 1999).
Cosmetics that have held an important place in human life since
the beginning of time are products that are not required to be sterile
microbiologically but have to be of proper quality in terms of con-
sumer health (Steinberg, 2006). It has been determined that these
products can be contaminated with microorganisms found in the
production environment or in raw materials, especially water, and
that the contamination can occur after production due to unhealthy
storage conditions or during consumer use (Underwood, 1998).
As a result of microorganism contamination of cosmetics prod-
ucts, unwanted changes may occur regarding the product smell,
color, viscosity, and performance, disintegrated skin may become
infected, the endotoxins and metabolites produced by microor-
ganisms may cause abrasion, irritation and allergies on the skin
(Smart and Spooner, 1972). Cosmetic products are suitable media
for microorganisms to grow in due to the materials in their com-
positions. In a study conducted, 8 of the 49 body moisturizers of
which 25 were cream and 24 were lotion were contaminated with
more than 103 microorganisms per gram or milliliter and that the
products contained genus of Escherichia coli, Pseudomonas, Staphy-
lococcus and Bacillus species (Okeke and Lamikanra, 2001). Given
the wide range of usage of cosmetic products it can be seen that
the microorganism contamination of the products may pose a sig-
nificant risk in terms of consumer health. Therefore, against the

0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2012.11.013
 A. Yorgancioglu, E.E. Bayramoglu / Industrial Crops and Products 44 (2013) 378–382
possibility of being contaminated, addition of some substances
of different chemical structures known to be preservatives in to
the cosmetic products were planned to prevent microbial growth
(Sasseville, 2004). While the upper limit on the number of non-
pathogenic microorganism in cosmetic products was determined
to be 103 in milliliter, it has been reported that they should not con-
tain pathogenic microorganisms that are a threat to human life like
E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella
species, Clostridium species and Candida albicans. (Naki Sivri, 2005).
Recent studies have identified that the most widely used preser-
vative agents in cosmetic products, parabens could cause contact
allergies (Charnock and Finsrud, 2007). It has been thought that
they have a connection with breast cancer especially in products
applied to the armpits (Darbre, 2003). Studies conducted in vivo and
in vitro have also shown that parabens adversely affect spermato-
genesis and the secretion of hormones, thus impairing the functions
of the reproductive system (Oishi, 2002). The United States Envi-
ronmental Protection Agency has reported that parabens used in
cosmetic products cause disruption to estrogen hormone by dis-
rupting the body’s endocrine system. In addition, parabens cause
skin irritation and dermatitis in individuals with paraben allergies
(Nagel et al., 1977).
In this study, cosmetic purpose formulations were prepared
through emulsifying collagen which is a skin protein by using oil
in water (O/W) technique, and adding natural essential oils which
are reported to have antimicrobial efficacy instead of synthetic
chemicals which are harmful for human health regarding protec-
tive purposes (Bayramoglu, 2005, 2007, 2010; Bayramoglu et al.,
2006, 2008, 2009; Sekeroglu et al., 2011). Antimicrobiocity of the
formulations were tested by using various essential oils and using
them in different proportions, and also the formulations prepared
were investigated in terms of pH, viscosity and the distribution of
particle size.
2. Experimental
2.1. Material
Collagen hydrolysate, and essential oils used in this study except
Origanum onites were obtained from Sigma from United States of
America. O. onites was obtained from Turer Tarim from Turkey.
2.2. Method
2.2.1. Formulation studies
In the emulsion formulation selected for this study, both water
and humectants and emollients that have skin lubricating features
were used together with occlusive agents that resoften and reelas-
ticise the skin. And, emulsion type was selected by taking the effects
of the products while being in use into account.
While W/O type emulsions provide more humidity in the skin,
they form a film layer which is more oily and viscous. Therefore,
they are not commonly preferred in cosmetic terms. And, O/W
type emulsions are more commonly used than W/O type emulsions
for they are very well tolerated by the users although they have
lower emollient and occlusive effects compared to the W/O type
emulsions (Choudhury et al., 1985). For these reasons mentioned
above, all emulsions in our study were prepared in O/W type. In
our study, it has been aimed at obtaining a formulation which has
a high moisturizing feature based on the emulsification of collagen
hydrolysate. Organoleptic features and the physical properties of
the product were evaluated in the preparatory studies performed,
and basic formulas one for each in the form of O/W type emul-
sions were selected. According to the essential oil proportions, 3
379
Table 1
Formulations.
Emulsion formulations
Formulation I Formulation II Formulation III
%2 stearyl alcohol %2 stearyl alcohol %2 stearyl alcohol
%2 isopropyl myristate %2 isopropyl myristate %2 isopropyl myristate
%2 Tween 60 %2 Tween 60 %2 Tween 60
%3 beewax %3 beewax %3 beewax
%3 lanolin %3 lanolin %3 lanolin
%1 borax %1 borax %1 borax
%3 glycerine %3 glycerine %3 glycerine
%0.5 essential oil %1 essential oil %2 essential oil
%1 collagen hydrolysate %1 collagen hydrolysate %1 collagen hydrolysate
% 82.5 distilled water %82 distilled water %81 distilled water
formulations were obtained for each through adding collagen
hydrolysate into these basic formulas.
2.2.1.1. Calculation of HLB value. HLB value is a scale involving val-
ues ranging from 0 to 20 and giving an opinion about the type of
emulsion to be formed by the emulsifying agents. As we want that
the emulsifying agent we selected in our study to form oil/water
emulsion, Tween 60 was selected as emulsifying agent. HLB value
of this emulsifying agent is 14.9. At the end of the studies, the
formulations’ HLB value in average was calculated to be 12.
2.2.1.2. Preparation of emulsions. In our study, 3 different formu-
lations were prepared for each essential oil including the control
group and these formulations are shown in Table 1.
The preparation of the emulsion type formulations are as in the
study of Yorgancioglu (2012). When proceeded to the cooling pro-
cess like in that study, as the temperature was lowered to 40 ◦ C,
collagen hydrolysate was added to the emulsion. Then, the tem-
perature was decreased to 30 ◦ C in a controlled manner in order to
add the essential oil. After the addition of essential oils in different
percentages, it was proceeded to the homogenization stage. At this
stage, the mixer was operated at constant speed of 1400 rev/min
(rpm) for 15 min. After mixing the composition obtained as a result
of this procedure until reaching the room temperature at a constant
speed in the same direction, it was placed in appropriate containers
while it was warm and thus it became ready to be studied.
Adding speed and mechanical mixing of the dispersed phase is
very important for the formation of the emulsion. So, it was affected
the viscosity and stability. The temperature at which the essen-
tials oils are added is also important with respect to the amount
of essential oils in the environment. Purohit and Ramdeen (1990)
have pointed out that essential oils to be added at temperatures
under 40 ◦ C in their cosmetic compositions. Ebrahimi et al. (2011)
suggested in their study that the amount of essential oils in the
environment decreased as environmental temperature increased.
2.2.2. Studies performed on formulations
On the emulsion type formulations, organoleptic assessments,
viscosity measurements and particle size analysis have been done.
In addition, pH measurements were performed in formulations.
Also, whether there was an antimicrobial efficacy or not in the
formulations produced was investigated.
2.2.2.1. Organoleptic assessments. In this stage, particular features
such as general appearance and consistence of the product, ease of
suffusing on the skin with respect to its coating features, ease of its
absorption from the skin, homogeneity of the product, occurrence
of phase separation and creamisation and leaving oily or non-oily
sensation on the skin were evaluated. Furthermore, certain changes
in the product emerging in short-term such as color and odor were
observed.
380 A. Yorgancioglu, E.E. Bayramoglu / Industrial Crops and Products 44 (2013) 378–382

Table 2 The effects of the formulations prepared following the prepara-

The criteria used in assessing the results of research.
Zone diameters (mm) Effectiveness of inhibitory
Diameter < 8.00 None inhibitory effect
9.00 < diameter < 14.00 Poor inhibitory effect
15.00 < diameter < 19.00 Medium inhibitory effect
20.00 < diameter High inhibitory effect
2.2.2.2. Viscosity measurements. Viscosities of the formulations
were measured at a fixed rotational speed of 1 rpm and with the
spindle 4, using Brookfield viscometer at the temperature of 20 ◦ C.
Viscosity values of the emulsions were expressed in (cP) (cen-
tipoise).
2.2.2.3. Particle size analysis. Particle size analysis of the emulsions
were performed by using the device Malvern Mastersizer 2000 par-
ticle size analyser.
2.2.2.4. pH measurement. Emulsion pHs were measured by using
standard pH paper.
2.2.2.5. Antimicrobial efficacy. Aspergillus fumigatus NRRL 5587,
E. coli, Enterococcus faecalis, S. aureus 6538-P, P. aeruginosa and
C. albicans which were used in our studies were taken from the
cultivational inventory of the Department of Basic and Industrial
Microbiology of the Division of Biology of the Faculty of Sci-
ence within the body of Ege University (Aegean University, Izmir).
Antimicrobial effect of the emulsions were determined by using
Disc Diffusion Method. The results of these determinations were
evaluated as in the studies of Roura et al. (Table 2).
3. Results and discussion
3.1. Effects of the cosmetic formulations prepared
Prepared cosmetic emulsions are shown in Table 3.
tory studies on microorganisms are given in Table 4.
Evaluation All microorganisms were growth in the control group formula-
− tion. While in all cosmetic formulations containing O. onites against
+ S. aureus it has been observed that inhibitory effect increased with
++ the increase of essential oil percentage (9 mm, 17 mm, 26 mm),
+++ zone formation of 16 mm in 1% Thymus vulgaris and 22 mm in 2%
T. vulgaris formulation has been observed. No effect against any
microorganism has been observed in all formulations containing
essential oils Eucalyptus globulus and Mentha piperita. Because it
is known from previous experiments that there has been some
effect, it is thought that by increasing the amounts of M. piperita
and E. globulus essential oils, inhibitory effect can be obtained.
No zone formation has been observed in any formulation against
P. aeruginosa. While a good inhibitory effect against E. coli was
observed in the formulation containing 2% O. onites essential oil,
zone formation of 27 mm in 0.5% T. vulgaris, 30 mm in 1% T. vulgaris
and 33 mm in 2% T. vulgaris has been observed. Only the formula-
tion containing the essential oil O. onites and all of its concentrations
showed an inhibitory effect against E. faecalis. It has been observed
that the inhibitory effect increased with the increase of O. onites
essential oil percentage in the formulation against A. fumigatus and
the formulation containing 2% essential oil of T. vulgaris also showed
a clear inhibitory effect. When the effect against C. albicans was
examined, zones of 11 and 16 mm in O. onites formulations con-
taining 1% and 2% essential oils and 17 and 21 mm in formulations
containing T. vulgaris.
Purohit has added 41 kinds of essential oils in to a cosmetic
composition and has investigated the antimicrobial effective-
ness against C. albicans, S. aureus and P. aeruginosa. At the
end of the tests using 0.5%, 1% and 2% essential oils, it has
been concluded that the best results were obtained from the
composition containing 2% essential oils (Purohit and Ramdeen,
1990). The fact that the 2% has been designated as the
most effective result, it is highly consistent with our own
findings.

Table 3
Cosmetic formulations.

No. Formulations No. Formulations

1 Control 8 %0.5 Eucalyptus globulus formulation

2 %0.5 Mentha piperita formulation 9 %1 Eucalyptus globulus formulation
3 %1 Mentha piperita formulation 10 %2 Eucalyptus globulus formulation
4 %2 Mentha piperita formulation 11 %0.5 Thymus vulgaris formulation
5 %0.5 Origanum onites formulation 12 %1 Thymus vulgaris formulation
6 %1 Origanum onites formulation 13 %2 Thymus vulgaris formulation
7 %2 Origanum onites formulation

Table 4
Antimicrobial effects of cosmetic formulations.

Inhibition zone diameters (mm)

No. Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Enterococcus faecalis Aspergillus fumigatus Candida albicans

1 − − − − − −
2 − − − − − −
3 − − − − − −
4 − − − − − −
5 + (9 mm) − − +++ + −
6 ++ (17 mm) − − +++ ++ + (11 mm)
7 +++ (26 mm) +++ − +++ +++ ++ (16 mm)
8 − − − − − −
9 − − − − − −
10 − − − − − −
11 − +++ (27 mm) − − − −
12 ++ (16 mm) +++ (30 mm) − − − ++ (17 mm)
13 +++ (21 mm) +++ (33 mm) − − +++ +++ (21 mm)
 A. Yorgancioglu, E.E. Bayramoglu / Industrial Crops and Products 44 (2013) 378–382 381

Table 5
Viscosity of formulations having antimicrobial properties.

Control formulation Formulations containing Origanum onites Formulations containing Thymus vulgaris

F1 F2 F3 F1 F2 F3

Viscosity (cP) 6148 5939 5446 4395 5439 4318 3759

When all the formulations were evaluated, the formulations Table 6

containing 2% T. vulgaris and O. onites essential oils were identified
as being most effective and it is believed that the inhibitory effect
will increase with the increasing of the essential oil percentage in
the formulation.
3.2. Organoleptic assessments
Formulations were assessed from the organoleptic point of view
for these considerations are very important with respect to the con-
sumers. Physical appearance of the emulsion formulations kept at
25 ◦ C was examined visually on time 0 (baseline), day 15 and day 30.
As results of the observations, formulations prepared were found
to be stable in structure, observed without any change in physical
appearance and were found to keep its clear, transparent and uni-
form appearance as of it had been primed. Any phase separation
was not observed in formulations assessed visually. None the less,
viscidity and lubricity were not observed in the formulations. The
formulations were found not to leave any residue following their
applications on the skin. When examined with respect to emitting
odor, the control group formulation was observed not to have any
irritating odor and essential oils used in other formulations were
found to emit odors inherent in themselves individually.
In addition, emulsions produced with cosmetic purposes were
subjected to rapid aging through keeping them in an oven at 37 ◦ C
temperature for 10 days for stability measurements. As a result of
this procedure, there observed no phase separation or creaming in
cream samples. Any decrease in odor intensity due to the time was
not observed in formulations kept in closed containers.
3.3. Viscosity measurements
French, in the studies performed with Brookfield viscometer,
demonstrated that viscosity of the emulsions had changed asso-
ciated with the particle size, and that decreased particle size had
increased the viscosity. As the particle size in average for emul-
sions decreases, total surface area of the particles increases. And,
the increase in the viscosity of the emulsions is due to this increase
in the surface area in total (French, 1958).
Viscosities of the formulations with antimicrobial efficacy out of
the formulations prepared with different essential oils were mea-
sured at a fixed rotational speed of 1 rpm and with the spindle 4,
using Brookfield viscometer at the temperature of 20 ◦ C. Viscosity
values of the formulations (cP) (centipoise) can be seen in Table 5.
According to the measurement done with the Brookfield vis-
cometer, it was observed that the viscosities of the emulsions had
decreased due to the increase in the proportion of essential oils.
Average particle size of formulations having antimicrobial properties.
Formulations Average particle size (␮m)
Control 179,241
%0.5 Origanum onites formulation 195,645
%1 Origanum onites formulation 228,430
%2 Origanum onites formulation 324,192
%0.5 Thymus vulgaris formulation 235,556
%1 Thymus vulgaris formulation 255,480
%2 Thymus vulgaris formulation 356,876
3.4. Particle size analysis
Droplet size in average or change in the size distribution of the
droplets is an important parameter in the evaluations of the emul-
sion stability. In a stable emulsion system, it may be observed a
rapid increase in droplet size at the beginning and this increase
may be followed by a slow change. Droplet size, droplet surface
area or droplet volume may alter in process of time. An increase in
droplet size of samples kept at different temperatures indicates an
impairment in stability (Rieger, 1986).
During the period of formulation preparation, it was intended
that the particle size of the emulsion would be small and within
a narrow distribution range. The purpose of the particles to be in
small size was that the droplets which would form in small size
could diffuse easily under the skin for the cream was a cosmetic
preparation. In addition, because the smaller the particle size the
greater the amount of the particles per unit area, the cream bright-
ness would also increase as the amount of light absorbed increases.
Particle sizes in average of the formulations with antimicrobial effi-
cacy are shown in Table 6. According to the results, particle size in
average was observed to increase as the proportion of essential oil
increased.
3.5. pH values
Skin pH is between 5 and 6 in normal conditions. pH values vary
due to race, skin tone or color, exposure to sunlight and diseases
(Flynn et al., 2001). Also, the products administered on the skin can
change skin pH. Skin pH is one of the most important biophysical
feature following sebum (Kim et al., 2006). pH of a skin preparation
is required to be in accord with the skin pH in order that the product
to be well tolerated (Hekimoğlu, 2004).
In the formulations prepared, pH measurements were per-
formed using pH papers in days 1, 3, 7 and 30. The stability of the
emulsion formulation is estimated by pH measurements. The pHs
of the prepared formulations are shown in Table 7. The pHs of the

Table 7
pH values of the cosmetic formulations.

Formulation pH Control Thymus vulgaris Origanum onites Eucalyptus globulus Mentha piperita

F1 F2 F3 F1 F2 F3 F1 F2 F3 F1 F2 F3

0 min 6–7 5–6 5–6 5–6 5–6 5–6 5 5–6 5 5 5–6 5–6 5–6
Day 3 6–7 5–6 5–6 5–6 5–6 5–6 5 5–6 5 5 5–6 5–6 5–6
Day 7 6–7 5–6 5–6 5–6 5–6 5–6 5 5–6 5 5 5–6 5–6 5–6
Day 30 6–7 5–6 5–6 5–6 5–6 5–6 5 5–6 5 5 5–6 5–6 5–6
382 A. Yorgancioglu, E.E. Bayramoglu / Industrial Crops and Products 44 (2013) 378–382
formulations ranged between 5 and 7. These rates are on the level
of being buffered by the skin.
4. Conclusion
In this study, instead of the compounds that are problematic
when used in cosmetic preparations, the availability of collagen,
which is a valuable protein in leather industry, that has been got-
ten antimicrobial features by using essential oils and that has been
emulsified was evaluated.
The pHs of the formulations obtained by the studies conducted
were found to be in extreme concordance with skin pHs. And, phase
separation or creaming which is one of the most common problems
encountered with in emulsion impairments were not observed in
any of the formulations.
It was seen that the viscosity of the formulations with antimi-
crobial features had decreased as the proportion of essential oil in
formulations had increased. While the formulation which did not
contain essential oil was found to be the formulation with the best
viscosity, the formulation with the lowest viscosity was recorded
as the formulation that contain %2 T. vulgaris essential oil. And,
as the particle size distribution of the formulations were exam-
ined, the lowest size distribution was found to be in the control
group formulation and the highest size distribution was found to
be in the formulation that contained the %2 T. vulgaris essential oil.
According to these results, it was demonstrated that the particle
size distribution in average increased as essential oil concentration
increased.
When the research results are analyzed, it is seen that essential
oils have inhibitory effects against pathogenic microorganisms that
develop in cosmetic products and can cause disease in humans. It is
thought that essential oils can be used in prevention of the develop-
ment of pathogenic microorganisms resulting from cosmetics but
because the inhibitory effect is weak in low concentrations, the con-
centration percentages have to be increased. In the formulations
produced for cosmetic purposes, only formulations containing O.
onites and T. vulgaris have shown good degrees of antibacterial and
antifungal effects. When the studies were evaluated, the highest
antimicrobial effect was seen in formulations containing O. onites
and the best concentration was the concentration containing 2% of
essential oil. It is believed that with the increase of concentration in
E. globulus and M. piperita essential oil formulations, antibacterial
effect may be seen.
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