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cosmetics
Article
Cosmetic Potential of Marine Fish Skin Collagen
Ana L. Alves 1,2 ID , Ana L. P. Marques 1,2, Eva Martins 1,2 ID , Tiago H. Silva 1,2 ,*ID and
Rui L. Reis 1,2
1
3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of
the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark—Parque
de Ciência e Tecnologia, 4805-017 Barco, Guimarães, Portugal; analves.bq@gmail.com (A.L.A.);
marques13@gmail.com (A.L.P.M.); eva.biotec@gmail.com (E.M.); rgreis@dep.uminho.pt (R.L.R.)
2ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal
*Correspondence: tiago.silva@dep.uminho.pt; Tel.: +351-253510900; Fax: +351-253510909
Received: 19 September 2017; Accepted: 2 October 2017; Published: 12 October 2017
Abstract:
Many cosmetic formulations have collagen as a major component because of its significant
benefits as a natural humectant and moisturizer. This industry is constantly looking for innovative,
sustainable, and truly efficacious products, so marine collagen based formulations are arising as
promising alternatives. A solid description and characterization of this protein is fundamental
to guarantee the highest quality of each batch. In the present study, we present an extensive
characterization of marine-derived collagen extracted from salmon and codfish skins, targeting
its inclusion as component in cosmetic formulations. Chemical and physical characterizations
were performed using several techniques such as sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE), Fourier Transformation Infrared (FTIR) spectroscopy rheology, circular
dichroism, X-ray diffraction, humidity uptake, and a biological assessment of the extracts regarding
their irritant potential. The results showed an isolation of type I collagen with high purity but with
some structural and chemical differences between sources. Collagen demonstrated a good capacity
to retain water, thus being suitable for dermal applications as a moisturizer. A topical exposure of
collagen in a human reconstructed dermis, as well as the analysis of molecular markers for irritation
and inflammation, exhibited no irritant potential. Thus, the isolation of collagen from fish skins for
inclusion in dermocosmetic applications may constitute a sustainable and low-cost platform for the
biotechnological valorization of fish by-products.
Keywords:
marine biomaterials; marine collagen; fish collagen; valorization of fish by-products
moisturizer; cosmetic application
1. Introduction
Collagen is the major structural protein of connective tissues such as skins, tendons, ligaments,
and bones, being the most prevalent component of the extracellular matrix (ECM) [
1
]. It represents
about one-fourth of the total protein content in most animals [
2
,
3
]. It is a fibrous protein formed
by three polypeptide
α
chains, arranged as a triple helix enfolded around each other. Each one is
composed of a set of amino acids with a repeated motif of Gly-X-Y, where X and Y are, predominantly,
proline and hydroxyproline [
4
]. Collagen can be extracted from several origins, with the bovine and
porcine by-products being the most prevalent in an industrial context. Associated with these sources
is the risk of the transference of zoonotic diseases such as BSE (bovine spongiform encephalopathy),
TSE (transmissible spongiform encephalopathy), and FMD (Foot and Mouth Disease) or even religious
constraints. Other sources such as marine collagen are arising as a relevant alternative to their
mammalian counterparts. Several organisms such as marine sponges [
5
,
6
], jellyfish [
7
,
8
], squid [
9
,
10
],
and fishes [
11
–
14
] have been exploited for the extraction of marine collagen. About 75% of a fish’s
Cosmetics 2017,4, 39; doi:10.3390/cosmetics4040039 www.mdpi.com/journal/cosmetics
Cosmetics 2017,4, 39 2 of 16
weight is discarded as skins, bones, fins, heads, and scales, and the scientific community has been
working on a sustainable exploration of marine products and its by-products as a valorization strategy.
Recent biotechnology advances have been made to discover, produce, or transform compounds
from marine sources to be incorporated as functional biomaterials or bioactive compounds [
15
–
18
].
Regarding collagen, it is being used/studied in the biomedical field by the incorporation of this
biopolymer in biomaterials for biomedical or pharmaceutical applications [
19
–
22
]. It can also be
used for personal care, as a component in cosmetic formulations (rather in its native form or as
gelatin) [
23
,
24
]. More than its contributions as an anti-aging and anti-wrinkling product [
25
,
26
],
collagen has long been known and used in the development of cosmetic formulations as a moisturizer
and natural humectant [
27
–
29
] component with a high substantivity to the skin [
29
]. Proteins of
higher molecular weight, such as collagen, cannot be absorbed by the stratum corneum of the skin;
they remain on the surface instead, working as water-uptake through hydration (keeping the skin
moisturized) [
28
,
30
] and as protectors against microbial infiltration in cases of wounded tissue [
31
].
Thereby, the public search for innovative, sustainable, and efficacious products to produce new
cosmetic formulations has brought marine collagen into cosmetic industry as a new, valuable, trendy
component. Marine collagen-based cosmetic formulations vary in their composition and properties,
species of animal, age, and catching origin. So, a good characterization practice and assessment of
quality is important to choose the right collagen for each formulation.
In the present study, we use salmon and codfish skins that are rich in type I collagen to archive
the moisturizing quality of marine collagen to be included as a component in cosmetic formulations.
We performed an extensive physical-chemical characterization of the isolated collagen and examined
both its moisture uptake capability and its irritant potential, considering the possible use of this marine
collagen in cosmetic preparations.
2. Materials and Methods
2.1. Materials
Atlantic Codfish (Gadus morhua) fresh skins (non-salted) were provided by the fish processing
industry (Frigoríficos da Ermida, Lda, Gafanha da Nazaré, Portugal), and Atlantic Salmon fresh skins
(Salmo salar) were provided by a local supermarket. The salmon and codfish skins were transported to
the laboratory facilities and stored at −20 ◦C until use.
2.2. Extraction and Purification of Collagen
All the procedures were performed at 4
◦
C for the salmon and codfish species. For both species,
the skins were cleaned by the removal of the remaining meat, fins, and scales. In the case of the salmon,
it was also necessary to remove the excess fats: the skins were plunged in 10% ethanol for 48 h, under
stirring with medium changes twice a day. Both skins were then rinsed with distilled water and cut
into small pieces to enhance collagen extraction. An acidic extraction was performed according to
the method of Senaratne et al. [
32
], with slight modifications. To remove non-collagenous proteins,
the skins were treated with 0.1 M NaOH (1:10 w/v) for 6 h under stirring. The solution was changed
every 2 h, and then the skins were cleaned several times with distilled water to remove the excess
NaOH solution, until the pH was close to 7. For the extraction, the skins were plunged in 0.5 M of
acetic acid solution (1:10 w/v) for 72 h under stirring, followed by centrifugation at 20,000 gfor 1 h
at 4
◦
C. The supernatants, containing Acid Soluble Collagen (ASC), were collected and saved at a
cold temperature (4
◦
C). In the case of the salmon, there was no need to perform a re-extraction since
there were no remaining skins. However, for the codfish, the rest of the skins needed to undergo a
re-extraction, following the same procedure. To precipitate the collagen, the supernatants were salted
out by adding NaCl to a final concentration of 0.7 M, followed by precipitation by adding NaCl to a
final concentration of 2.6 M in 0.05 M Tris–HCl (pH 7.5), and left overnight. The resultant precipitates
were separated by centrifugation at 20,000 gfor 1 h at 4
◦
C and resuspended in 0.5 M of acetic acid.
Cosmetics 2017,4, 39 3 of 16
These solutions were dialyzed against 0.1 M acetic acid for two days; then 0.02 M acetic acid for two
days; and finally against distilled water until pH 7. Then, the solutions were freeze-dried and stored at
room temperature until further use.
The extraction procedure was performed as previously described. The wet yield of ASC from
both fish skins was calculated using Equation (1):
Yield of collagen (wet) (%)=Weight of collagen (g)
Weight of wet skin (g)×100 (1)
2.3. Amino Acid Analysis
The amino acid content of the extracted collagen was determined by quantitative amino acid
analysis using a Biochrom 30 (Biochrom Ltd., Cambridge, UK) at Centro de Investigaciones Biológicas
of the Spanish National Research Council (CSIC), in Madrid (Spain). The samples were first hydrolyzed
and separated through a column of cation-exchange resin, following a procedure developed by
Spackman, More, and Stein in 1958. The column eluent was mixed with ninhydrin reagent and eluted
at a high temperature. This mixture reacted with the amino acids, forming colored compounds that
were analyzed at two different wavelengths: 440 and 570 nm. An internal standard of norleucine was
used for quantitative analysis. Three independent measurements for each sample were performed for
the quantification of the average amino acid contents.
2.4. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
SDS-PAGE was prepared using reagents from Sigma SDS-PAGE reagents and casted on a Biorad
Mini Protean II System. Freeze-dried collagen was mixed with 1X Laemmli buffer at 5 mg/mL
under stirring until complete dissolution. The samples were heated at 95
◦
C for 10 min into a digital
Thermoblock TD150P3 (FALC) until the denaturation of the proteins and were centrifuged at 10,000 g
for 1 min to sediment the eventual undissolved material. For the codfish-derived collagen, 20
µ
L of
the sample was loaded to the gel, and, for the salmon collagen, 20
µ
L and 40
µ
L were added. Also,
4
µ
L of protein marker was loaded along with the samples. The electrophoresis was carried out at
75 V for 15 min and then at 150 V until the frontline reached the lower part of the gel. After running,
the gels were stained in a Coomasie (0.125% Coomassie Blue R 250 (Biorad), 50% Methanol, 10%
Acetic acid) staining solution for 1 h and then soaked in destaining solution (5% Methanol, 7% Acetic
acid) overnight.
2.5. Fourier Transformation Infrared (FTIR) Spectroscopy
The lyophilized samples were mixed with Sigma potassium bromide (KBr) and grinded into a
powder. The compressed pellets were analyzed on a Shimadzu-IR Prestige 21 spectrometer in a spectral
region of 4000 to 800 cm
−1
with a resolution of 2 cm
−1
using 32 individual scans in absorbance mode.
2.6. Rheology
Oscillatory rheological experiments were assessed on a Kinexus Pro+ rheometer (Malvern
Instruments Ltd, Malvern, Worcestershire, UK) using the acquisition software rSpace. The measuring
system was composed of an upper stainless steel parallel plate of a 20 mm diameter (PU20 SR1740SS,
Malvern, UK) at a 1 mm gap. The oscillatory experiments were performed to obtain temperature
sweep curves, after Linear Viscoelastic Region (LVER) determination, as performed by Zhang et al. [
33
].
The 1% (w/v) collagen solutions were heated from 20 to 40
◦
C at a rate of 0.5
◦
C/min. The storage
modulus (G
0
), loss modulus (G
00
), complex viscosity (
η
*), and the loss tangent (tan
δ
= G
00
/G
0
) were
verified. All plots are the average of at least three experiments.
Cosmetics 2017,4, 39 4 of 16
2.7. Circular Dichroism
The secondary structure preservation of the collagen extracts (salmon and codfish) was evaluated
through Circular Dichroism (CD). The acquisition of the spectra was performed on a Jasco Model
J-1500 spectrometer (Jasco Corp., Tokyo, Japan) using a quartz cylindrical cuvette with a path length of
2 mm. For each reading, 600
µ
L of 0.1 mg/mL collagen solution in 50 mM of acetic acid was placed
into the cuvette. CD spectra were obtained by continuous wavelength scans (in triplicate) from 180 to
240 nm at a scan rate of 50 nm/min. The scans of the samples were recorded at 4
◦
C, 18
◦
C, and 30
◦
C.
2.8. X-ray Diffraction
The X-ray diffraction (XRD) patterns of codfish and salmon freeze-dried collagen were assessed
as described by Zhang et al. [
34
]. The patterns were obtained by an X-ray diffractometer (Bruker
D8 Discover, Karlsruhe, Germany) employing Cu–Ka radiation with a wavelength of
λ
= 1.5406 Å.
The scan was performed in a range of 2
θ
= 5
◦
to 30
◦
by a step of 0.02
◦
and a scanning speed of 2
◦
/min.
2.9. Humidity Regain
The capacity of collagen to retain water from the atmosphere was evaluated by assessing the
variation of the weight upon incubation in an atmosphere with controlled humidity. Lyophilized
collagen was pre-weighed and placed in a desiccator for more than 72 h. After that, the collagen
samples were transferred to a closed atmosphere system at room temperature in an environment of
32% constant relative humidity, maintained over a saturated CaCl
2
solution. The same procedure
was performed for a saturated atmosphere of 63% relative humidity, sustained with deionized water.
The samples were incubated for 48 h in those conditions and then reweighed. The humidity regains
were calculated and expressed as percentages of the dry weight.
2.10. Biological Assessment
2.10.1. Skin Test Irritation
The collagen potential to be an irritant to the skin was evaluated using an In Vitro Epiderm
™
Assay Kit, i.e., EpiDerm (EPI-200, MatTek Corporation, Ashland, MA, USA) [
35
]. This 3D reconstructed
human epidermal model is composed of human-derived epidermal keratinocytes and cultured on the
standing cell culture inserts, forming a multilayered and highly differentiated model of the human
epidermis. As recommended by the manufacturer, the tissues were preconditioned overnight at
37 ◦C and 5% CO2to overcome shipping stress. The collagen samples, positive control (PC) (Sodium
Dodecyl Sulfonate, SDS, 5% (w/v)), and negative control (NC) (sterile Dulbecco’s Phosphate Buffer
Saline (DPBS)) were added to the tissues the next day and incubated for 1h. Subsequently, all the
skin inserts were washed and transferred into fresh medium for 24h. The medium was recovered and
stored at
−
20
◦
C for cytokine analysis, and then the skin inserts were transferred to fresh plates for
MTT (3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazoliumbromide) assay. The tissues were exposed
for 3 h at 37
◦
C and 5% CO
2
to MTT reagent (1 mg/mL). After incubation, the tissues were washed to
remove the MTT medium and then placed in fresh plates. Isopropanol was added to each insert for
formazan extraction and incubated at room temperature for 2h with gentle shaking on a plate shaker
(120 rpm). Then the spectrophotometric analysis of the extracted formazan at 570 nm was carried out
by transferring 200
µ
L of extracted solution in a 96-well plate. The cytotoxicity was expressed as the
ratio of the cell viability, per treatment, to the maximum cell viability from the negative control, and the
collagen was then classified as either irritant or non-irritant based on the criteria in the given protocol.
2.10.2. Cytokine Analysis
Medium levels of IL-6 and IL-18 were measured by an enzyme-linked immunosorbent assay
(ELISA) technique. The cytokine analysis was performed using commercially available ELISA kits
Cosmetics 2017,4, 39 5 of 16
(IL-6 Human Instant ELISA
™
Kit and L-18 Human Instant ELISA
™
Kit from eBioscience, Vienna,
Austria) according to the manufacturer’s instructions. The assay sensitivities for the two kits were 0.92
pg/mL and 9.2 pg/mL, respectively. The absorbance was read at 450 nm using a microplate reader
(SYNERGY HT, BIO-TEK Instruments Inc., Winooski, VT, USA). A standard curve was generated
using a five-parameter curve-fit (Microsoft Office Excel) for each set of samples assayed. The values of
the samples were assigned in relation to the standard curve.
2.11. Statistical Analysis
All data values are presented as mean
±
Standard Deviation (SD). Statistical analysis was
performed using Graph Pad Prism 5.01 software (San Diego, CA, USA) Student’s t-test or One-way
ANOVA followed by the post-hoc Tukey
´
s test was used to perform the statistical analysis, and p-values
≤0.05 were considered statistically significant (*** <0.001; ** <0.002; * <0.05).
3. Results and Discussion
3.1. Collagen Extraction: Salmon and Codfish Skins
Among marine sources, fish skins are widely chosen for the extraction of type I collagen. Fish skins
are available at a large scale, have no risk of transmitting diseases, and have no religious constraints.
Considering that 75% of a fish’s weight is discarded as skins, scales, or fish bones [
32
], which is a result
of industrial processes, it can be used as a valuable source of collagen. Codfish (Gadus morhua) and
salmon (Salmo salar) skins were selected as raw materials for the isolation of type I collagen.
During ASC extraction, we observed that it was easier to solubilize collagen from salmon skin
in an acidic solution than from codfish skin: for the former, there was no need for re-extraction for
further solubilization of the collagen, since no skins remained after 72 h in the acetic acid solution.
Concerning the extraction yield of each species, for salmon skins, we obtained a yield of 19.6%,
while, for codfish skins, we obtained a yield of 10.9%. These values correspond only to the acidic
extraction and may indicate that collagen from Codfish skins is more resilient to this extraction and
that it may be necessary to use further enzymatic extraction [
36
]. Furthermore, these values are in
accordance with those observed in other studies with the same species or others [36,37].
3.2. Chemical and Physical Characterization of the Extracted Collagen
3.2.1. Amino acid Content of Collagen Extracts
There are some characteristics that make collagen unique. The amino acid analysis allows us to
understand the quantitative composition of collagen. Hydroxyproline (OHPro), for example, is an
amino acid present in collagen in a barely exclusive way, playing a key role on its stability. It accounts
for about 13% of collagen’s weight, being almost non-existent in other proteins. Another one that is
revealed to be in large quantities in collagen is Glycine (Gly); in every sequence of three amino acids
on each chain of the collagen triple helix, one of them is glycine (Gly-X-Y). The other two positions are
commonly for Proline (Pro, in position X or Y) and Hydroxyproline (OHPro, in position Y) [38].
Table 1shows the amino acid composition for codfish and salmon based collagen. These results
are very similar to those found by other researchers for the same species [
39
]. Slight differences can be
observed, but, as mentioned before, it is possible to validate the higher presence of glycine in both,
comprising up to 1/3 of the total residues, as well as alanine, as was expected [40].
The content of proline and hydroxyproline in collagen was 121/1000 for salmon and 146/1000
for codfish, which was similar to those reported by and Tylingo et al. [
41
] and Duan et al. [
42
],
respectively, with a hydroxylation degree of about 40% in both cases. It is known that the presence of
high hydroxyproline residues is related to higher denaturation temperatures [
43
] and an increase of
the stability of the triple helix of collagen due to the hydrogen bonds between the polypeptides [
44
].
We observed a higher content of hydroxyproline in codfish them in salmon. It is also expected
Cosmetics 2017,4, 39 6 of 16
that marine origin collagen has a lower hydroxyproline content, compared to its mammalian
counterparts [34,42,45]
, which is probably due to the marine environment that these two species
are from. Both are cold-water fishes, and that is reflected in their lower hydroxyproline content when
compared with other species from warmer waters [39,45].
Table 1. Amino acid content of collagen extracts obtained from salmon and codfish skins.
Amino Acid Salmon Collagen Codfish Collagen
moL %
Asp 47 51
Thr 16 23
Ser 56 67
Glu 71 71
Gly 365 332
Ala 121 106
Cys 3 5
Val 18 19
Met 28 17
Ile 6 11
Leu 24 21
Nleu 27 22
Tyr 1 4
Phe 16 12
OHLys 12 7
His 9 8
Lys 25 26
Arg 36 51
OHPro 48 55
Pro 73 91
Total 1000 1000
3.2.2. SDS-PAGE Analysis
The SDS-PAGE technique separates macromolecules such as nucleic acids and protein fragments
according to their molecular weight or size, based on differences in electrophoretic mobility under
an applied electrical field. Thus, this technique accesses the structural information, purity, and
breakdowns in collagen proteins.
In Figure 1, the SDS-PAGE patterns for both the salmon and codfish skin extracts show the
characteristic bands of collagen, namely the αand βchains.
For both species, salmon (a) and codfish (b) electrophoresis analysis demonstrated the presence
of
α1
and
α2
chains, correspondent to type I collagen [
1
,
46
], as well the presence of
β
dimer. The
β
component indicates that both collagens contain inter-molecular crosslinks. Similar patterns were
found for salmon and codfish by others [
40
,
41
,
47
], as well as for other types of fishes such as Japanese
sea-bass, bullhead shark, and chub mackerel [
48
]; brown backed toadfish [
32
]; Deep-sea Redfish [
49
];
European carp [
42
]; striped catfish [
50
]; and Indian carp and Mrigal carp [
51
]. Also, a
γ
trimer can be
also spotted in codfish, indicating that intra-molecular crosslinking is also present in the three chains
of collagen. This demonstrates the possibility of renaturing the native collagen [
52
]. A slightly shift in
the position of the
α
and
β
chains can be observed between the species. This may be probably due to
differences in the amino acid composition that lead to slight alterations in the molecular weight [37].
Cosmetics 2017,4, 39 7 of 16
Cosmetics 2017, 4, 39 7 of 16
(a) (b)
Figure 1. Electrophoresis analysis of salmon and codfish skin collagen. (a) Sodium Dodecyl Sulphate-
Polyacrylamide Gel Electrophoresis (SDS-PAGE) pattern of salmon collagen by Acid Soluble
Collagen (ASC) extraction; (b) SDS-PAGE pattern of Codfish collagen by ASC extraction.
3.2.3. FTIR Analysis
The extracted material from both marine sources was analysed through Fourier Transform
InfraRed spectroscopy (FTIR) to identify collagen by the presence of characteristic bands. Figure 2
showed the spectra for the ASC of salmon and codfish collagen. The overall spectra profile suggests
that the collagens extracted from salmon and codfish are comparable, indicating that both have
similar structural and chemical compositions, with visible the reference peaks for collagen being
characteristic of amide A, amide B, amide I, amide II, and amide III. The amide A band is relative to
the N-H stretching vibration typical of intermolecular hydrogen bonding and is commonly observed
in a range between 3000 and 3500 cm
−1
.
Figure 2. Fourier Transform InfraRed spectroscopy (FTIR) spectra of the ASC of salmon (red line) and
codfish (blue line) collagen.
Figure 1.
Electrophoresis analysis of salmon and codfish skin collagen. (
a
) Sodium Dodecyl
Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) pattern of salmon collagen by Acid Soluble
Collagen (ASC) extraction; (b) SDS-PAGE pattern of Codfish collagen by ASC extraction.
3.2.3. FTIR Analysis
The extracted material from both marine sources was analysed through Fourier Transform
InfraRed spectroscopy (FTIR) to identify collagen by the presence of characteristic bands. Figure 2
showed the spectra for the ASC of salmon and codfish collagen. The overall spectra profile suggests
that the collagens extracted from salmon and codfish are comparable, indicating that both have similar
structural and chemical compositions, with visible the reference peaks for collagen being characteristic
of amide A, amide B, amide I, amide II, and amide III. The amide A band is relative to the N-H
stretching vibration typical of intermolecular hydrogen bonding and is commonly observed in a range
between 3000 and 3500 cm−1.
Cosmetics 2017, 4, 39 7 of 16
(a) (b)
Figure 1. Electrophoresis analysis of salmon and codfish skin collagen. (a) Sodium Dodecyl Sulphate-
Polyacrylamide Gel Electrophoresis (SDS-PAGE) pattern of salmon collagen by Acid Soluble
Collagen (ASC) extraction; (b) SDS-PAGE pattern of Codfish collagen by ASC extraction.
3.2.3. FTIR Analysis
The extracted material from both marine sources was analysed through Fourier Transform
InfraRed spectroscopy (FTIR) to identify collagen by the presence of characteristic bands. Figure 2
showed the spectra for the ASC of salmon and codfish collagen. The overall spectra profile suggests
that the collagens extracted from salmon and codfish are comparable, indicating that both have
similar structural and chemical compositions, with visible the reference peaks for collagen being
characteristic of amide A, amide B, amide I, amide II, and amide III. The amide A band is relative to
the N-H stretching vibration typical of intermolecular hydrogen bonding and is commonly observed
in a range between 3000 and 3500 cm
−1
.
Figure 2. Fourier Transform InfraRed spectroscopy (FTIR) spectra of the ASC of salmon (red line) and
codfish (blue line) collagen.
Figure 2.
Fourier Transform InfraRed spectroscopy (FTIR) spectra of the ASC of salmon (red line) and
codfish (blue line) collagen.
Cosmetics 2017,4, 39 8 of 16
The FTIR profile can be observed proximally at 3473 cm
−1
for salmon and 3410 cm
−1
for codfish.
Also for salmon and codfish, amide B, relative to the asymmetrical and symmetrical stretch of CH
2
,
is found at 3074 cm
−1
and 2941 cm
−1
, respectively. The amide I peak, characteristic of the stretching
vibrations of C=O groups of proteins arises at 1660 cm
−1
and 1653 cm
−1
. For salmon and codfish, the
absorption band characteristic of amide II was very similar. The peaks arise at 1550 cm
−1
/1548 cm
−1
,
correspondent to the NH bending vibration coupled with CN stretching; 1450 cm
−1
, attributed to
CH
2
bending; and 1404 cm
−1
/1409 cm
−1
, derived from COO-symmetrical stretching, respectively.
Additionally, bands of amide III are also present at 1338 cm
−1
/1336 cm
−1
, correspondent to the NH
bending associated with CN stretching, and at 1240 cm
−1
/1244 cm
−1
, relative to C-O stretching,
respectively. The absorption ratio between the amide III and 1450 cm
−1
peaks was near to 1 (1.01 for
salmon and 0.97 for codfish), which was an indicator of the preservation of the triple helix structure
of collagen [
49
,
53
–
56
]. These findings are similar to other findings found in the literature for other
marine species [42,57–59], as well as the ones in this study [41].
3.2.4. Rheology
Changes in the temperature range affect the rheological properties of collagen in solution, which
can be translated as alterations in its structure. From LVER determination, at 1 Hz of frequency, a
shear strain of 5% was defined to be used on the oscillatory experiments. In Figure 3, the effect
of temperature on the structure of collagen was assessed by measuring the complex viscosity (
η
*)
and the loss angle (tan
δ
) through a range of temperatures (20 to 40
◦
C). The decrease of
η
* and a
concomitant rapid increase in tan
δ
reflect the fragility and further collapse of collagen’s triple helix
into a random coil [
33
]. The temperature at which these events occur is considered by some authors as
the denaturation temperature under dynamic rheological measurement [
33
,
60
]. However, from our
point of view, this event is characterized as the gelation point at which disorganized chains reorganize
to form a gel. That point is determined as the temperature at which the decrease of
η
* reached 50% of
its initial value or as the temperature at which the increase of tan δachieved a peak value.
Cosmetics 2017, 4, 39 8 of 16
The FTIR profile can be observed proximally at 3473 cm−1 for salmon and 3410 cm−1 for codfish.
Also for salmon and codfish, amide B, relative to the asymmetrical and symmetrical stretch of CH2 ,
is found at 3074 cm−1 and 2941 cm−1, respectively. The amide I peak, characteristic of the stretching
vibrations of C=O groups of proteins arises at 1660 cm−1 and 1653 cm−1. For salmon and codfish, the
absorption band characteristic of amide II was very similar. The peaks arise at 1550 cm−1/1548 cm−1,
correspondent to the NH bending vibration coupled with CN stretching; 1450 cm−1, attributed to CH2
bending; and 1404 cm−1/1409 cm−1, derived from COO-symmetrical stretching, respectively.
Additionally, bands of amide III are also present at 1338 cm−1/1336 cm−1, correspondent to the NH
bending associated with CN stretching, and at 1240 cm−1/1244 cm−1, relative to C-O stretching,
respectively. The absorption ratio between the amide III and 1450 cm−1 peaks was near to 1 (1.01 for
salmon and 0.97 for codfish), which was an indicator of the preservation of the triple helix structure
of collagen [49,53–56]. These findings are similar to other findings found in the literature for other
marine species [42,57–59], as well as the ones in this study [41].
3.2.4. Rheology
Changes in the temperature range affect the rheological properties of collagen in solution, which
can be translated as alterations in its structure. From LVER determination, at 1 Hz of frequency, a
shear strain of 5% was defined to be used on the oscillatory experiments. In Figure 3, the effect of
temperature on the structure of collagen was assessed by measuring the complex viscosity (η*) and
the loss angle (tan δ) through a range of temperatures (20 to 40 °C). The decrease of η* and a
concomitant rapid increase in tan δ reflect the fragility and further collapse of collagen’s triple helix
into a random coil [33]. The temperature at which these events occur is considered by some authors
as the denaturation temperature under dynamic rheological measurement [33,60]. However, from
our point of view, this event is characterized as the gelation point at which disorganized chains
reorganize to form a gel. That point is determined as the temperature at which the decrease of η*
reached 50% of its initial value or as the temperature at which the increase of tan δ achieved a peak
value.
Figure 3. Temperature dependence of η* and tan δ for 1.0% of the ASC of (a) salmon and (b) codfish
collagen.
For salmon collagen, that value is approximately 30 °C, and, for codfish collagen, it is about
28.5 °C. These values are slightly lower when compared, for example, with collagen from largefin
longbarbel catfish but are justified by taking into account the cold-water origin of the former fishes.
The different behaviors of these rheological patterns may be due to the difference in the viscosity of
collagen in 0.02 M acetic acid, in which salmon collagen kept a more gelified appearance than codfish
collagen.
20 22 24 26 28 30 32 34 36 38 40 42
0
10
20
30
40
50
60
0.24
0.26
0.28
0.30
0.32
0.34
* (Pa.s)
Tan
30 C
Salmon
(a)
Temperature (C)
* (Pa.s)
Tan
20 22 24 26 28 30 32 34 36 38 40 42
0
2
4
6
8
0.70
0.75
0.80
0.85
0.90
0.95
* (Pa.s)
Tan
28.5C
Codfish
Temperature (C)
* (Pa.s)
Tan
(b)
Figure 3.
Temperature dependence of
η
* and tan
δ
for 1.0% of the ASC of (
a
) salmon and (
b
)
codfish collagen.
For salmon collagen, that value is approximately 30
◦
C, and, for codfish collagen, it is about
28.5
◦
C. These values are slightly lower when compared, for example, with collagen from largefin
longbarbel catfish but are justified by taking into account the cold-water origin of the former fishes.
The different behaviors of these rheological patterns may be due to the difference in the viscosity
of collagen in 0.02 M acetic acid, in which salmon collagen kept a more gelified appearance than
codfish collagen.
3.2.5. Circular Dichroism
CD spectroscopy is typically used to assess the protein secondary structure through the differential
absorption of left and right handed circular polarized light in an asymmetric environment [
61
,
62
].
Cosmetics 2017,4, 39 9 of 16
In Figure 4, the results of the CD spectroscopy analysis of salmon and codfish collagen can be observed,
with clear differences between both marine species (Figure 4a). Regarding salmon collagen, a positive
band was observed at 222 nm, which is characteristic of triple helix, and a pronounced negative band
around 196 to 200 nm, typical of a random coil structure [
63
], while, for codfish, no positive band was
observed at 220 nm, indicating that the triple helix is preserved only in salmon collagen To evaluate
the effect of temperature on the triple helix structure, a range of temperatures were settled. At 4
◦
C
(Figure 4a), it is possible to observe the triple helical structure by the presence of a positive peak that
decreases with the increase of temperature from 4 to 18
◦
C and 30
◦
C (Figure 4b,c). At the maximum
temperature, the native collagen structure was completely destroyed.
Through a statistical analysis using a Nonlinear Curve Fit (Boltzmann function), which produces
a sigmoidal curve, the denaturation temperatures of both collagen extracts were calculated. For salmon
collagen, the denaturation temperature was around 27
◦
C. However, in the case of codfish, it was not
possible to determine.
Cosmetics 2017, 4, 39 9 of 16
3.2.5. Circular Dichroism
CD spectroscopy is typically used to assess the protein secondary structure through the
differential absorption of left and right handed circular polarized light in an asymmetric environment
[61,62]. In Figure 4, the results of the CD spectroscopy analysis of salmon and codfish collagen can
be observed, with clear differences between both marine species (Figure 4a). Regarding salmon
collagen, a positive band was observed at 222 nm, which is characteristic of triple helix, and a
pronounced negative band around 196 to 200 nm, typical of a random coil structure [63], while, for
codfish, no positive band was observed at 220 nm, indicating that the triple helix is preserved only in
salmon collagen To evaluate the effect of temperature on the triple helix structure, a range of
temperatures were settled. At 4 °C (Figure 4a), it is possible to observe the triple helical structure by
the presence of a positive peak that decreases with the increase of temperature from 4 to 18 °C and
30 °C (Figure 4b,c). At the maximum temperature, the native collagen structure was completely
destroyed.
Through a statistical analysis using a Nonlinear Curve Fit (Boltzmann function), which produces
a sigmoidal curve, the denaturation temperatures of both collagen extracts were calculated. For
salmon collagen, the denaturation temperature was around 27 °C. However, in the case of codfish, it
was not possible to determine.
4C
190 210 230 250
-8
-6
-4
-2
0
2
Salmon
Codfish
Wavelength (nm)
(a)
[mdeg]
18C
190 210 230 250
-8
-6
-4
-2
0
2
Wavelength (nm)
(b)
[mdeg]
Figure 4. Cont.
Cosmetics 2017,4, 39 10 of 16
Cosmetics 2017, 4, 39 10 of 16
Figure 4. CD spectra of ASC collagen from Salmon and Codfish in different range temperature (a) 4
°C; (b) 18 °C; and (c) 30 °C.
3.2.6. X-ray Diffraction
X-ray diffraction is often used to assess collagen fibril distribution and orientation in fish
mineralized tissues [34,64]. Figure 5 illustrates the X-ray spectra of lyophilized acid-soluble collagen
from Salmon (a) and Codfish (b) skins. The difference in organization between the structures of
Salmon and Codfish collagen were visible, with Salmon collagen spectrum presenting two peaks well
featured but, on the other hand, Codfish spectrum showed a flattened pattern. These two peaks are
characteristic of collagen molecule and can be seen as a signature. The first one and sharpest is related
to the triple helix conformation and distance between molecular chains, and the second peak is
related to the distance between the skeletons. The diffraction angles (2θ) of Salmon were about 7.70°
and 19.59° and using Bragg equation d(Å) = λ/2sinθ (λ = 1.54 Å) [34], the minimum values (d) of the
repeated spacings were calculated. The d corresponding to the sharp peak was 11.46 Å and for the
wide peak was 4.52 Å. These values are closed to those reported by Zhang et al. [34] corroborating
that our Salmon collagen has triple helix structure preserved, which is not detected on Codfish
collagen.
Figure 5. X-ray spectra of ASC collagen from (a) salmon and (b) codfish skins.
3.3. Humidity Regain Analysis
The ability for collagen to retain water is fundamental to its successful performance as cosmetic
component, with its main role being involved in on the control of skin moisture. Its capacity to be a
natural humectant was evaluated by measuring the relative water uptake from the atmosphere, using
freeze-dried samples under constant atmospheric humidity. From a perspective of biotechnological
30C
190 210 230 250
-8
-6
-4
-2
0
2(c)
[mdeg]
Wavelength (nm)
Salmon
510 15 20 25 30
0
1
2
3
Difraction angle 2 (degree)
Intensity (Counts)
(a)
Codfish
510 15 20 25 30
0
1
2
3
Difraction angle 2 (degree)
Intensity (Counts)
(b)
Figure 4.
CD spectra of ASC collagen from Salmon and Codfish in different range temperature (
a
) 4
◦
C;
(b) 18 ◦C; and (c) 30 ◦C.
3.2.6. X-ray Diffraction
X-ray diffraction is often used to assess collagen fibril distribution and orientation in fish
mineralized tissues [
34
,
64
]. Figure 5illustrates the X-ray spectra of lyophilized acid-soluble collagen
from Salmon (a) and Codfish (b) skins. The difference in organization between the structures of
Salmon and Codfish collagen were visible, with Salmon collagen spectrum presenting two peaks well
featured but, on the other hand, Codfish spectrum showed a flattened pattern. These two peaks are
characteristic of collagen molecule and can be seen as a signature. The first one and sharpest is related
to the triple helix conformation and distance between molecular chains, and the second peak is related
to the distance between the skeletons. The diffraction angles (2
θ
) of Salmon were about 7.70
◦
and
19.59
◦
and using Bragg equation d(Å) =
λ
/2sin
θ
(
λ
= 1.54 Å) [
34
], the minimum values (d) of the
repeated spacings were calculated. The d corresponding to the sharp peak was 11.46 Å and for the
wide peak was 4.52 Å. These values are closed to those reported by Zhang et al. [
34
] corroborating that
our Salmon collagen has triple helix structure preserved, which is not detected on Codfish collagen.
Cosmetics 2017, 4, 39 10 of 16
Figure 4. CD spectra of ASC collagen from Salmon and Codfish in different range temperature (a) 4
°C; (b) 18 °C; and (c) 30 °C.
3.2.6. X-ray Diffraction
X-ray diffraction is often used to assess collagen fibril distribution and orientation in fish
mineralized tissues [34,64]. Figure 5 illustrates the X-ray spectra of lyophilized acid-soluble collagen
from Salmon (a) and Codfish (b) skins. The difference in organization between the structures of
Salmon and Codfish collagen were visible, with Salmon collagen spectrum presenting two peaks well
featured but, on the other hand, Codfish spectrum showed a flattened pattern. These two peaks are
characteristic of collagen molecule and can be seen as a signature. The first one and sharpest is related
to the triple helix conformation and distance between molecular chains, and the second peak is
related to the distance between the skeletons. The diffraction angles (2θ) of Salmon were about 7.70°
and 19.59° and using Bragg equation d(Å) = λ/2sinθ (λ = 1.54 Å) [34], the minimum values (d) of the
repeated spacings were calculated. The d corresponding to the sharp peak was 11.46 Å and for the
wide peak was 4.52 Å. These values are closed to those reported by Zhang et al. [34] corroborating
that our Salmon collagen has triple helix structure preserved, which is not detected on Codfish
collagen.
Figure 5. X-ray spectra of ASC collagen from (a) salmon and (b) codfish skins.
3.3. Humidity Regain Analysis
The ability for collagen to retain water is fundamental to its successful performance as cosmetic
component, with its main role being involved in on the control of skin moisture. Its capacity to be a
natural humectant was evaluated by measuring the relative water uptake from the atmosphere, using
freeze-dried samples under constant atmospheric humidity. From a perspective of biotechnological
30C
190 210 230 250
-8
-6
-4
-2
0
2(c)
[mdeg]
Wavelength (nm)
Salmon
510 15 20 25 30
0
1
2
3
Difraction angle 2 (degree)
Intensity (Counts)
(a)
Codfish
510 15 20 25 30
0
1
2
3
Difraction angle 2 (degree)
Intensity (Counts)
(b)
Figure 5. X-ray spectra of ASC collagen from (a) salmon and (b) codfish skins.
3.3. Humidity Regain Analysis
The ability for collagen to retain water is fundamental to its successful performance as cosmetic
component, with its main role being involved in on the control of skin moisture. Its capacity to be a
natural humectant was evaluated by measuring the relative water uptake from the atmosphere, using
freeze-dried samples under constant atmospheric humidity. From a perspective of biotechnological
Cosmetics 2017,4, 39 11 of 16
valorization of fish by-products, the codfish is a more interesting species in the Portuguese context.
The industry exploration of this fish is very pronounced in our country, and the need for the valorization
of derived by-products is important for a sustainable exploitation of this resource, with environmental
and economic advantages. Therefore, we choose to use the codfish derived collagen for the next
experiments; the fact that codfish collagen did not show a preserved triple helical structure is not
relevant for cosmetic applications.
First, we exposed the dried samples to a relative humidity of 30%, using a saturated solution of
CaCl
2
to maintain it constantly. After 24 h, a slight increase in weight can be seen (Figure 6) due to the
water uptake, which is not very pronounced, mainly because of the lower water availability in the
environment. After 48 h of exposure, the codfish collagen did not present expressive differences in
weight because the system reached the equilibrium stage.
Cosmetics 2017, 4, 39 11 of 16
valorization of fish by-products, the codfish is a more interesting species in the Portuguese context.
The industry exploration of this fish is very pronounced in our country, and the need for the
valorization of derived by-products is important for a sustainable exploitation of this resource, with
environmental and economic advantages. Therefore, we choose to use the codfish derived collagen
for the next experiments; the fact that codfish collagen did not show a preserved triple helical
structure is not relevant for cosmetic applications.
First, we exposed the dried samples to a relative humidity of 30%, using a saturated solution of
CaCl2 to maintain it constantly. After 24 h, a slight increase in weight can be seen (Figure 6) due to
the water uptake, which is not very pronounced, mainly because of the lower water availability in
the environment. After 48 h of exposure, the codfish collagen did not present expressive differences
in weight because the system reached the equilibrium stage.
Figure 6. Measurement of the water uptake of codfish collagen under different percentages of relative
humidity. Values are Mean ± SD of at least three individual measurements. * p < 0.05 and ** p < 0.002
when the time was compared by one-way ANOVA, followed by Tukey’s test.
When the samples were exposed to 63% relative humidity, the water uptake increased
significantly, by about 6% of the sample’s weight. This result is in accordance with other
measurements performed on commercial collagens suitable for cosmetic applications [29]. Also, an
experiment with a fully saturated atmosphere (98% relative humidity) was performed (data not
shown), and the sample absorbed a considerable quantity of water, forming wet slurries. For both
conditions, the water uptake was kept constant after 24 h, an important indicator that collagen could
be able to retain water, keeping a moisturized skin.
3.4. Evaluation of Skin Irritation Potential
3.4.1. Effect over Viability of Keratinocytes on Reconstructed Skin Model
Assessing the irritant potential of a cosmetic component is of great importance when preparing
a formulation to apply to the skin. Although the induced irritation can be reversible in some cases,
there are others in which it can evolve into permanent damage. Reconstructed models of human
epidermis have been used to mimic human skin and then assess the tolerance of human skin to
potential irritants in cosmetic formulations [65,66]. In Figure 7, we can observe that the viability of
keratinocytes was not significantly affected by the presence of codfish collagen.
0 hours
24 hours
48 hours
0
2
4
6
8
63% Relative Humidity
30% Relative Humidity
**
*
*
**
% Relative Humidity uptake
Figure 6.
Measurement of the water uptake of codfish collagen under different percentages of relative
humidity. Values are Mean
±
SD of at least three individual measurements. * p< 0.05 and ** p< 0.002
when the time was compared by one-way ANOVA, followed by Tukey’s test.
When the samples were exposed to 63% relative humidity, the water uptake increased significantly,
by about 6% of the sample’s weight. This result is in accordance with other measurements performed
on commercial collagens suitable for cosmetic applications [
29
]. Also, an experiment with a fully
saturated atmosphere (98% relative humidity) was performed (data not shown), and the sample
absorbed a considerable quantity of water, forming wet slurries. For both conditions, the water uptake
was kept constant after 24 h, an important indicator that collagen could be able to retain water, keeping
a moisturized skin.
3.4. Evaluation of Skin Irritation Potential
3.4.1. Effect over Viability of Keratinocytes on Reconstructed Skin Model
Assessing the irritant potential of a cosmetic component is of great importance when preparing a
formulation to apply to the skin. Although the induced irritation can be reversible in some cases, there
are others in which it can evolve into permanent damage. Reconstructed models of human epidermis
have been used to mimic human skin and then assess the tolerance of human skin to potential irritants
in cosmetic formulations [
65
,
66
]. In Figure 7, we can observe that the viability of keratinocytes was not
significantly affected by the presence of codfish collagen.
The skin irritation potential of collagen is predicted if the mean relative tissue viability of three
individual tissues is bellow to 50%, when compared with the negative control (NC) [
67
], as clearly
observed in the presence of 5% SDS (PC). The codfish collagen shows a relative cell viability superior
to 50% and it is, thus, considered a non-irritant.
Cosmetics 2017,4, 39 12 of 16
Cosmetics 2017, 4, 39 12 of 16
Figure 7. MTT cytotoxicity assay of codfish collagen to assess skin irritation. Values are Mean ± SD of
at least three individual tissues. *** p < 0.0001, when compared with positive control (PC) by one-way
ANOVA, followed by Tukey’s test.
The skin irritation potential of collagen is predicted if the mean relative tissue viability of three
individual tissues is bellow to 50%, when compared with the negative control (NC) [67], as clearly
observed in the presence of 5% SDS (PC). The codfish collagen shows a relative cell viability superior
to 50% and it is, thus, considered a non-irritant.
3.4.2. Cytokine Analysis
The irritant and inflammatory potentials of codfish collagen were also evaluated by measuring
the expression levels of two key cytokines, IL-18 and IL-6. Interleukin (IL)-18 is a member of the IL-1
family of cytokines, which are expressed by many cells, including keratinocytes [68]. It is a marker
for irritability and is highly expressed in some skin diseases such as psoriasis, atopic dermatitis,
urticaria, contact dermatitis, and drug allergy [69]. Interleukin (IL)-6 is believed to be the central
regulator of the immunological processes and a well-known marker of inflammatory states [70].
The results for cytokine assays are derived from the same tissues that generate the relative
viability data, as well as the controls. For both IL-18 and IL-6, no evidence of the release of those
cytokines was detected for the time period of incubation with the collagen (1 h). Overall, this data
supports the idea that the tested codfish collagen can be considered a non-irritant, being safe for
dermocosmetic formulations.
4. Conclusions
To input value to by-products of marine species in a sustainable way, salmon and codfish skins
were used for collagen extraction. Collagen with high purity was extracted, as can be observed by
SDS-PAGE and FTIR, with the former displaying profiles compatible with type I collagen. The effect
of temperature on collagen stability was evaluated by rheology, which showed a gelation
temperature of 30 °C for salmon and 28.5 °C for codfish. Moreover, CD analysis revealed the presence
of a triple helix structure in salmon collagen, with a denaturation temperature of around 27 °C, while,
in codfish collagen, no triple helix was detected. Considering a biotechnological valorization of the
produced collagens, the cosmetic evaluation was processed using codfish collagen, which showed a
good moisturizing effect through water absorption, preventing skin dehydration without signs of
irritancy to the skin, as demonstrated by topical exposure and cytokine evaluation.
This study demonstrated that the common sources of collagen in cosmetic applications can be
substituted with marine collagen as a sustainable platform for the biotechnological valorization of
fish by-products.
Acknowledgments: The authors would like to acknowledge to European Union for the financial support under
the scope of European Regional Development Fund (ERDF) through the projects 0687_NOVOMAR_1_P
NC
PC
Codfish
0
50
100
150
***
***
ns
Relative viability (%)
Figure 7.
MTT cytotoxicity assay of codfish collagen to assess skin irritation. Values are Mean
±
SD of
at least three individual tissues. *** p< 0.0001, when compared with positive control (PC) by one-way
ANOVA, followed by Tukey’s test.
3.4.2. Cytokine Analysis
The irritant and inflammatory potentials of codfish collagen were also evaluated by measuring
the expression levels of two key cytokines, IL-18 and IL-6. Interleukin (IL)-18 is a member of the IL-1
family of cytokines, which are expressed by many cells, including keratinocytes [
68
]. It is a marker for
irritability and is highly expressed in some skin diseases such as psoriasis, atopic dermatitis, urticaria,
contact dermatitis, and drug allergy [
69
]. Interleukin (IL)-6 is believed to be the central regulator of the
immunological processes and a well-known marker of inflammatory states [70].
The results for cytokine assays are derived from the same tissues that generate the relative
viability data, as well as the controls. For both IL-18 and IL-6, no evidence of the release of those
cytokines was detected for the time period of incubation with the collagen (1 h). Overall, this data
supports the idea that the tested codfish collagen can be considered a non-irritant, being safe for
dermocosmetic formulations.
4. Conclusions
To input value to by-products of marine species in a sustainable way, salmon and codfish skins
were used for collagen extraction. Collagen with high purity was extracted, as can be observed by
SDS-PAGE and FTIR, with the former displaying profiles compatible with type I collagen. The effect of
temperature on collagen stability was evaluated by rheology, which showed a gelation temperature
of 30
◦
C for salmon and 28.5
◦
C for codfish. Moreover, CD analysis revealed the presence of a
triple helix structure in salmon collagen, with a denaturation temperature of around 27
◦
C, while,
in codfish collagen, no triple helix was detected. Considering a biotechnological valorization of the
produced collagens, the cosmetic evaluation was processed using codfish collagen, which showed
a good moisturizing effect through water absorption, preventing skin dehydration without signs of
irritancy to the skin, as demonstrated by topical exposure and cytokine evaluation.
This study demonstrated that the common sources of collagen in cosmetic applications can be
substituted with marine collagen as a sustainable platform for the biotechnological valorization of
fish by-products.
Acknowledgments:
The authors would like to acknowledge to European Union for the financial support under
the scope of European Regional Development Fund (ERDF) through the projects 0687_NOVOMAR_1_P (POCTEP
(Programa Operacional de Cooperação Transfronteiriça España-Portugal) 2007/2013) and 0302_CVMAR_I_1_P
(POCTEP 2014/2020) and the Structured Project NORTE-01-0145-FEDER-000021 (Norte2020) and under the
scope of the European Union Seventh Framework Programme (FP7/2007-2013) through grant agreement
ERC-2012-ADG 20120216-321266 (ERC Advanced Grant ComplexiTE). The Portuguese Foundation for Science
and Technology is also acknowledged for the grant of A.L.A (Ana Luísa Alves.) under Doctoral Programme Do*
Mar (PD/BD/127995/2016). The authors also thank Sandra Pina (3B’s Research Group) for her assistance with
Cosmetics 2017,4, 39 13 of 16
the XRD measurements, Cristiana Gonçalves (3B’s Research Group) for helping with the rheology results analysis,
and Cristina Thiebaut (Cobiosa Industrias Asociadas SL, Spain) for her discussion on the cosmetic relevance of
collagen. Frigoríficos da Ermida, Lda. and Pingo Doce Braga are also gratefully acknowledged for their offers of
codfish and salmon skins, respectively.
Author Contributions:
Tiago H. Silva and Rui L. Reis conceived the study; Ana L. Alves, Ana L. P. Marques,
Eva Martins
and Tiago H. Silva designed the experiments; Ana L. Alves, Ana L. P. Marques and Eva Martins
performed the experiments; Ana L. Alves, Eva Martins and Tiago H. Silva analyzed the data; Rui L. Reis
contributed reagents/materials/analysis tools; Ana L. Alves and Eva Martins wrote the manuscript;
Tiago H. Silva
and Rui L. Reis revise the paper.
Conflicts of Interest: The authors declare no conflicts of interest.
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