cosmetics

Article
Cosmetic Potential of a Recombinant 50 kDa Protein
Nesma Aly, Emilie Benoit, Jean-Luc Chaubard, Kavyasree Chintalapudi , Soojin Choung, Monique de Leeuw,
Matthew Diaz, Dan Dueppen, Bryce Ferraro, Valerie Fischetti, Evan Gassaway, Isabelle Hansenne-Cervantes,
Arjan Heeres, Christina Karas , Mohamed Khan, Jonathan M. Kral, Srujana Lam, Richel Lartey, Mencius Leonard,
Stanley W. Lue, Joshua McDaniel, Kevin Ramirez, Brenna Rauw, Kelly A. Raymond, Catherine Roggero-Lovisi,
Scott Rubin, Kristin Ruebling-Jass, Zoë Spiegelhoff, Monica Celise Stewart, Shashwat Vajpeyi, Alejandro Vicente,
Kathleen E. Vincent, Jing Wang, David Williamson, Zhihao Yu and Lixin Dai *

Modern Meadow, Inc., 111 Ideation Way, Suite 100, Nutley, NJ 07110, USA; naly@modernmeadow.com (N.A.);
EBenoit@modernmeadow.com (E.B.); jeanluc.chaubard@gmail.com (J.-L.C.);
kchintalapudi@modernmeadow.com (K.C.); schoung@modernmeadow.com (S.C.);
mdeleeuw@modernmeadow.com (M.d.L.); mdiaz@modernmeadow.com (M.D.);
ddueppen@modernmeadow.com (D.D.); ferrarobryce@gmail.com (B.F.); vfischetti@modernmeadow.com (V.F.);
egassaway@modernmeadow.com (E.G.); ihansennecervantes@modernmeadow.com (I.H.-C.);
arjanheeres@gmail.com (A.H.); CKaras@modernmeadow.com (C.K.); Mohamed.khan@rutgers.edu (M.K.);
jkral@modernmeadow.com (J.M.K.); anajurs92@gmail.com (S.L.); RLartey@modernmeadow.com (R.L.);
mleonard@modernmeadow.com (M.L.); slue@modernmeadow.com (S.W.L.);
jmcdaniel@modernmeadow.com (J.M.); KRamirez@modernmeadow.com (K.R.);
brauw@modernmeadow.com (B.R.); kraymond@modernmeadow.com (K.A.R.);
croggerolovisi@modernmeadow.com (C.R.-L.); SRubin@modernmeadow.com (S.R.);
krueblingjass@modernmeadow.com (K.R.-J.); zspiegelhoff@modernmeadow.com (Z.S.);
mstewart@modernmeadow.com (M.C.S.); shashwat36@gmail.com (S.V.); avicente@modernmeadow.com (A.V.);
kvincent@modernmeadow.com (K.E.V.); jwang@modernmeadow.com (J.W.);
dwilliamson@modernmeadow.com (D.W.); joeboinc00@gmail.com (Z.Y.)



* Correspondence: ldai@modernmeadow.com


Citation: Aly, N.; Benoit, E.;
Abstract: Collagen and its derivative proteins have been widely used as a major component for
Chaubard, J.-L.; Chintalapudi, K.;
cosmetic formulations as a natural ingredient and moisturizer. Most commercially available collagens
Choung, S.; de Leeuw, M.; Diaz, M.;
Dueppen, D.; Ferraro, B.; Fischetti, V.;
are animal-derived collagen type I and other forms of collagen, such as type III collagen, are far less
et al. Cosmetic Potential of a prevalent in animals, making extraction and purification extremely difficult and expensive. Here, we
Recombinant 50 kDa Protein. report the production of a 50 kDa protein produced in yeast that is 100% identical to the N-terminus
Cosmetics 2022, 9, 8. https://doi.org/ of the human type III collagen. This recombinant protein has a larger molecular weight than most
10.3390/cosmetics9010008 incumbent recombinant collagen proteins available for personal care applications. We report the
industrialization of both the fermentation and purification processes to produce a final recombinant
Academic Editor: Alina Sionkowska
protein product. This final protein product was shown to be safe for general applications to human
Received: 6 December 2021 skin and compatible with common formulation protocols, including ethanol-based formulations.
Accepted: 29 December 2021 This recombinant collagen type III protein was also shown to uniquely stimulate both collagen type I
Published: 5 January 2022
and type III production and secretion by primary human dermal fibroblasts. The unique combination
Publisher’s Note: MDPI stays neutral of biostimulation, compatibility with beauty product formulations and demonstrated commercial
with regard to jurisdictional claims in production, make this novel recombinant type III collagen a good candidate for broad application in
published maps and institutional affil- the cosmetics industry.
iations.

Keywords: collagen; collagen III; Pichia; formulation; recombinant; cosmetics

Copyright: © 2022 by the authors.

Licensee MDPI, Basel, Switzerland.
1. Introduction
This article is an open access article
distributed under the terms and Collagen is the most abundant protein in the human body, present in connective
conditions of the Creative Commons tissue such as cartilage, bones, tendons, ligaments and skin [1]. The collagen superfamily
Attribution (CC BY) license (https:// comprises twenty-eight members in which type I collagen is the most abundant form
creativecommons.org/licenses/by/ (~80–85% of total collagen) and the major protein in the extra-cellular matrix of human
4.0/).

Cosmetics 2022, 9, 8. https://doi.org/10.3390/cosmetics9010008 https://www.mdpi.com/journal/cosmetics

Cosmetics 2022, 9, 8 2 of 13

cells. It assembles into fibers that form the structural and mechanical scaffold (matrix) of
bone, skin, tendons, cornea, blood vessel walls and other connective tissues.
Collagen type III is the second most abundant collagen (~10–15% of total collagen). It
is primarily produced by young fibroblasts before tougher type I collagen is synthesized [2].
Collagen type III is found in granulation tissue, artery walls, skin, intestines and the
uterus [3–5]. Both collagen type I and III are fibrillar collagens that form higher order
structures (particularly fibrils, fibers and bundles), but they have slightly different structural
properties. Type I collagen tends to form densely packed fibrils and rigid triple helices for
structure building. On the other hand, type III collagen forms a more flexible triple helical
structure that assembles into small fibrils that are associated with the more rigid type I
collagen fibrils. As a result, collagen type III functions as a “modulator” of overall tissue
elasticity and function. Indeed, the ratio of collagen type I/III correlates with skin aging
and elasticity [6–9].
Type I collagen is frequently used as a supplement in cosmetics products. While most
commercial forms of collagen come from mammals, such as bovine or porcine, other animal
sources such as chicken, fish skin and jelly fish have also been reported [10–16]. Although
animal-derived collagen is generally abundant and cheap, it does suffer drawbacks such as
risk of infectious disease transmission, potential viral vector transmission from animals
to humans, causing allergic reaction within the human body and can have an unpleasant
smell and color [17]. Non-type I collagens, such as type III collagen, tend to be very rare
and expensive due to challenges with sourcing sufficient protein quantities and purifying
from natural sources [18].
In the last two decades, more and more effort has been put into recombinant collagen
production as part of the rapid development of genetic engineering technology. Recom-
binant collagen molecules of different sizes have been expressed in all major expression
platforms including mammalian cells, insect cells, yeast, bacteria and plant cells [19–37]. In
general, high quality full-length collagen proteins have been produced in eukaryotic hosts
but with very low productivity. Prokaryotic hosts, such as E. coli were also explored for pro-
duction of unmodified collagen; however, these recombinant proteins were generally either
very small (a few kDa up to 20 kDa) or differ extensively from wild-type human collagen
sequences. For example, it is well known that prokaryotic hosts lack post-translational
modification functions that are needed to generate the hydroxyproline amino acid residues
that are found in mature, animal-derived collagen [38–40]. As such, previously produced
recombinant collagen proteins generally only exhibit an unmodified collagen sequence.
To produce a novel collagen-based ingredient that is natural, safe, sustainable and
inexpensive for cosmetics application we developed a scalable platform that produced a
50 kDa protein in a eukaryotic host (Pichia pastoris) that is 100% identical to an N-terminal
portion of human type III collagen. We successfully increased production to an industrial
scale and the final protein preparation has been shown to be safe to human skin, being
neither allergenic nor mutagenic. It is also compatible with all formulation protocols we
tested. Unexpectedly, this protein stimulated collagen synthesis in primary human dermal
fibroblast assays making it an exciting new bioactive product for the cosmetics industry.

2. Materials and Methods

2.1. Formulation Compatibility Evaluation
The testing materials were each dissolved at 1% by weight concentration in deionized
water, with 1% glycols for preservation. The resulting solutions were added at 3% by weight
into seven base formulations to test compatibility and performance: gel (carbomer system);
serum (ammonium acryloyldimethyltaurate/VP copolymer and acrylates/C10-30; alkyl
acrylate crosspolymer system); cream emulsion (oil/water); hair conditioner (containing
cetrimonium chloride and behentrimonium methosulfate); shampoo/cleanser (sulfate-free);
toner (water-based system); toner (alcohol-based system). The result is repeatable in three
individual experiments. Skin fell evaluation was a blind analysis of each material in a
1% solution with a request for feedback regarding stickiness, tack, roll-up, irritation, or
Cosmetics 2022, 9, 8 3 of 13

any other feel-specific observation that the testers might comment on. The testers include
chemists and licensed aesthetician.

2.2. EpiOcular
An EpiOcular irritation test was performed using a previously disclosed protocol [41].

2.3. Bacterial Reverse Mutation Assay

A bacterial reverse mutation assay (Ames Test) was done according to a previously
used protocol [42]. The bacterial reverse mutation assay was used to evaluate the mutagenic
potential of the test sample at a single concentration of the test sample: 1%. Testing was
done with the appropriate solvent control and positive controls were plated with overnight
cultures of the test systems (Salmonella Typhimurium TA97a, Salmonella Typhimurium TA98,
Salmonella Typhimurium TA100, Salmonella Typhimurium TA102, Salmonella Typhimurium
TA1535) on the selective minimal agar in the presence and absence of the Acroclor-induced
rat liver S9. All dose levels of the test samples, solvent controls and positives were plated
in triplicates.

2.4. MTT Assay

Primary human dermal fibroblast cells (ThermoFisher Scientific, Waltham, MA USA)
were seeded in a petri dish. After a 2-day incubation, the cell culture medium was removed
and the fibroblasts were washed twice with PBS to remove any remaining test material.
After the final wash, 500 µL of DMEM supplemented with 0.5 mg/mL MTT was added to
each well and the cells were incubated for 1 h at 37 ◦ C and 5% CO2 . After the incubation,
the DMEM/MTT solution was removed and the cells were washed again once with PBS.
Isopropyl alcohol (0.5 mL) was added to the well to extract the purple formazin crystals.
Two hundred microliters of the isopropyl extracts were transferred to a 96-well plate and
the plate was read at 540 nm using isopropyl alcohol as a blank. The mean MTT absorbance
value for the negative control cells was calculated and used to represent 100% cell viability.
The individual MTT values from the cells undergoing the various treatments were then
divided by the mean value for the negative control cells and expressed as a percent to
determine the change in cell viability caused by each treatment.

2.5. Type I Collagen Assay

Human primary dermal fibroblasts were seeded into the individual wells of a 24-well
plate in 0.5 mL of Fibroblast Growth Media (FGM) and incubated overnight at 37 ± 2 ◦ C
and 5 ± 1% CO2 . On the following day the media was removed via aspiration to eliminate
any non-adherent cells and replaced with 0.5 mL of fresh FGM. The cells were grown until
confluent, with a media change every 48 to 72 h. Upon reaching confluency the cells were
treated for 24 h with DMEM supplemented with 1.5% FBS to wash out any effects from the
growth factors included in the normal culture media. After this 24 h wash out period the
cells were treated with the test materials at the specified concentrations dissolved in FGM
with 1.5% FBS. TGF-β (50 ng/mL) was used as a positive control for collagen. Untreated
cells (negative controls) just received DMEM with 1.5% FBS. The cells were incubated for
48 h and at the end of the incubation period cell culture medium was collected and either
stored frozen (−75 ◦ C) or assayed immediately. Materials were tested in triplicate.
A series of type I C-peptide standards were prepared ranging from 0 ng/mL to
640 ng/mL. Next, an ELISA microplate was prepared by removing any unneeded strips
from the plate frame followed by the addition of 100 µL of peroxidase-labeled anti-
procollagen type I-C peptide antibody to each well used in the assay. Twenty microliters of
either the collected tissue culture media or the standard was then added to appropriate
wells and the microplate was covered and allowed to incubate for 3 ± 0.25 h at 37 ◦ C.
After incubation, the wells were aspirated and washed three times with 400 µL of wash
buffer. The final wash was removed; 100 µL of a peroxidase substrate solution (hydrogen
peroxide + tetramethylbenzidine as a chromagen) was added to each well and the plate was
Cosmetics 2022, 9, 8 4 of 13

incubated for 15 ± 5 min at room temperature. After the incubation, 100 µL of stop solution
(1 N sulfuric acid) was added to each well and the plate was read using a microplate reader
at 450 nm.

2.6. Type III Collagen Assays

A series of standards were prepared and 100 µL of these standards or samples were
added to the wells of the type III collagen ELISA plates. The plates were then incubated
at 37 ◦ C for 1.5 h. After this incubation, the ELISA plates were washed twice with wash
buffer, followed by the application of 100 µL of detection antibody solution. The ELISA
plates were then incubated for 1 h at 37 ◦ C. After incubation, all the ELISA plates were
washed with wash buffer, followed by the addition of 100 µL of HRP conjugate solution and
incubated at 37 ◦ C for 30 min. After this incubation the ELISA plates were again washed
and 100 µL of substrate solution was added to each well and the plates were incubated for
10–30 min at room temperature to allow the color generation reaction to occur. At the end
of the color generation reaction, 100 µL of stop solution was added to each well and the
plates were read at 460 nm using a plate reader.

3. Results
3.1. Characterization of 50 kDa Protein
The production of recombinant 50 kDa protein is summarized in Figure 1A. We first
generated a plasmid containing a promoter, a secretion signal and N-terminus of the human
COL3A1 gene codon optimized for expression in Pichia pastoris. The circular plasmid was
linearized and transformed into Pichia pastoris cells and transformants were selected
Cosmetics 2022, 8, x FOR PEER REVIEW 5 of 15
on
YPD plates with antibiotics (Zeocin, 100 µg/mL).

Figure 1. (A) Workflow to produce 50 kDa protein. (B) SDS PAGE analysis of 50 kDa protein and
Figure
other 1. (A) Workflow
commercially to produce
available collagen50proteins.
kDa protein.
Lane(B) SDS PAGE
1, protein analysis
ladder; Lane 2of&3,
50 kDa protein
Modern and
Meadow
other commercially available collagen proteins. Lane 1, protein ladder; Lane 2 &3, Modern
50 kDa protein; Lane 4, commercially available collagen protein sample # 1 (marine collagen); Lane Meadow
50 kDa protein; Lane 4, commercially available collagen protein sample # 1 (marine collagen); Lane
5, commercially available collagen protein sample # 2 (recombinant collagen produced in bacteria).
5, commercially available collagen protein sample # 2 (recombinant collagen produced in bacteria).
(C)
(C)The
Thefinal
finalpurified
purified5050
kDa
kDaprotein prep
protein doesn’t
prep contain
doesn’t detectable
contain genetically
detectable modified
genetically DNA.
modified Lane
DNA.
1,Lane
DNA 1, DNA ladder; Lane 2, positive control PCR (10 ng plasmid DNA was used); Lane 3, PCRof
ladder; Lane 2, positive control PCR (10 ng plasmid DNA was used); Lane 3, PCR result
result
50 kDaof 50 kDasolution
protein proteinatsolution at 2%
2% (1 µL was(1used
μL was used as template).
as template).

After several rounds of screening, the best clone (PP1186) was promoted into fermen-
tation development. A three-step fermentation process was started by inoculating the best
clone in shake flasks, followed by a seed fermentation lasting approximately 23–27 h,
Cosmetics 2022, 9, 8 5 of 13

After several rounds of screening, the best clone (PP1186) was promoted into fermen-
tation development. A three-step fermentation process was started by inoculating the best
clone in shake flasks, followed by a seed fermentation lasting approximately 23–27 h, which
was then used to inoculate a production fermenter with BMGY media. The production
fermenter was run for about 72 h with a 24 h batch phase followed by a 48 h fed-batch phase.
At the end of fermentation, yeast cells were removed by centrifugation and the supernatant
containing target protein was processed to further purify the recombinant protein.
The purified protein was diluted to 1% by weight and resolved on SDS PAGE (Bis-Tris
4–20%). Due to the protein’s extended conformation, it migrated at ~65 kDa, which is larger
than the actual molecular weight of ~ 50 kDa (Figure 1B). Lanes 4 and 5 are two commercial
collagen samples at 1%. We also checked for any residual genetically modified DNA in the
final protein prep by PCR assay. As seen in Figure 1C, a PCR product between 500–750 bp
(lane 2) was detected when the PCR template was the plasmid used to transform yeast cells.
No band was detected under the same condition when 1 µL final protein prep was used as
template at 2% (lane 3).

3.2. Formulation Testing and Potential Applications

First, we assessed the appearance and rheology of this protein. It dissolved quickly
when mixing at 50 RPM, did not perceive any solubility issues. When dissolved in water,
it shows as a clear to slightly hazy liquid and very flowable with low viscosity. This
1% protein solution readily incorporated into deionized water and ethanol. We tested
various formulations including Gel, Serum, Cream, Hair conditioner, Shampoo, Toner (both
water-based and alcohol-based), Carbomer Gel System, Acrylates Polymer Gel System,
Emulsion System Hair Conditioner and Sulfate-free Cleanser and found this recombinant
50 kDa protein is compatible with all formulations within 10 min of mixing. Notably, the
bioactive protein exhibited good color and odor and could easily be incorporated into
certain formulas.
There was no negative skin feel observed for any of the 1% solutions at any of the eval-
uated levels. Enhanced elasticity, wrinkle reduction, smoothing, moisture-loss prevention,
firming and film forming are all potential benefits seen or expected when incorporating
the recombinant protein into typical formulations. As such, we believe this material could
be safely formulated into the products listed in Table 1, as well as many other products,
with a reasonable expectation that these formulations will be safe, stable and well suited
for topical formulations in the personal care market.

Table 1. Potential cosmetic applications of 50 kDa protein.

Major Category Specific Examples

Eye care—Cream, serum, mask (sheet, pad, cream, etc..), treatment
Eyebrow—serums, primer and treatment
Eyelash—serums, primer and treatment
Face care
• Sun Care Products, Day and/or night, tinted or not, with/without SPF
• Face, neck and decollete
Skincare Serum, lotion, cream, balm, gel, oil, oil in cream, toner, micellar water, face mist, face essence, BB
formula and CC formula, face mask (sheet, pad, cream, etc..), Self-tanning face care, face sunscreen,
cleansing oil
Lip care (tinted or not, with/without SPF)—lip oil, lip balm, lip mask, lip treatment
Body care
• Overall body, hand, feet
• Stretch mark treatment
• Tinted or not, with/without SPF
Cosmetics 2022, 9, 8 6 of 13

Table 1. Cont.

Major Category Specific Examples

Moisturizer (all forms lotion, cream, gel, balm, oil, oil in cream, mist), body butter, mask,
moisturizing cleanser, self-tanning body care, body sunscreen
• Face exfoliator, cleanser, peel and make up remover
• Lip scrub, exfoliator
• Body cleanser, scrub, exfoliator, bath/soak cleanser
• Cellulite treatment
Haircare—prewash hair treatment, hair mist, scalp treatment and/or oil, serum, primer, leave-in
conditioner, hair mask, hair toner, leave on spray, lotion, haircare shampoo and dry shampoo, wash
off conditioner, hair dye, scalp scrub and all haircare styling product
Shaving—Aftershave balm, razor burn and bumps relief, bear oil
• Shaving products—men/women—body/face/head—cream, foam, oil, lotion, gel -Deodorant
and anti-perspirant

• Foundation all forms—liquid, cream, powder (compact/loose)

• Concealer all forms—liquid, cream, stick
• Color corrector—cream, stick, etc.
• Make up Base, Make up primer, Setting spray/powder
• Eye primer
• Lipstick, lip gloss, lip oil, lip balm, lip stain
Makeup
• Highlight, bronzer
• Eye shadow
• Lip and eye liner
• Lip plumper
• Mascara
• Contour, blush in form of powder, cream, stick
Supplements/drinks

3.3. Safety Testing

We conducted ocular irritation test to verify the expected safety performance of this
50 kDa protein. Ocular irritation is defined as reversible damage to the eye following
application of a test substance, whereas ocular corrosion is defined as irreversible damage.
The data from EpiOcular test predicted that this protein to be classified as a GHS NC, with
an average viability score of 85.3% and passed quality control checks.
We also assessed the risk of the protein to induce bacterial reverse mutation using
the classical Ames test. This test was used to evaluate the mutagenic potential of the test
sample at a 1% concentration. In the Ames mutagenic potential evaluation, this protein
was found to be not cytotoxic to the test system and there was no detectable genotoxic
activity associated with this protein at the 1% concentration.

3.4. Collagen Synthesis Induction in Human Primary Dermal Fibroblast by 50 kDa Protein
We used a fibroblast cell culture model to assess the ability of the recombinant 50 kDa
protein to affect collagen synthesis. Fibroblasts are the main source of extracellular matrix
peptides, including the structural proteins collagen and elastin. Procollagen is a large
protein synthesized by fibroblasts in the dermal layer of the skin and is the precursor for
collagen. As the protein chain is processed to form a mature collagen protein, the pro-
peptide portion is cleaved off (forming a type I C-peptide). Both the mature collagen protein
and the type I C- peptide fragments are then released into the extracellular environment.
As collagen is synthesized, the type I C-peptide fragment accumulates into the tissue
culture medium. Since there is a 1:1 stoichiometric ratio between the two parts of the
procollagen peptide, assaying for type I C-peptide will reflect the amount of collagen
protein synthesized. Type 1 C-peptide can be assayed via an ELISA-based method.
Cosmetics 2022, 9, 8 7 of 13

In addition, the viability of cells after exposure to the protein prep was assessed by the
MTT assay. The MTT assay is a colorimetric analysis of the metabolic activity of the cell,
which reflects the number of viable cells.
As seen in Figure 2A, untreated cells were used as a negative control and set as 100%
and addition of 50 ng/mL TGFβ1 slightly increases cell viability by ~20%. The recombinant
Cosmetics 2022, 8, x FOR PEER REVIEW
50 kDa protein and other commercial samples did not decrease cell viability 8 of 15
significantly at
different concentrations.

Figure 2. MTT assay and collagen synthesis induction data. C, negative control with no treatment;
Figure 2. MTT assay and collagen synthesis induction data. C, negative control with no treatment;
T, TGFβ at 50 ng/mL final concentration; bar #1, 0.1%; bar #2, 0.05%; bar #3, 0.01%; bar #4, 0.005%;
T, TGFβ at 50 bar
bar #5, 0.001%; ng/mL final
#6, 0.1%; barconcentration;
#7, 0.05%; bar #8,bar #1, 0.1%;
0.01%; bar #9,bar #2, 0.05%;
0.005%; bar #10;bar
bar #3,
#11,0.01%;
1%; barbar #4, 0.005%;
#12, #5,
bar 0.5%; bar #13,bar
0.001%; 0.1%; bar0.1%;
#6, #14, 0.05%; bar0.05%;
bar #7, #15, 0.01%;
barSample #1 commercial
#8, 0.01%; collagen sample
bar #9, 0.005%; bar #10;#1 bar
in #11, 1%; bar
Figure
#12, 1B; Sample
0.5%; bar #13,#2 0.1%;
commercial collagen
bar #14, sample
0.05%; #2 in 0.01%;
bar #15, Figure 1B. All samples
Sample were first dissolved
#1 commercial collagen sample #1 in
in water at 2% and added into fibroblast growth media at different concentrations (A) MTT assay.
Figure 1B; Sample
(B) collagen #2 (C)
type I assay commercial
collagen typecollagen sample #2 in Figure 1B. All samples were first dissolved
III assay.
in water at 2% and added into fibroblast growth media at different concentrations (A) MTT assay.
(B) collagen type I assay (C) collagen type III assay. * p < 0.0001.
Cosmetics 2022, 9, 8 8 of 13

Both collagen type I and type III synthesis assays were performed under the same
conditions. While commercial marine collagen samples did not stimulate any collagen I
production, 50 kDa protein (0.1%) significantly increased collagen I production in fibroblast
to ~200% of baseline (Figure 2B). This collagen induction effect is comparable to TGFβ1,
which is well known as a potent collagen synthesis stimulator. This collagen induction
activity was not detected at lower concentrations of the 50 kDa protein (0.05% or lower).
Similarly, the recombinant 50 kDa protein stimulated collagen III production in fi-
broblast with an even higher potency. The collagen protein prepared at both 0.1% and
0.05% significantly increased soluble collagen III synthesis more effectively than the TGFβ1
control (Figure 2C). The commercial marine collagen samples had no or some negative
effect on collagen III synthesis at the same concentrations.

4. Discussion
Many previous studies have tried to produce recombinant collagen molecules in dif-
ferent systems including bacteria, yeast and plant [25,28–31,37,43–46]. These recombinant
proteins include full-length or fragments of collagen I, II, III and XXI at different hydroxy-
lation levels (unhydroxylated or hydroxylated). All hydroxylated collagen proteins were
produced intracellularly with co-expression of P4H enzymes resulting in low productivity
and recovery rate due to cell lysing. Some unhydroxylated collagen proteins were pro-
duced by secretory pathway but they only represent a very small portion of the full-length
collagen polypeptide (<30 kDa). In other cases, collagen-like protein (CLP) was used or
the wild-type collagen sequence was modified to enhance productivity and stability. This
study illustrated the production and characterization of a 50 kDa recombinant protein
from a eukaryotic host (yeast). Unlike many other recombinant collagen-like proteins, this
protein is 100% identical to human collagen type III alpha chain without any sequence
manipulation and mimics natural collagen III function. Given the close correlation between
aging and the collagen I/III ratio, this protein could well balance the collagen composition.
Because this protein is produced in a single-cell organism (yeast), it may be amenable
to scaling the fermentation process to industrial scale to reduce cost. We used simple
fermentation media and mostly physical separation to purify the final sample and the
resulting protein preparation is highly pure and free of harmful chemicals (strong acid or
base), which are often seen in animal-derived collagen preparations. Since Pichia pastoris is
a eukaryotic host, we also have the flexibility to add post-translational modifications on the
final protein, such as proline-4 hydroxylation.
Many collagen samples suffer from poor color, possess an odor, have poor solubility
and are difficult or impossible to formulate for use in personal care products. This 50 kDa fi-
nal protein prep is odorless and has only light color at high concentrations. When we tested
it in various formulation types, it was compatible with all formulations assessed, including
ethanol-based protocols. Furthermore, from a safety perspective the Occular Irritation and
Ames tests results verified the safety of the protein for personal care applications.
Collagen peptides have been shown to stimulate collagen I synthesis in human der-
mal fibroblast cultures, but only when they were mixed with antioxidant constituents
such as Carnosine, CoEnzyme Q10, Resveratrol (red wine powder extract), Borage seed
oil/Primrose oil Hyaluronic acid Piperine (Piper Nigrum seed extract), Lycopene (tomato
pulp extract), Acai berry extract or Pomegranate juice (concentrated) [47]. Unexpectedly,
this 50 kDa human collagen protein alone was able to enhance both collagen I and III
production in fibroblasts comparable to or even better than the TGFβ1 positive control [48].
A specific motif in collagen type I alpha 2 chain has been found to induce collagen I syn-
thesis in human dermal fibroblasts, but no such motif has been reported for collagen type
III induction [49].
All these properties make this 50 kDa protein a good candidate for personal care
product development. A panel of basic formulations have been tested for this protein and
collagen stimulation data were acquired by in vitro cell culture assay. Further studies to
Cosmetics 2022, 9, 8 9 of 13

better evaluate this novel recombinant protein include testing broader formulation testing
and collagen induction study in skin model and clinical studies.
There are many known uses for collagen in the cosmetics and skincare industry. For
example, skincare compositions that include collagen can be used to combat the effects of
ageing and environmental stress on the appearance, elasticity and thickness of skin [50].
Ageing and environmental factors can lead to dermatological conditions including, but
not limited to fine lines, wrinkles, dry skin, excessive pore size, skin dyschromia, reduced
elasticity, unwanted hair, skin thinning, purpura, actinic keratosis, pruritus, eczema, acne,
rosacea, erythema, telangiectasia, actinic telangiectasia, skin cancer and rhinophyma. Oral
collagen supplements/drinks have been shown to improve skin hydration and elasticity
for older people [51–54]. Although there are numerous skincare products on the market
to improve skin appearance, many consumers are hesitant to use chemically synthesized
products they perceive as being environmentally unfriendly or otherwise unsafe.
Therefore, this 50 kDa protein potentially can be used to treat a wide range of dermal
conditions and applied in a variety of different compositions. Since this protein was
produced in a natural yeast host and purified mostly by physical separation, it is a safer and
more environmentally friendly substitute for animal-derived collagen. To achieve these
goals, this recombinant protein can be formulated into various formulations described
herein (Table 2).

Table 2. Potential formulations of 50 kDa protein.

Ingredients: %wt/wt
Alcohol-Based Toner
Water (Aqua) Remainder Ingredient
Recombinant collagen
about 0.0005% to about 25%
fragment disclosed herein
Alcohol Denat. 10–20%
Pentylene Glycol 5–10%
Glycerin 1–5%
Gluconolactone 0.1–1%
Dipotassium Glycyrrhizate 0.1–1%
Sodium Citrate 0.1–1%
Sodium Benzoate 0.1–1%
Water-Based Toner
Water (Aqua) Remainder Ingredient
Recombinant collagen
about 0.0005% to about 25%
fragment disclosed herein
Niacinamide 1–5%
Pentylene Glycol 1–5%
Propanediol 1–5%
Glycerin 1–5%
Biosaccharide Gum-1 0.1–1%
Glyceryl Caprylate 0.1–1%
Sodium Anisate 0.1–1%
Sodium Hydroxide 0.1–1%
Caprylhydroxamic Acid 0.1–1%
Acrylates/C10-30 Alkyl
0.1–1%
Acrylate Crosspolymer
Sodium Levulinate 0.1–1%
Caprylyl Glycol 0.1–1%
Cream
Water (Aqua) Remainder Ingredient
Recombinant collagen
about 0.0005% to about 25%
fragment disclosed herein
Cosmetics 2022, 9, 8 10 of 13

Table 2. Cont.

Ingredients: %wt/wt
Cetearyl Alcohol 5–10%
Glycerin 1–5%
Squalane 1–5%
Butyrospermum Parkii (Shea) Butter 1–5%
Glyceryl Caprylate 1–5%
Microcrystalline Cellulose 1–5%
Glyceryl Stearate Citrate 0.1–1%
Tocopheryl Acetate 0.1–1%
Cetearyl Glucoside 0.1–1%
Sodium Stearoyl Glutamate 0.1–1%
Cellulose Gum 0.1–1%
Xanthan Gum 0.1–1%
Caprylhydroxamic Acid 0.1–1%
Sodium Phytate 0.01–0.1%
Gel
Water (Aqua) Remainder Ingredient
Recombinant collagen about 0.0005% to about
fragment disclosed herein 25%
Sodium Phytate 0.1–1%
Sodium Hydroxide 0.1–1%
Carbomer 0.1–1%
Phenoxyethanol 0.1–1%
Serum
Water (Aqua) Remainder Ingredient
Recombinant collagen fragment
about 0.0005% to about 25%
disclosed herein
Pentylene Glycol 1–5%
Niacinamide 1–5%
Dimethicone 1–5%
Propanediol 1–5%
Tocopherol 0.1–1%
Sodium Hyaluronate 0.1–1%
Linoleic Acid 0.1–1%
Ammonium
Acryloyldimethyltaurate/VP 0.1–1%
Copolymer
Acrylates/C10-30 Alkyl Acrylate
0.1–1%
Cross polymer
Caprylyl Glyceryl Ether 0.1–1%
Tetrasodium Glutamate Diacetate 0.01–0.1%
Sodium Hydroxide 0.01–0.1%
Phenoxyethanol 0.01–0.1%
Linolenic Acid 0.001–0.0
Shampoo
Water (Aqua) Remainder Ingredient
Recombinant collagen
about 0.0005% to about 25%
fragment disclosed herein
Cocamidopropyl Betaine 5–10%
Sodium Lauroyl Methyl
5–10%
Isethionate
Propanediol 1–5%
Sodium Methyl Oleoyl
1–5%
Taurate
Sodium Cocoyl Isethionate 1–5%
Trisodium Ethylenediamine
0.1–1%
Disuccinate
Cosmetics 2022, 9, 8 11 of 13

Table 2. Cont.

Ingredients: %wt/wt
Caprylhydroxamic Acid 0.1–1%
Panthenol 0.1–1%
Citric Acid 0.1–1%
Caprylyl Glycol 0.1–1%
Sodium Benzoate 0.1–1%
Conditioner
Water (Aqua) Remainder Ingredient
Recombinant collagen
about 0.0005% to about 25%
fragment disclosed herein
Cetearyl Alcohol 5–10%
Glyceryl Caprylate 1–5%
Behentrimonium
1–5%
Methosulfate
Glycerin 1–5%
Caprylhydroxamic Acid 0.1–1%
Panthenol 0.1–1%
Hydroxyethylcellulose 0.1–1%
Cocos Nucifera (Coconut)
0.1–1%
Oil
Citric Acid 0.01–0.1%

5. Conclusions
We reported the production of a recombinant 50 kDa protein in yeast at large scale.
This protein is safe to human skin, compatible with many different formulations, stimulates
collagen synthesis in human dermal fibroblast and we proposed its potential for broad
cosmetic applications.

Author Contributions: Conceptualization, L.D.; methodology, J.-L.C., K.C., M.D., M.K., R.L., S.L.,
M.L., K.R., Z.S., M.C.S. and Z.Y.; investigation, N.A., E.B., M.d.L., B.F., E.G., A.H., C.K., J.M.K., J.M.,
S.R., K.R.-J., S.V., A.V., K.E.V. and J.W.; data curation, V.F.; writing—original draft preparation, L.D.,
C.R.-L. and I.H.-C.; writing—review and editing, C.R.-L., I.H.-C., S.C. and D.W.; supervision, D.D.,
B.R., K.A.R. and S.W.L. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Paolo Giacomoni and Peter Foltis for critical review of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Ricard-Blum, S. The collagen family. Cold Spring Harb. Perspect. Biol 2011, 3, a004978. [CrossRef]
2. Fleischmajer, R.; MacDonald, E.D.; Perlish, J.S.; Burgeson, R.E.; Fisher, L.W. Dermal collagen fibrils are hybrids of type I and type
III collagen molecules. J. Struct. Biol. 1990, 105, 162–169. [CrossRef]
3. Kuivaniemi, H.; Tromp, G. Type III collagen (COL3A1): Gene and protein structure, tissue distribution, and associated diseases.
Gene 2019, 707, 151–171. [CrossRef] [PubMed]
4. Nielsen, M.J.; Karsdal, M.A. Type III Collagen. In Biochemistry of Collagens, Laminins and Elastin, 1st ed.; Karsdal, M.A., Ed.;
Academic Press: Cambridge, MA, USA, 2016; pp. 21–30.
5. Manturova, G.O.; Smirnova, V.A.; Stupin, E.V.; Silina, N.E. The ratio of collagen types I/III as a marker of skin aging and
prognosis of aesthetic facial surgery results. J. Pharm. Sci. Res. 2018, 10, 2543–2546.
6. Wang, C.; Rong, Y.; Ning, F.; Zhang, G. The content and ratio of type I and III collagen in skin differ with age and injury. Afr. J.
Biotechnol. 2011, 10, 2524–2529.
Cosmetics 2022, 9, 8 12 of 13

7. Parkin, J.D.; San Antonio, J.D.; Persikov, A.V.; Dagher, H.; Dalgleish, R.; Jensen, S.T.; Jeunemaitre, X.; Savige, J. The collagen III
fibril has a “flexi-rod” structure of flexible sequences interspersed with rigid bioactive domains including two with hemostatic
roles. PLoS ONE 2017, 12, e0175582. [CrossRef]
8. Liu, X.; Wu, H.; Byrne, M.; Krane, S.; Jaenisch, R. Type III collagen is crucial for collagen I fibrillogenesis and for normal
cardiovascular development. Proc. Natl. Acad. Sci. USA 1997, 94, 1852–1856. [CrossRef]
9. Wang, C.; Brisson, B.K.; Terajima, M.; Li, Q.; Hoxha, K.; Han, B.; Goldberg, A.M.; Sherry Liu, X.; Marcolongo, M.S.; Enomoto-
Iwamoto, M.; et al. Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage
and meniscus. Matrix Biol. 2020, 85–86, 47–67. [CrossRef]
10. Cliche, S.; Amiot, J.; Avezard, C.; Gariépy, C. Extraction and characterization of collagen with or without telopeptides from
chicken skin. Poult. Sci. 2003, 82, 503–509. [CrossRef] [PubMed]
11. Munasinghe, K.A.; Schwarz, J.G.; Nyame, A.K. Chicken Collagen from Law Market Value By-Products as an Alternate Source. J.
Food Process. 2014, 2014, 298295. [CrossRef]
12. Zhou, C.; Li, Y.; Yu, X.; Yang, H.; Ma, H.; Yagoub, A.E.A.; Cheng, Y.; Hu, J.; Out, P.N.Y. Extraction and characterization of chicken
feet soluble collagen. LWT Food Sci. Technol. 2016, 74, 145–153. [CrossRef]
13. Jafari, H.; Lista, A.; Siekapen, M.M.; Ghaffari-Bohlouli, P.; Nie, L.; Alimoradi, H.; Shavandi, A. Fish Collagen: Extraction,
Characterization, and Applications for Biomaterials Engineering. Polymers 2020, 12, 2230. [CrossRef] [PubMed]
14. O’Sullivan, A.; Shaw, N.B.; Murphy, S.C.; van de Vis, J.W.; van Pelt-Heerschap, H.; Kerry, J.P. Extraction of Collagen from Fish
Skins and Its Use in the Manufacture of Biopolymer Films. J. Aquat. Food Prod. Technol. 2006, 15, 21–32. [CrossRef]
15. Khong, N.M.H.; Yusoff, F.M.; Jamilah, B.; Basri, M.; Maznah, I.; Chan, K.W.; Armania, N.; Nishikawa, J. Improved collagen
extraction from jellyfish (Acromitus hardenbergi) with increased physical-induced solubilization processes. Food Chem. 2018, 251,
41–50. [CrossRef]
16. Coppola, D.; Oliviero, M.; Vitale, G.A.; Lauritano, C.; D’Ambra, I.; Iannace, S.; de Pascale, D. Marine Collagen from Alternative
and Sustainable Sources: Extraction, Processing and Applications. Mar. Drugs 2020, 18, 214. [CrossRef]
17. Browne, S.; Zeugolis, D.I.; Pandit, A. Collagen: Finding a solution for the source. Tissue Eng. Part A 2013, 19, 1491–1494. [CrossRef]
18. Asgari, M.; Latifi, N.; Heris, H.K.; Vali, H.; Mongeau, L. In vitro fibrillogenesis of tropocollagen type III in collagen type I affects
its relative fibrillar topology and mechanics. Sci. Rep. 2017, 7, 1392. [CrossRef] [PubMed]
19. Fertala, A. Three Decades of Research on Recombinant Collagens: Reinventing the Wheel or Developing New Biomedical
Products? Bioengineering 2020, 7, 155. [CrossRef]
20. Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R. Introduction of the human pro alpha
1(I) collagen gene into pro alpha 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I
collagen. Proc. Natl. Acad. Sci. USA 1987, 84, 764–768. [CrossRef]
21. Olsen, A.S.; Geddis, A.E.; Prockop, D.J. High levels of expression of a minigene version of the human pro alpha 1 (I) collagen
gene in stably transfected mouse fibroblasts. Effects of deleting putative regulatory sequences in the first intron. J. Biol. Chem.
1991, 266, 1117–1121. [CrossRef]
22. Specks, U.; Mayer, U.; Nischt, R.; Spissinger, T.; Mann, K.; Timpl, R.; Engel, J.; Chu, M.L. Structure of recombinant N-terminal
globule of type VI collagen alpha 3 chain and its binding to heparin and hyaluronan. EMBO J. 1992, 11, 4281–4290. [CrossRef]
[PubMed]
23. Mazzorana, M.; Gruffat, H.; Sergeant, A.; van der Rest, M. Mechanisms of collagen trimer formation. Construction and expression
of a recombinant minigene in HeLa cells reveals a direct effect of prolyl hydroxylation on chain assembly of type XII collagen. J.
Biol. Chem. 1993, 268, 3029–3032. [CrossRef]
24. Fertala, A.; Sieron, A.L.; Ganguly, A.; Li, S.W.; Ala-Kokko, L.; Anumula, K.R.; Prockop, D.J. Synthesis of recombinant human
procollagen II in a stably transfected tumour cell line (HT1080). Biochem. J. 1994, 298, 31–37. [CrossRef] [PubMed]
25. Olsen, D.; Yang, C.; Bodo, M.; Chang, R.; Leigh, S.; Baez, J.; Carmichael, D.; Perälä, M.; Hämäläinen, E.R.; Jarvinen, M.; et al.
Recombinant collagen and gelatin for drug delivery. Adv. Drug Deliv. Rev. 2003, 55, 1547–1567. [CrossRef] [PubMed]
26. Fertala, A.; Shah, M.; Hoffman, R.; Arnold, W.V. Designing recombinant collagens for biomedical applications. Curr. Tissue Eng.
2016, 5, 73–84. [CrossRef]
27. Bulleid, N.J.; John, D.C.; Kadler, K.E. Recombinant expression systems for the production of collagen. Biochem. Soc. Trans. 2000,
28, 350–353. [CrossRef]
28. Ruggiero, F.; Exposito, J.Y.; Bournat, P.; Gruber, V.; Perret, S.; Comte, J.; Olagnier, B.; Garrone, R.; Theisen, M. Triple helix assembly
and processing of human collagen produced in transgenic tobacco plants. FEBS Lett. 2000, 469, 132–136. [CrossRef]
29. Xu, X.; Gan, Q.; Clough, R.C.; Pappu, K.M.; Howard, J.A.; Baez, J.A.; Wang, K. Hydroxylation of recombinant human collagen
type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase. BMC Biotechnol. 2011, 11, 69.
[CrossRef]
30. Gellermann, P.; Schneider-Barthold, C.; Bolten, S.N.; Overfelt, E.; Scheper, T.; Pepelanova, I. Production of a Recombinant
Non-Hydroxylated Gelatin Mimetic in Pichia pastoris for Biomedical Applications. J. Funct. Biomater. 2019, 10, 39. [CrossRef]
31. Rutschmann, C.; Baumann, S.; Cabalzar, J.; Luther, K.B.; Hennet, T. Recombinant expression of hydroxylated human collagen in
Escherichia coli. Appl. Microbiol. Biotechnol. 2014, 98, 4445–4455. [CrossRef]
32. John, D.C.; Watson, R.; Kind, A.J.; Scott, A.R.; Kadler, K.E.; Bulleid, N.J. Expression of an engineered form of recombinant
procollagen in mouse milk. Nat. Biotechnol. 1999, 17, 385–389. [CrossRef] [PubMed]
Cosmetics 2022, 9, 8 13 of 13

33. Weber, S.C.; Herz, A.H. Method for Recombinant Yeast Expression and Isolation of Water-Soluble Collagen-Type Polypeptides.
Application No. 383,748; U.S. Patent 5,710,252, 20 January 1998.
34. Mashiko, T.; Takada, H.; Wu, S.H.; Kanayama, K.; Feng, J.; Tashiro, K.; Asahi, R.; Sunaga, A.; Hoshi, K.; Kurisaki, A.; et al.
Therapeutic effects of a recombinant human collagen peptide bioscaffold with human adipose-derived stem cells on impaired
wound healing after radiotherapy. J. Tissue Eng. Regen. Med. 2018, 12, 1186–1194. [CrossRef] [PubMed]
35. Shi, J.; Ma, X.; Gao, Y.; Fan, D.; Zhu, C.; Mi, Y.; Xue, W. Hydroxylation of Human Type III Collagen Alpha Chain by Recombinant
Coexpression with a Viral Prolyl 4-Hydroxylase in Escherichia coli. Protein J. 2017, 36, 322–331. [CrossRef] [PubMed]
36. Vuorela, A.; Myllyharju, J.; Nissi, R.; Pihlajaniemi, T.; Kivirikko, K.I. Assembly of human prolyl 4-hydroxylase and type III
collagen in the yeast pichia pastoris: Formation of a stable enzyme tetramer requires coexpression with collagen and assembly of
a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J. 1997, 16, 6702–6712. [CrossRef]
37. Báez, J.; Olsen, D.; Polarek, J.W. Recombinant microbial systems for the production of human collagen and gelatin. Appl. Microbiol.
Biotechnol. 2005, 69, 245–252. [CrossRef]
38. Sipilä, K.H.; Drushinin, K.; Rappu, P.; Jokinen, J.; Salminen, T.A.; Salo, A.M.; Käpylä, J.; Myllyharju, J.; Heino, J. Proline
hydroxylation in collagen supports integrin binding by two distinct mechanisms. J. Biol. Chem. 2018, 293, 7645–7658. [CrossRef]
39. Rappu, P.; Salo, A.M.; Myllyharju, J.; Heino, J. Role of prolyl hydroxylation in the molecular interactions of collagens. Essays
Biochem. 2019, 63, 325–335.
40. Pihlajaniemi, T.; Myllylä, R.; Kivirikko, K.I. Prolyl 4-hydroxylase and its role in collagen synthesis. J. Hepatol. 1991, 13 (Suppl. S3),
S2–S7. [CrossRef]
41. Kolle, S.N.; Rey Moreno, M.C.; Mayer, W.; van Cott, A.; van Ravenzwaay, B.; Landsiedel, R. The EpiOcular™ Eye Irritation Test is
the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations: Correlation Analysis of EpiOcular Eye
Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes. Altern. Lab.
Anim. 2015, 43, 181–198.
42. Ames, B.N.; Mccann, J.; Yamasaki, E. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-
microsome mutagenicity test. Mutat. Res. 1975, 31, 347–364. [CrossRef]
43. Myllyharju, J.; Nokelainen, M.; Vuorela, A.; Kivirikko, K.I. Expression of recombinant human type I–III collagens in the yeast
Pichia pastoris. Biochem. Soc. Trans. 2000, 28, 353–357. [CrossRef]
44. He, J.; Ma, X.; Zhang, F.; Li, L.; Deng, J.; Xue, W.; Zhu, C.; Fan, D. New strategy for expression of recombinant hydroxylated
human collagen α1(III) chains in Pichia pastoris GS115. Biotechnol. Appl. Biochem. 2015, 62, 293–299. [CrossRef]
45. Nokelainen, M.; Tu, H.; Vuorela, A.; Notbohm, H.; Kivirikko, K.I.; Myllyharju, J. High-level production of human type I collagen
in the yeast Pichia Pastoris. Yeast 2001, 18, 797–806. [CrossRef]
46. Ouzounov, N.; Lorestani, A.; Bhatia, M. Recombinant collagen and elastin molecules and uses thereof. U.S. Patent WO2019068018,
23 May 2019.
47. Edgar, S.; Hopley, B.; Genovese, L.; Sibilla, S.; Laight, D.; Shute, J. Effects of collagen-derived bioactive peptides and natural
antioxidant compounds on proliferation and matrix protein synthesis by cultured normal human dermal fibroblasts. Sci. Rep.
2018, 8, 10474. [CrossRef] [PubMed]
48. Chan, C.P.; Lan, W.H.; Chang, M.C.; Chen, Y.J.; Lan, W.C.; Chang, H.H.; Jeng, J.H. Effects of TGF-beta s on the growth, collagen
synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro. Arch. Oral Biol. 2005, 50, 469–479. [CrossRef]
[PubMed]
49. Hwang, S.J.; Ha, G.H.; Seo, W.Y.; Kim, C.K.; Kim, K.; Lee, S.B. Human collagen alpha-2 type I stimulates collagen synthesis,
wound healing, and elastin production in normal human dermal fibroblasts (HDFs). BMB Rep. 2020, 53, 539–544. [CrossRef]
[PubMed]
50. Bolke, L.; Schlippe, G.; Gerß, J.; Voss, W. A Collagen Supplement Improves Skin Hydration, Elasticity, Roughness, and Density:
Results of a Randomized, Placebo-Controlled, Blind Study. Nutrients 2019, 11, 2494. [CrossRef]
51. Lin, P.; Hua, N.; Hsu, Y.C.; Kan, K.W.; Chen, J.H.; Lin, Y.H.; Kuan, C.M. Oral Collagen Drink for Antiaging: Antioxidation,
Facilitation of the Increase of Collagen Synthesis, and Improvement of Protein Folding and DNA Repair in Human Skin
Fibroblasts. Oxid. Med. Cell. Longev. 2020, 2020, 8031795. [CrossRef] [PubMed]
52. Yamanaka, H.; Okada, S.; Sanada, H. A multicenter, randomized, controlled study of the use of nutritional supplements containing
collagen peptides to facilitate the healing of pressure ulcers. J. Nutr. Intermed. Metab. 2017, 8, 51–59. [CrossRef]
53. Asserin, J.; Lati, E.; Shioya, T.; Prawitt, J. The effect of oral collagen peptide supplementation on skin moisture and the dermal
collagen network: Evidence from an ex vivo model and randomized, placebo-controlled clinical trials. J. Cosmet. Dermatol. 2015,
14, 291–301. [CrossRef]
54. Lee, G.; West, V.; Parker, T.; Vollmer, D. A Multi-Type Collagen-Based Drink Supplement Significantly Improved Markers of
Aging, both in vitro and in a Human Clinical Study. J. Clin. Exp. Dermatol. Res. 2020, 11, 1000531.