SLIDE 1:

To our guests, co-students, friends and our esteemed faculty members, ma’am/sir, ladies and
gentlemen, GOOD MORNING.
In this session, we will be going to delve into how science can turn a thing into something. My Study is
entitled: “Waste No More: The Isolation and Characterization of Collagen from Viscera of Yellowfin Tuna
and Milkfish. With me is Dr. Hernando Bacosa, Dr. Ronald Bual, Job Pague And Engr. Zesreal Cain Bantilan

SLIDE 2:

The escalating demand for fish, coupled with the continuous growth of the fish processing
industry, has led to the generation of a substantial amount of waste. Globally, the fish processing
industries discard approximately 130 million metric tons of fish waste annually (Sayana K.S; Sirajudheen
T.K, 2017). This waste encompasses by-catch, on-board waste, home waste, and processing waste, all of
which contribute to the overall surge in waste production (Michael, S. and Catherine M., 2012).

SLIDE 3:

In the Philippines, the most common fishes produced, among others, are Yellowfin Tuna, and
Milkfish. “It is estimated that about 70% of the tuna’s weight is discarded in the production of canned
fillets (Garofalo et al 2023)” and “ more than 30% of milkfish are discarded as waste in milkfish
processing (Wulandari et al. 2019)

SLIDE 4:

The solid fish waste consists of a head, tail, skin, gut, fins, and frames. These byproducts can be
a great source of value-added products such as proteins, amino acids, collagen, gelatin, oil, enzymes, etc.

SLIDE 5:

One of the most important environment-friendly and profitable options for utilization of fish
waste for the recovery of marketable by-products include the production of animal feed, renewable
energy in the form of biodiesel and biogas, composting for the production of organic fertilizers, and
lastly the meat of this study the extraction of collagen

SLIDE 6:

Collagen is a protein found in the skin and bones that is fibrous and essential to life. The demand
for collagen has grown significantly across industries, driven by its versatile applications and unique
properties. These are mainly derived from land based animals such bovine and porcine, But due to the
concerns about bovine spongiform encephalopathy and foot-and-mouth disease make people nervous
about the safety of collagen from land-based animals (Jongjareonrak, Benjakul, Visessanguan, Nagai, &
al., 2013). Also, specific food applications cannot use collagen from pigs because of religious restrictions
(Sadowska M, 2003). In such cases, there is a need to find an alternative source for collagen.

SLIDE 7:

Collagen from marine animals is regarded as the best alternative source due to its high
availability, lack of disease transmission risk, and lack of religious restrictions.

SLIDE 8:

Though there were existing studies about the extraction of collagen, but they are mostly from
skin, fins, and bones, and there is limited knowledge about extracting collagen from the viscera of fish.
This leads to the purpose of this study, In this study, fish wastes such viscera will be utilized to create a
high-value-added product, specifically by extracting collagen, which aims to reduced waste, and promote
sustainable practices in the fishing industry.

SLIDE 9:

Viscera are organs that include the liver, intestines, spleen, and stomach, they are typically
discarded as byproducts of the fishing industry. Viscera present environmental concerns due to their
high moisture content, which can lead rapid decay and foul odour if improperly disposed of, contributing
to pollution, and attracting pests. Implementing sustainable practices such collagen extraction or
conversion into value-added products can address these challenges, reducing environmental impact and
maximizing resource efficiency in the fishing industry.

SLIDE 10:

This study is aligned with SDG 12: Responsible Consumption and Production. This goal
recognizes that unsustainable consumption and production practices, which involve the inefficient use of
resources and the generation of significant waste and pollution, have serious environmental, social, and
economic consequences. In this study, fish by-products such as viscera will be utilized to create a high-
value-added product like collagen. This not only reduces the environmental impact of the fishing
industry but also contributes to the overall sustainability goals

SLIDE 11:

The General objective of this study is to isolate and characterize collagen from the viscera of Yellowfin
Tuna, and Milkfish. Specifically, it aims to…

SLIDE 12: METHODOLOGY

SLIDE 13:

For the sample preparation the viscera were cleaned and sized reduced,and soaked in 0.9%
saline solution
SLIDE 14:

Then it was pretreated with 1:5 ratio 10% ethanol for 24 h in 4 degree celcius to removed fats, It
was neutralized with distilled cold water, then deproteinized with 0.1 M NaOH to removed non—
collagenous protein. Then it was extracted with 0.5 M acetic acid for 24 hrs. Then it was filtered using
cheesecloth and precipitated using 2.6 M NaCl

SLIDE 15:

To separate the solids from the solution it will centrifuged at 6000xg for 30 min, then it was
dissolved in 0.5 M acetic acid using a vortex mixer. Then it was Purified/dialyzed against 0.1 M Acetic acid
and distilled water. After, it was freezed dried to removed moisture. After 24 hours, Acid soluble collagen
is obtained and it undergoes Characterization processes like ATR-FTIR, UV-VIS, DSC, HPLC, and SEM

SLIDE 16: RESULT ( Yellowfin tuna are highly sought after by commercial fisheries for their meat,
particularly in the sushi and sashimi markets. Milkfish are also commercially important, especially in
aquaculture, where they are farmed extensively in various parts of the world for their tasty and nutritious
flesh.)

SLIDE 17: YIELD

 Milkfish has a greater yield than yellowfin tuna

 Such differences underscore the importance of meticulously optimizing extraction
methodologies tailored to the specific characteristics of each species to enhance both
the quality and yield of extracted collagen.
 Temperature Tolerance: Yellowfin tuna are highly migratory and can inhabit a wide
range of temperatures, typically preferring warmer waters between 20°C to 30°C (68°F to
86°F). Milkfish are eurythermal, meaning they can tolerate a wide range of temperatures
but thrive in tropical and subtropical waters.
 Habitat: Yellowfin tuna are pelagic fish found in the open ocean, often congregating near
structures such as reefs, seamounts, and floating objects. They undertake long migrations
and can be found in both coastal and offshore waters. Milkfish are primarily found in
coastal environments, including estuaries, lagoons, and tidal flats, often associated with
mangrove areas.

SLIDE 18: Amino Acid Profiling

The structure of collagen fibers involves several hierarchical levels of organization, that is
collagen fibers, fibrils, and collagen molecules where we can find the Amino Acid Chains which are
composed of Triple helics structure of Glycine, Proline and Hydroxyproline. In other words, collagen
should contain these three amino acids glycine, proline and hydroxyproline, but unfortunately, we are not
able to determine the amount of Hydroxyproline due to its unavailability in our standard solution.
SLIDE 19:
• In Amino Acid Profiling shows that Glycine appears to be the dominant amino acid in
Yellow fin tuna, similar to the ASC from the skin of tilapia and eel fish in previous studies of
(Veeruraj et al. 2013; Chen et al. 2016) and Proline emerges as the predominant amino acid in
milkfish.
• Villamil et al. (2017) stated that proline is a dominant amino acid because of its involvement in
diverse biological processes and its capacity to shield a species from environmental pressures.
Furthermore, it has been discovered that there is a negative correlation between it and serum
Immunoglobulin E (IgE) levels in individuals with high-IgE-mediated allergy, indicating its
potential as a biomarker for allergic diseases. Proline accumulation is commonly observed in
reaction to environmental stressors like high temperatures, which align with the preferred
temperature range of Milkfish, typically between 29-35 °C (Hassenrück et al. 2021).
• Furthermore, both ASC from the viscera of yellowfin tuna and milkfish were rich in hydrophobic
amino acids which is an indication that collagen could be used as an alternative source for
antioxidant peptides and antihypertensive peptides (Chen et al. 2016).
• Antioxidant peptides exert their effects by scavenging free radicals, by scavenging these free
radicals, antioxidant peptides help prevent oxidative damage to cellular components such as
lipids, proteins, and DNA.
• antihypertensive peptides Lowers blood pressure helps reduce the risk of hypertension-related
complications such as heart disease, stroke, and kidney damage.
SLIDE 20:
According to study conducted by Yung Kai lin and Cheng Liu 2005, entitled Comparison of
physical chemical properties of type I collagen from different species such Bird feet bovine skin, frog
skin, porcine skin, and shark skin. The UV-Vis absorption of these animals were
• According to Chinh et al. 2019 an absorption peak at 280 nm belongs to the absorption of
aromatic amino acids
• Phenylalanine, tyrosine, and tryptophan had absorption bands in the range of 250 to 270, 270 to
290, and 280 to 300 nm, respectively.
In other words, our desired result should not be 280 nm and beyond. The lesser the value the better. As
you can see, the bird feet and frog skin were beyond and almost close to 280 nm, which indicates that
sensitive chromopores which UV light such as Phenylalanine, tyrosine and tryptophan were not removed
in their methods.

SLIDE 21:
In our result, the collagen from the viscera of yellowfin tuna and milkfish has UV-vis absorption at 237
nm which shows better spectra compared to the references mentioned.
SLIDE 22:
This can also be supported by the low of Methionine, Phenylalanine, Histidine, and Tyrosine in
amino acid profiling which indicates procedures used in this study is effective in removing such sensitive
chromopores.

SLIDE 23:
A collagen should have functional of groups such Amide A&B, and Amide 1,2 and 3. In the FTIR
results the extracted collagen from viscera of yellowfin tuna and milkfish showed obvious
absorption peaks in the main wave number, which indicates that the extracted product is the
protein
- The observed lower frequency shift in the amide-A peaks of collagen from yellowfin tuna and milkfish
viscera suggests a significant role of hydrogen bonds in maintaining the triple helical structure. This
finding aligns with previous studies on collagen from different sources similar to the observations made
in collagen from the skin of Amur sturgeon, as reported by Wang et al. (2014). The consistent presence of
amide-B and amide-I peaks at specific wavenumbers reflects the characteristic vibrational modes
associated with alkenyl C-H2, NH3+, and peptide bonds, providing further evidence of the molecular
composition of the collagen samples. The distinct differences in the amide-II region, with yellowfin tuna
exhibiting a lower frequency peak and milkfish collagen showing a higher frequency peak, suggest
varying levels of molecular order and intermolecular crosslinks between the two species. The 1240
cm⁻¹/1450 cm⁻¹ band ratio close to 1.0 for both samples indicates the preservation of the collagen triple
helix. These findings collectively contribute to a comprehensive understanding of the molecular
distinctions in collagen from yellowfin tuna and milkfish viscera, shedding light on their unique structural
characteristics and potential applications in various fields.

SLIDE 24:
Differential Scanning Calorimetry is a analysis used to determine which certain temperature a
material will denature or degrade. In other words, the higher temperature the better. The result shows a
melting temperature of 101.8 degree Celsius and 105.89 degree Celsius for yellowfin tuna and milkfish
respectively.

-The achieved outcome surpassed the PSC of the skeletal and head components of the silvertip
shark in the study of jeevithan et al 2014
The yellowfin tuna prefers warm surface waters with temperatures ranging from 26 to 28 °C, At the same
time, the milkfish can tolerate a broader range of temperatures, from 24 to 35 °C The differences in
thermal stability could be attributed to the degree of hydration and the quantity and characteristics of
covalent cross-linkages in collagen (Wang et al. 2014). It could also result in the fragmentation of peptide
chains, leading to the degradation of materials (Nagai et al. 2008).
SLIDE 25: Morphological Properties
The interconnective structure evident in the morphology of ASCs of the two species aligns with
the soft and smooth textures observed in the naked eye and may be utilized as a strong potential for
biomedical use (Hamdan and Sarbon 2019). Architectural features, such as pore shape, pore wall
morphology, and interconnectivity of collagen, have also been suggested for use in cell seeding, growth,
gene expression, migration, mass transport, and new tissue formation (Hamdan and Sarbon 2019;
Jeevithan et al. 2014)

SLIDE 26: CONCLUSION

Collagen isolation from yellowfin tuna and milkfish viscera using acetic acid yielded structurally
sound collagen, primarily type I, as demonstrated by UV-Vis spectra and amino acid analysis. FTIR
spectra of both species revealed comparable primary and secondary structures. Despite variations in
yields and melting temperatures, the overall structural integrity of the isolated collagen from yellowfin
tuna and milkfish remained relatively stable. These findings suggest that acetic acid-soluble collagens
(ASCs) from yellowfin tuna and milkfish hold promise as substitutes for mammalian collagen. However,
further investigations are necessary to evaluate the cytotoxicity and biocompatibility of ASCs for
potential applications.