Title Collagen extraction from jellyfish: method optimization
Title (croatian) Ekstrakcija kolagena iz meduza: optimizacija metode
Author Natalija Štimec
Mentor Daniela Kalafatović (mentor)
Committee member Milan Mesić (član povjerenstva)
Committee member Jasminka Giacometti (predsjednik povjerenstva)
Committee member Daniela Kalafatović (član povjerenstva)
Granter University of Rijeka (Faculty of Biotechnology and Drug Development) Rijeka
Defense date and country 2024-10-23, Croatia
Scientific / art field, discipline and subdiscipline BIOTECHNICAL SCIENCES Biotechnology Molecular Biotechnology
Abstract Jellyfish are a novel source for collagen extraction, and it is derived from
multiple Medusozoa species, including Rhizostoma pulmo. Their collagen is
evolutionary older and simpler than mammalian collagens making them
compatible with human biology. Its specific response to macrophages - a
lower M1 macrophage response and a higher M2 macrophage response -
triggers regeneration in tissue, making jellyfish collagen a candidate
biomaterial for tissue engineering. To compare the collagen composition,
multiple sequence alignment was performed between different types of
human collagen and between murine collagen using SALIGN. Sequence
alignment of fibrillar collagens showed similarities in characteristic glycine
and proline regions, and in lysine sites in the sequence. Amino acid analysis
showed several differences in composition between different collagen
sources. The extraction of collagen from jellyfish consists of three main
steps: sample preparation, extraction, and recovery, and can last up to 7
days. This lengthy procedure can lead to collagen degradation and a
decrease in final yield. Current methods consist of tissue cutting and
chemical pretreatment during sample preparation, acid and enzymatic
extraction, and dialysis. The goal of this research was to see how changes
in the extraction steps affect collagen yield, purity, and the methods
duration. For this purpose, samples from Rhizostoma pulmo were first
lyophilized, followed by acid extraction with 0,5 M acetic acid, salting out
with NaCl, and purification with C18 cartridges. The obtained collagen
powder was analyzed using MALDI-TOF-MS to confirm the successful
extraction of collagen. In addition, FTIR and SDS-PAGE were performed to
further confirm the presence of collagen. The collagen yields that were
obtained are similar to those recorded in previous studies. Based on these
results, it can be concluded that the use of C18 cartridges can be an
alternative for dialysis and could shorten the extraction process.
Abstract (croatian) Meduze su nov izvor kolagena, a kolagen se dobiva iz nekoliko vrsta
Medusozoa, uključujući Rhizostoma pulmo. Kolagen iz meduza je evolucijski
stariji i jednostavniji od kolagena sisavaca, što ga čini kompatibilnim s
ljudskom biologijom. Njegov specifičan odgovor na makrofage - niži
odgovor M1 makrofaga i viši odgovor M2 makrofaga - potiče regeneraciju
tkiva, što čini kolagen meduza potencijalnim biomaterijalom u tkivnom
inženjerstvu. Da bi se usporedio sastav kolagena, višestruko poravnanje
sekvenci (multiple sequence alignment) je izvršeno između različitih tipova
ljudskog kolagena i između mišjeg kolagena koristeći SALIGN. Poravnanje
sekvenci fibrilarnih kolagena pokazalo je sličnosti u karakterističnim
glicinskim i prolinskim regijama te u mjestima na kojima su lizini u
sekvencama. Analiza aminokiselina pokazala je nekoliko razlika u sastavu
između različitih izvora kolagena.
Ekstrakcija kolagena iz meduza sastoji se od tri glavna koraka: pripreme
uzorka, ekstrakcije i oporavka, i može trajati do 7 dana. Ovaj dugotrajan
postupak može dovesti do degradacije kolagena i smanjenja konačnog
prinosa. Trenutne metode uključuju usitnjavanje tkiva i kemijsko tretiranje
tokom pripreme uzorka, kiselinsku i enzimsku ekstrakciju, kao i dijalizu. Cilj
ovog istraživanja bio je utvrditi kako promjene u koracima ekstrakcije utiču
na prinos, čistoću kolagena i trajanje metode. U tu svrhu, uzorci vrste
Rhizostoma pulmo su prvo liofilizirani, nakon čega je uslijedila ekstrakcija
kiselinom koristeći 0,5 M octenu kiselinu, isoljavanje pomoću NaCl i
pročišćavanje uz pomoć C18 kolona. Dobiveni kolagen je analiziran pomoću
MALDI-a kako bi se potvrdila uspješna ekstrakcija kolagena. Također, FTIR
i SDS-PAGE su odrađeni radi dodatne potvrde prisustva kolagena u izolatu.
Prinosi kolagena koji su dobiveni slični su onima zabilježenim u prethodnim
studijama. Na osnovu ovih rezultata, može se zaključiti da upotreba C18
kolona može biti alternativa za dijalizu i mogla bi skratiti proces ekstrakcije.
Keywords
collagen
jellyfish
Rhizostoma pulmo
collagen extraction
MALDI-TOF-MS
FTIR
Keywords (croatian)
kolagen
meduze
Rhizostoma pulmo
ekstrakcija kolagena
MALDI-TOF-MS
FTIR
Language english
URN:NBN urn:nbn:hr:193:142435
Study programme Title: Biotechnology in medicine Study programme type: university Study level: graduate Academic / professional title: magistar/magistra biotehnologije u medicini (magistar/magistra biotehnologije u medicini)
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Created on 2024-10-22 22:14:54