Development of serum alternative with fermented soybean residue (okara): towards cultured meat production application

• Main Author: Teng, Ting Shien
• Other Authors: Chen Wei Ning, William
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Engineering::Bioengineering
• Online Access: https://hdl.handle.net/10356/161609
• Institution: Nanyang Technological University

As global population continues to increase rapidly, the amount of meat consumed correspondingly rises. The world relies heavily on livestock for protein supply. However, the current livestock sector requires large amount of resources committed to supporting the system, and that heavily strains the environment. Land degradation, loss of biodiversity, intensive consumption of feedstock and drinkable water, and greenhouse gases (GHG) emission are some of the impacts commonly associated with livestock system. Excessive consumption of animal derived products has also been linked to health deterioration, rise in cancer incidence, and exposure to zoonotic diseases. To mediate the situation, cultured meat was proposed as a potential alternative to animal proteins. Cultured meat is the deliberate culturing of skeletal muscle cells, obtained from animals, in vitro to produce consumable meat products. However, such innovative solution to replace animal products is not without its disadvantage. At present, cultured meat production is greatly limited by technical challenges. One critical limitation lies in the use of foetal bovine serum (FBS) in culture medium. FBS uses have been beset by controversies, citing ethical, biosafety, scientific, cost and sustainability concerns. This prompted the search for alternatives to animal serum, and one promising replacement suggested was protein hydrolysates. Okara is the agro-industrial by-products obtained after the production of soymilk and tofu. It is often discarded at the end of the production process as high moisture content in the matter leads to quick putrefaction. However, okara is also high in protein content, measuring at approximately 20-30% dry matter. This make okara an economical source of protein for generating protein hydrolysate. In this work, fermentation by microorganisms Rhizopus oligosporus was selected as the hydrolytic method over enzymes and acid uses. The protein hydrolysates prepared from fermentation was subsequently tested on various cell lines to determine its effect at maintain the cell viability and stimulating cell proliferation. The work of this thesis begins by optimising the serum replacement parameters with fermented okara extract in HEK293 cultures. Based on the results, it was determined that fermented okara extract supplementation has a tight concentration range, where the optimal concentration was found to be approximately 1.0% w/v. Also, attempt to partially replace culture medium with the fermented okara extract found the optimal percentage to be less than 25%. Stepwise adaptation of cultures to lower serum levels did not help to improve cell viability. Metabolomics analysis by GC-MS was performed to elucidate the difference between fermented and unfermented okara samples. In the second part of the work, fermented okara extract was ultrafiltrated and added to cultures of HEK293 and HepG2 as serum substitute. The cell viability and proliferation were studied using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assays taken over 96 hours. Free amino nitrogen (FAN) assay was performed to determine the level of free amino nitrogen present in the extracts. Results showed that HEK293 and HepG2 cultures demonstrated an estimate of 70% relative growth efficiency (RGE) when cells were grown in serum-free medium, supplemented with fermented okara extracts. Gas chromatography-mass spectrometry (GC-MS) was used to study the metabolite profiles of the various ultrafiltrated extracts, as well as the changes in the metabolite compositions of the spent culture media taken from HEK293 and HepG2 cultures. The observed differences in the results suggested that cells may be stimulated by specific metabolites from the extract, and such interactions may vary from cell line to cell line. An in-depth study on the cellular metabolite interaction would be required to optimise the proliferative effects that the fermented okara extracts have on the cells. The last part of the work examines the applicational value of fermented okara extracts in actual cultured meat production – by culturing skeletal muscle cells in media supplemented with fermented okara extracts. C2C12 and immortalised porcine myoblast (IPM) cells were used in this study. The results demonstrated that serum presence is essential for cell growth. When fermented okara extracts were added to 5.0% serum media, cell viability of C2C12 was even higher than positive control. However, cultures of IPM did not observe similar effect when the cells were grown in presence of FBS and fermented okara extract. GC-MS analysis illustrated the changes in the metabolite profile from spent media taken from C2C12 and IPM cultures. The result pointed towards putrescine as a possible compound that regulates cell growth in the myoblast cultures. In conclusion, this work demonstrated the potential of utilising fermented okara extracts as possible substitute for use in cell cultures, as well as in cultured meat production.