Ameliorating nutritional properties of soybean waste (Okara) through biofermentation using Rhizopus oligosporus

• Main Author: Gupta Sulagna
• Other Authors: Chen Wei Ning, William
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Engineering::Chemical engineering
• Online Access: https://hdl.handle.net/10356/138687
• Institution: Nanyang Technological University

Okara is a type of soybean residue, that is generated as a major by-product during the production of tofu and soymilk. It is considered to be one of the prime agri-crop wastes. Given the rise of health consciousness, vegetarianism, veganism and lactose intolerance amongst the world population today, it is to be expected that the generation of this food waste will only increase further. Currently, okara is either disposed in landfills or incinerated. Since not much finances are diverted towards the management of industrial waste, an economical and environment friendly, yet viable solution for the management of such food wastes is the need of the hour for a sustainable approach. In this thesis, biofermentation has been suggested as a valorization technique to improve the nutritional properties of okara, to gainfully repurpose this food waste. This work was initiated with FDA-approved microbes Rhizopus oligosporus and Lactobacillus plantarum, but switched to the usage of only the former in the later stages of the study when it yielded better results than the latter. In the first stage of this study, a GC-MS based metabolomic analysis was conducted to understand the changes occurring due to the microbial biochemical processes. The detected metabolites were subsequently mapped to their respective biochemical pathways, to understand the pathways being triggered during the biofermentation process. A preliminary DPPH test showed that Rhizopus oligosporus fermented okara has a radical scavenging activity of 52.81%, as opposed to that 27.44% for raw okara. The results obtained in this stage justified that fermented okara may be a potential functional food. Next, an attempt was carried out to extricate a clean, green extract from fermented okara using a combination of water and ultrasound sonication. Upon subjecting these extracts to DPPH, FRAP, O2- and .NO antioxidant analyses, it was observed that the results were significantly better than those obtained by raw okara extracts. Subsequent erythrocyte lysis assays revealed that fermented okara extract is not only non-toxic to erythrocytes at concentrations as high as 4 mg/mL, but could also prevent AAPH induced haemolysis of erythrocytes at concentrations as low as 500 ng/mL. The study then endeavoured to use the fermented okara extract on cancer cell line HepG2, to check for antiproliferative activities. A parallel was simultaneously run on NIH 3T3 cell line, a non-cancer cell line, to check for toxicity. The experiments revealed that 48 hours incubation yields antiproliferative activities on HepG2 cell line in a dose-dependent manner. After 48 hours incubation, the highest tested concentration (100 mg/mL) of fermented okara extract could inhibit HepG2 cells by 48.47 ± 5.28%, which was significantly different from its effect on NIH 3T3 cells. To understand the compositional differences in the extracts leading to the bioactivities, a GC-MS metabolomics analysis was performed, which showed that the fermented okara extracts contained more amino acids and organic acids, and less sugars. The results obtained in this stage validated the in-vitro antioxidant and antiproliferative properties of fermented okara. Finally, the study focussed on the improvement of polyphenol content post fermentation, with an emphasis on aglycone isoflavones. TPC and TFC analyses showed that fermented okara extracts yield significantly better results than unfermented okara extracts. An LCQTOF/MS analysis followed by HPLC quantification revealed that ethanolic extracts of fermented samples contained 11.782 ± 0.325 µg/mL and 10.125 ± 1.028 µg/mL of daidzein and genistein respectively. In contrast, extracts of unfermented okara contained only 6.7 ± 2.42 µg/mL and 4.55 ± 0.316 µg/mL of daidzein and genistein, respectively. The results obtained in this stage established the phenolic enhancement of okara post biofermentation. Summing up, this thesis demonstrates a method to ameliorate the nutritional properties of okara via biofermentation using Rhizopus oligosporus, as a sustainable method of dealing with this agri-industrial food waste.