Soy whey protein isolate (SWPI) – Fenugreek gum conjugates were developed and their molecular characteristics and emulsifying properties were investigated. SWPI was extracted from soy whey of tofu processing. SWPI exhibited excellent emulsifying properties comparable to soy protein isolate. However, to improve the emulsifying properties of SWPI for some applications, it was conjugated to fenugreek gum. The extent of conjugation was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared (FTIR) and High performance size exclusion chromatography (HPSEC). The SDS-PAGE of the conjugates showed polydispersed bands at the top of the separating gel in the conjugates suggesting the formation of high molecular weight products. Refractive index spectrum of HPSEC profiles showed a reduction of protein peak of unconjugated mixture and shifted a peak to higher molecular weight of the conjugates. Ultraviolet spectrum of HPSEC showed an increase of protein peak intensity at polysaccharide region. FTIR spectrum showed an amide band I and II were still observed in the conjugates after the unreacted proteins were removed. 1D NMR spectra showed that fenugreek gum was covalently bound to proteins through interaction between the reducing end of mannose residue and lysine. The protein solubility of SWPI – Fenugreek gum conjugates improved as compared to SWPI and SWPI – Fenugreek gum mixture when assessed in the pH range 3 to 8 at 22oC, especially at isoelectric point of protein (pl). A 1:3 and 1:5 ratio of SWPI – Fenugreek gum gave rise to better emulsion stabilization compared to 1:1 ratio. Particle size analysis revealed that conjugation of SWPI – Fenugreek gum at 60oC for 3 days was enough to produce relatively small droplet sizes in oil-in-water emulsions. SWPI – Unhydrolyzed fenugreek gum conjugates exhibited better emulsifying properties compared to SWPI – Partially hydrolyzed fenugreek gum conjugates. The conjugates improved emulsifying properties of SWPI, particularly around the pl of protein. The emulsifying properties were greatly increased by heating the conjugates before emulsification. The conjugates also improved emulsion stability at high salt concentration compared to SWPI. In summary, incorporation of SWPI into fenugreek gum improved emulsifying properties of SWPI near the pl of protein and at high salt concentration. / No / No

Capillary electrophoresis mass spectrometry (CE-MS) is a versatile instrumental method for metabolomics, which allows for comprehensive metabolite profiling of volume-limited biological specimens in order to better understand the molecular mechanisms associated with chronic diseases, including an alarming epidemic of obesity worldwide. Multiplexed CE separations enable high-throughput metabolite screening with quality assurance to prevent false discoveries when combined with rigorous method validation, robust experimental designs, complementary statistical methods, and high-resolution tandem mass spectrometry (MS/MS) for unknown metabolite identification. In this thesis, multiplexed CE-MS technology is applied for both targeted and untargeted metabolite profiling of various biological fluids, including covalently bound thiol-protein conjugates, as well as free circulating metabolites in serum and plasma, and excreted/bio-transformed compounds in urine due to complex host-gut microflora co-metabolism. This work was applied to characterize aberrant metabolic responses of obese subjects in response to dietary challenges, and measure the benefits of dietary interventions that reduce adiposity without deleterious muscle loss. Chapter 2 presents, a simple, sensitive yet robust analytical protocol to expand metabolome coverage in CE-MS for the discovery of labile protein thiols in human plasma using a rapid chemical derivatization method based on N-tert-butylmaleimide (NTBM). Chapter 3 describes targeted metabolite profiling of serum and plasma to investigate the differential metabolic responses between healthy and unhealthy obese individuals before and after consumption of a standardized high-caloric meal, respectively. Chapter 4 of this thesis describes an untargeted metabolite profiling strategy for urine using multisegment-injection (MSI)-CE-MS for elucidating the effects of protein supplementation following a short-term dietary weight-loss intervention study. This work revealed six urinary metabolites that were classified as top-ranking treatment response biomarkers useful for discriminating between subjects consuming carbohydrate (control), soy, and whey supplemented diets. In summary, this thesis demonstrated the successful implementation of multiplexed CE-MS technology for biomarker discovery in nutritional-based metabolomic studies as required for more effective treatment and prevention of obesity for innovations in public health. / Thesis / Doctor of Philosophy (PhD)