Impact of ultrasound pretreatment on biological activity of peptides derived from lupin protein
Creators: name: Fadimu, Gbemisola
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300602
subject: Food chemistry and food sensory science
classificationCode: 300607
subject: Food technology
classificationCode: 300699
subject: Food sciences not elsewhere classified

description: Diabetes and hypertension are the major risk factors for coronary heart disease, and they have been recognised as serious medical and public health crises in the twenty-first century. Although the conditions have some genetic links, they are exacerbated by a sedentary lifestyle and excessive intake of calories. Conventionally, these conditions are managed using synthetic medications, known to cause other medical conditions in the body. Given the adverse effects associated with synthetic medications, scientists are looking for an alternative to these medications. So far, food protein-derived peptides have demonstrated physiological effects comparable to synthetic drugs.  Bioactive peptides may possess antihypertensive, antioxidant, antimicrobial, antidiabetic, antiviral, and antithrombotic effects and are usually prepared via enzymatic hydrolysis of food proteins. However, the peptides' biological activities depend on factors such as the protein source, type of protease used, and type of pre-treatment applied to the protein before or during enzymatic hydrolysis. This PhD thesis provides new information about the impact of ultrasound pre-treatment on the antidiabetic and antihypertensive effects of peptides derived from lupin protein and depicts the molecular mechanism of interaction between the peptides and the key enzymes involved in the management of diabetes and hypertension.

In the first experimental chapter, it is hypothesised that structural modifications caused by ultrasound pre-treatment could influence the bioactivity of hydrolysates from lupin protein. Lupin protein isolate was subjected to ultrasound treatment for 5, 10, and 20 min and hydrolysed using alcalase, flavourzyme, and protamex. The resulting hydrolysate samples were analysed for their physicochemical, structural, and antioxidant properties, using non-sonicated hydrolysate as control. Furthermore, a two-principal component model was performed using principal component analysis (PCA) to establish the influence of different enzymes and ultrasonication time on the studied parameters. Circular dichroism (CD) and FTIR analyses revealed that the secondary structure composition of the hydrolysate samples changed after ultrasonic treatment, suggesting that ultrasonic power tends to alter the structural configuration of lupin protein. In addition, spectroscopic and microscopic techniques such as scanning electron microscopy, UV-Vis, zeta potential, and X-ray diffraction measurement indicated a change in the protein configuration and that the level of structural alteration depends on the type of enzyme used and duration of ultrasound treatment. PCA showed that ultrasonication pre-treatment for 5 and 10 min altered the secondary and tertiary structures of LPH regardless of the enzyme used, while maximum antioxidant activity was also achieved after 10 min of ultrasonic treatment.

Following these positive results, the second and third experimental chapters focused on evaluating the effect of ultrasonication time and protease type on the amino acid composition, molecular weight pattern, thermal stability, antihypertensive, antioxidant, and antidiabetic activities of lupin protein hydrolysate. In these chapters, SDS-PAGE and MALDI-TOF analysis revealed that the duration of sonication and protease type greatly influenced the molecular weight distribution of LPH, which indicates that biological activities are likely to be affected by these processing parameters. The amino acids such as arginine, glutamic acid, leucine, and aspartic acid, known to contribute to antihypertensive and antidiabetic activities, were found to be the dominant amino acids in LPH. Still, their concentration varied with different proteases and ultrasonication times. Generally, ultrasonicated hydrolysates prepared with either alcalase or flavourzyme exhibited higher α-amylase, α-glucosidase, angiotensin-converting enzyme (ACE) inhibitory, and antioxidant activities than non-sonicated hydrolysates and those prepared with protamex. Loading plots and biplot analyses based on PCA carried out on the data revealed a strong correlation between the duration of ultrasound treatment and protease used. Furthermore, LPI ultrasonicated for 10 min and hydrolysed using either alcalase or flavourzyme could yield hydrolysates with potent antihypertensive and antidiabetic activities. Considering the substantial improvement recorded in the α-amylase, α-glucosidase, ACE inhibitory, and antioxidant activities of LPH, it is suggested that ultrasonic technology could be employed to prepare hydrolysate for different applications.

The last experimental chapters (chapters 5 and 6) explored the molecular docking analysis to predict the peptides responsible for the α-amylase, α-glucosidase, and ACE inhibitory effects observed in hydrolysates generated using alcalase and flavourzyme. Prior to this, hydrolysate samples from alcalase and flavourzyme enzymes prepared under 10 min of ultrasound pre-treatment were fractionated into three molecular weight fractions using the ultrafiltration technique. The IC50 values obtained showed that original hydrolysates from alcalase and flavourzyme displayed higher inhibitory potentials towards ACE, α-glucosidase, and α-amylase enzymes than their ultrafiltrated fractions. Then, the potent ACE, α-glucosidase, and α-amylase peptides were identified using LC-MS-QTOF. The in-silico structural-activity relationship and molecular mechanisms for interaction between the identified peptides and ACE, α-glucosidase, and α-amylase enzymes were investigated using molecular docking approach. Several ACE, α-glucosidase, and α-amylase inhibitory peptides were identified in alcalase and flavourzyme hydrolysates. These results suggest that lupin protein hydrolysates prepared using flavourzyme and alcalase under ultrasonication pre-treatment could be used as a functional ingredient in the development of nutraceuticals for hypertensive and diabetic patients. The findings of this thesis contribute to the current knowledge on the use of non-thermal techniques, such as ultrasound, to facilitate the release of novel antihypertensive and antidiabetic peptides from lupin protein. In addition, the thesis may serve as a template to guide nutraceutical industries about the use of non-thermal techniques to improve the biological activities of their product.