Abstract
The objectives of this PhD thesis were to investigate the nutritional composition of Huhu (Prionoplus reticularis) at various developmental stages, and to assess the protein functionalities, transcriptomics, and in silico prediction of bioactive peptides from Huhu. This thesis is divided into 3 parts.
The first part of the thesis presents an investigation of the major nutritional components, fatty acid profile, lipid nutritional indices, and mineral profile of Huhu at various development stages (small, medium, and large larvae and pupae). Analysis of major chemical components consisted of 58.7 and 75.2% moisture, 26.2 ̶ 30.5% protein, 32.1 ̶ 58.4% fat, and 1.5 ̶ 3.2% ash on a dry weight (DW) basis for the Huhu, demonstrating that Huhu larvae and pupae contain substantial amounts of nutrients. The ash and crude protein contents were similar across the development stages. The lipid content was highest in pupae (58.4% DW). Between 10 and 13 fatty acids were found in each of the four development stages. Palmitic acid (7.45 – 13.95 g/100g DW), oleic acid (22.04 – 40.54 g/100g DW), and linoleic acid (0.82 – 1.47 g/100g DW) were the most abundant saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty (PUFA) acid, respectively. The PUFA/SFA ratio ranged from 0.07 to 0.09, while the ω6/ω3 ratio ranged between 16.23 and 29.54. The hypocholesterolemic/hypercholesterolemic acid ratio ranged from 2.89 to 3.15. The atherogenicity indices ranged between 0.32 and 0.37, the thrombogenicity index range was 1.63 – 1.99, and the health-promoting index range was 0.07 – 0.11. Overall, the larvae and pupae development stages of the Huhu contain relatively high levels of lipids, but they differ in their fatty acid profile and lipid nutritional indices. A mineral profile analysis detected 28 minerals (11 essential, 13 non-essential and 4 heavy metals) in the Huhu. The most abundant minerals in large Huhu were manganese (37.5 mg/kg DW), magnesium (1306.7 mg/kg DW), phosphorus (3970.0 mg/kg DW), iron (28.0 mg/kg DW), copper (8.9 mg/kg DW), and zinc (53.6 mg/kg DW). The heavy metal content in all four developmental stages of Huhu was found to be below detection levels for arsenic and vanadium, but the cadmium concentration ranged from 0.28 – 0.68 mg/kg DW, and lead ranged from 0.016 – 0.023 mg/kg DW were detected, but at below toxicity levels.
The second part of the thesis presents an investigation of the functionalities of Huhu proteins. The amino acid profile, techno-functionalities (foaming stability/capacity, emulsion stability/capacity, solubility, and coagulation), and physicochemical characteristics (colour, particle size, surface hydrophobicity, Fourier-transform infrared spectroscopy, and differential scanning calorimetry) of protein extracts (PE) obtained from Huhu larvae (HLPE) and pupae (HPPE) were investigated. Total essential amino acid contents of 386.7 and 411.7 mg/g protein were found in HLPE and HPPE, respectively. The essential amino acid index (EAAI) was 3.3 and 3.4 for HLPE and HPPE, respectively, demonstrating their nutritional equivalence. A unique nitrogen-to-protein conversion constant, k, and the corresponding protein content of the extracts were 6.1 and 6.4, and 72.1% and 76.5%, respectively. HLPE (37.1 J/g) had a lower enthalpy than HPPE (54.1 J/g). HPPE (1% w/v) exhibited a foaming capacity of 50.7%, which was higher than that of HLPE (41.7%) at 150 min. The foaming stability was 75.3% for HLPE and 73.1% for HPPE after 120 min. Both protein extracts (1% w/v) had emulsifying capacities that were 96.8% stable after 60 min. Therefore, protein extracts from Huhu larvae and pupae are of a good nutritional quality (based on their EAAI) and have techno-functional properties, such as foaming and emulsification, that afford them potential for certain food technology applications.
The third part of the thesis presents a comparative investigation of the transcriptomes of the small larvae and large larvae of the Huhu to gain insights into the molecular mechanisms governing their early life cycles and development. Functional annotations and classifications were assigned to the transcripts through Gene Ontology (GO) and Clusters of Orthologous Groups (COG). The Swiss-Prot database resulted in the discovery of 100 Huhu proteins with high-quality manually annotated and non-redundant protein sequences. The GC content of Huhu larvae ranged from 36% to 39%. A total of 603,786 assembled transcripts were obtained, with an average length of 762 bp and an N50 value of 1378 bp. In total, 30,803 transcripts were found to be differentially expressed with DESeq2, 25,781 with edgeR, and 27,193 with limma which are popular R-based bioinformatics tools commonly used for differential expression analysis. When comparing small and large larvae, 17,775 common differentially expressed transcripts were identified across the three packages. EdgeR found 14,731 up-regulated, 387 down-regulated transcripts and 2657 non-significant transcripts. DESeq2 found 14,699 up-regulated, 2,655 down-regulated transcripts and 421 non-significant transcripts. Limma detected 14,515
up-regulated and 616 down-regulated transcripts and 2644 non-significant transcripts. Functional annotations and classifications conducted on the Transdecoder output were assigned to the transcripts through Gene Ontology (GO) and Clusters of Orthologous Groups (COG) analysis. The COG analysis identified functional categories, such as posttranslational modification, protein turnover, chaperones (825 transcripts), signal transduction mechanisms (703 transcripts), and translation, ribosomal structure, and biogenesis (530 transcripts). GO enrichment analysis highlighted key biological processes, cellular components, and molecular functions, including aminoglycan metabolic process (GO:0006022), structural constituent of chitin-based cuticle (GO:0040003), carbohydrate catabolic process (GO:0016052), and hydrolase activity, hydrolysing O-glycosyl compounds (GO:0004553). The results of this study contribute to a deeper understanding of the molecular processes underlying the early development of the Huhu and provide valuable information for further investigations into its bioactive properties.
The in silico evaluation of bioactive peptides, specifically their antioxidant, antidiabetic, and antihypertensive properties from the large Huhu proteins, were also investigated. A total of 68 proteins were obtained from the UniProtKB database, based on transcriptome analysis from Chapter 6 and after filtering using SDS-PAGE (i.e., MW between 10-80 kDa). These proteins were subjected to in silico simulated gastrointestinal digestion using the BIOPEP-UWM database. The resulting peptides were screened for potential bitterness, toxicity, and allergenicity. The bioactive properties of the screened peptides were predicted using MultiPep. Out of 1980 screened peptides, 1372 were identified as potential antihypertensive peptides, and 15 were predicted to have multiple bioactivities (i.e., antioxidative, anti-hypertensive, anti-diabetic, and ACE inhibitor). In silico physiochemical properties, drug-likeness, and pharmacokinetics of the 15 multifunctional Huhu peptides were analysed and compared to standard drugs such as acarbose, captopril, and aliskiren. Nine peptides were found to meet the Lipinski rule of five criteria and exhibited comparable bioavailability scores to the standard drugs. Two peptides (Val-Ala-Leu, Ile-Pro-Leu) demonstrated good oral bioavailability, indicating the potential of Huhu-derived peptides as orally active compounds. This chapter acknowledges the limitations of in silico prediction and emphasizes the need for in vitro or in vivo experimental validation of the findings. This thesis shows the high nutritional profile and protein functionalities of Huhu and in silico-derived peptides, confirming the suitability of Huhu for consumption.