Plant-based protein hydrolysates as a functional food ingredient from Azolla Pinnata fern

Globally, there is a growing interest for sustainable alternative protein due to the projected population increase to about 9.1 billion by 2050. Animal-derived proteins, commonly used in the food industry, often have negative environmental impacts, are expensive and not widely available in some regions. Moreover, overconsumption of animal-based diet has become a major global health concern due to the high risk of non-communicable diseases. Although synthetic compounds offer promising therapeutic strategies for managing these diseases, long-term use may have adverse health effects, leading to growing demand for natural and eco-friendly alternatives. Thus, food researchers conducting extensive studies for the development of natural compounds as functional food ingredients, particularly protein hydrolysates containing bioactive peptides. Therefore, the current work aimed to develop plant-based multifunctional protein hydrolysates from Azolla pinnata fern as functional food ingredients. Azolla fern protein concentrate (AFPC) with a maximum yield of 18.93% and recovery rate of 73.66% was obtained using alkaline extraction. The AFPC had met almost all the essential amino acids (EAA) recommended by FAO/WHO for an adult diet, except for lysine and histidine. AFPC had a compact structure with numerous surface pores, having a molecular weight ranging from 17 to 56 kDa and exhibiting good thermal and techno-functional properties. Then, AFPC was hydrolysed using alcalase, flavourzyme and papain at different extent of hydrolysis (DH) of 10% (partial), 20% (moderate) and 30% (extensive). During hydrolysis, as the DH increased the structural flexibility of all protein hydrolysates increased and significantly enhanced their solubility and foaming capacity, however reduced the thermal stability, water holding capacity, oil holding capacity, emulsifying activity index, emulsion stability index and foam stability. Overall, alcalase hydrolysates exhibited superior techno-functional properties across all DH and pH levels. Similarly, the bioactivities antihypertensive, antidiabetic and antioxidant were dependent on the protease types and DH, in which the extensively hydrolysed (30%) alcalase hydrolysate (AFPH-AE) displayed the most potent activities. The AFPH-AE exhibited distinct inhibition patterns, displaying an uncompetitive inhibition mode against ACE and α-glucosidase, a mixed inhibition mode against α-amylase and a noncompetitive inhibition mode against DPP-IV. The AFPH-AE demonstrated good stability at various pH, salt concentrations and temperatures The low molecular weight fraction (<3 kDa) was the most active, containing a total of 15 bioactive peptide sequences characterised by low molecular weights and short chain lengths. Molecular docking revealed that the bioactive peptides interacted with the target enzymes (ACE, DPP-IV, α-glucosidase, α-amylase & MPO) with a binding energy between -6.7 to -10.1 kcal/mol and were mainly mediated via hydrogen bonds and hydrophobic interactions. The AFPH-AE was investigated for its dual performance as emulsifier and nutraceutical ingredient in an oil/water emulsion system. The AFPH-AE stabilised emulsion (AFPH-AE-E) had excellent performance, characterised by small and uniformly dispersed oil droplets and resistance to creaming or phase separation under different food processing conditions. Moreover, the AFPH-AE-E demonstrated strong and stable multi biological activities under different food processing and gastrointestinal digestion conditions. Notably, the AFPH-AE-E demonstrated a significant shift in the gut microbiota towards a more "health-promoting" composition and exhibited no adverse effects on human colon cells. The AFPH-AE-E positively modulated the population of beneficial bacteria and their secondary metabolites. The current work reveals the potential of novel multifunctional Azolla pinnata fern protein hydrolysates as versatile candidates for functional foods that offer health-promoting benefits.

Text 122624.pdf Download (1MB) Official URL or Download Paper: http://ethesis.upm.edu.my/id/eprint/18625

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