Protease-Assisted Mild Extraction of Soluble Fibre and Protein from Fruit By-Products: A Biorefinery Perspective

Abstract

By-products from the fruit supply chain, especially seeds/kernels, have shown great potential to be valorised, due to their high content of macronutrients, such as lipids, protein, and fibre. A mild enzymatic assisted extraction (EAE) involving the use of a protease was tested to evaluate the feasibility of a cascade approach to fractionate the main fruit by-products components. Protease from Bacillus licheniformis (the enzyme used in the AOAC 991.43 official method for dietary fibre quantification) was used, and besides protein, the conditions of hydrolysis (60 °C, neutral pH, overnight) allowed us to dissolve a portion of soluble fibres, which was then separated from the solubilized peptide fraction through ethanol precipitation. Good protein extraction yields, in the range 35–93%, were obtained. The soluble fibre extraction yield ranged from 1.6% to 71% depending on the by-product, suggesting its applicability only for certain substrates, and it was found to be negatively correlated with the molecular weight of the fibre. The monosaccharide composition of the soluble fibres extracted was also diverse. Galacturonic acid was present in a low amount, indicating that pectin was not efficiently extracted. However, a predominance of arabinose and galactose monomers was detected in many fractions, indicating the isolation of a fruit soluble fibre portion with potential similarity with arabinogalactans and gum arabic, opening up perspectives for technological applications. The residual solid pellet obtained after protease assisted extraction was found to be an excellent fibre-rich substrate, suitable for being subjected to more “hard” processing (e.g., sequential pectin and hemicellulose extraction) with the objective to derive other fractions with potential great added economic value.

Oil and protein extraction from fruit seed and kernel by-products using a one pot enzymatic-assisted mild extraction

“…Also, the protein fraction of pumpkin seeds (48 ± 1%) resulted the highest in comparison with other nutrients and with that of other fruit seeds/kernels, in agreement with previous data on other pumpkin varieties ( Glew et al, 2006 ). Lemon seeds resulted quite good as protein source (8.87 ± 0.05%), as already reported ( Fuso et al, 2023 ). The protein percentage of lemon seeds was comparable with those of avocado (7 ± 1%) and litchi (7.1 ± 0.2%) seeds; stone fruit kernels and mango seeds were the lowest in proteins, especially peach kernels with a mean value of 2.95 ± 0.02%.…”

“…As previously documented ( Fuso et al, 2023 , Kumoro et al, 2020 ), fruit seeds and kernels are good sources of dietary fibres. Among the analysed fruit biomasses, the kernels of peach, cherry and apricot were found to be the richest in TDF, ranging between 77 ± 2% and 93 ± 2%.…”

“…The proximate composition analysis of each substrate was performed in triplicates by using the methods of AOAC International for vegetable matrices, slightly adapted ( Fuso et al, 2023 ). Briefly, moisture was determined according to AOAC 925.09 by weighing the sample before and after drying in oven at 105 °C for 24 h and determining the difference.…”

“…For the enzyme-assisted extraction (EAE) of nutrients from each fruit seeds/kernels, an extraction protocol previously published ( Fuso et al, 2023 ) based on Bacillus licheniformis protease (the enzyme used in the AOAC 991.43 official method for dietary fibre determination), was performed. Briefly, an enzyme/substrate ratio of 1:100 (w/w) was mixed with a phosphate buffer solution (10 mM Na 2 HPO 4 /NaH 2 PO 4 ) and hydrolysed at 60 °C and pH 7.5 overnight (16 h), then heated at 90 °C for 10 min to inactivate the enzyme.…”

“…This study addresses the valorisation of fruit biomasses and the application of mild enzyme technology integrated with biorefinery cascade processing, as a more sustainable and eco-friendly method to simultaneously recover oil and proteins from fruit seed and kernel waste. To this aim, fruit residues covering different fruit varieties (e.g., stone fruits, citrus and exotic fruits) were characterized for their proximate composition, then an enzymatic assisted extraction protocol, based on the use of a protease and previously tested for other fruit by-products ( Fuso, Viscusi, Larocca, Sangari, Lolli & Caligiani, 2023 ) was performed. Also, the evaluation of yields and chemical characterization of the oils (fatty acid and unsaponifiable composition) and proteins (amino acid profile and hydrolysis degree) resulting from the extraction and purification processes has been described.…”