Abstract
Microalgae have been regarded as a viable alternative protein source for food due to their high protein content and well-balanced amino acid profiles. However, the extraction of microalgae proteins is affected by the presence of robust cell walls which requires the application of disruption technologies. Although progress has been made in using various cell disruption techniques, there is still a need to increase cell disruption efficiency by considering time, cost, scalability and the morphological variety of microalgae. Moreover, there is currently a lack of information on the impact of the applied disruption process on the in vitro digestibility, characterisation, and profiling of the obtained proteins. This research aimed to investigate cell disruption strategies and their impact on protein extraction, in vitro digestibility, and modelling digestion kinetics of three microalgae species with different cell wall structures, Arthrospira platensis, Nannochloropsis sp., and Tetraselmis sp. Proteomics techniques were also used to study the protein profiles and putative allergenicity risk assessments in A. platensis species as a selected species with a higher amount of extracted proteins. The microalgae in the first investigation were subjected to high-pressure homogenisation (HPH) with various operational conditions. Each tested microalgae needed different operational strategies depending on its cell wall structure. HPH did not change the SDS-PAGE profile of protein extracts obtained from A. platensis compared to the control. On the contrary, based on the differences result of untreated and treated samples, it was clear that Nannochloropsis sp. and Tetraselmis sp. needed HPH to release proteins with different operational processing conditions.
In Nannochloropsis sp., the optimum yield was achieved using low biomass concentration (2% w/v) and two passes through the homogeniser with the highest loading pressure (1500 bar). HPH process at 600 bar was able to rupture the Tetraselmis sp. cell walls and release the proteins, suggesting less resistance of this species compared to Nannochloropsis sp. In both species, the application of intense operational conditions i.e., more passes (for Nannochloropsis sp. > 2) and higher pressures (for Tetraselmis sp. above 1200 bar), decreased the yield. This indicates that although the excessive pressures could increase cell rupture (as confirmed by fluorescent microscopy), they decrease the protein yield(as confirmed by the coulometric assay result). Since HPH is expensive equipment that is economically viable only for limited applications, in further study, the application of high-speed homogenisation (HSH) as a low-cost, simple, and scalable mechanical cell disruption was investigated.
The application of freeze-drying and HSH combined with pH treatment as another disruption method had different effects on the amount of soluble proteins, thus confirming variations in the cell structures. The rehydration process of the freeze-dried samples, referred to as "washout," resulted in the significant release of microalgae proteins, specifically measuring 25% of dry weight (DW), 15% of DW, and 50.23% of DW from A. platensis, Nannochloropsis sp., and Tetraselmis sp., respectively. The combined treatment of HSH and pH values indicated a synergic effect on extracting proteins from A. platensis and Tetraselmis sp. The combination of shearing at 23000 rpm for 3 minutes at pH 12 resulted in the highest protein yield (405.34 mg. g ā1 DW) for A. platensis. For Tetraselmis sp. species, the optimum conditions to reach the highest yield (9.89 mg. gā1 DW) were 13000 rpm for 15 min, combined with treatment at pH 12. However, this synergic effect was not observed in Nannochloropsis sp. The only efficient parameter was a high alkaline pH (pH 13), which remarkably led to the release of proteins (48.6 mg. g ā1 DW).
The assessment of in vitro protein digestibility further uncovered disparities among the microalgae samples and the treatment methods employed (overnight soaking and non-soaked) to explore the influence of sample processing on the digestibility of the samples.
The in vitro protein digestibility assessment also revealed differences in the microalgae samples. The in vitro protein digestibility of non-soaked A. platensis samples was higher than the two samples However, the protein hydrolysis patterns changed after the overnight soaking of the species, where the digestograms of A. platensis and Nannochloropsis sp The result indicated a remarkable improvement in the digestibility of Nannochloropsis sp. after soaking. This pre-treatment improved the in vitro digestibility of Tetraselmis sp., although the degree of the protein hydrolysis of this species (305.42 mg L-serine equivalent released per g DW) was still less than that of two other microalgae. The effect of HSH processing treatment (3000 to 23000 rpm) of A. platensis revealed a sharp decrease in the degree of protein hydrolysis due to increasing homogenisation speeds. At the highest speed (23000 rpm), the protein hydrolysis showed a 31.73% decline in the degree of hydrolysis. This decrease in the degree of protein hydrolysis could be attributed to the protein structure changes that limit the accessibility of hydrolysis enzymes.
The in-depth proteome characterisation of the A. platensis sample revealed the effect of freeze-drying (washout) and HSH combined with pH treatment in obtaining proteins with different predicted physicochemical properties. Overal, 699 proteins were identified, but the maximum number of exclusive proteins (79) was obtained at high-shear homogenisation and pH 2 treatment samples. Unsupervised hierarchical cluster analysis (HCA) and partial least square discrimination analysis (PLS-DA) analysis indicated a clear separation of the identified proteins obtained in pH 2 treatment compared to the entire identified proteome. The second separation was observed in proteins obtained from the rehydrating treatment (washout), which showed significant differences (p-value<0.05) in GRAVY the score and instability index compared to proteins obtained after shearing and pH treatment.
A. platensis proteins showed significant similarity with some proteins coded in the Allergome and Allergen Nomenclature databases. Allergenicity assessment of the A. platensis proteome resulted in 69 putative allergens. The most predominant food allergen was the C-phycocyanin beta subunit (P72508) found in all the extraction protein treatments.
This research increases our knowledge about protein extraction from microalgae and helps us understand that structural variations in morphology affect the extraction efficiency, final yield, and the in vitro digestibility of proteins from microalgae of various species. These findings can help manufacturers choose the proper disruption and extraction processing based on the microalgae species to obtain optimum protein yields or modulate microalgae protein digestibility. Also, the study highlighted the effect of the applied process on the protein profile of the various microalgae, suggesting that the efficiency and impacts of extraction methods need to be considered when microalgae proteins are to be used as ingredients in functional foods.