RESEARCH

Protein diversification: exploring the potential of seaweed for food

Authors: Stephen Haines, Alastair Ross, Ancy Thomas, Linda Samuelsson, Santanu Deb-Choudhury

Affiliation: AgResearch Limited, Lincoln Research Centre, 1365 Springs Road, Lincoln 7674, New Zealand

Introduction

All-year-round availability and relatively easy collection makes seaweed a potentially inexpensive food source [1]. Undaria pinnatifida is directly used in culinary applications in several Asian countries [2]. Seaweed in general is, however, also used exclusively for the extraction of food hydrocolloids such as agar, alginates and carrageenan. Marine algae are a viable protein source with an essential amino acid composition often on par with other food protein sources [3]. They are a rich source of nutrients including those that are insufficient in some Western diets, including fibre, micronutrient minerals, omega-3 fatty acids and other lipids, and proteins. In general seaweed proteins are rich in glycine, alanine, glutamic acid and arginine and, although limited in lysine and cysteine, contain all the essential amino acids. The amino acid score in Undaria species is determined to be 100, similar to that in animal-derived foods [4]. Red seaweed has the highest protein content, comparable to legumes at 30-40% on a dry weight basis, whereas brown seaweed such as Undaria has a protein content of 16% [5]. Amino acids such as aspartic and glutamic acid which impart flavour are present in higher quantities in brown compared to red seaweed [1]. Undaria pinnatifida or ‘Wakame’ is a kelp that has been studied extensively in New Zealand as an invasive species but the best approach for its management is yet to be reached and the ecological impact of Undaria has not been conclusively proven [6]. Undaria pinnatifida has a broad ecological niche and is highly productive at forming habitats. It inhabits rocky substrates up to a depth of 18 m and is widespread at depths of 1-3 m [7]. It is an important species for seaweed mariculture in China, Japan and Korea and therefore has significant economic value. Although Undaria pinnatifida may have an ecological impact it is not considered major as it does not appear to cause ecosystem change in most invaded regions. This is mainly due to its low natural dispersal ability and comparatively low rate of nutrient uptake and nitrate storage compared to other invasive brown macroalgae species [8]. The Ministry of Primary Industries (New Zealand) has a revised policy for the commercial use of Undaria and for its wild harvest from artificial substrates or when cast shore, in selected regions [6]. Nutrient availability

Blanched and salted Undaria pinnatifida is a major wakame product. For blanching, fresh wakame is plunged in water at 80°C for one minute and then cooled quickly using cold water. For the salted product, 1:3 ratio of salt to seaweed is used and the seaweed is treated for 24 hours after which it is stored at -10°C [9, 10]. Wakame is usually cooked before consumption. Although seaweed have poor protein digestibility in their raw and unprocessed form and therefore require adequate processing to improve their bioavailability in food, in vitro studies suggest that Undaria pinnatifida has 87% bioaccessibility expressed as a percentage of casein bioaccessiblity (100%) [3]. One of the major reasons for reduced algal protein digestibility is due to the fibre content of the algal cell wall [11]. Other contributing factors are a high level of cell wall anionic polysaccharides, which may vary according to species, the time of harvest [12] and the presence of phenolic compounds that react with amino acids, rendering them inaccessible [12]. Previous studies have shown different modes of cooking wakame and their effects on nutrient availability of potential bioactives such as carotenoids, polyphenols and polysaccharides and physical characteristics such as colour and texture [13]. There is little evidence of the effect of cooking on the protein profiles and their subsequent modifications. However, to understand cooking effects, robust procedures need to be established to first extract and then to analyse the proteins present in wakame. Proteins in marine algae are also affected by seasonal changes such as seawater temperature and nutritive salt concentration, which greatly affect the growth and maturation of seaweed. Proteomic and metabolomic studies can provide valuable information at a molecular level both on the seasonal changes in seaweed composition and on the effect of processing such as cooking on protein bioaccessibility in seaweed derived food.

Research project

Joint research between AgResearch, University of Otago and A*STAR (Singapore) funded through the MBIE Catalyst Strategic fund will be looking at the digestive and nutritional attributes of seaweed, as relevant to humans, using Undaria pinnatifida as an exemplar. The research will create new knowledge about flavour, digestibility, and health benefits of Undaria seaweed from New Zealand and Singapore, as a whole food. The project’s aim is exploring how these attributes can be modulated through cooking technologies, to create an alternative whole food protein source. As well as characterising the protein fraction of Undaria and how it is affected by cooking, we will also be investigating the small molecules (metabolites) present in the seaweed to determine the impact of cooking beyond proteins and get a wider understanding of potential functionality of seaweed constituents. This short paper reports some early results obtained during development of proteomic and metabolomic methods that will be applied to the detailed analysis of the Undaria.

Materials & methods

Samples of frozen Undaria and Ulva seaweeds, and of air-dried Undaria that had been rinsed with brine, were provided by the Department of Botany, University of Otago. Phenol protein extraction method

Dried Undaria was ground under liquid nitrogen with a mortar and pestle. The resultant powder was then sequentially extracted with cold (-20°C) 10% trichloroacetic acid in acetone, 0.1 M ammonium acetate in 80% methanol, and finally 80% acetone. The residue was air dried and then extracted at 4°C with Tris-buffered phenol (pH 8.0) and dense SDS buffer (2% SDS, 30% sucrose and 100 mM dithiothreitol in 100 mM Tris buffer, pH 8.0, containing Roche cOmplete™ protease inhibitor cocktail). Proteins were precipitated from the upper phenol phase by incubation with methanol containing 0.1 M ammonium acetate overnight in a -20°C freezer. The precipitated protein was sequentially washed with cold (-20°C) methanol and with acetone and was then allowed to air dry. Finally, the protein was resuspended in 2% SDS / 4 M urea in 50 mM Tris (pH 8). Protein concentration in the extract was determined using the DC Protein Assay (Bio-Rad). Protein digestion with trypsin

The filter-aided sample preparation (FASP) method [14] was used to digest the Undaria protein extract prior to liquid chromatographytandem mass spectrometry (LC-MS/MS). In brief, 50 µg of protein was added to a 10 kDa ultrafiltration device and was reduced by incubation for 1 h with dithiothreitol in 8 M urea/100 mM Tris buffer, pH 8 (‘UT buffer’). After three washes with UT buffer, the reduced proteins were alkylated with iodoacetamide in UT buffer for 60 min in the dark. Following washes with UT buffer and then 50 mM ammonium bicarbonate, the reduced and alkylated protein was finally digested with trypsin at 37°C and at an enzyme:substrate ratio of 1:25. The digest was recovered by centrifugation and was dried in a vacuum centrifuge before being resuspended in 0.1% formic acid for LC-MS/MS analysis. LC-MS/MS analysis of digested Undaria proteins

LC-MS/MS was performed on an Ultimate 3000 nano-LC (Thermo Scientific) connected to an impact II Q-TOF mass spectrometer by a CaptiveSpray interface fitted with a nanoBooster device (Bruker Daltonics). The tryptic digest was injected onto a PepMap100 C18 trap column (5 µm particle size; 0.3 x 5 mm; Thermo Scientific) and then separated on a ProntoSIL C18AQ column (15 cm, 100 µm i.d., 3 µm particle size and 200Å pore size; nanoLCMS solutions, Oroville, CA, USA) at a flow rate of 1 µL/min. Mobile phase A was 0.1% formic acid and mobile phase B was 0.1% formic acid in acetonitrile. A multistep linear gradient was used for separation of peptides, as is shown in Fig. 1. Peptide and protein identification

Peptides and proteins were identified using PEAKS Studio XPro (Bioinformatics Solutions Inc, Waterloo, Canada). This involved initial de novo sequencing followed by a search against a non-redundant database of Undaria, Ulva and Gracilaria sequences downloaded from UniProt (2021_03 release, 32,300 sequences). Finally, an exhaustive PEAKS PTM search was conducted to identify peptides containing amino acid modifications.

Figure 1: LC-MS chromatogram of a phenol protein extract of Undaria pinnatifida (blue). This is overlaid by the LC gradient (grey), which rose from 2% to 20% solvent B over 45 min and then to 45 % B over a further 15 min. The consistent spread of peaks across the chromatogram shows that seaweed digest contains a multiplicity of peptides of widely varying hydrophobic character, including some very abundant ones that are evidenced by the major peaks in the chromatogram

Figure 2: REIMS fingerprints of Undaria (top) and Ulva (bottom). The lines in the fingerprints are from metabolites and lipids present in the samples, with the scale based on relative amount of each metabolite feature in the sample. The X-axis is m/z, nominally equivalent to molecular weight. These spectra show that Undaria and Ulva have highly divergent metabolite profiles and provide further support for the diversity of seaweed and from this, different potential for food applications.

Results and discussion

Seaweed is a difficult matrix from which to extract protein due to the strength of the macroalgal cell wall and the presence of high levels of polysaccharides and polyphenols [15]. Global protein extraction for proteomics requires efficient disruption of the cell wall and removal of non-protein nitrogen compounds that can bind proteins and interfere with the analysis. Several methods have been developed to achieve this, with the most frequently employed being those based on the protocol of Contreras et al. [16]. This involves the phenol extraction of seaweed that has been pulverised under liquid nitrogen. In this pilot study we have tested the phenol extraction procedure on Undaria and evaluated the quality of the protein extract by LC-MS/MS. As may be seen in Fig. 1, the phenol extract of Undaria gave a complex LC-MS chromatogram that contained a multitude of peaks evenly spaced over the full range of the LC gradient. PEAKS Studio XPro software identified 927 peptides derived from 115 proteins in the sample, with the most abundant (based upon number of identified peptides) being ribulose bisphosphate carboxylase, ATP synthase, phosphoenolpyruvate carboxykinase and heat shock protein 70. These preliminary results confirm that the phenol extraction method works well with Undaria and is suitable for use in future detailed proteomic analyses that quantitatively reveal the impacts of environmental parameters and processing on the protein composition of the seaweed. We have also optimised the use of LA-REIMS to measure seaweed. REIMS is an instrument that detects a fingerprint of the small molecules (up to 1500 Daltons) present in a sample without the need for timeconsuming sample preparation [17]. Results obtained by application of the technique to two seaweeds, Undaria and Ulva, are presented in Fig. 2. REIMS fingerprinting of the two different types of seaweed found a major compositional difference between Ulva and Undaria (Fig. 2). While there is overlap for some metabolite features (e.g., m/z 255 and 309), there is a difference in the ratio, and a clearly higher proportion of high molecular weight features in Ulva, which correspond to phospholipids and triglycerides. Further work is required to confirm compound identifications. The potential impacts of the difference in metabolite and lipid content between different seaweeds for different food as well as potential relationships with the protein content will be investigated in the MBIE-funded programme. The results to date suggest that using LA-REIMS is a useful tool for getting a rapid overview of compositional differences between different species of seaweed as a first step to guide further detailed analyses.

Conclusion

Seaweed is a challenging material for analytical measurements and adjusting proteomic and metabolomic tools to measure the protein and metabolite content of macroalgae underpins the work with our research partners on how the diversity of composition is influenced by cooking and how these impact on culinary and nutritional aspects of seaweed. This work, together with other projects on seaweed, will help to fulfil the potential of an abundant New Zealand marine resource that is still locally underutilised as a domestic and export whole food and ingredient. Acknowledgements

This research was funded by the 2020 Catalyst Strategic - New Zealand Singapore Future Foods Research Programme (NZBN 9429038966224). Seaweed samples were kindly provided by Katja Schweikert, Department of Botany, University of Otago. References

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