Extraction and characterization of protein from Irish brown seaweed Ascophyllum nodosum

    Extraction and characterization of protein from Irish brown seaweed Ascophyllum nodosum ´ Shekhar U. Kadam, Carlos Alvarez, Brijesh K. Tiwari, Colm P. O’Donnell PII: DOI: Reference:

S0963-9969(16)30294-0 doi: 10.1016/j.foodres.2016.07.018 FRIN 6345

To appear in:

Food Research International

Received date: Revised date: Accepted date:

16 March 2016 14 July 2016 22 July 2016

´ Please cite this article as: Kadam, S.U., Alvarez, C., Tiwari, B.K. & O’Donnell, C.P., Extraction and characterization of protein from Irish brown seaweed Ascophyllum nodosum, Food Research International (2016), doi: 10.1016/j.foodres.2016.07.018

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Extraction and characterization of protein from Irish brown seaweed Ascophyllum nodosum a

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Shekhar U. Kadama, Carlos Álvarezb, Brijesh K. Tiwarib*, and Colm P. O’Donnella School of Biosystems and Food Engineering, University College Dublin, Dublin 4, Ireland Food Biosciences, Teagasc Food Research Centre, Dublin 15, Ireland

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*Corresponding Author Contact Details Phone: 0035318059785 E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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This study investigates traditional and non-conventional methods of extraction of protein from Irish brown seaweed A. nodosum. Acid, alkali, combined acid-alkali with and without ultrasound pretreatment were investigated for extraction of protein from A. nodosum. Molecular weight of protein was determined using high performance size exclusion chromatography and amino acid profiling was carried out using an amino acid analyzer. Combination of first acid and then alkali extraction was found to be the most efficient method of extraction among all methods investigated (59 % of recovery); followed by single step of alkali extraction assisted with ultrasound (68.4 µm) which was able to extract 57% of total protein. Alkaline extraction was shown to yield the best protein/algae liquefaction ratio (1.28). This can be attributed to the release of polysaccharide complexes first by acid and then solubilization of proteins by alkali solvent. The molecular weight of extracted protein was found to be relatively low, in the range of 2-4 kDa average MW. The alkali method of extraction was found to be optimum for extraction of amino acids from A. nodosum.

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Keywords

Protein, Ascophyllum nodosum, Seaweed, Acid and alkali, Extraction, Ultrasound

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Extraction of protein from A. nodosum using traditional and non-conventional methods Characterization of protein by molecular weight Amino acid profiling of proteins for investigation of nutritional aspects of protein Amino acid degradation varies depending on the extraction method employed

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Highlights

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Introduction Traditionally, seaweed has been exploited as a source of food in many Asian countries,

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whereas elsewhere it has been widely used as source of biochemicals for food, pharma and

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cosmetic applications. Seaweeds are an important source of biologically active compounds including polysaccharides, carotenoids, phycobilins, fatty acids, vitamins, sterols, tocopherol and

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phycocyanins among others (Kadam and Prabhasankar 2010). The popularity of seaweeds is

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attributed to their high polysaccharide content which is used for stabilizing and thickening applications in the food industry.- Seaweeds are also increasingly exploited as a source of

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proteins. However, to date, extraction and fractionation of macroalgae proteins, peptides and amino acids has mainly been performed at laboratory scale. One of the main disadvantages of

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using seaweed as a protein source is the large variability on protein content in the raw material,

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which can vary from 3 to 47% (Harnedy & FitzGerald, 2013), depending on species,

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geographical location and season of growth (Fleurence, 1999). Generally, due to a lower photosynthetic activity which results in carbohydrates being produced and stored at a lower rate, relative protein content is higher in winter. Also the protein content of brown seaweeds is lower compared to green or red seaweeds. The protein content of A. nodosum, employed in the present study, generally varies from 3-15% of dry weight (Fleurence, 1999) and contains acidic amino acids, ranging from 18 to 44% of protein content (Harnedy and FitzGerald 2011). Ascophyllum nodosum belongs to the class Phaeophyceae and is mainly confined to the north Atlantic basin where it dominates the rocky intertidal zones and can be easily harvested (Guiry 2013). It is widely employed as supplement in food and agricultural applications (Fan et al. 2011). In general, protein from seaweed is extracted by means of aqueous, acid and alkaline extraction or enzymatic hydrolysis from dried seaweed powders; the supernatant rich in proteins is collected after centrifugation and the proteins are recovered by ultrafiltration, precipitation using 3

ACCEPTED MANUSCRIPT ammonium sulfate or chromatographic techniques (Galland-Irmouli, Fleurence, Lamghari, Luçon, Rouxel, Barbaroux, et al., 1999). Enzymatic extraction involves the use of enzymes such

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as proteases, cellulases, amylases, glucanases or endoproteases (Kadam, Álvarez, Tiwari, &

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O’Donnell, 2015), which degrade the seaweed matrix and release the proteins. Chemical hydrolysis or subcritical water hydrolysis have been also investigated (Kadam, Álvarez, Tiwari,

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& O’Donnell, 2015). These conventional methods of protein extraction are time consuming and

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require large amount of solvents and the efficiency of extraction is limited. One of the most important factors influencing isolation and extraction of seaweed proteins is the complex

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seaweed matrix which supposes a physical barrier (Harnedy and FitzGerald, 2013). Proteins of seaweed species are bound to other non-protein components such as polysaccharides and

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polyphenols (Wijesinghe and Jeon 2012); which are believed to be the main components that obstruct seaweed protein extraction (Conde et al. 2013). Because of the above-mentioned

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reasons, non-conventional extraction techniques are currently being investigated and developed to improve the extraction yield while minimizing the time and resources required. Microwave assisted extraction (MAE) (Chemat & Cravotto, 2012), supercritical fluid extraction (SFE) (Liang & Fan, 2013; Sereewatthanawut, Prapintip, Watchiraruji, Goto, Sasaki, & Shotipruk, 2008), ultrahigh pressure extraction (UPE), pressurized fluid extraction (PFE) (Nobre, Marcelo, Passos, Beirão, Palavra, Gouveia, et al., 2006), pressurized liquid extraction (PLE) and ultrasound assisted extraction (UAE) can enhance the extraction rate and yield.

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techniques improve the mass transfer rate, increasing the interaction between solvent and solute, helping to reduce extraction challenges caused by complex seaweed matrices. In this study ultrasound was investigated as a tool for enhancing extraction from A. nodosum. When ultrasound is applied, rarefactions and compression occurs. If the pressure is

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ACCEPTED MANUSCRIPT higher than the tensile strength of the liquid vapor bubbles are formed. Such bubbles, under high ultrasound fields, collapse generating a cavitation effect (Vilkhu, Mawson, Simons, & Bates,

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2008). This process of cavitation leads to peeling, erosion, particle breakdown and degradation

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of the solid-liquid surfaces, which facilitates the release of the target compound. Besides, an enhanced mass transfer can be observed (Vilkhu, Manasseh, Mawson, & Ashokkumar, 2011).

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UAE has been used for extraction of protein from various sources including microalgae

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(Parniakov et al., 2015a); wheat germ (Zhu et al. 2009), defatted rice bran (Chittapalo and Noomhorm 2009), brewers spent grain (TaNg et al. 2010), rapeseed (Dong et al. 2011), sorghum

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(Bean et al. 2006) and perilla seed (Zhu and Fu 2012). However, to date UAE has not been reported for the extraction of protein from A. nodosum. The objective of this study was to

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investigate the effect of ultrasound assisted acid/alkali extraction of protein from A. nodosum on

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yield and amino acid degradation.

Materials and Methods

Seaweed samples

Brown seaweed A. nodosum was harvested at Silver Strand beach, Co. Galway, Ireland in June, 2014. Harvested seaweed was washed to remove impurities, chopped and hot air oven dried at 40 °C for 12 h. Dried seaweed was powdered using a hammer mill (Retsch SM100, GmbH, Germany) and sieved through a 0.5 mm mesh. Samples were stored at 4 °C prior to extraction studies.

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Extraction of protein

All chemicals (NaOH, HCl, molecular weight markers for HPSEC, trichloroacetic acid,

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phosphate salts and ultrapure water) employed in this work were supplied by Sigma Aldrich (Ireland, Vale Rd, Arklow, Co. Wicklow). All chemicals were grade reagent ACS.

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Protein extraction from A. nodosum was carried out using a modification of the method

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of Harnedy and FitzGerald (2013) (Figure 1). Dried seaweed samples of 2 g were dissolved in 40

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ml of distilled water and incubated at 4 °C for 16 h. The solution was centrifuged at 9,000 rpm for 20 min at 4 °C (Sigma 2-16PK, United Kingdom). After centrifugation, the pellets obtained

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were treated with acid (HCl) and alkali (NaOH) at concentrations of 0.1, 0.2, 0.3, and 0.4 M. A solid to solvent ratio of 1:15 was used and solutions were stirred for 1 h at 4 °C and then

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centrifuged at 9,000 rpm for 20 min at 4 °C. The resultant pellets were dried at 40 °C for 18 h

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and analyzed for protein content and total liquefaction. Protein content was measured in the

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supernatant obtained in first extraction step combined with the supernatant from the second extraction step.

The second type of extraction investigated was sequential extraction where first 0.1 M HCl was added to the pellet obtained after rehydration step, followed by centrifugation and the pellet obtained from the first extraction was subsequently treated with 0.1 M NaOH. Supernatants obtained from both acid and alkali assisted extractions were mixed and dried for protein yield estimation. In a similar manner, extraction was also carried out by reversing the order of solvent addition, namely first adding 0.1 M NaOH solvent followed by 0.1 M HCl addition. A solid to solvent ratio of 1:15 was used for both extractions. In the third type of extraction, a ultrasound pretreatment was carried out. Pellet from rehydration was suspended in either 0.1M HCl or 0.1M NaOH buffer using the same solvent to solids ratio of 1:15; then the mixtures were sonicated for 10 min. A probe type of ultrasound 6

ACCEPTED MANUSCRIPT equipment with 750 W capacity and 20 kHz frequency was employed (Sonics VCX750, Newton USA); amplitude levels of 22.8 and 68.4 µm were used. The probe diameter was 13 mm.

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Additionally, an ultrasonic water bath was employed to perform a 60 min pretreatment.

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Ultrasound bath is low power equipment, to have similar impact as that of probe equipment bath treatment was given for 60 min. For water bath treatment, similar 1:15 solid to solvent ratio was

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used. Once the pretreatment was completed the supernatant and pellet were collected as

Protein determination

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previously described.

Protein content of all the samples was determined using a LECO FP628 (LECO Corp., MI,

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USA) protein analyzer based on the Dumas method according to the AOAC method 992.15

A. nodosum liquefaction

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(1990). A sample extract of 0.25 g was used for protein estimation.

After protein extraction and centrifugation for supernatant recovery, the pellet obtained was carefully collected and completely dried (105 °C, 48h). The final weight was recorded and then compared with the initial amount of dry raw material employed for extraction. %solubilized material was calculated as in Eq. 1: Eq. 1 Where W0 is the initial amount of sample (typically 2 g), and W1 is the weight of the dried pellet after extraction.

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High performance size exclusion chromatography (HPSEC) Proteins were isolated from A. nodosum extracts samples using a Waters® Alliance

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HPLC High Throughput (HT) System (Waters, Milford, MA, USA) equipped with a sample

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manager, column heater and photodiode array detector at an absorbance wavelength of 214 nm.

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A ZORBAX (GF-250/450, particle size (4-6 µm) and pore size of 150-300 Å, (Agilent Technologies, USA) macroporous HPLC column was used. Phosphate buffer at pH of 7.5 was

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used as mobile phase. Ten µl of sample was injected into the system. A flow rate of 0.85 ml/min

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was maintained in the column at 40 °C for 25 min (Ojha et als., 2016). A calibration curve was prepared using albumin (66kDa), carbonic anhydrase (29 kDa), citocrome C (12.4 kDa),

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aprotinin (6.5 kDa), angiotensin II acetate, (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe; 1046 Da) and

Amino acid profiling

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leucine enkephalin, (Tyr-Gly-Gly-Phe-Leu; 555 Da).

Selected protein samples extracted with acid, alkali and combination of first acid and then alkali were used for amino acid profiling. Samples were hydrolysed using 6 M HCl at 110°C for 23 h prior to amino acid profiling (Hill 1965). Samples were deproteinised by mixing equal volumes of 24% (w/v) trichloroacetic acid (TCA) and sample. The mixture was allowed to stand for 10 min before centrifuging at 14400 x g (Microcentaur, MSE, UK) for 10 min. Supernatants were removed and diluted with 0.2 M sodium citrate buffer at pH 2.2 to give approximately 250 nMol of each amino acid residue. Samples were then diluted using a 1 in 2 dilution with the norleucine internal standard, to give a final concentration of 125 nmol/l and subsequently derivatized with ninhydrin. Amino acids were quantified using a Jeol JLC-500/V amino acid analyser (Jeol Ltd., Garden city, Herts, UK) fitted with a Jeol Na+ high performance cation

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ACCEPTED MANUSCRIPT exchange column and detected at 690 nm according to manufacturer instructions. Examples of obtained chromatograms have been supplied as supplementary material.

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2.7 Ultrafiltration of supernatants

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Selected supernatants were ultrafiltered using a Macrosep Advance Centrifugal device (Pall Life Sciences Ireland, Cork, Ireland); a molecular weight cut off (MWCO) of 1 kDa was

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employed.. 20 mL of the supernatant were placed in such a devices and centrifuged at 3500 g for

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30 and 60 minutes. The volume of the retentate was recorded and protein content was evaluated by means of HPSEC. Relation between peak areas of initial supernatant and ultrafiltered samples

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Results and discussion

Protein content, recovery and yield of extraction

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were used to calculate the fold concentration.

The total protein content of A. nodosum was 7.133 ± 0.015% on a dry weight basis which

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is in the range of 3-15 % previously reported by Fleurence (1999). It is also comparable to reported values for other seaweeds species such as Caulerpa veravelensis (7.77 ± 0.59%), Caulerpa scalpelliformis (10.50 ± 0.91%), and Laminaria japonica (9.1%), Palmaria palmata (13.5%) (Suresh Kumar, Ganesan, Selvaraj, & Subba Rao, 2014). During this study chemical methods (alkaline and acid extraction at different HCl or NaOH concentration) were studied for their efficiency of extraction. Then, ultrasound was applied to determine its effect on extraction yield. Table 1 outlines the protein yield obtained using the range of extraction methods investigated. The protein recovery (%) was found to increase with an increase in acid concentration from 7.97±0.26% (0.1 M HCl) to 16.90±0.32% (0.4 M HCl). The pH of these samples was found to be 1.25 and 0.64 respectively. For alkali extractions, protein recovery (%) ranged from 49.57±1.27% (0.3 M NaOH, pH = 14.03) to 56.35±1.96% (0.4 M NaOH, pH = 14.32); however no statistical differences (p>0.05) were found in the yield when 0.1, 0.2 or 0.3 M of NaOH was employed. Higher recovery of proteins using alkali was observed compared to acid assisted extractions at comparable concentrations. 9

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alkaline conditions facilitating the solubilization of water

insoluble hydrophobic seaweed proteins (Barbarino & Lourenço, 2005). The effect of pH on protein extraction from Nannochloropsis sp. microalgae was more remarkable, an increase in the

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pH from 8.5 to 11 resulted in double the amount of proteins extracted when assisted by US (Parniakov et al. 2015b)

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A sequential extraction using acid and alkali was found to be most efficient among all

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methods investigated, even those involving ultrasounds. The percentage of recovery for protein from A. nodosum has been found to be 59.75±2.44 % for first acid and then alkali extraction

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method, whereas 51.07±1.63% of recovery was found for method using first alkali and then acid extraction. The acid used before alkali may have helped release the polysaccharides to protein or

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to biological matrix of cell wall, thus allowing protein molecules to get solubilized easily in the alkaline solution. However with the alkaline solution no benefit was observed in the release of polysaccharides from the cell wall, hence lower recovery and yields were obtained.

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Ultrasound at amplitude levels of 22.8 µm and 68.4 µm, and US bath were investigated

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as a non-conventional pre-treatment technique to enhance protein extraction yield. US assisted extraction at ultrasound amplitude of 68.4 µm using acid and alkali treatments alone yielded a

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recovery of 43.13±2.21% and 57.23±2.31% respectively; which supposes an increment of 540% and 27% in recovery yield respectively. When PEF was applied for protein extraction from microalgae, similar yield increases were observed, ranging from 13% to 27% depending on the species (Barba, Grimmi and Vorobiev, 2015). This remarkable improvement on acid hydrolysis after US application, leads to think that the cell wall couldn’t be eroded by simply acid attack. While these recovery yields are lower compared to sequential extraction using first acid and then alkali addition, application of ultrasound facilitates a reduction in the concentration of acid or alkali required and the extraction processing time is greatly educed from 60 to 10 minutes. Thus ultrasound can be considered as a green/eco-friendly technology to enhance traditional acid and alkali based extraction methods. The effect of ultrasound can be attributed to bubble cavitation which facilitates the disruption of biological matrices (Kadam et al. 2013) and subsequently release of proteins. Ultrasound assisted extraction has been reported to enhance the extraction yield of bioactive compounds (laminarin, fucose, uronic acid and phenolics) from A. nodosum (Kadam et al. 2015b; Kadam et al. 2015a).

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ACCEPTED MANUSCRIPT In the case of US bath pretreatment, the extraction time was similar to that in stirred acid or alkali extraction (i.e., 60 minutes); no significant differences were found when both processes are compared. The cavitation process and matrix degradation is more intense when US is applied

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by means of a probe; consequently the efficacy of the extraction is higher.

A. nodosum liquefaction

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The difference in mass between original sample (2g) and remaining pellet after extraction was measured. This analysis was carried to find out the amount of material that was solubilized

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(including proteins, carbohydrates, phenols and other seaweed components) after each treatment. The reported chemical composition of A. nodosum on a dry matter basis is: mineral residue: 17-

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34%, carbohydrates: 45-60%, proteins: 5-13% and lipids: 2-4% (Baardseth, E. 1970). It was determined that total material solubilized after acid/alkaline treatment was 71.01±2.58% or

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64.77±2.37 in the alkali/acid extraction (see Table 1), meaning that compounds other than proteins were solubilized, mainly carbohydrates and minerals, which are the most abundant

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compounds found in this macroalgae. When acid or alkaline treatment was carried out, it was

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found that liquefaction was remarkably lower, being in these cases 30.78±2.43% (0.4 M of HCl) and 61.08±3.01% (0.1M NaOH). While an increase in acid concentration promoted a higher liquefaction; the opposite effect was observed when alkaline concentration was increased. Taking into account, as it was stated previously, that higher alkaline concentrations lead to higher protein recovery; the use of alkaline extraction enables a higher purity of protein to be obtained in the supernatant. In Figure 3, it can be observed that highest ratios of protein extracted compared to total liquefaction are 1.28 and 1.27, which correspond to extracts obtained after 0.3 and 0.4M NaOH stirring extraction. This means that proteins are more prone to be extracted at alkaline pH, whereas other seaweed compounds are more easily extracted at an acidic pH. In Figure 3 the ratio of protein extracted/total liquefaction is shown. Ratios obtained employing HCl are the lowest, ranging from 0.26 to 0.51; which means that other compounds than proteins were preferentially extracted. This ratio almost doubles if US pre-treatment at 68.4 µm of amplitude is employed, reaching a value of 0.92. Highest ratios were obtained after alkaline extraction, demonstrating that protein extraction is better under such conditions.. For US pretreatment under alkaline conditions, the ratio of protein/liquefaction is lower than when no pretreatment is employed, however the percentage of proteins extracted are slightly higher using 11

ACCEPTED MANUSCRIPT US. It has been reported that ultrasounds can effectively increase the liquefaction of complex carbohydrates such as lignose and cellulose (Kunaver, Jasiukaitytė, & Čuk, 2012). These authors reported that ten minutes of US treatment was sufficient to fully liquefy raw lignocellulosic

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material; reducing the time required to complete liquefaction by a factor of up to nine compared with control. These findings are in agreement with the liquefaction of A. nodosum results after

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ultrasound treatment observed in this study, since twice the amount of raw material was

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solubilized when compared to the stirring experiments, even when the processing time is significantly shorter. These results indicate that in addition to the extracted proteins; many other

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compounds have been extracted, so a further step of purification has to be carried out in order to obtain a final extract rich in protein. It has been suggested that chromatographic techniques, salt precipitation or ultrafiltration can be used for this purpose (Galland-Irmouli, et al., 1999).

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A linear relation (r2 = 0.73) was observed between percentage of proteins extracted and percentage of total compounds solubilized (Fig 4). These results indicate that proteins are linked

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or clustered by other biomolecules in the native matrix, since is not possible to extract proteins

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without matrix liquefaction by means of the techniques applied in this work. Finally, the total quantity of liquefied material using acid or alkaline extraction separately is 74.45±0.66% (g

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solids liquefied/g of dry sample), which adjusts at the value obtained after the combined extraction process (71.01±2.58). These results suggest that specific and different compounds are being liquefied depending on the pH of the solvent used for extraction.

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Molecular weight of extracted proteins Table 1 lists the molecular weights of protein obtained after extraction methods

investigated. The molecular weight ranged from 2.6 kDa to 3.8 kDa. These values are low compared to reported molecular weights for P. palmata which ranged from 14.8 kDa to 55 kDa (Harnedy and FitzGerald, 2013). The highest molecular weight of protein identified was 3.8 kDa which was obtained using first acid and then alkali extraction. However, in the chromatograms (Figure 2) it can be observed that the retention time at the starting point of the peaks corresponds to proteins in the range of 40 kDa (15 min of elution). After this point, an increased amount of smaller proteins was detected. It can be observed that when acid extraction is performed, even after US pre-treatment, a peak corresponding to very large molecular weight compound (more 12

ACCEPTED MANUSCRIPT than 106 kDa) was detected; such a peak was not found when alkaline extraction was done, although it was detected when sequential extraction was done. This result supports the theory that different molecules are solubilized depending on the pH employed for the process. Under

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acidic conditions, carbohydrates are easier to extract.

The use of acid or alkali could lead to protein hydrolysis, thus proteins recovered are in

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form of shorter peptides, resulting in lower molecular weight of proteins in the extracts. Harnedy

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and FitzGerald (2011) showed that alkaline soluble proteins had a lower average molecular size and the bands corresponding to water soluble proteins larger than 20 kDa were not present.

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Moreover, it was reported that molecular weight of whey proteins after ultrasound treatment was significantly reduced to various sonochemical reactions and physical effects (Jambrak et al.

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2014). However, in this work, the use of ultrasounds produced a similar chromatographic profile; with no significant differences between all the extraction processes carried out.

Protein concentration by ultrafiltration (UF)

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Since the average molecular weight of extracted proteins was found to be higher than 2

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kDa, the use of membrane filtration (MWCO of 1kDa) was employed for protein purification and concentration. It was expected that small soluble compounds (small carbohydrates, phenols and salts) solubilized in the extraction process were removed with permeate; and thus yielding a final retentate richer in proteins and other large molecular weight compounds. An experiment was conducted employing supernatants from US pre-treated samples (22.8 and 68.4 µm) under 0.1M NaOH extraction solvent. Filtrate volume obtained after 30 min of centrifugation was 8 mL in every case; and 15 mL after one hour of centrifugation. Analysis of the chromatogram peak area, when compared to unfiltered sample, shows that the protein concentration increased by 4.1 and 3.9 in samples pretreated at 22.8 and 68.4 µm. The concentration of protein in the retentate after UF was 33 (22.8 µm) and 26 (68.4 µm) times higher than in the permeate obtained after 60 min. The percentage of protein loss during this process was 8.98% for protein extracted using 22.8 µm US pre-treatment and 11.58% using 68.4 µm US pre-treatment. No differences in the molecular weight distribution were observed between initial supernatant and final retentate. These results indicate that ultrafiltration is a suitable technology to concentrate the level proteins extracted from seaweeds which at the same time removing undesirable small compounds such as salts, carbohydrates and phenols. 13

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Amino acid profile of proteins

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To the best of our knowledge this is the first investigation of the amino acid profile of A. nodosum. Since the protein content of brown seaweeds is low compared to other red and green

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seaweeds, the quantity of amino acid is correspondingly lower, as well. (Fleurence, 1999). Table 2 shows the amino acid profile of protein obtained from control, acid, alkali and acid/alkali

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combined extraction methods. The results show that Arg, Ile, Leu and Tyr are completely degraded regardless of the extraction method employed. Alkaline extraction resulted in higher

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retention of Phe, Ser, Gly and Val compared to acid extraction; meanwhile Thr was degraded under alkaline extraction conditions only. Essential amino acids (His, Thr, Val and Phe) were

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found to be present in extracted samples (Table 2). It has been reported that protein hydrolysis under strong alkaline or acid hydrolysis leads to complete degradation of certain amino acids (Ravindran and Bryden 2005; Fountoulakis and Lahm 1998; Álvarez et al. 2013) into other

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Conclusion

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nitrogenous compounds.

Among the different methods of extraction investigated, it was found that sequential extraction (acid treatment followed by alkaline treatment) yielded the highest protein extraction of 59.76%; protein extracted had an average molecular weight of 3.27 kDa. This study demonstrated that the application of ultrasound facilitates a reduction in processing time (from 60 to 10 minutes) in both acid or alkali extraction processes. It was found that ultrasound assisted extraction improves the liquefaction of A. nodosum dry powder. This work demonstrates a strong correlation between protein extracted and total compounds liquefied. Finally ultrafiltration was demonstrated to be a suitable technique for concentration and purification of extracted proteins. 5

Acknowledgements

The authors wish to acknowledge financial support for this study received the from the Irish Research Council Embark Initiative.. This work has also been supported by the Marine Functional Foods Research Initiative (NutraMara project) which is a program for marine based functional food development. This project (Grant-Aid Agreement No. MFFRI/07/01) was carried

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ACCEPTED MANUSCRIPT out under the Sea Change Strategy with the support of the Marine Institute and the Department of Agriculture, Food and the Marine, funded under the National Development Plan 2007 – 2013. 6

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Harnedy, P.A., FitzGerald, R.J. (2011). Bioactive proteins, peptides, and amino acids from macroalgae Journal of Applied Phycology, 47(2), 218-232. Harnedy, P.A., FitzGerald, R.J. (2013). Extraction of protein from the macroalga Palmaria palmata. LWT - Food Science and Technology, 51 (1), 375-382. Hill, R.L. (1965). Hydrolysis of proteins. In: C.B. Anfinsen MLAJTE, Frederic MR (eds) Advances in Protein Chemistry, vol Volume 20. Academic Press, Massachusetts, USA, pp 37-107. Jambrak, A.R., Mason, T.J., Lelas, V., Paniwnyk, L., Herceg, Z. (2014). Effect of ultrasound treatment on particle size and molecular weight of whey proteins. Journal of Food Engeneering, 121(0), 15-23. Kadam, S., Donnell, C., Rai, D., Hossain, M., Burgess, C., Walsh, D., Tiwari, B. (2015a) Laminarin from Irish Brown Seaweeds Ascophyllum nodosum and Laminaria hyperborea: Ultrasound Assisted Extraction, Characterization and Bioactivity. Marine Drugs, 13(7), 4270-4280. Kadam, S.U., Álvarez, C., Tiwari, B.K., O’Donnell, C.P. (2015). Chapter 9 - Extraction of biomolecules from seaweeds. In: Troy BKTJ, editor. Seaweed Sustainability. San Diego: Academic Press. p 243-269. Kadam, S.U., Prabhasankar, P. (2010) Marine foods as functional ingredients in bakery and pasta products. Food Research International, 43(8), 1975-1980. Kadam, S.U., Tiwari, B.K., O'Donnell, C.P. (2013). Application of novel extraction technologies for bioactives from marine algae. Journal of Agriculture and Food Chemistry, 61(20), 4667-4675. Kadam, S.U., Tiwari, B.K., Smyth, T.J., O’Donnell, C.P. (2015b). Optimization of ultrasound assisted extraction of bioactive components from brown seaweed Ascophyllum nodosum using response surface methodology. Ultrasonic Sonochemistry, 23, 308-316. Khan, W., Zhai, R., Souleimanov, A., Critchley, A.T., Smith, D.L., Prithiviraj, B. (2012). Commercial extract of Ascophyllum nodosum improves root colonization of alfalfa by its bacterial symbiont sinorhizobium meliloti. Commun Soil Sci Plant Anal, 43 (18), 24252436. Kunaver, M., Jasiukaitytė, E., Čuk, N. (2012). Ultrasonically assisted liquefaction of lignocellulosic materials. Bioresource technology, 103(1), 360-366. Liang, X., Fan, Q. (2013). Application of sub-critical water extraction in pharmaceutical industry. Journal of Materials Science and Chemical Engineering, 1(05), 1. Moulton, K., Wang, L. (1982). A Pilot-plant study of continuous ultrasonic extraction of soybean protein. Journal of Food Science, 47(4), 1127-1129. Nobre, B., Marcelo, F., Passos, R., Beirão, L., Palabra, A., Gouveia, L., Mendes, R. (2006). Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from the microalga Haematococcus pluvialis. European Food Research and Technology 223(6):787-790. Ojha, K. S., Alvarez, C., Kumar, P., O'Donnell, C. P., & Tiwari, B. K. (2016). Effect of enzymatic hydrolysis on the production of free amino acids from boarfish (Capros aper) using second order polynomial regression models. LWT-Food Science and Technology, 68, 470-476. Parniakov, O., Apicella, E., Koubaa, M., Barba, F. J., Grimi, N., Lebovka, N., ... & Vorobiev, E. (2015a). Ultrasound-assisted green solvent extraction of high-added value compounds from microalgae Nannochloropsis spp. Bioresource technology, 198, 262-267. 16

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Parniakov, O., Barba, F. J., Grimi, N., Marchal, L., Jubeau, S., Lebovka, N., & Vorobiev, E. (2015b). Pulsed electric field and pH assisted selective extraction of intracellular components from microalgae Nannochloropsis. Algal Research, 8, 128-134. Ravindran, G., Bryden, W.L. (2005). Tryptophan determination in proteins and feedstuffs by ion exchange chromatography. Food chemistry, 89(2), 309-314. Sereewatthanawut, I., Prapintip, S., Watchiraruji, K., Goto, M., Sasaki, M., Shotipruk, A. (2008). Extraction of protein and amino acids from deoiled rice bran by subcritical water hydrolysis. Bioresource technology, 99(3), 555-561. Suresh-Kumar, K., Ganesan, K., Selvaraj, K., Subba Rao, P.V. (2014). Studies on the functional properties of protein concentrate of Kappaphycus alvarezii (Doty) Doty – An edible seaweed. Food Chemistry, 153(0), 353-360. TaNg, D.-S., TiaN, Y.-J., He, Y.-Z., Li, L., Hu, S.-Q., Li, B. (2010). Optimisation of ultrasonicassisted protein extraction from brewer's spent grain. Czech Journal of Food Science, 28(1), 9-17. Vilkhu, K., Manasseh, R., Mawson, R., Ashokkumar, M. (2011). Ultrasonic recovery and modification of food ingredients. Ultrasound Technologies for Food and Bioprocessing: Springer. p 345-368. Vilkhu, K., Mawson, R., Simons, L., Bates, D. (2008). Applications and opportunities for ultrasound assisted extraction in the food industry — A review. Innovative Food Science & Emerging Technologies, 9(2), 161-169. Wijesinghe, W.A., Jeon, Y.J. (2012). Enzyme-assistant extraction (EAE) of bioactive components: a useful approach for recovery of industrially important metabolites from seaweeds: a review. Fitoterapia, 83 (1), 6-12. Zhu, J., Fu, Q. (2012). Optimization of ultrasound-assisted extraction process of perilla seed meal proteins. Food Science and Biotechnology, 21(6), 1701-1706. Zhu, K.-X., Sun, X.-H., Zhou, H.-M. (2009). Optimization of ultrasound-assisted extraction of defatted wheat germ proteins by reverse micelles. Journal of Cereal Science, 50(2), 266271.

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ACCEPTED MANUSCRIPT Tables Table 1: Protein extraction yield, liquefied material and average molecular weight of A. nodosum extracts after different extraction processes. % raw material

Average molecular

extracted

solubilized

weight (kDa)

0.1 M HCl

7.97±0.26

23.12±1.66

2.81

0.2 M HCl

7.71±0.51

23.96±1.41

0.3 M HCl

15.47±0.11

30.16±1.03

3.27

0.4 M HCl

16.90±0.32

0.1 M NaOH

51.80±1.74

0.2 M NaOH 0.3 M NaOH

3.04

30.78±2.43

2.62

61.08±3.01

2.87

50.82±0.99

51.46±2.00

2.82

49.57±1.36

38.15±0.88

2.82

56.35±1.96

43.67±1.96

2.82

59.76±2.44

71.01±2.58

3.27

0.4 M NaOH -> 0.4 M HCl

51.07±1.63

64.77±2.37

3.81

0.1 M HCl -US-22.8 µm

18.94±1.02

41.13±2.11

3.28

0.1 M HCl -US-68.4 µm

43.13±2.21

45.95±1.99

2.72

0.1 M NaOH -US-22.8 µm

26.27±1.74

50.43±2.67

2.89

0.1 M NaOH -US-68.4 µm

57.23±2.31

73.48±3.32

2.99

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% protein Extraction

ACCEPTED MANUSCRIPT Table 2: Amino acid profile of proteins extracted from A. nodosum. Amino acid

Method of extraction Acid

Alkali

Acid and

PT

(% of total amino acid)

Native seaweed

alkali

12.68±0.17

2.74±0.17

3.04±0.11

Taurine (Tau)

--

--

25.17±0.32

--

Aspartic acid (Asp)

25.36±0.36

13.24±0.53

15.73±0.77

14.01±1.01

Threonine (Thr)

6.29±0.11

--

0.21±0.07

5.09±0.55

Serine (Ser)

--

0.60±0.05

--

4.49±0.41

Glutamic acid (Glu)

24.04±0.88

41.02±0.98

25.19±1.02

24.21±0.99

Glycine (Gly)

--

2.41±0.45

--

5.66±0.25

Alanine (Ala)

6.58±0.51

5.17±0.21

4.71±0.16

6.05±0.41

13.11±0.27

16.75±0.70

14.69±0.33

0.44±0.07

--

0.21±0.03

0.99±0.04

5.85±0.17

Isoleucine (Ile)

--

--

--

3.96±0.11

Leucine (Leu)

--

--

--

6.16±0.40

--

--

--

2.75±0.12

Phenylalanine (Phe)

--

1.18±0.14

--

5.24±0.26

Histidine (His)

7.78±0.68

1.59±0.09

3.22±0.10

2.40±0.09

Lysine (Lys)

5.33±0.14

5.14±0.16

7.35±0.86

5.91±0.14

Arginine (Arg)

--

--

--

4.73±0.31

Valine (Val)

Tyrosine (Tyr)

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Cysteine (Cys)

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11.46±0.21

SC

Cysteic acid (CA)

profile

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Figure 1 Flow-chart for extraction of protein

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Figure 2 HP-SEC chromatogram of protein profile obtained after different extraction methods. Arrows point the molecular weight size of main peaks. Figure 3: Ratio of percentage of protein extracted and percentage of liquefaction.

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Figure 4: Relation between protein extracted and material liquefied under different extraction process. Circles: acid extraction; squares: alkaline extraction; rhombus: sequential acid/alkaline extraction. Hollow figures correspond to ultrasound assisted extraction. Line represents equal protein extraction and liquefied material.

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Figure 2

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Figure 3

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Figure 4

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Graphical abstract

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ACCEPTED MANUSCRIPT Highlights

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Extraction of protein from A. nodosum using different traditional and novel methods Characterization of protein by molecular weight Amino acid profiling of proteins for investigation of nutritional aspects of protein

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