Protein and solute release from the red seaweed, Gracilaria sp., using a high voltage pulsed electric field and pH shift

Protein and solute release from the red seaweed, Gracilaria sp., using a high voltage pulsed electric field and pH shift | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protein and solute release from the red seaweed, Gracilaria sp., using a high voltage pulsed electric field and pH shift Mrinal Kashyap, Supratim Ghosh, Klimentiy Levkov, Yoav D. Livney, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4131953/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract This study explores the potential of Gracilaria sp., a red seaweed species, as a source of protein ingredients. We investigated the effects of high voltage pulsed electric field (PEF) pre-treatment, followed by either water-mediated extraction or pH shift, on the total-solute (“dry matter”) and protein release, and the content of essential and branched-chain amino acids (BCAA) in the extracts. The PEF was administered using a custom-made generator within a specially designed electroporation cell with sliding electrodes. The treatment parameters comprised 1000 V, a 50 µs pulse duration, 200 pulses at a 3 Hz frequency, and an inter-electrode gap ranging from 3.9 to 4.4 mm. The pH shift extraction was executed through combination of fractions extracted sequentially at pH-shift steps spanning the pH range of 1 to 12. Both PEF and pH played pivotal roles in the fractionation of Gracilaria sp., enhancing protein release compared to water-mediated extraction, PEF pre-treatment, or pH shift alone. The combination of PEF and sequential pH washes (pH 1 to 12) resulted in a total solute release of 9.61±0.04% and protein release of 28.02±0.2% with essential amino acid (EAA) content of 63.6±5.3%, and BCAA content of 3.47±0.06%. It is noteworthy that PEF significantly increased the EAA and BCAA content of the protein extracts, irrespective of pH shifts. These findings underscore the promising potential of PEF pre-treatment in producing functional food ingredients derived from macroalgal sources. Macroalgae Pulsed electric field (PEF( Protein functional food ingredients essential amino acids branched amino acids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Alternative proteins play a pivotal role in achieving the UN Sustainable Development Goals (SDGs) by addressing concerns related to climate change and food insecurity (Fanzo 2019). Conventional animal-based protein production is unsustainable due to its adverse environmental impact, low plant-protein to animal-protein conversion efficiency, and high ecological footprints (Geada et al. 2021). While plant-based alternatives are more environmentally friendly, they still impose strain on freshwater and agricultural land resources (De Boer and Aiking 2011). Seaweeds present a promising solution to these challenges, as they can be cultivated in seawater, not requiring freshwater or land, and yield valuable biomass with various prospects, especially as alternative protein sources (Hua et al. 2019; Øverland et al. 2019). Among seaweeds, red macroalgae, including Gracilaria sp., have gained attention due to their relatively high protein content, with reported content ranging from 2.7% to 47% (Nan et al. 2017; Rawiwan et al. 2022). However, commercializing these proteins remains challenging due to variability of chemical composition, lack in suitable harvesting technologies and in environmentally responsible extraction processes. Innovative approaches are necessary to unlock the full potential of Gracilaria sp. and other macroalgae as sustainable protein sources. The commercialization of seaweed proteins has been hindered by technological challenges and associated costs. The production of proteins from macroalgae involves four main steps: cultivation, harvesting, extraction, and purification. Harvesting and extraction, in particular, are costly and pose significant obstacles. Various techniques, such as ultrasonication, microwave-assisted extraction, pH-shift methods, and high-pressure processing, have been explored for protein extraction from Gracilaria sp. However, the presence of agar in its cell walls makes protein extraction challenging in this species, as it reduces protein release yield (Juul et al. 2021; Kazir et al. 2019; Magnusson et al. 2019; O’Connor et al. 2020). One promising technology for biomass conversion into value-added compounds is pulsed electric fields (PEF) (Golberg et al. 2016). PEF treatment involves applying voltage pulses across biological membranes, leading to electro permeabilization and extraction of biomolecules from cells (Pereira et al. 2022; Pereira et al. 2023). We recently reported the use of a continuous PEF device to extract water-soluble proteins from Gracilaria sp. biomass (Kashyap et al. 2022). The current study aims to further enhance protein extraction by combining PEF with pH shift. PH shift is a promising technology for seaweed protein extraction, used previously on Porphyra umbilicalis , Ulva lactuca , and Saccharina latissimi (Harrysson et al. 2018), and some other species like Ulva fenestrate (Juul et al. 2021), but not on Gracilaria sp . While previous studies have explored protein extraction from various Gracilaria species ( Table 2 ) (Bozdemir et al. 2022; Fleurence et al. 1995; Kazir et al. 2019), none have utilized PEF pretreatment methods except our previous study on the Gracilaria sp. using a continuous PEF device (Kashyap et al. 2022). The present study represents the first report of combining PEF and pH shift to improve total solute and protein release from Gracilaria sp. We also examined the composition of the released proteins in terms of essential and branched amino acid composition. We show how process parameters significantly affect the release yield and composition of Gracilaria sp. proteins, with regards to their potential food application. 2. Materials and methods 2.1. Gracilaria sp. biomass production and harvesting Gracilaria sp. biomass was grown in a 700 L polyvinylchloride (PVC) tank with running seawater and continuous aeration at Israel Oceanographic and Limnological Research (IOLR), Haifa, Israel, in 2021. Twice a week, 0.1 mM PO 4 and 1.0 mM NH 4 were added to the seaweed tank. The biomass utilized as a stock was 1.5 kg FW m -3 and harvested after it grew to 3-4 kilogram FW m -3 in around 5 weeks. The harvested biomass was cleansed in a 20L tank filled with clean tap water. A batch centrifuge was used to remove surface water and the "fresh weight" (FW) biomass of Gracilaria sp. was obtained. 2.2 Description of the high voltage PEF system The electrical circuit of the High Voltage Pulse Generator (HVPG) used in this investigation is shown in Figure 1a and was described in detail in our previous publications (Levkov et al. 2020; Prabhu et al. 2020; Prabhu et al. 2019). The sliding Press-Electrode Device ( Figure 1b ) was created especially for handling biomass while applying pressure during PEF-induced electroporation. To exert this pressure, one electrode is allowed to move freely and vertically while being driven by the weight placed on the load-receiving platform. The sliding Press-Electrode Device consists of an Electroporation Cell (EPC), an Electroporation Cell Retainer (EPCR), a load-receiving platform, a node of the moving electrode, a holder, and a sensor for detecting the inter-electrode gap. Guides at the base of the holder ensure precise alignment of the installed EPCR. The EPC ( Figure 1c ), which consists of a cylinder with one electrode at the bottom and another at the lower end of the sliding electrode assembly's rod, acts as the working volume for electroporation and pressing. The liquid fraction can exit the cylinder through slit-like apertures in the side walls, and a ring-shaped groove renders it convenient to drain the liquid component into a storage container. The high-voltage input of the HVPG is attached to the operational electrode of the EPC via a male connector. 2.3 Experimental design Using a spinner, 20 g of FW Gracilaria sp. biomass was dewatered until less than 1 g of surface water remained. The PEF apparatus described above was then used to treat 20 g of FW biomass in three replicates using the following parameters: frequency=3 Hz; pulse duration=50 µs; number of pulses=200; pulse voltage=1000 V; inter-electrode gap was varied in the range of 3.9 to 4.4 mm; the 20 g of biomass was treated with 10 PEF cycles by feeding 2 g/cycle (step 1 in Figure 2 ). The PEF treatment steps were not used in the control group. Following PEF, the treated biomass was suspended in 100 mL of distilled water, agitated at 32°C and 150 RPM overnight in a shaking incubator (Benchmark Scientific, Incu-shaker small, USA), and then separated with a batch centrifuge (Yingtai Instruments, TGL 18, China) at 3750 g for 10 min (steps 2 and 3 in Figure 2 ). The biomass (solids 3b, Figure 2 ), was further treated with different pH treatments: 1) sequential pH 12 to 1 extraction, 2) sequential pH 1 to 12 extraction ( Table 1, steps 4 and 5 in Figure 2 ). The compositions of buffers used appear in Table 2 . To establish the total solutes (dry matter) released and protein released, all the liquid fractions from the sequential extractions were combined. The residual biomass was dried in an oven at 40°C ( step 6, Figure 2 ). Table 1. Protein extraction from Gracilaria sp. biomass using various process steps; process description based on the experimental design shown in Figure 2. Process stage Equipment Process parameters Control (without PEF) 1) Pressing 5 kg weight, gravitational PEF device 20 g of biomass was pressed with 10 cycles by feeding 2 g/cycle, no PEF applied. 2) Water-mediated extraction Shaker incubator Condition 1: 32°C, 150 RPM for 1 hr 3 and 5) centrifugation Batch centrifuge 3750 xg for 10 min 4) pH treatment Shaker incubator Conditions 2 and 3: Sequential pH 1 to 12 and 12 to 1 treatments: 2 g FW biomass from step 3 (b), Figure 2 was treated with solutions at pH levels ranging from 1 to 12 (condition 2) and 12 to 1(condition 3). The same biomass was transferred from one falcon tube to another falcon tube with 10 mL of solution of a different pH and incubated at 32°C and 150 RPM for 1 hr at each pH . Then it was centrifuged, and separated. The protein content of all liquid (supernatant) fractions was combined for each condition. 6) Drying Oven 40°C PEF treatment (with electric fields) 1) Pressing 5 kg weight, gravitational PEF device Fresh biomass (20 g) was treated with 10 PEF cycles by feeding 2 g/cycle. PEF parameters: Frequency=3 Hz, pulse length=50 µs, number of pulses=200, pulse voltage=1000 V, inter-electrode gap varied from 4.4 to 3.9 mm because of the biomass compression and liquid extraction. 2) Aqueous extraction Shaker incubator Condition 4 : 32°C, 150 RPM for 1 hr 3 and 5) centrifugation Batch centrifuge 3750 xg for 10 min 4) pH treatment Shaker Incubator Conditions 5 and 6: Sequential pH 1 to 12 and 12 to 1: 2 g FW biomass from step 3 (b) was treated with solutions at pH levels ranging from 1 to 12 (condition 5), and from 12 to 1 (condition 6) The same biomass was transferred from one falcon tube to another falcon tube with 10 mL of different pH solution and incubated at 32°C and 150 RPM for 1 hr in each pH . Then it was centrifuged, and separated. The protein content of all liquid (supernatant) fractions was combined for each condition. 6) Drying Oven At 40°C Table 2. Volumes and molar strengths of the salts, acids, and bases used for preparation of pH solution 1 to 12 used in the current study. pH solution Composition of the solution Molar Concentration (M) pH1 50 mL of 0.2 M KCl + 134 mL of 0.2 M HCl 3.68 pH2 50 mL of 0.2 M KCl + 13 mL of 0.2 M HCl 1.26 pH3 100 mL of 0.1 M C 8 H 5 KO 4 + 44.6 mL of 0.1 M HCl 1.44 pH4 100 mL of 0.1 M C 8 H 5 KO 4 + 0.2 mL of 0.1 M HCl 1.02 pH5 100 mL of 0.1 M C 8 H 5 KO 4 + 45.2 mL of 0.1 M NaOH 1.45 pH6 100 mL of 0.1 M KH 2 PO 4 + 11.2 mL of 0.1 M NaOH 1.11 pH7 100 mL of 0.1 M KH 2 PO 4 + 58.2 mL of 0.1 M NaOH 1.58 pH8 100 mL of 0.1 M KH 2 PO 4 + 93.4 mL of 0.1 M NaOH 1.93 pH9 100 mL of 0.1 M tris (hydroxymethyl) amino methane + 11.4 mL 0.1 M HCl 1.11 pH10 100 mL of 0.05 M NaHCO 3 + 21.4 mL of 0.1 M NaOH 0.62 pH11 100 mL of 0.05 M NaHCO 3 + 45.4 mL of 0.1 M NaOH 0.96 pH12 50 mL of 0.2 M KCl + 12 mL of 0.2 M NaOH 1.04 2.4 Protein quantification The protein content in the initial biomass was quantified after cell wall and membrane disruption by bead-beating under alkaline conditions, and after drying in an oven at 40°C until reaching a constant weight. Gracilaria sp. dry biomass (15 mg) and 1.5 mL of 2 M NaOH were mixed. Zirconium beads (2mm, Sarstedt) were added to one-third of the bead-beater tubes containing a mixture of biomass and 2 M NaOH. The tubes containing the mixture were then put into a Mini Bead-Beater-16, Model 607 EUR, Biospec, OK, and subjected to three cycles of 60 seconds each, separated by 10-minute intervals, at 25°C. After bead beating, the tubes were centrifuged at 19,500 g for 20 min in an Eppendorf 5424 centrifuge (Germany), and protein was quantified in the supernatant (Prabhu et al. 2020). The protein content of the various liquid fractions collected throughout the investigation was determined using Lowry's technique. To create the linear calibration curve, several dilutions corresponding to concentrations ranging from 0-500 µg/mL were prepared, using a bovine serum albumin (BSA) stock solution of 1 mg/mL (Lowry et al. 1951). To measure protein concentration, 100 µL of samples or standard (BSA, 0-500 g/mL) were mixed with 200 µL of Biuret reagent (500 µL of 1% cupric sulfate, 500 µL of 2% sodium potassium tartrate, in 50 mL of 2% sodium carbonate in 0.1 N NaOH). After thoroughly blending, the mixture was equilibrated at room temperature for 15 min. 20 µL of 1 N Folin and Ciocalteu's reagent (Sigma-Aldrich, St. Louis, USA) were added to the reaction mixture and it was incubated for 30 min at room temperature. After the incubation, absorbance at 650 nm was measured using a microplate reader (TECAN Infinite 200-Pro, Tecan, Switzerland). 2.5 Amino acid profiling The protein was extracted from the PEF-treated samples and control from step 3a) and step 5a) ( Figure 2 ) in aqueous solution and following sequential pH changes from 1 to 12 and from 12 to 1. The protein-rich sediment was precipitated using 35% and 85% ammonium sulfate salting out processes (Duong-Ly and Gabelli 2014). The precipitated proteins were then dialyzed using a SnakeSkin™ dialysis bag (3.5 kDa, ThermoFisher Scientific, USA). The dialyzed proteins were then lyophilized and analyzed for amino acid profile through high-pressure anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) as follows. One mg of biomass was added to 1 mL of 6 M HCl and heated at 112 °C for 16 h in a dry bath (Bio-Base, China). The samples were then dried using nitrogen. The dried samples were suspended in 1 mL of ultrapure ion chromatography grade water and left at rest for 1 h (Kazir et al. 2019). The samples were then filtered using a 0.22 μm syringe filter. The filtered samples were separated through HPAED-PAD Dionex ICS-5000 (Dionex, Thermo Fischer Scientific, MA, USA). Amino-pack 10 analytical grade column was used for amino acid separation. Signal detection was done using a gold AAA electrochemical detector with an AgCl reference electrode. Separation was achieved with gradient elution of the following parameters: 0.250 mL/min, column temperature 30°C, auto-sampler temperature 5°C, and a 25 μL injection loop volume. An amino acid standard mix (AAS18, Sigma-Aldrich, MO, USA) with the following dilutions 1/5, 1/10, 1/15, 1/20, and 1/25 was used to validate the separation program. The following amino acids were detected in the amino acid mix: alanine, arginine, aspartate, cysteine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. The calibration curve formed from these dilutions (R 2 >99% for each amino acid) was then incorporated into the program to quantify the amino acids in the unknown samples. 2.6 Release yield and content calculations. Solute (dry matter) release yield (DMRY %) was calculated according to Equation 1 : where the dry matter (DM(g)) of the initial biomass was measured by drying in the oven at 40 0 C to constant weight and the DM (g) of the extract was measured by drying all the extracts combined for each of the conditions in Table 1 in the oven at 40 0 C to constant weight. Protein release yield (PRY%) was calculated using Equation 2 : where protein (g) in the extracts (Protein extract ) was measured by Lowry and protein in the initial biomass (Protein initial_biomass )was measured by Lowy after bead beading in alkaline solution. PRY% of the pH shift experiments is the combined yield of water and all pH extracts (1 to 12) in the specific experiment. Essential Amino Acid content (EAA %) was calculated using Equation 3 : Where Y EAA (g) is the content of essential amino acids detected with HPIC in the Protein extract and Y TAA are the total content of amino acids detected in the Protein extract . EAA determined were histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine and total amino acids (TAA) determined were alanine, arginine, aspartate, cysteine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. Branched Amino Acid content (BCAA%) was calculated using Equation 4 : Where Y(g) is the content of amino acids detected with HPIC in the P extract . BCAA determined were leucine, isoleucine, and valine. 2.7 Statistical analysis Student’s t-test was performed to analyze the significance of the difference between data sets obtained in the study. The p-values were calculated using the Student’s t-test in a paired manner with 0.05 as the maximal p value indicating statistical significance. 3. Results 3.1 Gracilaria sp. biomass fractionation using PEF and pH shifts. The wet Gracilaria sp. biomass was treated under different conditions and fractionated into soluble and insoluble fractions ( Table 1 ). The control and PEF-treated biomass samples were further subjected to water-mediated extraction or to sequential pH extraction to estimate the amount of dry matter and protein released. In addition, the essential amino acid content was determined and the branched amino acid content (BCAA) of the protein fraction was calculated. The mass balance analysis of each condition described in Figure 1 and Table 1 appears in Tables S1 to S6 and is discussed below. 3.1.1. Water-mediated extraction process of PEF treated biomass. The raw biomass had an initial protein content of 15.58±0.25% DW . The water-mediated extraction alone yielded 7.5±0.04% DMRY and 7.22±0.3% of PRY ( Table S1 ), with 4.4±2.4% EAA, and 1.4±0.5% BCAA content. PEF treatment before the water-mediated extraction resulted in 7.5±0.04% DMRY and 8.1±0.1% of PRY, with 54.5±5.9% EAA, and 2.8±1.3% BCAA content ( Table S2 ). PEF pre-treatment did not significantly impact DMRY (p-value = 1), and BCAA content (p-value = 0.29), but significantly increased the PRY (p-value= 0.009) and the fraction of EAA (p-value = 0.009). 3.1.2 Gracilaria fractionation process using PEF pre-treatment and pH shift. Sequential pH-shift extraction (12 to 1) led to 9.62±0.04% DMRY and 14.45±0.1% of PRY ( Table S3 ) with 14.12±1.4% EAA and 0.06±0.03% of BCAA content. Sequential pH extraction (12 to 1), which followed PEF, led to 9.64±0.02% DMRY and 24.1±0.03% of PRY ( Table S4 ), with 62.1±5.5% EAA and 3.1±0.7% BCAA content in the extract. PEF pre-treatment did not change the DMRY (p-value = 0.68) but significantly increased the PRY (1.7 fold, p-value = 3·10 -8 ) and the content of EAA (4.4 fold, p-value = 0.007) and branched amino acids (51.6 fold, p-value = 0.03). Sequential pH-shift extraction (1 to 12) led to 9.61±0.02% DMRY and 17.33±0.8% of PRY ( Table S5 ), with 31.1 ±2.2% EAA and 0.75±0.09% BCAA content. Sequential pH-shift extraction with PEF (1 to 12) led to of 9.61±0.04% DMRY, and 28.02±0.2% of PRY ( Table S6 ), 63.6±5.3% EAA and 3.47±0.06% BCAA ( Table S7 ). PEF pre-treatment did not change the DMRY (p-value = 0.99) but significantly increased the PRY (1.6 fold, p-value = 3·10 -5 ) and the content of EAA (2 fold, p-value = 0.02) and BCAA (4.6 fold, p-value = 8·10 -4 ). In experiments without PEF, although the order of pH shift sequence (1 to12 vs. 12 to 1) did not impact the DMRY, (p-value = 0.42), the order significantly affected the PRY (1.19 fold p-value = 0.004), EAA content (2.2 fold change, p-value = 0.01) and BCAA content (12.5 fold change, p-value= 0.009). The order of pH shifts (1 to 12 vs. 12 to 1) following PEF, did not impact the DMRY, (p-value = 0.25), EAA content (p-value = 0.85) and BAA content (p-value = 0.5). However, the PRY was impacted significantly (1.16 fold change, p-value = 9·10 -6 ) when PEF pre-treated samples further treated with pH 1 to 12, and pH 12 to 1 were compared. Thus, regardless of PEF pretreatment, the PRY was impacted by the order of the pH shifts. In both cases the samples treated with pH 1 to 12 showed higher PRY compared to those treated with pH 12 to 1. 3.2 Determination of the impact of process parameters on Gracilaria sp. biomass fractionation 3.2.1 The impact of pH on solute and protein release yield and content of essential and branched amino acids of the extracts. We investigated how pH shifting impacts the Gracilaria fractionation efficiency and quality of the products regardless of PEF pre-treatment. The addition of pH shifts in comparison to water-mediated extraction (without pH shifts), impacted the DMRY (Figure 3a , fold change 1.28, p-value = 7.7·10 ‑6 ) and PRY ( Figure 3b , 1.64 fold change, p-value = 4.8·10 -6 ). However, it did not significantly affect EAA, and BCAA content ( Figure 3c and Figure 3d , p-value = 0.28, and 0.73) of the extracts. 3.2.2 The impact of PEF pre-treatment on solute and protein release yield, and on essential and branched amino acids contents of the extracts We also investigated if addition of PEF pre-treatment increases the Gracilaria biomass fractionation efficiency and quality of the products. There was no significant difference in the DMRY between PEF treated and untreated samples ( Figure 4a, p-value=0.99). However, an addition of PEF significantly increased the PRY% ( Figure 4b, 1.54 fold change, p-value=0.03), EAA fraction ( Figure 4c, 3.62 fold change, p-value=8.0 . 10 -5 ) and BCAA fraction ( Figure 4d, 4.27 fold change, p-value=1.3·10 -4 ). 3.2.3 Combined effect of pH and PEF pre-treatment on solute and protein release yields and on essential and branched amino acids contents of the extracts. Next, we tested whether the combined effect of pH and PEF significantly improves the Gracilaria sp. biomass fractionation. Combined PEF and pH-shift treatments did not improve the DMRY in comparison with pH shifts alone ( Figure 5a, p-value=0.44 ), but showed higher DMRY in comparison with PEF pre-treatment alone ( Figure 5a, 1.28 fold change, p-value= 2.8 . 10 -7 ). A combined PEF and pH-shift treatment led to higher protein release yield, higher EAA and BCAA contents than PEF or pH-shift alone ( Figure 5b, p-value=0.00017, and 0.0003, Figure 5c, p-value=0.02, and 0.04 Figure 5d, p-value=0.0077, and 0.05 respectively) with a fold change of 3.23, and 1.97 in protein release yield, 1.15, and 2.71 in EAA, 1.18, and 13.12 in BCAA respectively. The %DMRY, %PRY, %EAA, and %BCAA under different conditions used in the study are summarized in Table 3 . Table 3 : A summary of %DMRY, %PRY, %EAA, and %BCAA obtained under different conditions in the current study. Condition DMRY(%) PRY (%) EAA (%) BCAA (%) Control Biomass (No PEF) Water-mediated extraction 7.5±0.04 7.22±0.3 4.4±2.4 1.4±0.5 pH shift (pH 1 to 12) 9.61±0.02 17.33±0.8 31.1 ±2.2 0.75±0.09 pH shift (pH 12 to 1) 9.62±0.04 14.45±0.1 14.1±1.4 0.06±0.03 PEF Treated Biomass Water-mediated extraction 7.5±0.04 8.1±0.1 54.5±5.9 2.8±1.3 pH shift (pH 1 to 12) 9.61±0.04 28.02±0.2 63.6±5.3 3.47±0.06 pH shift (pH 12 to 1) 9.64±0.02 24.1±0.03 62.1±5.5 3.1±0.7 4. Discussion Protein solubility and release from the algal biomass strongly depend on pH (which determines protein surface charge), on membrane and cell wall integrity, and on protein interactions with the algal polysaccharides, some of which make the cell walls. In our current study, we explored both the impact of PEF, and of sequentially shifting pH through a range of pH values during pH-shift experiments to increase protein extraction yield. A recent study on Gracilaria dura sheds light on how some of these factors impact the behaviour of proteins (Bozdemir et al., 2022). Notably, when re-dissolved in water, the “powdered” Gracilaria dura exhibited a negative surface charge (zeta potential), likely attributed to its cell wall composition primarily comprising negatively charged polysaccharides (Bozdemir et al., 2022). The minimal solubility of Gracilaria dura proteins occurred at pH 4.0, and from that point it increased with increasing pH, at least up to pH 13 (Bozdemir et al., 2022). Recent investigations into microalgal species have revealed intriguing patterns of protein solubility. Notably, one study demonstrated an impressive ~84% solubility within a pH range of 2-12 (Grossmann et al. 2019). In contrast, research on Tetraselmis spp. highlighted a unique behaviour: protein solubility remained unaffected by the ionic strength of the solution, even up to 0.5 M (Schwenzfeier et al. 2011). Remarkably, proteins from Tetraselmis spp. exhibited complete solubility above pH ≥ 5.5. Our current study underscores the importance of broad pH exploration during protein extraction. By encompassing a wide pH range, we aimed to enhance overall protein release yield, a critical consideration for optimizing protein extraction protocols. In some earlier articles (Harnedy and FitzGerald 2013; Kumar et al. 2014), the topic of protein isolation using pH alterations in red macroalgae was discussed. A study on Kappaphycus alvarezii show that the minimal solubility of nitrogenous compounds was found at pH 4, and the authors suggested that this pH may be the isoelectric point of the protein concentrate of Kappaphycus alvarezii (Kumar et al. 2014). Proteins acquire net positive and net negative charge, below and above their pI respectively, that lead to repulsion between the molecules and enhance protein solubility (Seena and Sridhar 2005). The attractive interactions between positively charged proteins and polyanions can lower the apparent pI, by shifting the pH of minimal solubility of the protein to a lower pH (Zimet and Livney 2009), where charge-neutralized complexes form with the polyanions (Williams 2009). The mechanisms underlying protein release yield under various pH and pre-treatment conditions depends on this insight. The pH shift approach is based on the fact that each protein has a unique isoelectric pH, at which it is least soluble, hence collecting the soluble proteins during each step of the pH shift process and combining them, should significantly increase the protein extraction yield (compared to collecting the soluble proteins at a certain pH where some of the proteins have zero zeta potential, and are hence insoluble). It should also help avoiding loss of protein due to complex formation with the anionic polysaccharides, which, as discussed above, also has a strong pH dependence. We treated the biomass in two ways: 1) Starting with the highest pH 12 and progressively adjusting to each subsequent pH level until reaching pH 1. 2) Starting with the lowest pH 1 and progressively adjusting to each subsequent pH level until reaching pH 12. We found that the second way, i.e. when pH is gradually increased, was more effective in increasing protein release, ( Table 3 ) leading to a higher PRY 17.33±0.8 vs. 14.45±0.1. A likely reason for this is the presence of the anionic cell wall polysaccharides, like agar, which are uncharged at very low pH (much below the pk a of its carboxylic groups, which is around 5 (Carisma et al. 2020). As pH rises, and cell wall polysaccharides become more negatively charged, they bind positively charged proteins (up to the pI of each protein, or slightly above it, if local positive patches exist) and form electrostatic complexes with them. This complexation decreases protein release. Above the pI, solubility of the proteins and their consequent release increase, as they become more negatively charged, and repulsed from the polysaccharides. When starting at high pH, the cell wall polysaccharides are very negative during most of the process, binding much proteins below their pIs, and decrease their release. In contrast, when starting at low pH, as the cell wall polysaccharides are uncharged, most of the proteins are released in the beginning, as they are very positively charged, and the uncharged cell wall polysaccharides interfere less with their release. The scientific literature highlights the impact of pulsed electric field (PEF) treatment on macroalgal cells, increasing membrane permeability/porosity, thereby leading to loss of turgor pressure within the cells, dehydration and release of salts and small proteins (Robin et al., 2018; Polikovsky et al., 2016; Prabhu et al., 2019). In our current study, we improved protein extraction by combining PEF with pH shifts. This combination synergizes: (1) the increased membrane porosity, (2) the pH-dependent protein release, partly hampered by cell-wall polysaccharides, and (3) the chemical potential gradient between the cell interior and its surrounding medium. Consequently, at each pH step (pH solutions, as detailed in Table 2), there exists a motive force for the diffusive release of distinct proteins, which is facilitated by the loss of membrane-barrier efficacy by PEF, and escaping cell-wall polysaccharide complexation by shifting the pH. PEF both increases the permeability of the cell membrane and removes salts, which increases protein release, thereby leading to higher protein release yield (Robin et al. 2018) A combination of PEF and sequential pH-shifting from 1 to 12 led to the highest fractionation increasing the DMRY by 28.3% (from 7.5±0.04% to 9.61±0.04%), PRY by 288.1% (from 7.22±0.3% to 28.02±0.2%), and EAA content by 1282.6% (from 4.6±2.4% to 63.6±5.3%), and BCAA content by 147.9% (from 1.4±0.5% to 3.47±0.06%w) in comparison to aqueous extraction alone. These results are higher than previous reports on Gracilaria protein extraction ( Table 2 ), probably due to the hurdle effects and the synergy of PEF and pH-shifting, where PEF pre-treatment facilitates the following protein release yield at various pH conditions. We observed that the combination of PEF and pH shift method can provide much better yields than enzymatic methods, ultrasonication, chemical methods, and PEF alone ( Table 4 ). The most interesting impact of the PEF pre-treatment is the enhancement in the EAA and BCAA in the extracts obtained from PEF pre-treated biomass. The PEF pre-treated samples followed by aqueous extraction had highest enhancement in EAA 54.5±5.9% (p-value=0.02), and BCAA 2.8±1.3% (p-value=0.009), respectively compared to samples treated only with sequential pH (pH 1 to 12) that had 31.1 ±2.2%, and 0.75±0.09% of EAA, and BCAA respectively. This is a new and interesting observation, which is in line with our previous works on Ulva sp. biomass, in which we reported selective protein extraction by PEF in comparison to aqueous extraction alone (Polikovsky et al. 2016). This observed enhancement in the EAA, and BCAA extraction in PEF-treated biomass with or without pH shifts, suggests that the PEF treatment aids in the release of EAA and BCAA. The mechanisms of such selective extraction are still unknown, and motivate further study. Table 4. Summary of protein extraction from Gracilaria sp. using various protocols. Species Technology Conditions Protein extraction yield References Gracilaria verrucosa Enzymatic extraction Agarase (pH-6) and cellulase (pH-3.8), temperature-50°C to 55°C for 2 h with 0.1 M MesNaOH and oxalate NaOH buffer 6.3%dw (Fleurence et al. 1995) Gracilaria sp. Continuous PEF device Voltage-400V, Frequency-5Hz, Pulse duration (µs)-50, number of pulses -1000, inter-electrode gap-4mm, sample feeding rate-100g/10 min 4.75% from 8.4%dw initial protein content (Kashyap et al. 2022) Gracilaria sp. Ultrasonication Treatment with NaOH (10% w/v) and then ultrasonication for 2 h 19.84% algal protein concentrate from 25%dw initial protein content (Kazir et al. 2019) Gracilaria dura Ultrasonication Ultrasonication time-257.57s, time for extraction-22.61h 2.09% from 11.97±0.05%dw initial protein content (Bozdemir et al. 2022) Gracilaria sp. Batch PEF device (gravitational-PEF device) and pH shift (sequential pH 1 to 12) treatment. Voltage-1000V, Frequency-3Hz, Pulse duration (µs)-50, number of pulses-200, inter-electrode gap-4mm. 28.02±0.2% from initial protein content This study Conclusion Both PEF and pH play crucial roles in the fractionation of Gracilaria sp. biomass. Utilizing a combination of PEF and sequential pH shifts (pH 1 to 12) resulted in notable outcomes, including a solute (dry matter) release yield of 9.61±0.04%, protein release yield of 28.02±0.2%, EAA content of 63.6±5.3%, and BCAA content of 3.47±0.06%. It is noteworthy that PEF significantly increased EAAs and BCAAs extraction efficiency, as demonstrated by samples treated with PEF alone, which exhibited 54.5±5.9% EAA and 2.8±1.3% BCAA. In contrast, samples treated solely with pH shifts (pH 1 to 12) and without PEF showed EAA and BCAA levels of 31.1±2.2% and 0.75±0.09%, respectively. This underscores the cooperative effect of combining PEF and pH-shift treatments (particularly when gradually raising pH from 1 to 12), highlighting their synergistic impact in amplifying protein release yield and particularly essential amino acids and branched amino acids in Gracilaria sp. biomass extracts. Declarations Acknowledgements Funding The study in the present manuscript was funded by Good Food Institute, Israel and Israel Ministry of Health. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Conflict of Interest AG and KL have patent applications on devices and use of PEF technologies for seaweed processing with Ramot, Tel Aviv University. AG has interest in Genesea Advanced Technologies Ltd, which focuses on seaweed protein production. Authors contribution statement Mrinal Kashyap . Writing original manuscript draft, execution of experiments, data interpretation and analysis. Supratim Ghosh . Supportive role in execution of experiments, Klimentiy Levkov . Hardware, software development, experiment Yoav D Livney. Review and editing of the original manuscript. Alvaro Israel . Review and editing of the original manuscript. Alexander Golberg. Conceptualization Data analysis, editing of the manuscript . References Bozdemir A, Şensu E, Okudan EŞ, Özçelik B, Yücetepe A (2022) Ultrasound‐assisted enzymatic extraction of proteins from Gracilaria dura : Investigation of antioxidant activity and techno‐functional properties. Journal of Food Processing and Preservation 46 (8):e16803 Carisma NAS, Gonzales RYE, Lazaro-Llanos N (2020) An Investigation on Zinc Biosorption with Agar Extraction Waste from Gracilaria tenuistipitata . Kimika 31 (2):11-26. doi:10.26534/kimika.v31i2.11-26 Cavonius LR, Albers E, Undeland I (2015) pH-shift processing of Nannochloropsis oculata microalgalbiomass to obtain a protein-enriched food or feed ingredient. Algal research 11:95-102 Carisma, N. A. S., Gonzales, R. Y. E., & Lazaro-Llanos, N. (2020). An Investigation on Zinc Biosorption with Agar Extraction Waste from Gracilaria tenuistipitata . Kimika, 31 (2), 11-26. doi: 10.26534/kimika.v31i2.11-26 De Boer J, Aiking H (2011) On the merits of plant-based proteins for global food security: Marrying macro and micro perspectives. Ecological economics 70 (7):1259-1265 Duong-Ly KC, Gabelli SB (2014) Salting out of proteins using ammonium sulfate precipitation. In: Methods in enzymology, vol 541. Elsevier, pp 85-94 Fanzo J (2019) Healthy and Sustainable Diets and Food Systems: the Key to Achieving Sustainable Development Goal 2? Food Ethics 4 (2):159-174. doi:10.1007/s41055-019-00052-6 Fleurence J, Le Coeur C, Mabeau S, Maurice M, Landrein A (1995) Comparison of different extractive procedures for proteins from the edible seaweeds Ulva rigida and Ulva rotundata . Journal of Applied Phycology 7:577-582 Geada P, Moreira C, Silva M, Nunes R, Madureira L, Rocha CM, Pereira RN, Vicente AA, Teixeira JA (2021) Algal proteins: Production strategies and nutritional and functional properties. Bioresource technology 332:125125 Golberg A, Sack M, Teissie J, Pataro G, Pliquett U, Saulis G, Stefan T, Miklavcic D, Vorobiev E, Frey W (2016) Energy-efficient biomass processing with pulsed electric fields for bioeconomy and sustainable development. Biotechnology for biofuels 9:1-22 Grossmann L, Hinrichs J, Weiss J (2019) Solubility and aggregation behavior of protein fractions from the heterotrophically cultivated microalga Chlorella protothecoides . Food Research International 116:283-290 Harnedy PA, FitzGerald RJ (2013) Extraction of protein from the macroalga Palmaria palmata . LWT-Food Science and Technology 51 (1):375-382 Harrysson H, Hayes M, Eimer F, Carlsson N-G, Toth GB, Undeland I (2018) Production of protein extracts from Swedish red, green, and brown seaweeds, Porphyra umbilicalis Kützing, Ulva lactuca Linnaeus, and Saccharina latissima (Linnaeus) JV Lamouroux using three different methods. Journal of Applied Phycology 30:3565-3580 Hua K, Cobcroft JM, Cole A, Condon K, Jerry DR, Mangott A, Praeger C, Vucko MJ, Zeng C, Zenger K (2019) The future of aquatic protein: implications for protein sources in aquaculture diets. One Earth 1 (3):316-329 Juul L, Danielsen M, Nebel C, Steinhagen S, Bruhn A, Jensen S, Undeland I, Dalsgaard T (2021) Ulva fenestrata protein–Comparison of three extraction methods with respect to protein yield and protein quality. Algal Research 60:102496 Kashyap M, Ghosh S, Steinbruch E, Levkov K, Israel Al, Bala K, Livney Y, Golberg A (2022) Extracting Water-Soluble Proteins from the Red Macroalgae Gracilaria sp. with Pulsed Electric Field in a Continuous Process. ACS Food Science & Technology 3 (4):562-575 Kazir M, Abuhassira Y, Robin A, Nahor O, Luo J, Israel A, Golberg A, Livney YD (2019) Extraction of proteins from two marine macroalgae, Ulva sp. and Gracilaria sp., for food application, and evaluating digestibility, amino acid composition and antioxidant properties of the protein concentrates. Food Hydrocolloids 87:194-203 Kumar KS, Ganesan K, Selvaraj K, Rao PS (2014) Studies on the functional properties of protein concentrate of Kappaphycus alvarezii (Doty) Doty–An edible seaweed. Food chemistry 153:353-360 Levkov K, Linzon Y, Mercadal B, Ivorra A, González CA, Golberg A (2020) High-voltage pulsed electric field laboratory device with asymmetric voltage multiplier for marine macroalgae electroporation. Innovative Food Science & Emerging Technologies 60:102288 Lowry O, Rosebrough N, Farr AL, Randall R (1951) Protein Measurement with the Folin Phenol Reagent. Journal of Biological Chemistry 193 (1):265-275. doi:10.1016/s0021-9258(19)52451-6 Magnusson M, Glasson CR, Vucko MJ, Angell A, Neoh TL, de Nys R (2019) Enrichment processes for the production of high-protein feed from the green seaweed Ulva ohnoi. Algal Research 41:101555 Nan F, Feng J, Lv J, Liu Q, Fang K, Gong C, Xie S (2017) Origin and evolutionary history of freshwater Rhodophyta: further insights based on phylogenomic evidence. Scientific reports 7 (1):2934 O’Connor J, Meaney S, Williams GA, Hayes M (2020) Extraction of protein from four different seaweeds using three different physical pre-treatment strategies. Molecules 25 (8):2005 Øverland M, Mydland LT, Skrede A (2019) Marine macroalgae as sources of protein and bioactive compounds in feed for monogastric animals. Journal of the Science of Food and Agriculture 99 (1):13-24 Pereira RN, Avelar Z, Pereira SG, Rocha CM, Teixeira JA (2022) Pulsed electric fields for the extraction of proteins and carbohydrates from marine resources. In: Innovative and Emerging Technologies in the Bio-marine Food Sector. Elsevier, pp 173-195 Pereira SG, Pereira RN, Rocha CM, Teixeira JA (2023) Electric fields as a promising technology for the recovery of valuable bio compounds from algae: Novel and sustainable approaches. Bioresource Technology Reports:101420 Polikovsky M, Fernand F, Sack M, Frey W, Müller G, Golberg A (2016) Towards marine biorefineries: Selective proteins extractions from marine macroalgae Ulva with pulsed electric fields. Innovative Food Science & Emerging Technologies 37:194-200 Prabhu MS, Israel A, Palatnik RR, Zilberman D, Golberg A (2020) Integrated biorefinery process for sustainable fractionation of Ulva ohnoi (Chlorophyta): process optimization and revenue analysis. Journal of Applied Phycology 32:2271-2282 Prabhu MS, Levkov K, Livney YD, Israel A, Golberg A (2019) High-voltage pulsed electric field preprocessing enhances extraction of starch, proteins, and ash from marine macroalgae Ulva ohnoi . ACS Sustainable Chemistry & Engineering 7 (20):17453-17463 Rawiwan P, Peng Y, Paramayuda IGPB, Quek SY (2022) Red seaweed: A promising alternative protein source for global food sustainability. Trends in Food Science & Technology 123:37-56 Robin A, Sack M, Israel A, Frey W, Müller G, Golberg A (2018) Deashing macroalgae biomass by pulsed electric field treatment. Bioresource technology 255:131-139 Schwenzfeier A, Wierenga PA, Gruppen H (2011) Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp. Bioresource technology 102 (19):9121-9127 Seena S, Sridhar K (2005) Physiochemical, functional and cooking properties of Canavalia. Journal of Food Chemistry 32:406-412 Vilg JV, Undeland I (2017) pH-driven release yield and isoelectric precipitation of proteins from the brown seaweed Saccharina latissima —effects of osmotic shock, water volume and temperature. Journal of Applied Phycology 29:585-593 Williams PA (2009) Molecular interactions of plant and algal polysaccharides. Structural Chemistry 20 (2):299-308. doi:10.1007/s11224-009-9420-5 Zimet P, Livney YD (2009) Beta-lactoglobulin and its nanocomplexes with pectin as vehicles for ω-3 polyunsaturated fatty acids. Food Hydrocolloids 23 (4):1120-1126. doi:10.1016/j.foodhyd.2008.10.008 Additional Declarations Competing interest reported. AG and KL have patent applications on devices and use of PEF technologies for seaweed processing with Ramot, Tel Aviv University. AG has interest in Genesea Advanced Technologies Ltd, which focuses on seaweed protein production. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 Apr, 2024 Reviews received at journal 26 Mar, 2024 Reviewers agreed at journal 25 Mar, 2024 Reviewers invited by journal 25 Mar, 2024 Editor assigned by journal 20 Mar, 2024 Submission checks completed at journal 20 Mar, 2024 First submitted to journal 19 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4131953","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281728330,"identity":"50b7e06a-d641-4e16-a255-850980819f2a","order_by":0,"name":"Mrinal Kashyap","email":"","orcid":"","institution":"Tel Aviv University","correspondingAuthor":false,"prefix":"","firstName":"Mrinal","middleName":"","lastName":"Kashyap","suffix":""},{"id":281728331,"identity":"b678629b-feca-4978-a3ae-8bfc1c40b9a6","order_by":1,"name":"Supratim Ghosh","email":"","orcid":"","institution":"Tel Aviv University","correspondingAuthor":false,"prefix":"","firstName":"Supratim","middleName":"","lastName":"Ghosh","suffix":""},{"id":281728332,"identity":"c4a03245-09de-4f01-ab53-bf2dab850b0a","order_by":2,"name":"Klimentiy Levkov","email":"","orcid":"","institution":"Tel Aviv University","correspondingAuthor":false,"prefix":"","firstName":"Klimentiy","middleName":"","lastName":"Levkov","suffix":""},{"id":281728333,"identity":"408b79db-80e5-42ff-a764-4b5f2d3bafab","order_by":3,"name":"Yoav D. 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(PBC), The National Institute of Oceanography","correspondingAuthor":false,"prefix":"","firstName":"Álvaro","middleName":"","lastName":"Israel","suffix":""},{"id":281728335,"identity":"207f6a4b-61f2-495f-bf06-84b22c1d9647","order_by":5,"name":"Alexander Golberg","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYHACxsMgkh9NVEIGnx6wFsk2qFKYFh6CWgyOoWphwKmFv3/xgcMFFffsNt9vfibxsY2hjn9GAuOHHwwWOLVI3HiWcHjGmeLkbcfYzCRnnGGQkLiRwCzZg89hN84YHOZtS0g2O8ZgJs1TAXTYjQQGaXx+kQdr+ZeQbNzG/k36jwGDhDzQlt/4tBic7wFqaUiwM2DjMZNmANpicCOBDa8thjfYgH45lpAgcSyn2LLnjITkxjMP2yx7DHBrkTt/+ODjgpoEe/7m4xtv/Gyz4Zc7nnz4xo+KOjmc3pdIAFOJDVAuEDMC2QY4NQAj5gCYssejZBSMglEwCkY6AABgA1Fxw78pLwAAAABJRU5ErkJggg==","orcid":"","institution":"Tel Aviv University","correspondingAuthor":true,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Golberg","suffix":""}],"badges":[],"createdAt":"2024-03-19 16:39:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4131953/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4131953/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53217949,"identity":"a1f245f5-6777-4136-845c-0df89e36144e","added_by":"auto","created_at":"2024-03-22 04:00:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":565402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLaboratory PEF system for macroalgae processing\u003c/strong\u003e. (a) Schematic diagram of the electric circuit of the high voltage pulse generator (b) Press electrode batch device for biomass electroporation (c) Electroporation chamber.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4131953/v1/0b2b67303b0a3c8af563f804.png"},{"id":53217514,"identity":"d14af504-cbf0-4b7b-b521-b3ae3e307e63","added_by":"auto","created_at":"2024-03-22 03:52:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63272,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design for the \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass treatment using PEF device. Red arrows show an alternative process, which included sequential washes with solutions of various pH values (1 to 12 or 12 to 1).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4131953/v1/57677b5994e598e94effe50a.png"},{"id":53217517,"identity":"b2e51d3d-19b2-44e1-bae8-5ee2abd45ae4","added_by":"auto","created_at":"2024-03-22 03:52:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":80624,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of pH-shifting on (a) Solute (dry matter) release yield (DMRY%) (b) Protein release yield (PRY%) (c) Essential amino acids content of the extract (d) Branched amino acids content of the extract. p-values are indicated in the figure between the compared groups (n=3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4131953/v1/a8c6cbbb97a4987cb6cebd6f.png"},{"id":53217515,"identity":"85e7d5d6-a467-4f6e-aee4-6bd89ef948b6","added_by":"auto","created_at":"2024-03-22 03:52:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90808,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of PEF pre-processing step on (a) Solute (dry matter) release yield (DMRY%) (b) Protein release yield (PRY%) (c) Essential amino acids content (EAA%) of the extract (d) Branched amino acids content (BCAA%) of the extract. **p-values are indicated in the figure between the compared groups (n=3).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4131953/v1/b92951aae3985a8895555de3.png"},{"id":53217518,"identity":"82405842-83f9-4e80-bcf4-9556405bd1a3","added_by":"auto","created_at":"2024-03-22 03:52:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104256,"visible":true,"origin":"","legend":"\u003cp\u003eThe combined impact of PEF and pH on (a) Solute (dry matter) release yield (DMRY%) (b) Protein release yield (PRY%) (c) Essential amino acids content (EAA) of the extract (d) Branched amino acids (BCAA%) content of the extract. **p-values are indicated in the figure between the compared groups (n=3).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4131953/v1/887b1ca3a778eae848be3d6d.png"},{"id":53218098,"identity":"959d1a8b-6357-419b-bb6d-abbbf22415a4","added_by":"auto","created_at":"2024-03-22 04:09:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1528799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4131953/v1/3379b0f2-81aa-460d-967f-4a111dd43fe1.pdf"}],"financialInterests":"Competing interest reported. AG and KL have patent applications on devices and use of PEF technologies for seaweed processing with Ramot, Tel Aviv University. AG has interest in Genesea Advanced Technologies Ltd, which focuses on seaweed protein production.","formattedTitle":"Protein and solute release from the red seaweed, Gracilaria sp., using a high voltage pulsed electric field and pH shift","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAlternative proteins play a pivotal role in achieving the UN Sustainable Development Goals (SDGs) by addressing concerns related to climate change and food insecurity (Fanzo 2019). Conventional animal-based protein production is unsustainable due to its adverse environmental impact, low plant-protein to animal-protein conversion efficiency, and high ecological footprints (Geada et al. 2021). While plant-based alternatives are more environmentally friendly, they still impose strain on freshwater and agricultural land resources (De Boer and Aiking 2011). Seaweeds present a promising solution to these challenges, as they can be cultivated in seawater, not requiring freshwater or land, and yield valuable biomass with various prospects, especially as alternative protein sources (Hua et al. 2019; \u0026Oslash;verland et al. 2019). Among seaweeds, red macroalgae, including \u003cem\u003eGracilaria\u003c/em\u003e sp., have gained attention due to their relatively high protein content, with reported content ranging from 2.7% to 47% (Nan et al. 2017; Rawiwan et al. 2022). However, commercializing these proteins remains challenging due to variability of chemical composition, lack in suitable harvesting technologies and in environmentally responsible extraction processes. Innovative approaches are necessary to unlock the full potential of \u003cem\u003eGracilaria\u003c/em\u003e sp. and other macroalgae as sustainable protein sources.\u003c/p\u003e\n\u003cp\u003eThe commercialization of seaweed proteins has been hindered by technological challenges and associated costs. The production of proteins from macroalgae involves four main steps: cultivation, harvesting, extraction, and purification. Harvesting and extraction, in particular, are costly and pose significant obstacles. Various techniques, such as ultrasonication, microwave-assisted extraction, pH-shift methods, and high-pressure processing, have been explored for protein extraction from \u003cem\u003eGracilaria\u003c/em\u003e sp. However, the presence of agar in its cell walls makes protein extraction challenging in this species, as it reduces protein release yield (Juul et al. 2021; Kazir et al. 2019; Magnusson et al. 2019; O\u0026rsquo;Connor et al. 2020).\u003c/p\u003e\n\u003cp\u003eOne promising technology for biomass conversion into value-added compounds is pulsed electric fields (PEF) (Golberg et al. 2016). PEF treatment involves applying voltage pulses across biological membranes, leading to electro permeabilization and extraction of biomolecules from cells (Pereira et al. 2022; Pereira et al. 2023). We recently reported the use of a continuous PEF device to extract water-soluble proteins from \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass (Kashyap et al. 2022). The current study aims to further enhance protein extraction by combining PEF with pH shift. PH shift is a promising technology for seaweed protein extraction, used previously on \u003cem\u003ePorphyra umbilicalis\u003c/em\u003e, \u003cem\u003eUlva lactuca\u003c/em\u003e, and \u003cem\u003eSaccharina latissimi \u003c/em\u003e(Harrysson et al. 2018), and some other species like \u003cem\u003eUlva fenestrate \u003c/em\u003e(Juul et al. 2021), but not on \u003cem\u003eGracilaria \u003c/em\u003esp\u003cem\u003e. \u003c/em\u003eWhile previous studies have explored protein extraction from various \u003cem\u003eGracilaria\u003c/em\u003e species (\u003cstrong\u003eTable 2\u003c/strong\u003e) (Bozdemir et al. 2022; Fleurence et al. 1995; Kazir et al. 2019), none have utilized PEF pretreatment methods except our previous study on the \u003cem\u003eGracilaria\u003c/em\u003e sp. using a continuous PEF device (Kashyap et al. 2022). The present study represents the first report of combining PEF and pH shift to improve total solute and protein release from \u003cem\u003eGracilaria\u003c/em\u003e sp. We also examined the composition of the released proteins in terms of essential and branched amino acid composition. We show how process parameters significantly affect the release yield and composition of \u003cem\u003eGracilaria\u003c/em\u003e sp. proteins, with regards to their potential food application. \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass production and harvesting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGracilaria\u003c/em\u003e sp. biomass was grown in a 700 L polyvinylchloride (PVC) tank with running seawater and continuous aeration at Israel Oceanographic and Limnological Research (IOLR), Haifa, Israel, in 2021. Twice a week, 0.1 mM PO\u003csub\u003e4\u003c/sub\u003e and 1.0 mM NH\u003csub\u003e4\u003c/sub\u003e were added to the seaweed tank. The biomass utilized as a stock was 1.5 kg FW m\u003csup\u003e-3\u003c/sup\u003e and harvested after it grew to 3-4 kilogram FW m\u003csup\u003e-3\u003c/sup\u003e in around 5 weeks. The harvested biomass was cleansed in a 20L tank filled with clean tap water. A batch centrifuge was used to remove surface water and the \u0026quot;fresh weight\u0026quot; (FW) biomass of \u003cem\u003eGracilaria\u003c/em\u003e sp. was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Description of the high voltage PEF system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe electrical circuit of the High Voltage Pulse Generator (HVPG) used in this investigation is shown in \u003cstrong\u003eFigure 1a\u003c/strong\u003e and was described in detail in our previous publications (Levkov et al. 2020; Prabhu et al. 2020; Prabhu et al. 2019). The sliding Press-Electrode Device (\u003cstrong\u003eFigure 1b\u003c/strong\u003e) was created especially for handling biomass while applying pressure during PEF-induced electroporation. To exert this pressure, one electrode is allowed to move freely and vertically while being driven by the weight placed on the load-receiving platform.\u003c/p\u003e\n\u003cp\u003eThe sliding Press-Electrode Device consists of an Electroporation Cell (EPC), an Electroporation Cell Retainer (EPCR), a load-receiving platform, a node of the moving electrode, a holder, and a sensor for detecting the inter-electrode gap. Guides at the base of the holder ensure precise alignment of the installed EPCR. The EPC (\u003cstrong\u003eFigure 1c\u003c/strong\u003e), which consists of a cylinder with one electrode at the bottom and another at the lower end of the sliding electrode assembly\u0026apos;s rod, acts as the working volume for electroporation and pressing. \u0026nbsp;The liquid fraction can exit the cylinder through slit-like apertures in the side walls, and a ring-shaped groove renders it convenient to drain the liquid component into a storage container. The high-voltage input of the HVPG is attached to the operational electrode of the EPC via a male connector.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing a spinner, 20 g of FW \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass was dewatered until less than 1 g of surface water remained. The PEF apparatus described above was then used to treat 20 g of FW biomass in three replicates using the following parameters: frequency=3 Hz; pulse duration=50 \u0026micro;s; number of pulses=200; pulse voltage=1000 V; inter-electrode gap was varied in the range of 3.9 to 4.4 mm; the 20 g of biomass was treated with 10 PEF cycles by feeding 2 g/cycle (step 1 in \u003cstrong\u003eFigure 2\u003c/strong\u003e). The PEF treatment steps were not used in the control group.\u003c/p\u003e\n\u003cp\u003eFollowing PEF, the treated biomass was suspended in 100 mL of distilled water, agitated at 32\u0026deg;C and 150 RPM overnight in a shaking incubator (Benchmark Scientific, Incu-shaker small, USA), and then separated with a batch centrifuge (Yingtai Instruments, TGL 18, China) at 3750 g for 10 min (steps 2 and 3 in \u003cstrong\u003eFigure 2\u003c/strong\u003e). The biomass (solids 3b, \u003cstrong\u003eFigure 2\u003c/strong\u003e), was further treated with different pH treatments: 1) sequential pH 12 to 1 extraction, 2) sequential pH 1 to 12 extraction (\u003cstrong\u003eTable 1,\u0026nbsp;\u003c/strong\u003esteps 4 and 5\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ein\u003cstrong\u003e\u0026nbsp;Figure 2\u003c/strong\u003e). The compositions of buffers used appear in \u003cstrong\u003eTable 2\u003c/strong\u003e. To establish the total solutes (dry matter) released and protein released, all the liquid fractions from the sequential extractions were combined. The residual biomass was dried in an oven at 40\u0026deg;C (\u003cstrong\u003estep 6, Figure 2\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Protein extraction from \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass using various process steps; process description based on the experimental design shown in Figure 2.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"577\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProcess stage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEquipment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProcess parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl (without PEF)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e1) Pressing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e5 kg weight, gravitational PEF device\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e20\u0026nbsp;g of biomass was pressed with 10\u0026nbsp;cycles by feeding 2\u0026nbsp;g/cycle, no PEF applied.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e2) Water-mediated extraction\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eShaker incubator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCondition 1:\u0026nbsp;\u003c/strong\u003e32\u0026deg;C, 150 RPM for 1 hr\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e3 and 5) centrifugation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eBatch centrifuge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e3750\u0026nbsp;xg for 10\u0026nbsp;min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e4) pH treatment\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eShaker incubator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConditions 2 and 3: Sequential pH 1 to 12 and 12 to 1 treatments:\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e2 g FW biomass from step \u003cstrong\u003e3 (b), Figure 2\u003c/strong\u003e was treated with solutions at pH levels ranging from 1 to 12 (condition 2) and 12 to 1(condition 3).\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThe same biomass was transferred from one falcon tube to another falcon tube with 10 mL of solution of a different pH and incubated at 32\u0026deg;C and 150\u0026nbsp;RPM for 1 hr at each pH\u003cstrong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e Then it was centrifuged, and separated. The protein content of all liquid (supernatant) fractions was combined for each condition.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e6) Drying\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eOven\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e40\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePEF treatment (with electric fields)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e1) Pressing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e5 kg weight, gravitational PEF device\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eFresh biomass (20 g) was treated with 10 PEF cycles by feeding 2 g/cycle.\u0026nbsp;\u003cbr\u003e\u0026nbsp;PEF parameters: Frequency=3 Hz, pulse length=50 \u0026micro;s, number of pulses=200, pulse voltage=1000 V, inter-electrode gap varied from 4.4 to 3.9 mm because of the biomass compression and liquid extraction.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e2) Aqueous extraction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eShaker incubator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCondition 4\u003c/strong\u003e: 32\u0026deg;C, 150 RPM for 1 hr\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e3 and 5) centrifugation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eBatch centrifuge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e3750\u0026nbsp;xg for 10\u0026nbsp;min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e4) pH treatment\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eShaker Incubator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConditions 5 and 6: Sequential pH 1 to 12 and 12 to 1:\u0026nbsp;\u003c/strong\u003e2 g FW biomass from step 3 (b) was treated with solutions at pH levels ranging from 1 to 12 (condition 5), and from 12 to 1 (condition 6)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThe same biomass was transferred from one falcon tube to another falcon tube with 10 mL of different pH solution and incubated at 32\u0026deg;C and 150 RPM for 1 hr in each pH\u003cstrong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e Then it was centrifuged, and separated. \u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eThe protein content of all liquid (supernatant) fractions was combined for each condition.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.10225303292894%\" valign=\"top\"\u003e\n \u003cp\u003e6) Drying\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eOven\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.44887348353553%\" valign=\"top\"\u003e\n \u003cp\u003eAt 40\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eVolumes and molar strengths of the salts, acids, and bases used for preparation of pH solution 1 to 12\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eused in the current study.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"584\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003epH solution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eComposition of the solution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMolar Concentration (M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e50 mL of 0.2 M KCl + 134 mL of 0.2 M HCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e3.68\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e50 mL of 0.2 M KCl + 13 mL of 0.2 M HCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e1.26\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e100 mL of 0.1 M\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eKO\u003csub\u003e4\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;+\u0026nbsp;\u003c/strong\u003e44.6 mL of 0.1 M HCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e1.44\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e100 mL of 0.1 M\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eKO\u003csub\u003e4\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;+\u0026nbsp;\u003c/strong\u003e0.2 mL of 0.1 M HCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e1.02\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e100 mL of 0.1 M\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eC\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eKO\u003csub\u003e4\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;+\u0026nbsp;\u003c/strong\u003e45.2 mL of 0.1 M NaOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e1.45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e100 mL of 0.1 M\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;+\u0026nbsp;\u003c/strong\u003e11.2 mL of 0.1 M NaOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e1.11\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e100 mL of 0.1 M\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;+\u0026nbsp;\u003c/strong\u003e58.2 mL of 0.1 M NaOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e1.58\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e100 mL of 0.1 M\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003cstrong\u003e\u0026nbsp;+\u0026nbsp;\u003c/strong\u003e93.4 mL of 0.1 M NaOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e1.93\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e100 mL of 0.1 M tris (hydroxymethyl) amino methane + 11.4 mL 0.1 M HCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e1.11\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e100 mL of 0.05 M NaHCO\u003csub\u003e3\u003c/sub\u003e + 21.4 mL of 0.1 M NaOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e0.62\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e100 mL of 0.05 M NaHCO\u003csub\u003e3\u003c/sub\u003e + 45.4 mL of 0.1 M NaOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e0.96\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.554794520547945%\" valign=\"top\"\u003e\n \u003cp\u003epH12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.33561643835616%\" valign=\"top\"\u003e\n \u003cp\u003e50 mL of 0.2 M KCl + 12 mL of 0.2 M NaOH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.10958904109589%\" valign=\"top\"\u003e\n \u003cp\u003e1.04\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Protein quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein content in the initial biomass was quantified after cell wall and membrane disruption by bead-beating under alkaline conditions, and after drying in an oven at 40\u0026deg;C until reaching a constant weight. \u003cem\u003eGracilaria\u003c/em\u003e sp. dry biomass (15 mg) and 1.5 mL of 2 M NaOH were mixed. Zirconium beads (2mm, Sarstedt) were added to one-third of the bead-beater tubes containing a mixture of biomass and 2 M NaOH. The tubes containing the mixture were then put into a Mini Bead-Beater-16, Model 607 EUR, Biospec, OK, and subjected to three cycles of 60 seconds each, separated by 10-minute intervals, at 25\u0026deg;C. After bead beating, the tubes were centrifuged at 19,500 g for 20 min in an Eppendorf 5424 centrifuge (Germany), and protein was quantified in the supernatant (Prabhu et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe protein content of the various liquid fractions collected throughout the investigation was determined using Lowry\u0026apos;s technique. To create the linear calibration curve, several dilutions corresponding to concentrations ranging from 0-500 \u0026micro;g/mL were prepared, using a bovine serum albumin (BSA) stock solution of 1 mg/mL (Lowry et al. 1951). To measure protein concentration, 100 \u0026micro;L of samples or standard (BSA, 0-500 g/mL) were mixed with 200 \u0026micro;L of Biuret reagent (500 \u0026micro;L of 1% cupric sulfate, 500 \u0026micro;L of 2% sodium potassium tartrate, in 50 mL of 2% sodium carbonate in 0.1 N NaOH). After thoroughly blending, the mixture was equilibrated at room temperature for 15 min. 20 \u0026micro;L of 1 N Folin and Ciocalteu\u0026apos;s reagent (Sigma-Aldrich, St. Louis, USA) were added to the reaction mixture and it was incubated for 30 min at room temperature. After the incubation, absorbance at 650 nm was measured using a microplate reader (TECAN Infinite 200-Pro, Tecan, Switzerland).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Amino acid profiling\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein was extracted from the PEF-treated samples and control from step 3a) and step 5a) (\u003cstrong\u003eFigure 2\u003c/strong\u003e) in aqueous solution and following sequential pH changes from 1 to 12 and from 12 to 1. The protein-rich sediment was precipitated using 35% and 85% ammonium sulfate salting out processes (Duong-Ly and Gabelli 2014). The precipitated proteins were then dialyzed using a SnakeSkin\u0026trade; dialysis bag (3.5 kDa, ThermoFisher Scientific, USA). The dialyzed proteins were then lyophilized and analyzed for amino acid profile through high-pressure anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) as follows. One mg of biomass was added to 1 mL of 6 M HCl and heated at 112 \u0026deg;C for 16 h in a dry bath (Bio-Base, China). The samples were then dried using nitrogen. The dried samples were suspended in 1 mL of ultrapure ion chromatography grade water and left at rest for 1 h (Kazir et al. 2019). \u0026nbsp;The samples were then filtered using a 0.22 \u0026mu;m syringe filter. The filtered samples were separated through HPAED-PAD Dionex ICS-5000 (Dionex, Thermo Fischer Scientific, MA, USA). Amino-pack 10 analytical grade column was used for amino acid separation. Signal detection was done using a gold AAA electrochemical detector with an AgCl reference electrode. Separation was achieved with gradient elution of the following parameters: 0.250 mL/min, column temperature 30\u0026deg;C, auto-sampler temperature 5\u0026deg;C, and a 25 \u0026mu;L injection loop volume. An amino acid standard mix (AAS18, Sigma-Aldrich, MO, USA) with the following dilutions 1/5, 1/10, 1/15, 1/20, and 1/25 was used to validate the separation program. The following amino acids were detected in the amino acid mix: alanine, arginine, aspartate, cysteine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. The calibration curve formed from these dilutions (R\u003csup\u003e2\u003c/sup\u003e\u0026gt;99% for each amino acid) was then incorporated into the program to quantify the amino acids in the unknown samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Release yield and content calculations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSolute (dry matter) release yield (DMRY %) was calculated according to \u003cstrong\u003eEquation 1\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"686\" height=\"96\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere the dry matter (DM(g)) of the initial biomass was measured by drying in the oven at 40\u003csup\u003e0\u003c/sup\u003eC to constant weight and the DM (g) of the extract was measured by drying all the extracts combined for each of the conditions in \u003cstrong\u003eTable 1\u003c/strong\u003e in the oven at 40\u003csup\u003e0\u003c/sup\u003eC to constant weight.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtein release yield (PRY%) was calculated using \u003cstrong\u003eEquation 2\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"693\" height=\"98\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere protein (g) in the extracts (Protein\u003csub\u003eextract\u003c/sub\u003e) was measured by Lowry and protein in the initial biomass (Protein\u003csub\u003einitial_biomass\u003c/sub\u003e)was measured by Lowy after bead beading in alkaline solution. PRY% of the pH shift experiments is the combined yield of water and all pH extracts (1 to 12) in the specific experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEssential Amino Acid content (EAA %) was calculated using \u003cstrong\u003eEquation 3\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"612\" height=\"103\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere Y\u003csub\u003eEAA\u003c/sub\u003e(g) is the content of essential amino acids detected with HPIC in the Protein\u003csub\u003eextract\u003c/sub\u003e and Y\u003csub\u003eTAA\u003c/sub\u003e are the total content of amino acids detected in the Protein\u003csub\u003eextract\u003c/sub\u003e. EAA determined were histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine and total amino acids (TAA) determined were alanine, arginine, aspartate, cysteine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine.\u003c/p\u003e\n\u003cp\u003eBranched Amino Acid content (BCAA%) was calculated using \u003cstrong\u003eEquation 4\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"617\" height=\"84\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere Y(g) is the content of amino acids detected with HPIC in the P\u003csub\u003eextract\u003c/sub\u003e. BCAA determined were leucine, isoleucine, and valine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudent\u0026rsquo;s t-test was performed to analyze the significance of the difference between data sets obtained in the study. The p-values were calculated using the Student\u0026rsquo;s t-test in a paired manner with 0.05 as the maximal p value indicating statistical significance.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass fractionation using PEF and pH shifts.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wet \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass was treated under different conditions and fractionated into soluble and insoluble fractions (\u003cstrong\u003eTable 1\u003c/strong\u003e). The control and PEF-treated biomass samples were further subjected to water-mediated extraction or to sequential pH extraction to estimate the amount of dry matter and protein released. In addition, the essential amino acid content was determined and the branched amino acid content (BCAA) of the protein fraction was calculated. The mass balance analysis of each condition described in \u003cstrong\u003eFigure 1\u003c/strong\u003e and \u003cstrong\u003eTable 1\u003c/strong\u003e appears in \u003cstrong\u003eTables S1 to S6\u003c/strong\u003e and is discussed below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.1. Water-mediated extraction process of PEF treated biomass.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw biomass had an initial protein content of 15.58\u0026plusmn;0.25%\u003csub\u003eDW\u003c/sub\u003e. The water-mediated extraction alone\u0026nbsp;yielded 7.5\u0026plusmn;0.04%\u0026nbsp;DMRY and 7.22\u0026plusmn;0.3% of PRY (\u003cstrong\u003eTable S1\u003c/strong\u003e), with 4.4\u0026plusmn;2.4% EAA, and 1.4\u0026plusmn;0.5% BCAA content. PEF treatment before the water-mediated extraction resulted in 7.5\u0026plusmn;0.04% DMRY and 8.1\u0026plusmn;0.1% of PRY, with 54.5\u0026plusmn;5.9% EAA, and 2.8\u0026plusmn;1.3% BCAA content (\u003cstrong\u003eTable S2\u003c/strong\u003e). PEF pre-treatment did not significantly impact DMRY (p-value = 1), and BCAA content (p-value = 0.29), but significantly increased the PRY (p-value= 0.009) and the fraction of EAA (p-value = 0.009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eGracilaria\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;fractionation process using PEF pre-treatment and pH shift.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequential pH-shift extraction (12 to 1) led to 9.62\u0026plusmn;0.04% DMRY and 14.45\u0026plusmn;0.1% of PRY (\u003cstrong\u003eTable S3\u003c/strong\u003e) with 14.12\u0026plusmn;1.4% EAA and 0.06\u0026plusmn;0.03% of BCAA content. Sequential pH extraction (12 to 1), which followed PEF, led to 9.64\u0026plusmn;0.02% DMRY and 24.1\u0026plusmn;0.03% of PRY (\u003cstrong\u003eTable S4\u003c/strong\u003e), with 62.1\u0026plusmn;5.5% EAA and 3.1\u0026plusmn;0.7% BCAA content in the extract. PEF pre-treatment did not change the DMRY (p-value = 0.68) but significantly increased the PRY (1.7 fold, p-value = 3\u0026middot;10\u003csup\u003e-8\u003c/sup\u003e) and the content of EAA (4.4 fold, p-value = 0.007) and branched amino acids (51.6 fold, p-value = 0.03).\u003c/p\u003e\n\u003cp\u003eSequential pH-shift extraction (1 to 12) led to 9.61\u0026plusmn;0.02% DMRY and 17.33\u0026plusmn;0.8% of PRY (\u003cstrong\u003eTable S5\u003c/strong\u003e), with 31.1 \u0026plusmn;2.2% EAA and 0.75\u0026plusmn;0.09% BCAA content. Sequential pH-shift extraction with PEF (1 to 12) led to of 9.61\u0026plusmn;0.04% DMRY, and 28.02\u0026plusmn;0.2% of PRY (\u003cstrong\u003eTable S6\u003c/strong\u003e), 63.6\u0026plusmn;5.3% EAA and 3.47\u0026plusmn;0.06% BCAA (\u003cstrong\u003eTable S7\u003c/strong\u003e). PEF pre-treatment did not change the DMRY (p-value = 0.99) but significantly increased the PRY (1.6 fold, p-value = 3\u0026middot;10\u003csup\u003e-5\u003c/sup\u003e) and the content of EAA (2 fold, p-value = 0.02) and BCAA (4.6 fold, p-value = 8\u0026middot;10\u003csup\u003e-4\u003c/sup\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn experiments without PEF, although the order of pH shift sequence (1 to12 vs. 12 to 1) did not impact the DMRY, (p-value = 0.42), the order significantly affected the PRY (1.19 fold p-value = 0.004), EAA content (2.2 fold change, p-value = 0.01) and BCAA content (12.5 fold change, p-value= 0.009).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe order of pH shifts (1 to 12 vs. 12 to 1) following PEF, did not impact the DMRY, (p-value = 0.25), EAA content (p-value = 0.85) and BAA content (p-value = 0.5). However, the PRY was impacted significantly (1.16 fold change, p-value = 9\u0026middot;10\u003csup\u003e-6\u003c/sup\u003e) when PEF pre-treated samples further treated with pH 1 to 12, and pH 12 to 1 were compared.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThus, regardless of PEF pretreatment, the PRY was impacted by the order of the pH shifts. In both cases the samples treated with pH 1 to 12 showed higher PRY compared to those treated with pH 12 to 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Determination of the impact of process parameters on \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass fractionation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.1 The impact of pH on solute and protein release yield and content of essential and branched amino acids of the extracts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated how pH shifting impacts the \u003cem\u003eGracilaria\u003c/em\u003e fractionation efficiency and quality of the products regardless of PEF pre-treatment. The addition of pH shifts in comparison to water-mediated extraction (without pH shifts), impacted the DMRY \u003cstrong\u003e(Figure 3a\u003c/strong\u003e, fold change 1.28, p-value = 7.7\u0026middot;10\u003csup\u003e‑6\u003c/sup\u003e) and PRY (\u003cstrong\u003eFigure 3b\u003c/strong\u003e, 1.64 fold change, p-value = 4.8\u0026middot;10\u003csup\u003e-6\u003c/sup\u003e). However, it did not significantly affect EAA, and BCAA content (\u003cstrong\u003eFigure 3c\u003c/strong\u003e and \u003cstrong\u003eFigure 3d\u003c/strong\u003e, p-value = 0.28, and 0.73) of the extracts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.2 The impact of PEF pre-treatment on solute and protein release yield, and on essential and branched amino acids contents of the extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also investigated if addition of PEF pre-treatment increases the \u003cem\u003eGracilaria\u003c/em\u003e biomass fractionation efficiency and quality of the products. There was no significant difference in the DMRY between PEF treated and untreated samples (\u003cstrong\u003eFigure 4a,\u0026nbsp;\u003c/strong\u003ep-value=0.99). However, an addition of PEF significantly increased the PRY% (\u003cstrong\u003eFigure 4b,\u0026nbsp;\u003c/strong\u003e1.54 fold change,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ep-value=0.03), EAA fraction (\u003cstrong\u003eFigure 4c,\u0026nbsp;\u003c/strong\u003e3.62 fold change, p-value=8.0\u003cstrong\u003e\u003csup\u003e.\u003c/sup\u003e\u003c/strong\u003e10\u003csup\u003e-5\u003c/sup\u003e) and BCAA fraction (\u003cstrong\u003eFigure 4d,\u0026nbsp;\u003c/strong\u003e4.27 fold change, p-value=1.3\u0026middot;10\u003csup\u003e-4\u003c/sup\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.3 Combined effect of pH and PEF pre-treatment on solute and protein release yields and on essential and branched amino acids contents of the extracts.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we tested whether the combined effect of pH and PEF significantly improves the \u003cem\u003eGracilaria sp.\u0026nbsp;\u003c/em\u003ebiomass fractionation. Combined PEF and pH-shift treatments did not improve the DMRY in comparison with pH shifts alone (\u003cstrong\u003eFigure 5a, p-value=0.44\u003c/strong\u003e), but showed higher DMRY in comparison with PEF pre-treatment alone (\u003cstrong\u003eFigure 5a,\u0026nbsp;\u003c/strong\u003e1.28 fold change, p-value= 2.8\u003csup\u003e.\u003c/sup\u003e10\u003csup\u003e-7\u003c/sup\u003e). A combined PEF and pH-shift treatment led to higher protein release yield, higher EAA and BCAA contents than PEF or pH-shift alone (\u003cstrong\u003eFigure 5b,\u0026nbsp;\u003c/strong\u003ep-value=0.00017, and 0.0003,\u003cstrong\u003e\u0026nbsp;Figure 5c,\u0026nbsp;\u003c/strong\u003ep-value=0.02, and 0.04\u003cstrong\u003e\u0026nbsp;Figure 5d,\u0026nbsp;\u003c/strong\u003ep-value=0.0077, and 0.05 respectively) with a fold change of 3.23, and 1.97 in protein release yield, 1.15, and 2.71 in EAA, 1.18, and 13.12 in BCAA respectively. The %DMRY, %PRY, %EAA, and %BCAA under different conditions used in the study are summarized in \u003cstrong\u003eTable 3\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e: A summary of %DMRY, %PRY, %EAA, and %BCAA obtained under different conditions in the current study.\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.227642276422763%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCondition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.772357723577235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDMRY(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePRY (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEAA (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBCAA (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.227642276422763%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl Biomass\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;(No PEF)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.772357723577235%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.227642276422763%\" valign=\"top\"\u003e\n \u003cp\u003eWater-mediated extraction\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.772357723577235%\" valign=\"top\"\u003e\n \u003cp\u003e7.5\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e7.22\u0026plusmn;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e4.4\u0026plusmn;2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e1.4\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.227642276422763%\" valign=\"top\"\u003e\n \u003cp\u003epH shift (pH 1 to 12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.772357723577235%\" valign=\"top\"\u003e\n \u003cp\u003e9.61\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e17.33\u0026plusmn;0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e31.1 \u0026plusmn;2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e0.75\u0026plusmn;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.227642276422763%\" valign=\"top\"\u003e\n \u003cp\u003epH shift (pH 12 to 1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.772357723577235%\" valign=\"top\"\u003e\n \u003cp\u003e9.62\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e14.45\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e14.1\u0026plusmn;1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e0.06\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.227642276422763%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePEF Treated Biomass\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.772357723577235%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.227642276422763%\" valign=\"top\"\u003e\n \u003cp\u003eWater-mediated extraction\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.772357723577235%\" valign=\"top\"\u003e\n \u003cp\u003e7.5\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e8.1\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e54.5\u0026plusmn;5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e2.8\u0026plusmn;1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.227642276422763%\" valign=\"top\"\u003e\n \u003cp\u003epH shift (pH 1 to 12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.772357723577235%\" valign=\"top\"\u003e\n \u003cp\u003e9.61\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e28.02\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e63.6\u0026plusmn;5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e3.47\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.227642276422763%\" valign=\"top\"\u003e\n \u003cp\u003epH shift (pH 12 to 1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.772357723577235%\" valign=\"top\"\u003e\n \u003cp\u003e9.64\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e24.1\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e62.1\u0026plusmn;5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\" valign=\"top\"\u003e\n \u003cp\u003e3.1\u0026plusmn;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eProtein solubility and release from the algal biomass strongly depend on pH (which determines protein surface charge), on membrane and cell wall integrity, and on protein interactions with the algal polysaccharides, some of which make the cell walls. In our current study, we explored both the impact of PEF, and of sequentially shifting pH through a range of pH values during pH-shift experiments to increase protein extraction yield.\u003c/p\u003e\n\u003cp\u003eA recent study on \u003cem\u003eGracilaria dura\u003c/em\u003e sheds light on how some of these factors impact the behaviour of proteins (Bozdemir et al., 2022). Notably, when re-dissolved in water, the \u0026ldquo;powdered\u0026rdquo; \u003cem\u003eGracilaria dura\u003c/em\u003e exhibited a negative surface charge (zeta potential), likely attributed to its cell wall composition primarily comprising negatively charged polysaccharides (Bozdemir et al., 2022). The minimal solubility of \u003cem\u003eGracilaria dura\u0026nbsp;\u003c/em\u003eproteins occurred at pH 4.0, and from that point it increased with increasing pH, at least up to pH 13 (Bozdemir et al., 2022).\u003c/p\u003e\n\u003cp\u003eRecent investigations into microalgal species have revealed intriguing patterns of protein solubility. Notably, one study demonstrated an impressive ~84% solubility within a pH range of 2-12 (Grossmann et al. 2019). In contrast, research on \u003cem\u003eTetraselmis spp.\u003c/em\u003e highlighted a unique behaviour: protein solubility remained unaffected by the ionic strength of the solution, even up to 0.5 M (Schwenzfeier et al. 2011). Remarkably, proteins from \u003cem\u003eTetraselmis spp.\u003c/em\u003e exhibited complete solubility above pH \u0026ge; 5.5. Our current study underscores the importance of broad pH exploration during protein extraction. By encompassing a wide pH range, we aimed to enhance overall protein release yield, a critical consideration for optimizing protein extraction protocols.\u003c/p\u003e\n\u003cp\u003eIn some earlier articles (Harnedy and FitzGerald 2013; Kumar et al. 2014), the topic of protein isolation using pH alterations in red macroalgae was discussed. A study on \u003cem\u003eKappaphycus alvarezii\u0026nbsp;\u003c/em\u003eshow that\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe minimal solubility of nitrogenous compounds was found at pH 4, and the authors suggested that this pH may be the isoelectric point of the protein concentrate of \u003cem\u003eKappaphycus alvarezii\u003c/em\u003e (Kumar et al. 2014). Proteins acquire net positive and net negative charge, below and above their pI respectively, that lead to repulsion between the molecules and enhance protein solubility (Seena and Sridhar 2005). The attractive interactions between positively charged proteins and polyanions can lower the apparent pI, by shifting the pH of minimal solubility of the protein to a lower pH (Zimet and Livney 2009), where charge-neutralized complexes form with the polyanions (Williams 2009). The mechanisms underlying protein release yield under various pH and pre-treatment conditions depends on this insight.\u003c/p\u003e\n\u003cp\u003eThe pH shift approach is based on the fact that each protein has a unique isoelectric pH, at which it is least soluble, hence collecting the soluble proteins during each step of the pH shift process and combining them, should significantly increase the protein extraction yield (compared to collecting the soluble proteins at a certain pH where some of the proteins have zero zeta potential, and are hence insoluble). It should also help avoiding loss of protein due to complex formation with the anionic polysaccharides, which, as discussed above, also has a strong pH dependence.\u003c/p\u003e\n\u003cp\u003eWe treated the biomass in two ways: 1) Starting with the highest pH 12 and progressively adjusting to each subsequent pH level until reaching pH 1. 2) Starting with the lowest pH 1 and progressively adjusting to each subsequent pH level until reaching pH 12. We found that the second way, i.e. when pH is gradually increased, was more effective in increasing protein release, (\u003cstrong\u003eTable 3\u003c/strong\u003e) leading to a higher PRY 17.33\u0026plusmn;0.8 vs. 14.45\u0026plusmn;0.1. A likely reason for this is the presence of the anionic cell wall polysaccharides, like agar, which are uncharged at very low pH (much below the pk\u003csub\u003ea\u003c/sub\u003e of its carboxylic groups, which is around 5 (Carisma et al. 2020). As pH rises, and cell wall polysaccharides become more negatively charged, they bind positively charged proteins (up to the pI of each protein, or slightly above it, if local positive patches exist) and form electrostatic complexes with them. This complexation decreases protein release. Above the pI, solubility of the proteins and their consequent release increase, as they become more negatively charged, and repulsed from the polysaccharides. When starting at high pH, the cell wall polysaccharides are very negative during most of the process, binding much proteins below their pIs, and decrease their release. In contrast, when starting at low pH, as the cell wall polysaccharides are uncharged, most of the proteins are released in the beginning, as they are very positively charged, and the uncharged cell wall polysaccharides interfere less with their release.\u003c/p\u003e\n\u003cp\u003eThe scientific literature highlights the impact of pulsed electric field (PEF) treatment on macroalgal cells, increasing membrane permeability/porosity, thereby leading to loss of turgor pressure within the cells, dehydration and release of salts and small proteins (Robin et al., 2018; Polikovsky et al., 2016; Prabhu et al., 2019).\u003c/p\u003e\n\u003cp\u003eIn our current study, we improved protein extraction by combining PEF with pH shifts. This combination synergizes: (1) the increased membrane porosity, (2) the pH-dependent protein release, partly hampered by cell-wall polysaccharides, and (3) the chemical potential gradient between the cell interior and its surrounding medium. Consequently, at each pH step (pH solutions, as detailed in Table 2), there exists a motive force for the diffusive release of distinct proteins, which is facilitated by the loss of membrane-barrier efficacy by PEF, and escaping cell-wall polysaccharide complexation by shifting the pH. PEF both increases the permeability of the cell membrane and removes salts, which increases protein release, thereby leading to higher protein release yield (Robin et al. 2018)\u003c/p\u003e\n\u003cp\u003eA combination of PEF and sequential pH-shifting from 1 to 12 led to the highest fractionation increasing the DMRY by 28.3% (from 7.5\u0026plusmn;0.04% to 9.61\u0026plusmn;0.04%), PRY by 288.1% (from 7.22\u0026plusmn;0.3% to 28.02\u0026plusmn;0.2%), and EAA content by 1282.6% (from 4.6\u0026plusmn;2.4% to 63.6\u0026plusmn;5.3%), and BCAA content by 147.9% (from 1.4\u0026plusmn;0.5% to 3.47\u0026plusmn;0.06%w) in comparison to aqueous extraction alone. These results are higher than previous reports on \u003cem\u003eGracilaria\u0026nbsp;\u003c/em\u003eprotein extraction (\u003cstrong\u003eTable 2\u003c/strong\u003e), probably due to the hurdle effects and the synergy of PEF and pH-shifting, where PEF pre-treatment facilitates the following protein release yield at various pH conditions. We observed that the combination of PEF and pH shift method can provide much better yields than enzymatic methods, ultrasonication, chemical methods, and PEF alone (\u003cstrong\u003eTable 4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe most interesting impact of the PEF pre-treatment is the enhancement in the EAA and BCAA in the extracts obtained from PEF pre-treated biomass. The PEF pre-treated samples followed by aqueous extraction had highest enhancement in EAA 54.5\u0026plusmn;5.9% (p-value=0.02), and BCAA 2.8\u0026plusmn;1.3% (p-value=0.009), respectively compared to samples treated only with sequential pH (pH 1 to 12) that had 31.1 \u0026plusmn;2.2%, and 0.75\u0026plusmn;0.09% of EAA, and BCAA respectively. This is a new and interesting observation, which is in line with our previous works on \u003cem\u003eUlva\u0026nbsp;\u003c/em\u003esp. biomass, in which we reported selective protein extraction by PEF in comparison to aqueous extraction alone (Polikovsky et al. 2016). This observed enhancement in the EAA, and BCAA extraction in PEF-treated biomass with or without pH shifts, suggests that the PEF treatment aids in the release of EAA and BCAA. The mechanisms of such selective extraction are still unknown, and motivate further study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u0026nbsp;\u003c/strong\u003eSummary of protein extraction from \u003cem\u003eGracilaria\u003c/em\u003e sp. using various protocols.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"603\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.11295681063123%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.943521594684384%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTechnology\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.906976744186046%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConditions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.93687707641196%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein extraction yield\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.099667774086377%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eReferences\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.11295681063123%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGracilaria verrucosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.943521594684384%\" valign=\"top\"\u003e\n \u003cp\u003eEnzymatic extraction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.906976744186046%\" valign=\"top\"\u003e\n \u003cp\u003eAgarase (pH-6) and cellulase (pH-3.8), temperature-50\u0026deg;C to 55\u0026deg;C for 2 h with 0.1 M MesNaOH and oxalate NaOH buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.93687707641196%\" valign=\"top\"\u003e\n \u003cp\u003e6.3%dw\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.099667774086377%\" valign=\"top\"\u003e\n \u003cp\u003e(Fleurence et al. 1995)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.11295681063123%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGracilaria\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.943521594684384%\" valign=\"top\"\u003e\n \u003cp\u003eContinuous PEF device\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.906976744186046%\" valign=\"top\"\u003e\n \u003cp\u003eVoltage-400V, Frequency-5Hz, Pulse duration (\u0026micro;s)-50, number of pulses -1000, inter-electrode gap-4mm, sample feeding rate-100g/10 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.93687707641196%\" valign=\"top\"\u003e\n \u003cp\u003e4.75% from 8.4%dw initial protein content\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.099667774086377%\" valign=\"top\"\u003e\n \u003cp\u003e(Kashyap et al. 2022)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.11295681063123%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGracilaria\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.943521594684384%\" valign=\"top\"\u003e\n \u003cp\u003eUltrasonication\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.906976744186046%\" valign=\"top\"\u003e\n \u003cp\u003eTreatment with NaOH (10% w/v) and then ultrasonication for 2 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.93687707641196%\" valign=\"top\"\u003e\n \u003cp\u003e19.84% algal protein concentrate from 25%dw initial protein content\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.099667774086377%\" valign=\"top\"\u003e\n \u003cp\u003e(Kazir et al. 2019)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.11295681063123%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGracilaria dura\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.943521594684384%\" valign=\"top\"\u003e\n \u003cp\u003eUltrasonication\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.906976744186046%\" valign=\"top\"\u003e\n \u003cp\u003eUltrasonication time-257.57s, time for extraction-22.61h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.93687707641196%\" valign=\"top\"\u003e\n \u003cp\u003e2.09% from 11.97\u0026plusmn;0.05%dw \u0026nbsp;initial protein \u0026nbsp;content\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.099667774086377%\" valign=\"top\"\u003e\n \u003cp\u003e(Bozdemir et al. 2022)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.11295681063123%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGracilaria\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.943521594684384%\" valign=\"top\"\u003e\n \u003cp\u003eBatch PEF device (gravitational-PEF device) and pH shift (sequential pH 1 to 12) treatment.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.906976744186046%\" valign=\"top\"\u003e\n \u003cp\u003eVoltage-1000V, Frequency-3Hz, Pulse duration (\u0026micro;s)-50, \u0026nbsp; \u0026nbsp; \u0026nbsp;number of pulses-200, inter-electrode gap-4mm.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.93687707641196%\" valign=\"top\"\u003e\n \u003cp\u003e28.02\u0026plusmn;0.2% from initial protein \u0026nbsp;content\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.099667774086377%\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n\u003c/table\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBoth PEF and pH play crucial roles in the fractionation of \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass. Utilizing a combination of PEF and sequential pH shifts (pH 1 to 12) resulted in notable outcomes, including a solute (dry matter) release yield of 9.61\u0026plusmn;0.04%, protein release yield of 28.02\u0026plusmn;0.2%, EAA content of 63.6\u0026plusmn;5.3%, and BCAA content of 3.47\u0026plusmn;0.06%. It is noteworthy that PEF significantly increased EAAs and BCAAs extraction efficiency, as demonstrated by samples treated with PEF alone, which exhibited 54.5\u0026plusmn;5.9% EAA and 2.8\u0026plusmn;1.3% BCAA. In contrast, samples treated solely with pH shifts (pH 1 to 12) and without PEF showed EAA and BCAA levels of 31.1\u0026plusmn;2.2% and 0.75\u0026plusmn;0.09%, respectively. This underscores the cooperative effect of combining PEF and pH-shift treatments (particularly when gradually raising pH from 1 to 12), highlighting their synergistic impact in amplifying protein release yield and particularly essential amino acids and branched amino acids in \u003cem\u003eGracilaria\u003c/em\u003e sp. biomass extracts.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study in the present manuscript was funded by Good Food Institute, Israel and Israel Ministry of Health.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAG and KL have patent applications on devices and use of\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp;PEF\u0026nbsp;technologies for seaweed processing with Ramot, Tel Aviv University. AG has interest in Genesea Advanced Technologies Ltd, which focuses on seaweed protein production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMrinal Kashyap\u003c/strong\u003e. Writing original manuscript draft, execution of experiments, data interpretation and analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupratim Ghosh\u003c/strong\u003e. Supportive role in execution of experiments,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKlimentiy\u003c/strong\u003e\u003cstrong\u003eLevkov\u003c/strong\u003e. Hardware, software development, experiment\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYoav D Livney.\u0026nbsp;\u003c/strong\u003eReview and editing of the original manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlvaro Israel\u003c/strong\u003e. Review and editing of the original manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlexander Golberg.\u0026nbsp;\u003c/strong\u003eConceptualization Data analysis, editing of the manuscript\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBozdemir A, Şensu E, Okudan EŞ, \u0026Ouml;z\u0026ccedil;elik B, Y\u0026uuml;cetepe A (2022) Ultrasound‐assisted enzymatic extraction of proteins from \u003cem\u003eGracilaria dura\u003c/em\u003e: Investigation of antioxidant activity and techno‐functional properties. Journal of Food Processing and Preservation 46 (8):e16803\u003c/li\u003e\n \u003cli\u003eCarisma NAS, Gonzales RYE, Lazaro-Llanos N (2020) An Investigation on Zinc Biosorption with Agar Extraction Waste from \u003cem\u003eGracilaria tenuistipitata\u003c/em\u003e. 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Food Hydrocolloids 23 (4):1120-1126. doi:10.1016/j.foodhyd.2008.10.008\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Macroalgae, Pulsed electric field (PEF(, Protein, functional food ingredients, essential amino acids, branched amino acids","lastPublishedDoi":"10.21203/rs.3.rs-4131953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4131953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study explores the potential of \u003cem\u003eGracilaria\u003c/em\u003e sp., a red seaweed species, as a source of protein ingredients. We investigated the effects of high voltage pulsed electric field (PEF) pre-treatment, followed by either water-mediated extraction or pH shift, on the total-solute (“dry matter”) and protein release, and the content of essential and branched-chain amino acids (BCAA) in the extracts. The PEF was administered using a custom-made generator within a specially designed electroporation cell with sliding electrodes. The treatment parameters comprised 1000 V, a 50 µs pulse duration, 200 pulses at a 3 Hz frequency, and an inter-electrode gap ranging from 3.9 to 4.4 mm. The pH shift extraction was executed through combination of fractions extracted sequentially at pH-shift steps spanning the pH range of 1 to 12. Both PEF and pH played pivotal roles in the fractionation of \u003cem\u003eGracilaria\u003c/em\u003esp., enhancing protein release compared to water-mediated extraction, PEF pre-treatment, or pH shift alone. The combination of PEF and sequential pH washes (pH 1 to 12) resulted in a total solute release of 9.61±0.04% and protein release of 28.02±0.2% with essential amino acid (EAA) content of 63.6±5.3%, and BCAA content of 3.47±0.06%. It is noteworthy that PEF significantly increased the EAA and BCAA content of the protein extracts, irrespective of pH shifts. These findings underscore the promising potential of PEF pre-treatment in producing functional food ingredients derived from macroalgal sources.\u003c/p\u003e","manuscriptTitle":"Protein and solute release from the red seaweed, Gracilaria sp., using a high voltage pulsed electric field and pH shift","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 03:52:54","doi":"10.21203/rs.3.rs-4131953/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-08T00:33:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-26T22:42:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c4ec77f0-0160-44ae-966f-0c7b5429b182","date":"2024-03-25T13:48:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-25T12:55:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-20T09:20:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-20T09:17:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2024-03-19T16:38:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5f902aca-fc5e-4864-adde-3a46663aacf9","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-03T14:01:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-22 03:52:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4131953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4131953","identity":"rs-4131953","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}