Recovery of a protein-rich biomass from shrimp (Pandalus borealis) boiling water: A colloidal study

Food Chemistry 302 (2020) 125299

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Recovery of a protein-rich biomass from shrimp (Pandalus borealis) boiling water: A colloidal study

T

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Bita Forghania, , Romain Bordesb, Anna Strömb, Ingrid Undelanda a b

Food and Nutrition Science, Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden Applied Chemistry, Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden

A R T I C LE I N FO

A B S T R A C T

Keywords: Shrimp boiling water Protein Biomass recovery Flocculation Waste water Polyelectrolyte Polysaccharide

Flocculation and sedimentation of a protein-rich biomass from shrimp boiling water (SBW) using food grade polysaccharides (carrageenan, alginate and carboxymethyl cellulose (CMC)) as flocculants was investigated at different pH-values. The effect of flocculant concentration on particle size and viscosity of SBW was also evaluated. Flocculation with carrageenan (0.45 g/L) at pH = 4 exhibited the most efficient protein sedimentation; protein concentration of the upper phase was here reduced by 77%, allowing 86% protein to be sedimented from SBW. Flocculation by alginate and CMC at pH = 4 showed 67% and 60% protein reduction of the upper phase at concentrations of 0.5 and 0.2 g/L, respectively. Contrary to alginate and CMC, carrageenan concentration affected the size distribution of flocs. Finally, carrageenan at 0.45 g/L and pH = 4 was successfully tested in a scaled up trial (5L) providing 78.5% protein recovery and a biomass with 75% protein on dry weight basis.

1. Introduction

2016; Jarrault et al., 2017). In that context, application of membrane technology has been evaluated to e.g. recover proteins from slaughter house process waters (Castro-Muñoz & Ruby-Figueroa, 2019), or natural flavoring compounds from shrimp cooking juice (Jarrault et al., 2017). However, for the recovery of protein- and lipid-enriched biomass, membrane technology has not fully reached successful commercial implementation due to drawbacks associated with for instance back pressure and membrane fouling. A common practice which is in place in many seafood factories is the coagulation-flocculation of proteins using lime, ferric chloride and alum (Fahim, Fleita, Ibrahim, & El-Dars, 2001) or flocculation with synthetic flocculants (Taskaya & Jaczynski, 2009). The destabilization of the suspended particles/flocs reached during such treatment usually enables an efficient dewatering by e.g. flotation, filtration or centrifugation. With the mentioned coagulants and flocculants, there are however safety concerns regarding the residual content of metal ions or synthetic/nonfood grade polymers in the water and in the recovered biomass. This leads to a significant waste of nutrients from seafood processing industries every year, which is indeed a non-sustainable handling of valuable raw materials. Finding suitable food grade flocculation systems to combine with cost-efficient dewatering techniques could allow the recovery of a completely new type of biomass during seafood process water remediation, which could then be used as a protein ingredient in feed or food production. Such an approach is well

Water is a cornerstone in seafood plants and is used in every step during the processing of the raw material, e.g. for storage, peeling, cutting and transportation. Currently, water is discharged with minimal or no food grade pre-treatment and is often a source of extra cost as the remediation of the process water has to be handled. As an example of a seafood trade ranking very high in terms of water consumption, shrimp processing factories generate large amounts of process water during boiling, peeling and transportation of the shrimps; often approaching 50 m3/ton of final peeled shrimp. This water contains significant amounts of high-value compounds such as protein, fatty acids and phosphate, which today are not recovered (Jarrault, Dornier, Labatut, Giampaoli, & Lameloise, 2017; Rodríguez, Pilar, Anxo, & Antonio, 2016; Rosa & Nunes, 2004). Our own preliminary studies have revealed that loss of protein and fatty acids into process waters is ∼89 and 17 kg, respectively per ton of peeled shrimp (Forghani et al., unpublished data). Based on the significant value of marine proteins and lipids on the market, these are incentives to find cost efficient recovery techniques which can return these compounds to the food chain. This task is however rendered difficult by the relatively low concentration of biomolecules in the shrimp process waters; e.g. ∼0.8–2.0 wt% protein in boiling waters, with variations being due e.g. to harvest season and post mortem age of the shrimps (Amado, González, Murado, & Vázquez,

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Corresponding author. E-mail address: [email protected] (B. Forghani).

https://doi.org/10.1016/j.foodchem.2019.125299 Received 7 February 2019; Received in revised form 4 July 2019; Accepted 29 July 2019 Available online 30 July 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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Flocculant stock solutions of 1% were prepared in milli-Q water obtained from a MilliPore system.

established for dairy protein, for which interaction with negatively charged polysaccharides has been studied extensively (De Kruif & Tuinier, 2001; Dickinson, 1998). Negatively charged polysaccharides interact with protein at pH-values where the net charge of the proteins is close to neutral and/or positive. At such pH, the addition of increasing amounts of polysaccharides will at first force the protein system to flocculate via bridging or patch-wise flocculation resulting in increased floc size, before reaching complete coverage leading to reduced aggregate size (Borkovec & Papastavrou, 2008; Dickinson, 1998; Glahn & Rolin, 1994; Ye, 2008). The amount required, or the effectiveness of specific polysaccharides, to create bridging flocculation of a protein slurry depends on several factors, e.g. their charge density, the molecular weight of the polysaccharide or the presence of carboxylic or sulfate groups. The application of polysaccharides as a strategy to flocculate and recover proteins from seafood process waters in a food and feed grade manner has so far only been reported for surimi process waters and fish meal stick water (Hao et al., 2016; Savant & Torres, 2003; Wibowo, Savant, Cherian, Savage, & Torres, 2005; Wibowo, Velazquez, Savant, & Torres, 2005, 2007). Savant and Torres (2003) used chitosan and chitosan complexed with alginate, pectin and carrageenan for surimi process water and reported on 81–90% recovery of proteins after 24 h incubation followed by centrifugation. Hao et al. (2016) flocculated fish meal stick water using carrageenan, guar, chitosan and alginate and reached protein recovery rates from 55 to 70%. In the study by Guerrero, Omil, Méndez, and Lema (1998) coagulation-flocculation with sodium polyacrylate and chitosan at pH 4 and 7.2–7.8, respectively followed by centrifugation removed 97% and 75% total suspended solids (TSS). However, there is to the best of our knowledge no previous report on the application of food grade polysaccharides to recover a protein-enriched biomass from shrimp processing water. In this study, the aim was to investigate the flocculation of protein and other biomolecules like lipids and astaxanthin in shrimp boiling water (SBW) using alginate, carrageenan and carboxymethyl cellulose (CMC). Alginate and carrageenan are polysaccharides derived from brown and red marine seaweed, respectively, containing different functional groups; carboxyl in alginate and sulfate groups in carrageenan. CMC is a cellulose derivative containing carboxyl groups, the number depending on its degree of substitution (DS). Here, we evaluated the effect of pH and polysaccharide concentration on flocculation and sedimentation of protein in SBW. We also monitored the resulting floc size and SBW viscosity. Finally, the most efficient flocculation conditions were used in a scaled up trial.

2.2. Proximate analysis of shrimp boiling water (SBW) 2.2.1. Fatty acid measurement Fatty acid analysis of crude SBW was performed after lipid extraction according to Lee, Trevino, and Chaiyawat (1995) and subsequent methylation according to Lepage and Roy (1986) with some modifications. Extraction was performed using chloroform:methanol (1:2). After addition of C:17 as internal standard and vortexing for 10 s, 0.5% NaCl was added to obtain a ratio between 0.5% NaCl and solvent mixture of 1:2.75 (V/V). Following phase separation, the chloroform phase was recovered and evaporated at 40 °C. Methylation was conducted by addition of 2 mL of toluene and 2 mL of methanol:acetylchloride mixture (1:10) and the solution was incubated at 60 °C for 120 min. One mL milli-Q water and 2 mL petroleum ether was added to the tubes which were vortexed for 10 s and centrifuged at 2500×g for 5 min. The upper phase was transferred to new tubes and evaporated under nitrogen at 40 °C. Evaporated samples were then dissolved in 0.5 mL isooctane. Identification and quantification of fatty acids were carried out by GSMS using an Agilent technologies 7890 A GC system connected to Agilent technologies 5975 inert MSD (Kista, Sweden) as described elsewhere (Cavonius, Carlsson, & Undeland, 2014). Total fatty acids content was calculated as the sum of all measured fatty acids in the sample minus the internal standard. 2.2.2. Protein measurement The protein content of the SBW was measured before and after flocculation (Section 2.4.2) following the method of Lowry, Rosebrough, Farr, and Randall (1951) modified by Markwell, Haas, Bieber, and Tolbert (1978) using bovine serum albumin as standard in the concentration range of 10–100 µg/mL. Absorbance was read at 660 nm using a Cary60 BIO UV–vis spectrophotometer (Varian Australia Pty Ltd., Victoria, Australia). 2.2.3. Trace element analysis Amount of copper, iron, zinc, nickel, cobalt and manganese were measured in crude SBW following the method of Fredrikson, Carlsson, Almgren, and Sandberg (2002). In short, 10 mL of SBW was freeze dried. The dried samples were subjected to microwave-aided acid digestion in Teflon vials. After the treatment the sample was decanted to a 10 mL marked test tube. The Teflon vial was rinsed with Milli-Q water and the rinsing water was transferred to the same 10 mL test tube where the solution was diluted to a final volume of 10 mL with milli-Q water. Ion chromatography was then conducted for analysis according to Fredrikson et al. (2002). Calcium and Magnesium content measurements were performed on the same samples using an HPLC system (Dionex BioLc system; Thermo Scientific, Sunnyvale, CA) which consisted of a GS50 gradient pump, CD20 conductivity detector and autosampler (Emmen, The Netherland). Ions were eluted through an IonPac CG14, 4 mm guard column and resolved in IonPac CS14 (Thermofisher Scientific, USA) by an isocratic flow of 0.9 mM methanesulfonic acid at 0.7 mL/min for 20 min. Calcium and magnesium were quantified based on standards in the concentration of 1–10 mg/L.

2. Materials and methods 2.1. Raw materials and chemicals SBW (Fig. 1) was collected at Räkor & Laxgrossisten AB, Gothenburg, Sweden in February 2016. Alginate (Manucol® DM) and λ carrageenan (Viscarine® GP-109NF) were provided by FMC Food and Nutrition (PA, USA). Carboxymethyl cellulose (CMC) (product number: C-5678) was purchased from Sigma–Aldrich (St. Louis. MO, USA).

2.2.4. Polypeptide profile analysis before and after centrifugation at different pHs Polypeptide profile of the native SBW and of supernatants obtained after adjusting the pH from the native pH (8.7) down to pH 3 and applying centrifugation at 10,000×g for 20 min was analyzed using SDSPAGE. SDS-PAGE was performed according to the method of Laemmli (1970) using Mini-protean TGX 4–20% pre-cast gels (Bio-Rad Laboratories, USA). Briefly, SBW samples were mixed 1:1 (v/v) with the loading dye and approximately 20 µg protein were loaded into each well. The protein molecular standard was of broad range, 5–250 kDa.

Fig. 1. Shrimp boiling water (SBW). 2

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internal pumping system of the device that was operated at 2000 rpm. The diffraction data was analyzed using the version 2.19 of the Malvern software and were reported in terms of particle size distribution and average size, D(3,2).

Protein bands were stained by Coomassie Brilliant blue G-250. 2.2.5. Particle charge analysis Particle charge density measurements of SBW as a function of polysaccharide concentration or pH were performed using a PCD 02 particle charge detector (BTG Mütek GmbH, Germany). When titrating with the three polysaccharides in order to find the zero charge point, portions of 150 µL polysaccharide were gradually added in until the charge was almost zero. When studying particle charge as a function of pH, the range of pH 2–9 was evaluated. 15 mL SBW was introduced in the measurement chamber and pH was adjusted using 1 N HCl and 1 N NaOH.

2.5.2. Viscosity measurements The viscosity of the whole SBW samples ( ± flocculant) and their supernatants obtained after 1 h of sedimentation was determined using a rheometer Physica 300 (Anton-Paar, Germany). The viscosity of the dispersions was determined between shear rates of 1 s−1 and 1000 s−1 at 20 °C. The temperature was controlled by a Peltier system. The geometry used was a parallel plate with a diameter of 50 mm and a gap of 1 mm.

2.3. Flocculation treatments of SBW 2.6. Scale-up trial to verify efficiency of the most promising flocculation treatment

To 20 mL of SBW, various amounts of 1% solution of alginate, carrageenan and CMC were added while the solution was first stirred at 200 rpm for 2 min and then at 100 rpm for 2 min. Flocculation and gradual sedimentation were allowed to continue without stirring at room temperature for 1 h. For each flocculant, first step was to find the pH at which the flocculant performed the best. In this trial, the pH of SBW was adjusted to 4, 5 and 7 before addition of flocculant at room temperature. Flocculant concentration was set at 0.5, 0.45 and 0.2 g/L, for alginate, carrageenan and CMC, respectively, which was determined based on PCD measurements (see Section 2.2.5). To evaluate the efficiency of the flocculation treatment, relative sediment height as well as turbidity and residual protein in the upper phase (“supernatant”) were analyzed (see below). In a second set of experiments, flocculation efficacy at different concentrations of flocculant were analyzed at the pH performing the best. The concentration ranges 0.05–5, 0.04–4.5, and 0.02–2 g/L of alginate, carrageenan and CMC, respectively, were selected. In these trials, viscosity of the whole suspension and upper phase as well as particle size of the whole suspension were analyzed, in addition to turbidity and protein measurements of upper phase.

Scale-up flocculation experiment was performed on 5L of SBW using carrageenan at 0.45 g/L prior to SBW acidified to pH 4. Flocculation tests were done in duplicates. The upper-phase was discarded while the sedimented flocs were recovered by centrifugation at 3000×g, 15 min. Protein, fat, ash, and moisture content were measured in SBW and recovered biomass. Protein content was evaluated following method described in Section 2.2.2 and fat content measured according to gravimetric method by Lee et al. (1995) described in Section 2.2.1. The moisture content was determined based on the gravimetric method. The pre-weighed samples were dried in a 110 °C oven (Electrolux, Stockholm, Sweden) until a constant weight was obtained. Moisture content was calculated using the following formula:

Moisture content (%) =

wet weight (g) − dried weight (g) × 100 wet weight (g)

Ash content was measured using pre-weighed sample heated in a muffle furnace (Paragon Industries, L.P., Texas, USA) to 550 °C for 5 h, samples were allowed to cool in the furnace to < 100 °C and placed in desiccator afterwards. The ash content was determined based on samples weight and combusted weight.

2.4. Responses used to follow the flocculation efficiency in both set of experiments

Ash content (%) = 100 × (1 −

2.4.1. Turbidity of upper phase Turbidity of the upper phase after sedimentation was measured as optical density at 400 nm on a Cary60 BIO UV–vis spectrophotometer (Varian Australia Pty Ltd., Victoria, Australia) against distilled water as blank.

sample weight (g ) − weight after combustion (g ) ) sample weight (g )

2.7. Statistical analysis Statistical differences among sample means of analyses was studied by analysis of variance (ANOVA) at p ≤ 0.05 using MINITAB release 16. The values are reported as mean values ± SD. Except for protein determinations which were carried out in triplicate, the rest of the analyses were performed in duplicate.

2.4.2. Analysis of residual protein in upper phase The protein concentration of the supernatant was analyzed as described in Section 2.2.2 after 1 h of sedimentation. The resulting protein concentration was than related to the protein concentration of the initial SBW.

3. Results and discussion 2.4.3. Relative sediment height Relative sediment height obtained after treatment with the different flocculants was analyzed after 1 and 3 h and calculated as the ratio between the height of the sediment formed at the bottom of tube and the total height of the solution.

3.1. Composition of shrimp boiling water (SBW) The shrimp boiling water (SBW) had an opaque orange-pink appearance (see Fig. 1) attributed to the presence of astaxanthin. In a preliminary study we have documented 2.8–4.1 mg astaxanthin/L in SBW. The protein content of SBW was found to be 12.5 ± 0.4 g/L (n = 3), which was in agreement with other studies where the reported protein content of shrimp boiling waters was in the range of 3.0–26.2 g/ L (Amado et al., 2016; Pérez-Santín, Calvo, López-Caballero, Montero, & Gómez-Guillén, 2013; Vandanjon, Cros, Jaouen, Quéméneur, & Bourseau, 2002). The total fatty acid content was found to be 1.36 ± 0.01 g/L (n = 2) with LC n-3 PUFA contributing to 25% of total fatty acids (0.35 ± 0.01 g/L, n = 2). Data on the lipid content of SBW

2.5. Responses to further analyze the flocculation efficacy in the second set of experiments 2.5.1. Particle size distribution analysis Variations in particle size as a function of flocculant concentration was measured using a MasterSizer MicroPlus (Malvern, UK) laser diffractometer. The flocs obtained in presence of the different flocculants (see Section 2.3) were injected in the measurement chamber using the 3

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from Northern pink shrimp (P. borealis) has not been reported earlier, however the lipid content of P. borealis muscle and its shell by-product with head were reported as 4 and 6 g/kg, respectively. LC n-3 PUFA comprised 25% of the total fatty acids in the muscle (Heu, Kim, & Shahidi, 2003). In the present study the lipids in SBW could originate from both muscle and head. The cooking juice from Penaeus spp. contained higher amount of lipids, 9.5 g/L (Pérez-Santín et al., 2013). Among the minerals analyzed, Ca2+ and Mg2+ were by far the two most predominant with concentration of 111.60 ± 3.44 and 59.93 ± 1.21 mg/L respectively, followed by Cu2+, Fe2+, Zn2+ and Ni2+ at 4.65 ± 0.07, 3.95 ± 0.10 and 1.21 ± 0.08, 0.17 ± 0.01 mg/L, respectively. Co2+ and Mn2+ could not be detected. Zn2+ and Cu2+ content found in the present study was 8-fold and 2-fold, respectively, lower compared to those reported in P. borealis muscle, 10.2 mg/L (Heu et al., 2003). Jarrault et al. (2017) also reported on high content of Ca2+ and Mg2+ in shrimp cooking juice, 0.1 and 0.154 g/L, respectively. Ca2+ and Mg2+ ions could originate from the tap water used for boiling shrimps or the shrimp peel. Other researchers reported that calcium, magnesium and sodium were the dominant ions in shrimp peel, and reported on 5.8 g of Mg2+/kg in dried shrimp peel (Rødde, Einbu, & Vårum, 2008).

Fig. 3. Overall charge of the SBW proteins as a function of pH as measured using a particle charge detector (PCD). Data are shown as mean values ± SD (n = 2).

3.2. Polypeptide profile of crude SBW and its soluble phase as a function of pH

Leakage of protein during boiling of shrimps was most likely initiated by the high temperature in the steaming process, and since the shrimps were whole at this stage, proteins were potentially released from both peel and muscle. Martinez, Jakobsen Friis, and Careche (2001) reported that water-, low-salt and high-salt-soluble proteins were extracted from abdominal muscles of P. Borealis during ice storage. At day zero, the water-soluble polypeptides gave rise to bands around 25–100 kDa when subjected to SDS-PAGE.

The polypeptide profile of the crude SBW and of the supernatants obtained after centrifugation at the different pHs is shown in Fig. 2. SBW at its native pH (8.7) contained polypeptides ranging from 10 to 90 kDa with four clear bands corresponding to molecular weights (MW) of ∼90, ∼80, ∼35, and ∼22 kDa. The band at ∼90 kDa tentatively corresponds to paramyosin and the band at ∼22 kDa to the myosin light chain (Ayuso et al., 2008; Gómez-Estaca, Montero, & GómezGuillén, 2014). The polypeptide profile of the SBW was unchanged after centrifugation at 10,000×g at the native pH, demonstrating that all detected polypeptides were soluble. The polypeptide profile of the supernatant was also unchanged when performing the centrifugation at pH 8 and 7. At pH 6 a clear change in the supernatant was however observed, and polypeptides of higher molecular weights (∼90 and ∼80 kDa) precipitated indicating that their isoelectric point (pI) was located between pH 6–7. At pH 5.5–6, the intensity of the bands at ∼35 and 22 kDa decreased, and they disappeared completely at pH 4–5. They then re-appeared at pH 3, indicating a pI is between pH 4 and 5.

3.3. Protein charge of SBW as a function of pH To more carefully measure the isoelectric point of SBW proteins, SBW was titrated as function of pH while monitoring their streaming potential using a particle charge detector (PCD). The PCD potential relates to the overall protein charges, even though its absolute value does not refer to a true potential. In practice, PCD potential can be used in a titration to determine the pI. Fig. 3 shows the evolution of the overall charge of SBW as the pH was changed from pH 8 to pH 3. The PCD potential passed zero at pH 4.7, thus confirming the above results

kDa 250 150 100 75 50 37 25 20 15 10 5 pH 3

4

4.5

5

5.5

6

7

8

EaƟǀĞ EaƟǀĞ ĞŶƚƌŝĨƵŐĞĚ

Fig. 2. Polypeptide profile of SBW before and after subjecting to pH-adjustment in the range of 3–8 and centrifugation at 10,000×g. pH of untreated “native” SBW was 8.7. ‘Native centrifuged’ is the supernatant after centrifugation of SBW at native pH at 10,000×g. Each well was loaded with 20 µg protein. 4

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(b) 16

(a) 2.4

native pH pH7 pH5 pH4 A A A A A A

14 A A

12

Protein concentration in upper phase (g/L)

Turbidityof the upper phase

2.0

native pH pH7 pH5 pH4 A A A A A A A A

1.6 1.2 0.8 0.4

B

B B

0.0

No Treatment

Carrageenan

B

B

A A A

10 8 6

B

B B

B B

4 C

2

B

Alginate

A

0

CMC

No Treatment

Carrageenan

Flocculating agent

Alginate

CMC

Flocculating agent

(c) 1.2

Relative sediment height

A

pH5 - 1h pH5 - 3h

pH4 - 1h pH4 -3h

A

A A

A

A

A

A

0.8

A

B A

B

B B

B

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0.4

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Carrageenan

Alginate

CMC

Flocculating agent Fig. 4. SBW flocculation with carrageenan (0.45 g/L), alginate (0.5 g/L) and CMC (0.2 g/L). Turbidity in upper phase (400 nm) (a), Residual protein in upper phase (g/L) (b), relative sediment height (c), Results in A and B were measured after 1 h. Bars show mean values ± SD (n = 2).

and 4, in the absence of any flocculant. This indicates that the SBW suspension remained stable and protein aggregates did not form, or were held in suspension, during the time tested (here 1 h). However, the addition of carrageenan, alginate or CMC led to a significant reduction in turbidity at pH 5 and 4, and a clear sediment was observed at these pHs. There were no important differences between the performances of the three flocculants at pH 4 and pH 5 when it came to reducing turbidity. At the native pH of the SBW or at pH 7, no change in turbidity was observed when the flocculants were added. Fig. 4B displays the residual protein measured in the upper phase after sedimentation of the flocs for 1 h, in the absence and presence of alginate, carrageenan and CMC at pH 4, 5, 7 and 8.7. Upon addition of flocculants, reduction in residual protein was only observed at pH 4 and 5, in agreement with the turbidity results. At pH 4, carrageenan was significantly more efficient to induce flocculation compared to alginate and CMC. No significant difference between the three polysaccharides was observed at pH 5 and pH 7. The results showed that lowering pH from 7 to 4 in the presence of carrageenan reduced protein concentration of the upper phase from 10.9 to 2.4 g/L, i.e. by 78%. For alginate and CMC, the corresponding reductions in concentration were 67% and 62%, respectively. A lowering in the SBW pH from 7 to only pH 5 enabled a 60% reduction in protein concentration of the upper phase, for all the polysaccharides tested. The relative sediment height at pH 4 and 5 resulting from the addition of flocculants was measured as function of time. Fig. 4C shows

regarding placement of the pI of the SBW proteins between pH 4 and 5. Fig. 3 further shows that the protein carries an overall positive charge at pH < 4.5 and an overall negative charge at pH > 5.0. Ortea, Cañas, Calo-Mata, Barros-Velázquez, and Gallardo (2010) studied pI of watersoluble proteins of Pandalus borealis (Northern shrimp) and reported values of 4.58–4.89, on an average of 6 samples. 3.4. Flocculant efficacy as function of pH The efficacy of alginate, carrageenan and CMC to act as flocculants was tested at a fixed polysaccharide concentration at pH 4, 5, 7 and 8.7 (the latter being native pH of SBW). Control sample was the SBW alone at the different pH values, without added flocculant. The polysaccharides used as flocculants are weak acids with pKa of ∼3.5 in the case of alginate (Draget, Bræk, & Smidsrød, 1994), 4.3 for CMC (AlAchi, Gupta, & Stagner, 2013) and < 2 for carrageenan (Vleugels, Ricois, Voets, & Tuinier, 2018), meaning that their negative charge is reduced when changing the pH from 7 to pH 4. We expected that the shrimp proteins would be slightly positively changed at pH 4 and negatively charged in the pH range 5–7. The flocculating efficacy of each polysaccharide is illustrated in terms of turbidity of the upper-phase (Fig. 4A), concentration of residual protein in the upper phase (Fig. 4B) and the height of the sediment (Fig. 4C). Fig. 4A shows that no significant change in turbidity was observed as pH was reduced from the native pH of the native SBW (8.7) to 7, 5 5

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concentration range of 0.04–4.5 g/L, alginate 0.05–5 g/L and CMC 0.02–2 g/L. These concentrations were based on the results obtained by the PCD titration (0.45 g/L of carrageenan, 0.5 g/L of alginate and 0.2 g/L of CMC) and were selected so that the range studied passed the charge neutrality of the proteins and polysaccharides. In general, the change in protein concentration of the upper phase, along with data on sediment height, showed that small amounts of polysaccharides (i.e. 0.45 and 0.5 g/L for carrageenan and alginate, respectively) were sufficient to reach a relatively high degree of sedimentation in the SBW. For example, a concentration of 0.45 g/L of carrageenan was enough to reduce the protein concentration of the upper phase by 77%. Ten-fold increase in carrageenan concentration improved the flocculation only with 3 per cent units based on the protein concentration of upper phase (data not shown). Concentrations of alginate, ≥2.5 g/L led to gelation of the system, probably related to the calcium and other multivalent ions present in the SBW. For this reason, we did not further study alginate at elevated concentrations. In the case of CMC, the best protein sedimentation, shown as relative sediment height and change in protein concentration of upper phase, was seen at 0.2 g/L of CMC (0.58 and 74%, respectively). Above this concentration, no further improvement of protein sedimentation was observed. The effect of CMC was thus maximized where the charge ratio of polysaccharide to protein was close to 1.

the results after 1 h and 3 h which revealed similar trends. However, as expected, the effect was more pronounced after 3 h. If using sedimentation as a method to recover proteins under industrial settings, such a long waiting time would, however, be impractical and a shorter time span is likely to be used. Within this constraint, we observed that lowering the pH to 4 led to a lower relative sediment height for carrageenan and CMC at 1 h compared to alginate and CMC. That the sediment packs more tightly indeed leads to facilitated separation from the liquid phase. At 3 h, the relative sediment height was the same at pH 4 and pH 5 for SBW containing carrageenan and CMC while for SBW with alginate, the sediment height was significantly lower at pH 4. After 3 h, all flocculated SBWs showed similar sediment height at pH 4, ∼20%. Without adding any flocculant, the reduction in relative sediment height due to pH alone was minor, i.e. from 4% to 14%. Using the data of both of Fig. 4B and C, it is possible to calculate the relative amount of protein being sedimented, and also, the protein concentrating factor of the sediments compared to the original SBW. These calculations revealed that the combination of carrageenan and pH 4 allowed the highest protein recovery (86%) from SBW using sedimentation, and the sediment was 1.3 times more concentrated than the SBW. The second and third most promising conditions were alginate at pH 4 and carrageenan at pH 5 with protein recoveries of 74 and 68%, respectively. The flocculation efficacy of a polymer is related to the charge density and length of the polymer chain as well as the nature of the functional group (Dickinson, 1998). While carrageenan has sulfate moieties, alginate and CMC are polysaccharides functionalized with carboxylic groups. Sulfate groups have a low pKa value, around 2 or below, while the pKa of the carboxyl groups is of 3.5 (McClements, 2014; Smith, 1991). Under the settings of the current study, we assumed that carrageenan was charged over the whole pH range studied (4–8.7) whereas the charge of alginate varied, having a small charge density around pH 4 that increased to be high at pH 7 and at the native pH 8.7. It was somewhat surprising that carrageenan did not provide a better flocculating capacity at pH 5 than alginate and CMC, as it has been shown to stabilize dairy protein at pH’s slightly above the pI of the dairy protein. The efficiency of carrageenan to interact with dairy proteins, even though they carry a net negative charge, has been related to its sulfate content (Dickinson, 1998; Galazka, Smith, Ledward, & Dickinson, 1999). Some studies in which carrageenan and alginic acid were used as flocculants for wine containing 0.8 g protein/L, reported on around 60% protein adsorption to the polysaccharide. Carrageenan was then used in the concentration range 0.01–0.025%, which was almost 16 fold higher compared to the concentration of alginic acid used (CabelloPasini, Victoria-Cota, Macias-Carranza, Hernandez-Garibay, & MuñizSalazar, 2005). In another study, surimi wash water treated with a complex of chitosan and polysaccharides such as alginate, pectin and carrageenan at pH 6 which exhibited 78–94% protein adsorption to the polysaccharides (Savant & Torres, 2003). However, the differences in the protein concentration of the sample flocculated in our study (12.5 g/L) and these previous studies (0.8 g/L) make it difficult to directly compare the flocculation efficacy of the polysaccharides reported. Generally, however, it is clear that the amount of polysaccharide used is dependent on both the protein concentration and protein charge, the latter being dependent on the pH used thus large impact from pH on flocculation efficiency of the three polysaccharides.

3.5.2. Effect of polymer concentration on particle size distribution Floc size plays an important role in the ability of flocs to be sedimented. This is of practical relevance for large scale applications as it facilitates the dewatering of aggregates. Fig. 5 displays the changes in floc/particle size distribution for different concentrations of carrageenan. Native SBW had an initial bimodal particle size distribution, as a result of the presence of natural aggregates and flocs. The addition of carrageenan strongly affected this distribution, by reducing the number of particles within the SBW, leading to monomodal distribution. Flocculation of SBW with different concentrations of carrageenan changed the size distribution to bigger floc sizes, and at both 2.25 and 4.5 g/L, 100% of the flocs were in the range of 40–190 µm (Fig. 5). In the low concentration range, an increase in carrageenan concentration from 0.04 to 0.09 g/L resulted in almost identical percentage of floc with sizes over 40 µm, reaching 59% for both concentrations. However, flocculation of SBW with 0.45 g/L of carrageenan exhibited 80% of size distribution over 40 µm. Flocculation of SBW with alginate ranging from 0.05 to 0.5 g/L resulted in that 56–63% of the flocs size were over

3.5. Investigation of the effect of polysaccharide concentration on flocculation efficacy 3.5.1. Effect of polysaccharide concentration on protein in upper phase and relative sediment height The effect of polysaccharide concentration on flocculation efficiency was tested at a fixed pH of 4 as this was the pH when the polysaccharides performed the best. Carrageenan was tested in the

Fig. 5. Particle size distributions as affected by carrageenan concentration at pH of 4. 6

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followed by centrifugation and freeze drying (Wibowo et al., 2005). This ingredient gave the same protein efficiency ratio as casein in a subsequent rat feeding trial where it was included at 10 and 15% in the feed.

40 µm for all tested concentrations (data not shown). In agreement with alginate, the flocculation of SBW with CMC in the concentration range of 0.02–2 g/L did not yield any major differences in the floc size distributions. Flocs over 40 µm fluctuated around 56–57% for all CMC concentrations (data not shown), showing that higher concentrations of CMC did not lead to formation of larger flocs. The differences in functional groups, charge density and/or molecular weight could be the reason for different floc size behavior among the three flocculants. Based on the increase in floc size observed over the tested range of carrageenan concentrations, it is assumed that the sulfate side groups of carrageenan interact more efficiently with proteins than the carboxyl groups of CMC and alginate.

3.7. Industrial perspective To recover a protein enriched biomass from highly diluted shrimp process waters for food or feed purposes, upstream flocculation treatments using food grade polysaccharides appears crucial as a pretreatment prior to the application of a final dewatering technique like e.g. filtration, flotation or sedimentation/centrifugation to recover the flocculated biomass. Under industrial settings, the seasonal or batch to batch variations of shrimp proximate composition as well as the freshness of the shrimps should however be considered as these parameters will affect nutrient leakage into SBW; and thereby the efficiency of the downstream biomass recovery process. Considering that residuals of the natural polysaccharides will not distress the environment when the outlet water is discharged, this process is more environmentally friendly than process using synthetic hazardous coagulant-flocculants for water remediation. However, to make food grade flocculation implementable on an industrial scale, it must indeed be profitable to the shrimp peeling companies, which will depend on factors such as the cost of the polysaccharides, value of the recovered shrimp biomass as a food or feed ingredient, and strictness level of regional-environment regulations regarding water discharge costs. As this is a new type of biomass, market values are difficult to estimate; but comparisons could be made with conventional surimi (1.5–3 Euro/kg wet weight), or protein hydrolysates (10–25 Euro/kg dry weight). To estimate a potential profit, the cost of the recovery process, the shrimp boiling water and the flocculant must indeed be considered. Production costs are too early to estimate properly since this work was done batch-wise in lab scale. The cost of the boiling water is currently zero, or even negative, due to payments for waste water handling in many countries. Regarding carrageenan, alginate and CMC, market prices varies from 10 to 100 Euro/kg, and are thus on the high end for a food or feed protein ingredient. Based on the present proof-of-concept, future studies should thus investigate polysaccharides that are cheaper than alginate, carrageenan and CMC, and also evaluate the technofunctional and sensorial properties of the recovered shrimp biomass since such properties govern the application possibilities in different types of food or feed products. Overall, we foresee that this type of new protein-rich marine biomass with a pronounced shellfish flavor and appealing red colour could become a suitable ingredient e.g. soups, sauces, burgers, nutrient drinks, spreads or pates, while on the feed side, it would be promising as an aquafeed ingredient providing nutrients as well as potential appetizer and pigment enhancing properties.

3.5.3. Effect of polymer concentration on the viscosity and average particle size (D(3,2)) of the flocculated systems Both the addition of polysaccharides to SBW and the increase in size of the flocs will influence the viscosity of the system. The viscosity was therefore determined both on the SBW suspension as a whole, and on the supernatants obtained after 1 h sedimentation. Shear rate sweeps were performed and showed that all samples exhibited shear thinning. The viscosity of the different samples was compared at a shear rate of 80 s−1, and as above, at a pH of 4. Noticeable changes in viscosity of the SBW suspension or of the upper phases were measured only when the flocculants were carrageenan or alginate. Conversely, the addition of CMC did not induce any increase in viscosity of the full suspension nor of the supernatant even at a concentration as high as 2 g/L CMC (data not shown). The D[3,2] values and viscosity of carrageenan and alginate were plotted as a function of polysaccharide concentration (Fig. 6). With the addition of carrageenan from 0.45 to 2.25 g/L, the viscosity of the full suspension increased more than two-fold (from 11.2 to 26.7 mPa s). Meanwhile the average particle size, D[3,2], also increased by a factor two. When using the highest concentration of carrageenan, 4.5 g/L, we observed an increase in viscosity of the upper phase indicating that at such concentration and above, the free polysaccharide, i.e. not its complex with protein, contributes to the upper phase viscosity. Therefore, at lower concentrations of carrageenan, we hypothesize that the increase in viscosity of the SBW was primarily related to the increase in particle size, as carrageenan clearly is flocculating the proteins (Fig. 6A). The trends were less clear in the case of alginate, which could be related to the formation of a gel at concentrations > 1 g/L which largely increased both viscosity and particle size (Fig. 6B). However, a small increase in particle size was observed as alginate was added also at lower concentrations. 3.6. Protein recovery using centrifugation under the best conditions for flocculation To better estimate the efficiency of the most promising flocculation system to recover proteins from SBW, a larger quantity of SBW (5L) was treated with carrageenan (0.45 g/L, pH 4) followed by centrifugation at a scalable g-force (3000g). Data revealed that 78.5% protein could be recovered as sediment (Supplementary data – Table 1), which is similar to the protein recovery reported when applying chitosan complexes and centrifugation to surimi wash water (81–90%) (Savant & Torres, 2003). The protein content of the SBW biomass reached 10.2% on a wet weight basis, and thus, was increased by a factor of 10 compared to the crude SBW. The dry matter increased from 2% in the SBW to 13.6%, and total lipids were concentrated by a factor of ∼10 in the biomass, reaching 2,9%. The ash content went from 0.2 to 0.3%. If considering that the biomass would be dried; protein and lipid content would be 75.0 and 21.3%, respectively, which would make the product highly interesting as a feed or food protein ingredient, especially since the lipids are rich in LC n-3 PUFA. Previous work on recovering protein from surimi wash water yielded an ingredient with 61.4 and 73.1%, protein (dw basis) using chitosan or chitosan-alginate flocculated water, respectively,

4. Conclusion Carrageenan, alginate and CMC were evaluated as flocculants to recover a protein-enriched biomass from SBW. It was found that pH 4 was optimal to flocculate proteins with both carrageenan and alginate, while CMC gave equal results at pH 4 and 5. Carrageenan at 0.45 g/L was the most efficient flocculant in terms of protein sedimentation (≤86% of total SBW proteins), which also correlated with a build-up of large flocs and increased viscosity. When the results with 0.45 g/L carrageenan at pH 4 were confirmed in 5L-scale, 78.5% protein could be recovered using low g-force centrifugation, and a biomass with 75% protein on a dry weight basis was obtained. Future studies on charge density and molecular weight of the three polysaccharides used, in relation to flocculation is necessary to further describe their behavior in SBW.

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Fig. 6. Viscosity and average particle size (D[3,2]) of SBW at pH 4 as affected by flocculation using carrageenan and alginate at different concentrations. Carrageenan flocculation (A); Alginate flocculation (B).

Nordic Innovation (Project Mar 14322) for the financial support. We also wish to thank Räkor & Laxgrossisten AB for the process water provided for the analyses.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data Acknowledgement

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125299.

This study was part of the NoVAqua project and we wish to thank 8

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