Novel desalting method for protein recovery from fat rendering waste water. Functional properties of recovered proteins. Carlos Álvarez1, Liana Drummond1, Anne M. Mullen1* 1 Department of Food Chemistry & Technology, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland.

Contents Background of research approach Approach Results and discussion Conclusions http://www.revalueprotein.com

Background of ReValue Protein project (Mullen, Álvarez et al. 2015) Up to 263 kg/head of bovine or 35 kg/head of porcine of non-meat material is generated Many of this products currently have lower, neutral or even negative added-value High environmental impact

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Background of the project Several by-products and waste streams have been identified as potential sources of recoverable proteins within the project. Each one presents different properties, hence different approach has to be carried out Internal organs: ISP, hydrolysis, EPF Blood: ethanol precipitation, hydrolysis Brine solutions: ultrafiltration, electro-dialysis Exudates from meat curing: ultrafiltration, precipitation, electro-dialysis Cleaning water: centrifugation, decantation Process water from fat rendering, known as glue water: ultrafiltration, electro dialysis or novel gel desalting process.

Selected waste: Characterisation Glue water is the waste obtained after fat rendering by thermal treatment. Such glue water is currently considered a waste; and no characterisation based on its protein content, techno-functional applications or amino acid profile has been carried out. Proximate composition raw material: Moisture: 97.40±0.24% Total solids: 2.46±0.08% Protein content: 1.15±0.14% Ashes: 1.08±0.17% Fat :<0.01% pH: 7.06±0.15% SDS-PAGE profile: proteins from 150 to less than 5 kDa. Amino acid profile: High content in glycine and proline: protein come mainly from collagen, however other source of proteins are present. Free amino acids: 23.34±3.2% Proximate composition of freeze dried powder Moisture: 10.61±1.67% Protein: 37.66±3.67 Ashes: 48.88±5.58

Functional properties of freeze dried glue water Water holding capacity (g H2O/ g protein) Oil holding capacity (g oil/ g protein) Least gel concentration (% protein) Average value 3 batches 13.24±1.00 4.71±0.52 2.25±0.20

Gel is stable submerged in water and salt is released Gelling properties of freeze dried glue water Freeze dried protein from “glue water” is able to generate a thermo-reversible gel, which does not require a heating step to be formed. To reach the least gel concentration from raw glue water several techniques of concentration can be used: rotatory evaporation or hot air oven. Freeze dried glue water: 48% ashes 37% protein Solution 10% w/V: 4.8% ashes 3.7% protein At room temperature produces a hydrogel Gel is stable submerged in water and salt is released

Objectives Desalting to obtain a higher purity in recovered proteins b) Explore a novel methodology of salt removal based on the gelling properties

Material and methods Desalting by gel submersion. 15 gr of gel from 8 to 16% solid content were prepared. Gels were submerged in miliQ water. Volume ratio of 1:10. Water changes performed 3 times when conductivity value of water reached a plateau. Protein loss and ash content was evaluated. SDS-PAGE profile Amino acids profile Diafiltrafiltration at 1, 3 and 10 kDa MWCO. -10 ml of reconstituted freeze dried samples (2% total solids) were UF by means of centricons. -Centrifugation for 3 hours at 5000 rpm. Sample volume was restored after adding miliQ water. Repeated thee times -Protein loss and ash content was evaluated in both permeates and retentate -SDS-PAGE profile -Amino acid profile Rheological properties: -Ramp temperatures 5-45-5 °C -Plate to plate, 1 mm gap -G’ and G’’ recorded (gel strength) -Melting and gelling temperature determined .

Contents Background of research approach Approach Results and discussion Conclusions http://www.revalueprotein.com

Protein desalting by diafiltration Depending on the MWCO employed, the amount of protein recovery ranged from 60 to 80%. SDS-PAGE profiles showed that employing 10kDa MWCO membranes, proteins in the range of 6 kDa permeated. Amino acid profile suggested that main losses were due to permeation of free amino acids and short peptides. Regardless the MWCO employed a salt reduction higher than 90% was achieved. Retentate Permeate 10 kDa 3 kDa 1Kda 10 kDa 3 kDa 1Kda Protein loss after 10 kDa UF

Results from gel desalting process at different gel protein concentrations from one batch Solid content (protein content) Total solids after desalting (protein content) Protein recovery (%) Protein purity % of non protein solids removed 8% (2.13%) 2.91±0.07 (1.96±0.09) 90.32±2.3a 67.49±1.3a 81.82±0.29a 10% (3.55%) 3.06±0.10 (2.28±0.08) 86.56±0.31a 65.78±2.12a 82.48±2.9a 12% (4.27%) 3.76±0.54 2.55±0.35 85.94±0.11a 67.88±0.44a 84.49±2.43a 14% (4.98%) 4.08±0.27 (2.45±0.12) 79.12±1.07b 60.35±0.80b 82.19±1.54a 16% (5.69%) 4.79±0.09 (2.90±0.01) 80.35±0.16b 60.62±0.87b 81.87±0.72a Before desalting After desalting We used FD samples from one single batch to generate these gels. The effect of total solids and protein concentration on protein recovery and amount of salt removed was evaluated In spite of lower amounts of salt were removed when compared to diafiltration, higher amount of protein recovered. For this reason this method deserves further investigation. Proteins 15-30 kDa size SDS-PAGE profile of glue water and desalted hydrogel. Small size proteins are lost after desalting by submersion *The weigh of the gels employed was 14.59±0.45g. After desalting the weight was 19.72±1.73

Results from gel desalting process at different gel protein concentrations Lower salt removal, but higher protein recovery. Further investigation is deserved In terms of water consumption. Protein recovery is up to 4 times higher for the same amount of water volume employed. Std before desalting after desalting Solid content (protein content) Weigh of gel before (g) Protein% after desalting Total solids after desalting Protein recovery (%) Protein purity % of ash removed 8% (2.13%) 15.00 14.85 1.88±0.01 2.05±0.03 2.84±0.13 2.98±0.01 90.32±2.3a 67.49±1.3a 81.82±0.29a 10% (3.55%) 14.27 14.79 2.36±0.03 2.20±0.03 3.16±0.14 2.96±0.45 86.56±0.31a 65.78±2.12a 82.48±2.9a 12% (4.27%) 15.16 14.78 2.20±0.02 2.90±0.02 3.22±0.07 4.30±0.24 85.94±0.11a 67.88±0.44a 84.49±2.43a 14% (4.98%) 13.62 14.42 2.33±0.01 2.58±0.01 3.81±0.05 4.35±0.01 79.12±1.07b 60.35±0.80b 82.19±1.54a 16% (5.69%) 14.14 14.87 2.89±0.03 2.91±0.04 4.88±0.07 4.70±0.12 80.35±0.16b 60.62±0.87a,b 81.87±0.72a Proteins 15-30 kDa size SDS-PAGE profile of glue water and desalted hydrogel. Small size proteins are lost after desalting by submersion

Rheological properties of raw material AS the protein content increases, gels obtained were stronger and showed higher melting and gelling temperatures Good correlation between rheological parameters and protein concentration. %solids % protein G‘ (Pa) G'‘ (Pa) Tg (⁰C) Tm (⁰C) 6 2,133 7 0,55 5 25,03 8 2,844 48 1,44 10,27 26,49 10 3,555 120 3,18 13,58 28,38 12 4,266 190 5,04 16,66 30,24 14 4,977 282 8,46 16,9 29,53 16 5,688 626 18,91 24,7 30,93 Since gelling properties were the most promising for the proteins found in the glue water, a rheological characterisation was carried out

Rheological properties of desalted proteins After desalting, it was found that the gels formed are remarkably stronger (higher G´ and G´´). Besides, Tg and Tm were found to be slightly higher as well. It can be explained because the salt remove impeded to create intermolecular interactions between the proteins. Good correlation between protein content and rheological parameters. Within the range of protein concentrations studied, protein concentration has the potential to be used as predictor of rheological behaviour. % Protein G´ G´´ Tg Tm 1,88 4,53 0,92 5,00 14,17 2,05 2,67 0,68 15,12 2,20 13,64 1,97 6,96 19,14 2,33 26,33 2,21 6,21 24,53 2,36 48,53 2,77 10,03 23,64 2,57 54,73 2,95 8,14 24,83 2,89 135,62 5,79 14,29 27,43 2,90 154,25 6,51 15,47 28,85 2,91 142,10 5,66 15,71 27,19

Potential application as food coating A protein solution (8%) was placed in a water bath at 25 ºC after being melted at 45 ºC. Subsequently, a refrigerated and cylindrical piece of meat was submerged in this solution. Immediately a coat was formed. A control and the sample were stored fro 5 days in a fridge and then the coat was peeled.

Contents Background of research approach Approach Results and discussion Conclusions http://www.revalueprotein.com

Conclusions A waste stream from meat industry has been selected as a potential source of good functional proteins Gel desalting performed better than diafiltration for protein recovery, and there is still room to improve the amount salt removed Gels made with desalted proteins were stronger, and showed higher gelling and melting temperatures.

Acknowledgments “This work forms part of the ReValueProtein Research Project (Grant Award No. 11/F/043) supported by the Department of Agriculture, Food and the Marine (DAFM) under the National Development Plan 2007–2013 funded by the Irish Government.” Anne Maria Mullen Liana Drummond Principal Investigator Project Manager Anne.mullen@teagasc.ie Liana.drummond@teagasc.ie

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