ZEEVIVO: Seaweed protein in fish feed Extraction of protein for fish feed

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1 ZEEVIVO: Seaweed protein in fish feed Extraction of protein for fish feed Tom Wijers Researcher at University of Applied Sciences Van Hall Larenstein

2 Extraction of protein for fish feed The ZEEVIVO project Replacement of fishmeal/soy protein Seaweed protein extraction and purification Effect of species and preservation on extraction embrane filtration as purification step The use of seaweed protein as fish feed ingredients Page 2

3 ZEEVIVO: Seaweed protein in fish feed Seaweed cultivation Biorefinery Tsjippie Visser Optimize growth, protein content and amino acid composition Extract high quality protein to replace fish meal Fish production Culture healthy fish with as much seaweed protein as possible Page 3

4 Replacement of fishmeal with seaweed protein & + >60% protein Rainbow Trout (Oncorhynchus mykiss) ± 45% protein Page 4

5 Replacement of soy with seaweed protein & + >30% protein Nile tilapia (Oreochromis niloticus) ± 35% protein Page 5

6 Seaweed does not contain 35% protein Ascophyllum nodosum Saccharina latissima Fucus serratus Ulva lactuca 13.4% protein 8.4% protein 15.3% protein 9.6% protein Chondrus crispus 17.9% protein Protein measurements: Kjeldahl nitrogen x 5.0 according to Angell 2016 Page 6

7 Seaweed protein extraction Biomass water ratio 1:30, turrax mixing for 5 minutes at rpm, 1 hour extraction, shaking at 140 rpm Extraction Centrifuge Pellet Supernatant Supernatant Pellet Extract Seaweed Preservation technique (5 techniques) Homogenize Fresh seaweed (5 species) Page 7

8 Effect of preservation and species Preservation and species: Determine extraction efficiency (protein % in supernatant of total) Compare literature studies Process bulk supply of seaweed Preservation and extraction Fresh, demi-water at room temperature Fresh, 0.1 NaOH at 50 C Frozen, 0.1 NaOH at 50 C Freeze dried, 0.1 NaOH at 50 C Air dried at 70 C, 0.1 NaOH at 50 C Air dried at 40 C, 0.1 NaOH at 50 C Species Ascophyllum nodosum Saccharina latissima Fucus serratus Ulva lactuca Chondrus crispus Pagina 8

9 Extraction efficiency Preservation and species affects protein extractability Supernatant 80% 70% 60% 50% Increasing Species Up to 70% andph of preservation the andprotein temperature can technique beassists extracted have protein a big extraction effect Preservation and extraction method Fresh, demi-water at room temperature Fresh, 0.1 NaOH at 50 C Frozen, 0.1 NaOH at 50 C Freeze dried, 0.1 NaOH at 50 C Air dried at 70 C, 0.1 NaOH at 50 C Air dried at 40 C, 0.1 NaOH at 50 C 40% 30% 20% 10% 0% C. crispus F. serratus A. nodosum S. latissima U. lactuca Page 9

10 Purity (Protein % in supernatant) Purity of protein in supernatant Supernatant 25% 20% 15% Purification step is needed to get to >30% protein purity 17.9% 13.4% 15.3% Preservation and extraction method Fresh, demi-water at room temperature Fresh, 0.1 NaOH at 50 C Frozen, 0.1 NaOH at 50 C Freeze dried, 0.1 NaOH at 50 C Air dried at 70 C, 0.1 NaOH at 50 C Air dried at 40 C, 0.1 NaOH at 50 C 10% 8.4% 9.6%% 5% 0% C. crispus F. serratus A. nodosum S. latissima U. lactuca Page 10

11 Purity of pellet (Protein % in D) Purity in pellet increased in some cases Pellet Extraction efficiency of U. lactuca remains low ( %) ost of the proteins remain in the pellet 20% 18% 16% 14% 12% 16.2% Preservation and extraction method Fresh, demi-water at room temperature Fresh, 0.1 NaOH at 50 C Frozen, 0.1 NaOH at 50 C Freeze dried, 0.1 NaOH at 50 C Air dried at 70 C, 0.1 NaOH at 50 C Air dried at 40 C, 0.1 NaOH at 50 C Purity in some pellets increased almost 2 fold 10% 8% 6% 9.6% Still not the >30% protein needed Purify the supernatant 4% 2% 0% U. lactuca Page 11

12 Supernatant purification with membrane filtration U. lacuca: kg dried - Protein: 11.6% kg protein % extracted Supernatant C C C C P P C P C P P P C C P C C P C P C Supernatant: - Purity of 9.3% kg (14.3%) P Protein C Carbohydrates inerals UF membrane Concentrate C P P P C P P P CP P P C Concentrate: - Purity of 31.2% kg (7.0%) C C C C P C C P C C C Permeate: - Purity of 7.1% kg (7.3%) From 11.6% to 31.2% purity! Increased protein purity 3 fold! But Not efficient, a lot of loss Permeate Page 12

13 Seaweed protein concentrate for fish trials Extract as much non-protein substances as possible. Use the pellet with increased protein purity. Possible if you start with a high protein seaweed (>20%). Extract as much protein as possible and purify the supernatant. Works with membrane filtration but with low efficiency. Precipitation of proteins might be a possible solution. Red and green seaweed concentrates provided by external company. Page 13

14 The use of seaweed protein as fish feed ingredients Performance trial 25% of soy protein replaced Ad libitum feeding ix of Red, Green or Red and Green Tanks with 35 fish (n=3) Results: No difference in growth performance Higher feed conversion ratio in Green seaweed protein and reference diet compared to Red seaweed protein Preliminary data on courtesy of Jeroen Kals, Fedor den Elzen and Luca Hertfelder Nordic Seaweed Conference 2018 Page 14

15 The use of seaweed protein as fish feed ingredients Digestibility trial 20% of reference diet replaced Restricted feeding ix of Red, Green or Red and Green Tanks with 35 fish (n=3) Results: Next step: Dose response tests with Green seaweed protein concentrate. How much protein can we replace? Protein digestibilty lower in al seaweed protein diets compared to reference diet, but small differences Higher body weight gain and specific growth rate in Green seaweed protein and reference diet Preliminary data on courtesy of Jeroen Kals, Fedor den Elzen and Luca Hertfelder Nordic Seaweed Conference 2018 Page 15

16 To sum everything up Biorefinery of seaweed is necessary for the use of feed. Protein extractability is highly depended on species and preservation techniques. Encouraging results feeding trials: Green seaweed protein concentrate shows positive results. Seaweed is an interesting source of high quality protein for the use of fish feed. Page 16

17 Thank you for your attention! Project partners: I would like to thank Klaas Timmermans, Alwin Hylkema, Tsjippie Visser, Dirk Jan Vos, Tristan Speerstra, Jeroen Spruit, Ilona Bruggen, Jeroen Kals, Fedor den Elzen and Luca Hertfelder Contact: Tom.Wijers@hvh.nl The ZEEVIVO project is partially funded by SIA, part of the Netherlands Organization for Scientific Research (NWO)

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19 Purity of pellet (Protein % in DW) Purity of protein in pellet Pellet 25% 20% 15% 17.9% 15.3% Preservation and extraction method Fresh, demi-water at room temperature Fresh, 0.1 NaOH at 50 C Frozen, 0.1 NaOH at 50 C Freeze dried, 0.1 NaOH at 50 C Air dried at 70 C, 0.1 NaOH at 50 C Air dried at 40 C, 0.1 NaOH at 50 C 13.4% 9.6%% 10% 8.4% 5% 0% C. crispus F. serratus A. nodosum S. latissima U. lactuca Page 19

20 Extraction and purification Extraction Dissolve as much protein from the seaweed in water as possible Pellet Purification (Ultrafiltration membrane) Separates on size and charge to concentrate the protein inerals and sugars Extraction Centrifuge Supernatant UF Permeate Seaweed 8-18% protein Protein concentrate >35% protein Page 20

21 Protein % (of total) Protein % (of total) Effect of ph and temperature Supernatant 60% U. lactuca S. latissima 60% U. lactuca S. latissima 50% 50% 40% 40% 30% 30% 20% 20% 10%? 10% 0% ph 0% 20 C 30 C 40 C 50 C Temperature Page 21