Ultrasound assisted extraction of proteins from fish processing by-products.
Carlos Álvarez and Brijesh K. Tiwari
Department of Food Biosciences, Teagasc Food Research Centre, Dublin 15, Ireland.
The growing fishery industry needs to find new green-processes in order to find out a solution to the huge amount of wastes and by-products that such industrial activity produces. Currently, around a
40% of the total weight of the mackerel is considered a by-product, because just the fillets are used in the food market. Isoelectrical precipitation and solubilization (ISP) method has been revealed as a
useful tool for protein recovering, however the yield of this process is far away for the results obtained with enzymatic methods. In present work the use of sequential acid/alkaline extraction and ISP
extraction assisted by ultrasounds have been assayed in order to increase the yield of protein recovery. It has been proved how (i) sequential ISP extraction is able to recover practically 100% of total
protein, and (ii) alkaline extraction assisted by ultrasounds is possible to recover more than 95% of total protein from mackerel by-products. Extracted proteins were characterized according to their size,
and the amino acid profile of final product was determined as well.
Figure 1: Flow chart of sequential extraction assisted by ultrasounds
Figure 2: Schematic diagram of ultrasound assisted extraction assembly with probe system of ultrasound equipment,
illustrating mechanism of bubble cavitation (A – Ultrasound generator, B – transducer, C – ultrasound cylinder probe, D –
beaker with sample and solvent of extraction, E – bubble cavitation phenomena, F – thermocouple, G – data recorder)
Material and methods
Whole mackerel By-products (40%) Fillets (60%)
% of initial protein Mass balance (%)
Not recovered protein Recovered protein
HCl 0.1 44.84±0.39a 49.48±0.84a 94.32
HCl 0.2 43.99±0.38a 48.08±0.04a 92.07
HCl 0.3 57.11±0.67b 42.20±0.17b 99.31
HCl 0.4 77.63±0.51c 19.30±0.26c 96.93
NaOH 0.1 33.61±5.85d 64.05±0.09d 97.66
NaOH 0.2 33.8±0.15d 65.45±0.06e 99.25
NaOH 0.3 33.08±3.87d 63.06±0.08d 96.14
NaOH 0.4 23.2±0.16e 74.25±0.16f 97.45
Table 1: percentage of recovered protein, and not recovered protein (pellet) after ISP extraction
using different concentrations of NaOH or HCl. Different small superscripts (a to f) denote significant
Acid-Alkaline extraction Alkaline-Acid extraction
HCl 0.1 M NaOH 0.1 M NaOH 0.1 M HCl 0.1 M
49.48±0.84a 51.23±1.51a 100.6%a 64.05±0.09d 19.27±1.19b 83.3%c
US 20% 10 min
60.31±0.66b 41.27±8.18a 101.5%a 87.59±3.3e 4.86±0.80d 92.5%b
US 60% 10 min
74.66±5.25c 19.00±3.49b 93.6%b 94.71±0.82f 2.62±2.30d 97.3 %a
US Bath 1h 69.34±2.82d 36.21±0.82c 105.5%a 78.95±0.88c n.d. n.d.
Table 2: Percentage of protein recovered using ISP extraction process assisted by ultrasounds a t two different
amplitudes by means of a submerged probe and using an ultrasound bath. Different small superscripts (a to d) denote
significant differences (p<0.05).
Free amino acids and total amino acids, % of total
Total NNDSRa Leu et Als,
1981 NEAA Free
Total NNDSRa Leu et Als,
Asp 1.50 9.28 10.72 11.06 Ile 1.75 5.05 4.83 5.15
Tyr 2.37 0.89 3.53 3.28 Leu 2.76 9.39 8.51 8.81
Ser 1.93 3.47 4.27 4.22 Met 0.50 3.42 3.10 2.53
Glu 9.71 15.02 15.63 14.81 Lys 7.35 9.42 9.61 7.40
Gly 5.93 4.33 5.03 5.62 Thre 1.88 4.21 4.59 5.34
Ala 10.84 6.12 6.34 7.22 Phe 2.78 4.40 4.09 3.94
Cys 2.91 0.43 1.36 - Val 4.69 5.78 5.40 7.31
Arg 2.60 6.24 6.26 7.12 His 42.18 6.23 3.08 4.22
Pro - 3.01 3.70 1.41
37.79 45.78 53.14 53.33 Total 63.89 47.9 43.21 44.7
Table 3: proportion of amino acids found in protein extracted after sequential alkaline-acid. a: National nutrient
database for standard reference , USA.
Figure 3: SEC profile of extracted proteins: (a) HCl 0.1M (stirring); (b) NaOH 0.1 M
(stirring); (c) HCl 0.1 M (ultrasound assisted, 20% amplitude); (d) NaOH 0,1 M
(ultrasound assisted, 20% amplitude); (e) second supernatant after sequential acid-
alkali extractions (ultrasound assisted, 20% of amplitude) and (f) second
supernatant after alkali-acid (ultrasound assisted, 20% of amplitude).
Results and Discussions
It is possible to increase the recovery 97 % of proteins using 60% of amplitude for ten minutes in 0.1M NaOH buffer. Alkaline extraction is able to solubilize
a broader range of proteins, including a non-identified 13 kDa protein which can explain the differences in yield between acid and alkaline extractions.
From a nutritional point of view, the proteins recovered by the methods explored in this work, are suitable for food purposes due to its high content in
essential and non-essential amino acids.
That implies that sequential alkaline/acid extraction and ISP-US assisted extraction is a promising tool in order to develop greener technologies for using
fish by-product s, since the amount of water and reagents can be decreased; however further research work in order to optimize the process has to be
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This work has also been supported by the Marine Functional Foods Research Initiative (NutraMara project) which is a programme for marine based
functional food development. This project (Grant-Aid Agreement No. MFFRI/07/01) is carried out under the Sea Change Strategy with the support of
the Marine Institute and the Department of Agriculture, Food and the Marine, funded under the National Development Plan 2007–2013.