Waste and Biomass Valorization

https://doi.org/10.1007/s12649-020-01029-x

ORIGINAL PAPER

Enzymatic Hydrolysis of Fish Waste as an Alternative to Produce High

Value‑Added Products
J. Araujo1 · P. Sica2,3 · C. Costa1 · M. C. Márquez1

Received: 24 October 2019 / Accepted: 16 March 2020

© Springer Nature B.V. 2020

Abstract
Purpose  Fish waste was studied as a raw material for the simultaneous production of protein hydrolysates, collagen and fish
oil. Enzymatic hydrolysis was selected for recovering these by-products with high value-added.
Methods  Alcalase 2.4 L was used to hydrolyze fish waste in a batch reactor under controlled conditions (180 min, 50 °C
and pH 8). The influence of hydrolysis degree on by-products recovery was analyzed for different enzyme and substrate
concentrations.
Results  Results suggested that the enzyme/substrate ratio was the main factor controlling the hydrolysis rate. Linear rela-
tionships were found between the degree of hydrolysis and the amount of each of the obtained by-products. From these
relationships, the amounts of by-products with high added value can be predicted by only knowing the degree of hydrolysis
reached. In optimal conditions (DH = 25%), 430 g of protein hydrolysate, 10 g of collagen and 350 g of oil could be obtained
from 1000 g of fish waste. The use of fish waste as raw material for by-product fabrication resulted in a 79% reduction of
waste disposed to landfill.
Conclusion  Therefore, this study shows the enzymatic hydrolysis of fish waste as a feasible solution to obtain high value-
added products and an alternative to landfilling disposal.
Graphic Abstract

Keywords  Enzymatic hydrolysis · Alcalase · Fish protein hydrolysate · Fish collagen · Fish oil

Statement of Novelty

Global fish consumption has more than doubled in the past

* M. C. Márquez
[email protected] 50 years. As a consequence, a significant amount of fish
waste (rich in nutrients) is discarded each year and disposed
1
Department of Chemical Engineering, Faculty of Chemical of in the landfill and in the ocean. Against this background,
Sciences, Universidad de Salamanca, Plaza de los Caídos
this study develops an innovative treatment for the trans-
1‑5, 37008 Salamanca, Spain
2
formation of fish waste in new products with higher added
Department of Crop & Soil Sciences, 4111 Miller Plant
value and a large demand: protein hydrolysates, collagen and
Sciences Building, University of Georgia, Carlton St, Athens,
GA 30602, USA oil. The proposed treatment is an original recycling method
3 using enzymatic hydrolysis that allows the simultaneous pro-
Dipartimento di Agronomia, Animali, Alimenti, Risorse
Naturali e Ambiente, Università degli Studi di Padova, Vialle duction of the desired products.
Dell’Università, 16, 35020 Legnaro, PD, Italy

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 Waste and Biomass Valorization

Introduction hydrolysis could be the high price of some enzymes. For

these reasons, the enzymatic hydrolysis is an alternative
In the last decades, world fish consumption has been for the recovery of proteins and generating products with
increasing every year. In the 1960s, the per capita con- the potential to be applied in the previously cited areas.
sumption worldwide was about 9.0  kg. This amount The wide variety of applications of hydrolyzed protein
nearly doubled by the year 2013, when it reached more from fish occurs mainly due to their excellent balance of
than 20 kg per person [1]. The European Union also fol- amino acids, good digestibility, and its rapid uptake, as
lows this trend. It is estimated that from 1998 to 2030 well as good functional properties such as high solubility
the per capita consumption will increase from 22 to 24 kg in water [15, 16].
among its members; it is worth mentioning the countries Collagen is one of the main proteins present in fish waste.
of the Iberian Peninsula—Spain and Portugal—that are It is an insoluble fibrous protein, and its hydrolysis is more
expected to consume 39 and 57 kg of fish per person in difficult than for globular proteins existing in these wastes.
2030, respectively [2]. The low collagen hydrolysis is advantageous because it
The increasing of global fish consumption can be allows the recovery of the collagen for commercial uses:
explained by many factors such as an increasing popula- biomedical applications, cosmetics manufacturers, etc.[17,
tion, rising incomes and urbanization, plus the increas- 18]. Currently, most of the collagen is extracted from bovine
ing number of fishing industries and new and more mod- and porcine bones and skins; however, it is not acceptable by
ern ways of distribution of the industrialized frozen fish Hinduism, Islam, and Judaism because of religious restric-
around the world [1]. About 70% of the fish consumed tions [19]. In addition, collagen derived from prion-contam-
worldwide is processed in the industry before being sold. inated bovine supplies might be a potential source of expo-
After processed, from 20 to 80% of the weight of the fresh sure to bovine spongiform encephalopathy [20]. Therefore,
fish is sent to the market [3]; this value varies according fish waste is an interesting source for collagen production in
to the level of processing and the kind of fish [4]. In the the substitution of mammalian sources.
processing for white fish fillet production, for example, As previously indicated, fish oil can also be separated
for each 1000 g of processed fish, from 490 to 640 g are by enzymatic hydrolysis [21, 22]. The demand for fish oil
discarded: 240–340 g of bones and viscera, 210–250 g of has been increasing in the recent past due to the findings
heads and the rest are skins [5]. Some of these residues of the health benefits provided by omega-3 fatty acids. It
are used as ingredients in the production of animal feed is applied in cosmetic, pharmaceutical [23] and biodiesel
[6] but, most of the fish processing waste is dumped in industries [24].
the sea and landfills [7]. However, fish waste has a pro- Despite the growing demand for these three products
tein concentration very similar to the parts of fish used (protein hydrolysates, collagen, and fish oil), to the best of
for human consumption. It may be destined for a nobler our knowledge, no studies have been carried out to use the
application since fish proteins are superior when compared fish waste as a source for the simultaneous production of
to the proteins originate from plants, and have a better them. Against this background, this study aimed to deter-
amino acids balance when compared to other sources of mine the efficiency of the simultaneous production of protein
animal protein [8]. hydrolysates and recovery of collagen and oil from the fish
Protein hydrolysates providing mainly di- and tripep- waste using proteases as catalysts for the hydrolysis. Enzyme
tides are superior to intact (whole) proteins and free amino concentration, fish waste concentration and enzyme/sub-
acids to be applied in several areas, such as nutrition [9, strate ratio were analyzed to establish a relationship between
10], biotechnology [11, 12] and cosmetics industries [13]. the degree of hydrolysis and the production of by-products
Therefore, the production of protein hydrolysates is an allowing to predict easily the amounts of products that can
option to generate more income for the fish processing be obtained under any operating conditions.
plant. The enzymatic hydrolysis has several characteris-
tics that can facilitate its adoption in the treatment of fish
waste: it is considered a quick and easily reproducible Materials and Methods
method; it can separate not only the peptide fractions but
also oils from the insoluble solids; prevents extreme physi- Materials
cal and chemical treatments; compared to chemical hydrol-
ysis, it has the advantage of avoiding generating chemical Fish waste was collected from a local fish store. The residue
waste, besides being more easily controlled, thus reducing was predominantly composed of medium-sized fish heads
unwanted reactions that destroy high-value components of (like salmon), skins, viscera, mangled muscles of fish,
the proteins [14]. However, a disadvantage of enzymatic small fishes, as well as mollusks such as squid and mus-
sels. Fish waste was transported to the laboratory, ground

13
Waste and Biomass Valorization

to size ≤ 1 mm, homogenized and frozen in small portions at 550 °C to a constant weight [26]. The crude fiber was
at – 20 °C. Prior to the hydrolysis process, a portion of the determined by H ­ 2SO4 and KOH—hydrolysis followed by
frozen waste was thawed overnight in a refrigerator at 4 °C. calcination at 475 °C to a constant weight (Fibertec_ M6
In order to select the best protease to hydrolyze the fish Foss Tecator), according to the European Community leg-
waste, the material was independently hydrolyzed in previ- islation, Commission Regulation (EC) No 152/2009 [31].
ous studies by Alcalase 2.4 L, Flavourzyme 1000 L, Neu- Carbohydrate was calculated by difference: it was deter-
trase 0.8 L, Pancreatic Trypsin 6.0 S and Protamex from mined by subtracting the sum percentage of moisture, pro-
Novozymes A/S (Bagsvaerd, Denmark), respectively. The tein, fat, ash and crude fiber [32]. The number of analyses
Alcalase gave the highest degree of hydrolysis of all hydro- for each determination was chosen to obtain the highest
lysates, which agrees with the results of other studies [25]. accuracy of the methods. The results were expressed as
Therefore, Alcalase 2.4 L was employed in this research. mean ± standard deviation.
This enzyme is an endopeptidase from Bacillus licheni-
formis with a declared activity of 2.4 AU/g and a density
of 1.17 g/ml that fulfills the specifications of purity for Enzymatic Hydrolysis
food-grade enzymes recommended by the Joint FAO/WHO
Expert Committee on Food Additives (JECFA) and the Food Hydrolysis were carried out in 0.5 l cylindrical jacketed
Chemical Codex (FCC). glass reaction vessels with magnetic stirring, temperature
The reagents used in the experiments were of analytical control and pH control, as shown in Fig. 1. A measured
or food-grade quality. amount of fish waste was added to the reaction vessels con-
taining distilled water to make a substrate concentration
Analytical Methods ranging from 5 to 45 g/l. Before hydrolysis, the solution
was preheated to the reaction temperature and adjusted the
Fish waste samples were analyzed for moisture, crude pro- pH using 2 N NaOH. The reaction was initiated by add-
tein, collagen, ether extract, crude fiber and ash. Moisture ing the enzyme (0.94–4.68 AU/1). During the process, the
was determined by drying the sample to a constant weight temperature was controlled using a thermostatic bath and
in an oven at 105 °C [26]. Crude protein was determined pH was maintained by the addition of 2 N NaOH.
by the analysis of the nitrogen content according to the The controlled hydrolysis conditions were: pH = 8,
semi-micro Kjeldhal technique (Kjeltec-System Foss temperature = 50  °C, time = 180  min and stirring
Tecator) [27]; the protein content was calculated by mul- speed = 300  rpm. Optimal pH and temperature values,
tiplying the N content by a factor of 6.25 [28]. Total col- corresponding to the maximum enzymatic activity and
lagen content was measured by hydroxyproline determi- stability of Alcalase, were recommended by the enzyme
nation according to the method of Bonnet and Kopp [29]. supplier. The hydrolysis time was fixed at 180 min because
Fat in fish waste was determined as lipid content by petro- longer times do not cause a significant increase in the
leum ether extraction, HCl–hydrolysis and re-extraction degree of hydrolysis. A stirring speed of 300 rpm was
(Soxtec_ HT-6 Foss Tecator) [30]. Ash was determined chosen in order to keep homogeneous the reaction mixture
by pre-combustion on a hot plate, followed by calcination and avoid vortices formation in the wells.

Fig. 1  Experimental set-up: (1)

jacketed reactor, (2) thermome-
ter, (3) burette, (4) pH electrode,
(5) pH-meter, (6) stirrer bar, (7)
magnetic stirrer and (8) thermo-
static bath

13
 Waste and Biomass Valorization

Hydrolysis was monitored by measuring the extent of pro- Experimental Strategy

teolytic degradation using the degree of hydrolysis (DH)
according to the pH–stat method [28]: The enzymatic hydrolysis of fish waste protein has not been
used so far to produce hydrolysates, oil, and collagen simul-
Number of peptide bonds cleaved
DH (%) = × 100 (1) taneously. However, hydrolysis of food proteins has already
Total number of peptide bonds
been addressed by different authors and hence the variables
DH can be determined by using the following equation: that influence the kinetics of the reaction are well known:
temperature, pH, the initial enzyme concentration (Eo) and
B ⋅ Nb the initial substrate concentration (So) [28]. Accordingly, in
DH (%) = × 100 (2)
Mp ⋅ 𝛼 ⋅ htot this work, pH and temperature were fixed at 8 and 50 °C,
respectively, corresponding to the maximum enzymatic
where B is the alkali consumption during the hydrolysis (ml activity and stability of Alcalase, and the influences of Eo,
or l), N is the normality of the alkali, Mp is the initial mass So and Eo/So ratio on the by-products recovery were studied.
of protein in the reactor (g or kg), α is the average degree Four sets of analyses were carried out aimed to evaluate
of dissociation of the α-NH2 groups released during the the performance of Alcalase at different initial concentra-
hydrolysis and htot is the total number of peptide bonds in tions of enzyme Eo and protein So in the reactor. In addition,
the protein substrate (8.6 meqv/g or eqv/kg for fish protein, tests were conducted to demonstrate the enzyme–substrate
[28]), α varies with pH and temperature: at pH 8.0 and 50 °C ratio (Eo/So) influence on the by-products recovery effi-
of temperature, 1/α = 1.1 [28]. ciency and the relationship between the degree of hydrolysis
After the enzymatic hydrolysis, samples were placed and the by-products recovery.
into boiling water at 95–97 °C for 20 min with the purpose
of deactivating the enzyme and pasteurizing the mixture
(78 °C and 25 min are sufficient for pasteurization of the Results
mixture [33]). Then, samples were centrifuged at 6000 rpm
for 20 min and separated in three phases: lower phase (solid Characterization of Samples
residue) containing the collagen, intermediate phase (super-
natant) containing the protein hydrolysate, and the upper The fish waste used in this study had high moisture content
phase containing the separated oil. The residue was esti- (71.3 ± 0.6% w/w), in accordance with the literature where
mated for collagen recovery, the supernatant was estimated it is proved that fishes and shellfishes show a great con-
for solubilized protein (hydrolyzed protein) and upper-layer tent of water [34]. On a dry matter basis, waste was rich in
was estimated for oil recovery. proteins and lipids. Fish waste consisted of heads, skins,
Collagen in residue and protein in the supernatant were bones, viscera, shells and small whole fishes causing it to
analyzed as indicated in the section of “Analytical Methods”. have high protein content (44.7 ± 0.6% w/w); these results
Oil was determined by measuring its volume; by multiplying agree with other studies showing that the protein content
the obtained volume by the fish oil density (0.919 g cm−3), in fish waste varies between 35.9% w/w [35] and 57.9%
the weight of recovered oil was calculated. w/w [5]. The main fishes observed in the waste were blue
All the tests were duplicated, and student’s t-test was per- fishes with high-fat content that justifies the high percent-
formed (significant level p ≤ 0.05). age of lipids in samples (35.1 ± 2.1% w/w); according to the
Products recovery was calculated as the percentage of total literature reviewed, fat content for fish waste is comprised
content in fresh fish waste according to the following equations: between 19.1% w/w [5] and 52.5% w/w [35] which agrees
[
Collagen in the residue (g)
] with the obtained result. Although protein and lipids were
Collagen recovery (%) =
Initial collagen in reactor (g)
× 100 the major components, the ash content reported was also
(3) high (19.6 ± 0.6% w/w), agreeing with literature data [35]. In
fact, it is known that fish is an important source of minerals:
Hydrolysed protein recovery (%) heads and bones are an excellent source of many essential
minerals such as calcium, phosphorus, potassium, iodine,
[ ]
Protein in the supernatant (g)
= × 100
Initial protein in reactor (g) (4) selenium, zinc, iron, etc.[36]. Nevertheless, the content of
fiber (0.0% w/w) and carbohydrates (0.6 ± 0.0% w/w) were
Oilrecovery (%) negligible as expected at such waste [34]. Results obtained
[ ] for total collagen content reported 5.9 ± 0.1% (w/w); this
Oilinupperphaseafterthecentrifugation(g)
= × 100 is coherent with the composition of fish waste since fish
Initialfatinthereactor(g) muscles have low collagen content [37] but fish skins are
(5) rich in it [38].

13
Waste and Biomass Valorization

Influence of Alcalase Concentration The reduction of the molecular size of resulting peptides and
the increase of newly exposed ionizable amino and carboxyl
The gradual increase of the initial concentration of Alcalase groups increase the hydrophilicity of the hydrolysates [40].
(Eo from 0.94 AU/1 to 4.68 AU/1)—at a constant initial
substrate concentration (So = 25 g of protein per liter)—
increased the degree of hydrolysis values, and the oil and Influence of Protein Concentration
hydrolyzed protein recovery but not the collagen recovery
(Fig. 2). For a constant 0.84 UA/l concentration of Alcalase, the
It was noted that a one-unit increase in the activity of influence of the initial concentration of substrate in the range
Alcalase implied an increase of six-units in the degree of of 5–45 g/l was studied (Fig. 3). It was found that the num-
hydrolysis (Fig. 2a). This increase in the degree of hydroly- ber of broken peptide bonds increased with the concentra-
sis corresponds to an increase in the concentration of hydro- tion of substrate (as deduced from the increase of the alkali
lyzed protein (Fig. 2c) and a decrease in the amount of colla- consumption); however, due to the higher initial number of
gen in the residue (Fig. 2d). This result is consistent because peptide bonds, the degree of hydrolysis decreased as the So
the presence of enzyme favors the hydrolysis, which gener- increased (Fig. 3a). This is due to the enzyme concentration
ates an increase in the amount of produced hydrolysate (a remained constant during the experiments. At a constant
one-unit increase in the enzyme activity means an increase Alcalase concentration and a lower concentration of the sub-
of 75% in the production of hydrolyzed). Similar results strate, the substrate concentration is the limiting factor and
were reported for the enzymatic hydrolysis of proteins from the enzyme reaction rate increases as the initial substrate
yellowfin tuna wastes using Alcalase [39]. On the other concentration increases. However, at higher concentrations
hand, collagen is more difficult to be hydrolyzed making as used in this study, the substrate often acts as a dead-end
another part remain not hydrolyzed in the solid phase so that inhibitor by binding to the incorrect sites [41]. The hydro-
the decrease in collagen by hydrolysis in that same range of lyzed protein recovery decreased gradually as the initial
enzyme activity was only 37%. substrate concentration increased. Comparing the reaction
Regarding the oil recovery, an increase in the solubil- with 5–45 g/L, the hydrolyzed protein recovery decreased
ity of the protein after hydrolysis favors the separation of 43% (Fig. 3c). However, there was a rise of 86.19% in the
oil whose amount in the upper phase increases by 30% in collagen amount that remained in the solid phase (Fig. 3d).
the range of enzyme concentration studied, reaching a value Moreover, as consequence of the lower protein solubiliza-
higher than 75% of the oil contained in the waste (Fig. 2b). tion, the amount of released oil decreased by 49% (Fig. 3b).
Higher solubility of hydrolysates in comparison to the solu-
bility of waste is due to the modification in the secondary
structure of fish protein during the enzymatic hydrolysis.

Fig. 2  Influence of Alcalase
concentration on: a degree of
hydrolysis [DH], b oil recovery
[OR], c hydrolyzed protein
recovery [HPR] and d collagen
recovery [CR], at an initial
substrate concentration of 25 g
of protein per liter

13
 Waste and Biomass Valorization

Fig. 3  Influence of substrate
concentration on: a degree of
hydrolysis [DH], b oil recovery
[OR], c hydrolyzed protein
recovery [HPR] and d collagen
recovery [CR], at an initial
Alcalase concentration of 0.84
UA/l

Fig. 4  Degree of hydrolysis and

by-products recovery, at con-
stant 0.19 UA/g enzyme/sub-
strate ratio, for different enzyme
and substrate concentrations

Influence of Enzyme/Substrate Ratio the substrate is the most influential variable in the process
for the studied operating conditions.
The fact that the increase of the initial concentration of To check the influence of Eo/So ratio on by-products
substrate, keeping enzyme concentration constant, did not recovery, experiments were performed at different ratios,
favor the recovery of by-products seems to indicate that the from 0.03 to 0.20 AU/g (Fig. 5). As was expected, based
enzyme/substrate ratio is a significant process variable. To on previous experiments in sections entitled “Influence of
verify this assertion, experiments with different initial con- Alcalase concentration” and “Influence of protein con-
centrations of enzyme and substrate were carried out, keep- centration”, the amounts of the recovered by-products are
ing constant the Eo/So ratio. Results obtained at the same closely related to the enzyme/substrate ratio. The increas-
Eo/So ratio showed identical values as much for the degree ing of the ratio (obtained either from a higher enzyme con-
of hydrolysis as for the protein hydrolysate, collagen and oil centration or a lower substrate concentration) resulted in a
recoveries (Fig. 4). It proves that the ratio between the initial hydrolysate with a higher degree of hydrolysis. For a rise
concentration of the enzyme and the initial concentration of of only 0.045 AU/g, the hydrolysis degree increased 41.4%

13
Waste and Biomass Valorization

the reliability of the relationship obtained to determine

the amount of unhydrolyzed collagen from the degree of
hydrolysis.
According to the linear regression equations (Fig. 6), the
optimal yield for the by-products recovery could be achieved
with a degree of hydrolysis of 25% for which 430 g of hydro-
lyzed protein, 10 g of recovered collagen and 350 g of fish
oil could be obtained per kg of fish waste. Considering the
average market prices of protein hydrolysates, collagen and
fish oil, and the main operating costs (corresponding to
energy and enzyme costs), a sale revenue of 10.6 €/kg of
fish waste could be generated. Table 1 shows the detailed
data for calculating the sale revenue. Moreover, the use of
Fig. 5  Influence of different enzyme/substrate ratios on: a degree of fish waste as raw material for by-product recovery resulted
hydrolysis [DH], b oil recovery [OR], c hydrolyzed protein recovery
[HPR] and d collagen recovery [CR] in a 79% reduction in waste disposed to landfills.
For this degree of hydrolysis, the average peptide chain
(Fig. 5a), the hydrolyzed protein increased 100% (Fig. 5c), length, PCL, estimated as PCL = 100/DH [28], was four cor-
the collagen recovery decreased 59.7% (Fig. 5d) and the responding to peptides of about 500 Da of average molecular
oil recovery increased 56.2% (Fig. 5b). It is explained by
Silva et al. [42], who reported that, at higher relative enzyme
concentrations, there were more active sites available on the
enzyme to hydrolyze the substrate, resulting in greater cleav-
age of the peptide bonds, and consequently higher dissolu-
tion of the proteins.

Relationship Between Degree of Hydrolysis

and Recovery of By‑Products

Linear regression analysis was applied to assess the rela-

tionships between the degree of hydrolysis and the recov-
ery of oil, hydrolyzed proteins and collagen (Fig. 6). A
direct linear relationship between the degree of hydrolysis
and the formation of protein hydrolysates and oil recov-
ery was observed. The determination coefficients near 1
for both hydrolysate (0.991) and oil (0.991) indicate that
hydrolyzed protein and oil recovery had a general ten-
dency to increase as the degree of hydrolysis increased.
As it can be seen in Fig. 6a, b, the regression lines have an
ordinate at the origin of 0. It is what it would be logically
expected since it is evident that for DH = 0 the production
of hydrolysate and the recovery of oil are not possible.
This fact indicates that the obtained relationships are an
accurate adjustment set for calculating the causal effect of
DH on hydrolysate and oil production.
However, an inverse linear relationship can be observed
between the degree of hydrolysis and the collagen content in
the residue. In this case, the determination coefficient near
1 implies that collagen recovery had a clear tendency to
decrease as the degree of hydrolysis decreased. As expected,
now the regression line has an ordinate at the origin of
100, meaning that for DH = 0 all collagen remains in the Fig. 6  Correlation between the degree of hydrolysis [DH] and: a
solid part since it is not hydrolyzed. This result confirms hydrolyzed protein recovery [HPR], b oil recovery [OR] and c col-
lagen recovery [CR]

13
 Waste and Biomass Valorization

Table 1  Income and cost of production It was proved that the enzyme/substrate ratio was the main
Income a factor controlling the hydrolysis rate at the assayed condi-
tions. Linear relationships between the degree of hydrolysis
 Protein hydrolysate
and the obtained products were achieved. These relation-
  kg/kgfish waste 0.43
ships allow the prediction of the amounts of by-products that
  €/kg 22.11
can be obtained from fish waste at different operating con-
  €/kgfish waste 9.51
ditions by only knowing the degree of hydrolysis reached.
 Collagen
Thus, enzymatic hydrolysis is a promising technology
  kg/kgfish waste 0.01
  €/kg 16.01
with great potential to increase the fish processing plant
  €/kgfish waste 0.16
revenue, by obtaining simultaneously protein hydrolysates,
 Oil
collagen and oil from fish waste, and to reduce the amount of
  kg/kgfish waste 0.35
organic matter disposed of in landfills, making these indus-
  €/kg 4.99
tries more sustainable.
  €/kgfish waste 1.74
 Total (€/kgfish waste) 11.41
Costb
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