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May | June 2014

Use of a heat-stable protease in salmonid
feeds - Experiences from Canada and Chile

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The International magazine for the aquaculture feed industry

FEATURE

Use of a heat-stable protease in salmonid feeds

- Experiences from Canada and Chile
by M.A. Kabir Chowdhury, PhD, Jefo Nutrition Inc., Saint-Hyacinthe, Quebec, Canada
Dr Pedro Cardenas Villarroal, Alinat Chile, Chile

olatility of supply, price and quality of commonly-used

ingredients and lack of proper characterisation of their
components are forcing aquaculture feed manufacturers to
use high safety margins for nutrients while formulating a feed.

Techniques such as cooking, conditioning, soaking and finally, using

enzymes for various components are increasingly used to improve the
quality of ingredients in feed or to reduce the variations in their quality.
Besides phytase (for phosphorus) and some carbohydrases, dietary
proteolytic enzymes are gaining attention in recent years, mainly
because of the need for better utilisation of proteins from existing
sources.
Protease breaks down large, indigestible and insoluble proteins to
highly digestible smaller peptides and some free amino acids. These
small chain peptides may also contain some bioactive properties
influencing ingestion, digestion, absorption, and assimilation of nutrients
in animals.
These intrinsic properties of protease enzymes are encouraging for
nutritionists and feed formulators as they allow them to include more
low-digestible protein ingredients without compromising the quality of
the feed.

The influence of exogenous protease

In the intestine of animals, polypeptides are digested to smaller

peptides and amino acids by several enzymes derived from pancreas
or secretory cells of the intestinal epithelium in slightly alkaline environment achieved by pancreatic secretion of bicarbonates and bile acids
from the gall bladder (see Figure 1).
The absorption of nutrients occurs in the intestine by optimising
the intestinal surface area within the constraints of the coelomic cavity.
Presence of exogenous protease can influence the rate of reactions
in the intestine enhancing nutrient utilisation efficiency of the animals.
Effects of protease in aquaculture feed can be manifested in more
digestible proteins in feed, improved digestibility of nutrients in an
ingredient, better mucosal health, growth and feed conversion of the
farmed aquatic animals.
Trials with shrimp, crab, salmonids, carps, tilapia, pangasius, seabream
and other species have shown significant improvement in growth, feed
conversion or nutrient utilisation efficiency. In studies with salmonids
species, addition of protease in feed not only improved the protein
30 | INTERNATIONAL AQUAFEED | March-April 2014

FEATURE
quality of the feed but also stimulated
gut health, growth, and feed conversion helping the bottom line of feed
manufacturers and producers.

Table 1. Growth performance and intestinal villi height of rainbow trout fed diets containing graded level
(0, 175, 250 ppm) of Jefo protease
Treatments

Improving protein quality

Initial
body
weight
(g)

Final
body
weight
(g)

Specific
growth
rate
(SGR, %)

Thermalunit Growth
Coefficient
(TGC)

FCR

Villi size
(m)

In several in-vitro and in-vivo studies with the Jefo protease, a marked
Control
390
850a
0.92a
2.52a
1.43b
630a
improvement in protein digestibility
Control + 175 ppm protease
402
971b
1.05b
2.94b
1.35a
663b
of ingredient and feed was observed.
Control + 250 ppm protease
399
987b
1.07b
3.03b
1.33a
737b
In a study conducted at the
Notes: Different letters in a column denote significant differences (P<0.05) among the treatments
University of Saskatchewan of Canada,
addition of the protease to a coThe protein digestibility of a feed was then determined using the
extruded canola-pea based diets resulted in significant improvement
in apparent digestibility of crude protein, energy, lipid and dry matter following equation:
Protein Digestibility (%) = 100 x (Initial CP Final CP)/Initial CP
(P<0.05) in rainbow trout (see Figure 2A) (Drew et al. 2005).
The protein digestibility was analysed in three different hydrolysing
The improvement was less pronounced in the co-extruded flax-pea
conditions (temperature and pH). In all three cases, significantly more
based diets.
Availability of more digestible nutrients also resulted in improved digestible protein was reported in feeds containing the protease than
feed conversion and growth of rainbow trout fed diets containing with in those without (see Figure 3).
the protease (see Figures 2B and 2C).
In another in-vivo study conducted at the Universidad Catolica de Growth performance and intestinal health
Temuco with three species of salmonids (coho salmon, Atlantic salmon
Several growth and digestibility trials conducted in Canada and
and rainbow trout), both protein and carbohydrate digestibility were Chile showed significant improvement in performance of the test
improved significantly in fish fed the treatment diets containing the animals fed diets containing the protease compared to those fed the
protease than those fed the control diets (Chowdhury 2012).
control diets (see Table 1).
In an in-vitro digestibility study at the Universidad de Concepcion of
Similarly, height (m), density and structure of intestinal villi also
Chile, protein digestibility of commercially extruded (extrusion temp. showed a marked improvement in fish fed the protease diets (see
120oC) salmonids feeds with and without protease was determined Figure 4).
using the HCl-Pepsin method. The method involved grinding of the
Increased availability of nutrients coupled with increased intestinal
feed samples followed by HCl-Pepsin digestion for 16 hours and then, nutrient absorption capacity resulted in the better growth and feed
separation of solids.
conversion in treatment animals.
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March-April 2014 | INTERNATIONAL AQUAFEED | 31

FEATURE
FEATURE

Challenges for using a protease enzyme

position of shrimp was detected among the utilization were calculated from energy and of the mixed feeds was more apparent.
Issues with heat-stability have been a major protein gained in relation to energy and Gross energy retention efficiency was 15.1
hindrance
different treatments (Table 4).
for the efficiencies of energy and protein protein consumed. Here, the superiority percent for L. vannamei fed the 60%
The use of enzymes in aquafeed.
Very few enzymes in the market today are truly heatpolychaete meal, which was significantly
stable.
higher than the energy efficiency of shrimp
In addition, it is difficult for feed manufacturers to
on the fish meal and polychaete meal only
compare efficacy of various enzymes to improve the
diets (Figure 1). Similarly the crude proprotein quality of their feed using traditional or prescribed
tein retention efficiency was significantly
enzymatic activity assays. Traditional or prescribed enzyhigher for L. vannamei fed the 60 percent
matic assays rely on specific substrate, which may not be
polychaete at 22.7 percent compared to
suitable for a feed.
shrimp fed the single ingredient diets
Feedmills must be able to rapidly and accurately
(Figure 1). No significant difference was
test complete feeds for the presence of a protease
observed for energy or protein retention
as part of their QA/QC process. The in-vitro protein
efficiency for shrimp fed the polychaete
digestibility assays provide a solution to this problem
only diet compared to the 100 percent
enabling feed manufacturers to test the effects of an enzyme not Conclusion
control fishmeal diet.
by measuring activity but in real term, the quality of proteins.
Apart from their availability and poor nutrient characThis innovative solution should be standardised and utilised as a tool terisation, imbalanced amino acid profiles, poor digestibility
Conclusion
to compare effects of different enzymes on a particular feed.
of nutrients, presence Polychaete meal inclusion in the diets has
of various anti-nutritional factors of
Preference to multi-enzyme containing protease-complex has also been limiting the use of some supported equal growthaquaculL. vannamei novel ingredients in perforbeen a rising phenomenon.
ture feed.
mance
Fig. 1: Protein and energy retention efficiency in shrimp fed the experimental feeds
All enzymes are proteins and adding a protease in the cocktail creUsing a protease enzyme wouldefficiency compared to a standard
and feed therefore be a useful solution to
ates a situation where other enzymes become the nearest substrate address these unknownfish meal diet. Freeze-dried polychaete meal
factors.
Table 4: Proximate composition of juvenile L. vannamei fed diets containing polychaete meal at
for the protease. While itweight).
It can be assumed could in the near future, substitution phytase,
that thus serve as a total similar to for fishincreasing levels (per g wet is acceptable to use all the carbohydrases
together, using protease in a cocktail usually reduces the efficacy of protease enzymes would become an essential component of feed
meal. The final decision however is dependent
Dietary
30%
60%
100%
Initial
Fishmeal
othertreatment
enzymes.
as
upon to improve price of the product.
Polychaete
Polychaete a cost-effective solution availability and the quality of salmonids
Polychaete
Several published and unpublished trials with carps, shrimp and feeds.
salmonids showed lower beneficial effects of multi-enzyme compared
Dry matter, g
210
230 6.6
233 8.4
244 9.1
232 11.6
to a single protease or a protease-complex.
References:
More InforMatIon:
Ash, g
30.0
29.3 protease either separately or 0.7
27.1 2.0
28.3
28.0 0.5
If intended, it is recommended to use1.8
Chowdhury, M.A.K. 2012. Aquafeed:Lupatsch, PhD,Processing & Formulation,
Ingrid Advances in
in a protected form 144 multi-enzyme 3.7
in a
Protein, g
162 cocktail to prevent hydrolysis 8.2
162 6.5
170 Autumn161 8.9
Email: i.lupatsch@swansea.ac.uk
Issue.
of other enzymes. 3.92
Energy, kJ
4.61 0.1
4.81 0.3
5.13 0.2
4.84 0.3
Drew et al. 2005. Animal Feed Science and Technology, 119:117-128

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