Volatility 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.

1. May | June 2014
Use of a heat-stable protease in salmonid
feeds - Experiences from Canada and Chile
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2. V
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 environ-
ment 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
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
30 | INTERNATIONAL AQUAFEED | March-April 2014
FEATURE

3. quality of the feed but also stimulated
gut health, growth, and feed conver-
sion helping the bottom line of feed
manufacturers and producers.
Improving protein quality
In several in-vitro and in-vivo stud-
ies with the Jefo protease, a marked
improvement in protein digestibility
of ingredient and feed was observed.
In a study conducted at the
University of Saskatchewan of Canada,
addition of the protease to a co-
extruded canola-pea based diets resulted in significant improvement
in apparent digestibility of crude protein, energy, lipid and dry matter
(P<0.05) in rainbow trout (see Figure 2A) (Drew et al. 2005).
The improvement was less pronounced in the co-extruded flax-pea
based diets.
Availability of more digestible nutrients also resulted in improved
feed conversion and growth of rainbow trout fed diets containing with
the protease (see Figures 2B and 2C).
In another in-vivo study conducted at the Universidad Catolica de
Temuco with three species of salmonids (coho salmon, Atlantic salmon
and rainbow trout), both protein and carbohydrate digestibility were
improved significantly in fish fed the treatment diets containing the
protease than those fed the control diets (Chowdhury 2012).
In an in-vitro digestibility study at the Universidad de Concepcion of
Chile, protein digestibility of commercially extruded (extrusion temp.
120oC) salmonids feeds with and without protease was determined
using the HCl-Pepsin method. The method involved grinding of the
feed samples followed by HCl-Pepsin digestion for 16 hours and then,
separation of solids.
The protein digestibility of a feed was then determined using the
following equation:
Protein Digestibility (%) = 100 x (Initial CP – Final CP)/Initial CP
The protein digestibility was analysed in three different hydrolysing
conditions (temperature and pH). In all three cases, significantly more
digestible protein was reported in feeds containing the protease than
in those without (see Figure 3).
Growth performance and intestinal health
Several growth and digestibility trials conducted in Canada and
Chile showed significant improvement in performance of the test
animals fed diets containing the protease compared to those fed the
control diets (see Table 1).
Similarly, height (µm), density and structure of intestinal villi also
showed a marked improvement in fish fed the protease diets (see
Figure 4).
Increased availability of nutrients coupled with increased intestinal
nutrient absorption capacity resulted in the better growth and feed
conversion in treatment animals.
Table 1. Growth performance and intestinal villi height of rainbow trout fed diets containing graded level
(0, 175, 250 ppm) of Jefo protease
Treatments
Initial
body
weight
(g)
Final
body
weight
(g)
Specific
growth
rate
(SGR, %)
Thermal-
unit Growth
Coefficient
(TGC)
FCR
Villi size
(μm)
Control 390 850a 0.92a 2.52a 1.43b 630a
Control + 175 ppm protease 402 971b 1.05b 2.94b 1.35a 663b
Control + 250 ppm protease 399 987b 1.07b 3.03b 1.33a 737b
Notes: Different letters in a column denote significant differences (P<0.05) among the treatments
March-April 2014 | INTERNATIONAL AQUAFEED | 31
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4. Challenges for using a protease enzyme
Issues with heat-stability have been a major hindrance
for the use of enzymes in aquafeed.
Very few enzymes in the market today are truly heat-
stable.
In addition, it is difficult for feed manufacturers to
compare efficacy of various enzymes to improve the
protein quality of their feed using traditional or prescribed
enzymatic activity assays. Traditional or prescribed enzy-
matic assays rely on specific substrate, which may not be
suitable for a feed.
Feedmills must be able to rapidly and accurately
test complete feeds for the presence of a protease
as part of their QA/QC process. The in-vitro protein
digestibility assays provide a solution to this problem
enabling feed manufacturers to test the effects of an enzyme not
by measuring activity but in real term, the quality of proteins.
This innovative solution should be standardised and utilised as a tool
to compare effects of different enzymes on a particular feed.
Preference to multi-enzyme containing protease-complex has also
been a rising phenomenon.
All enzymes are proteins and adding a protease in the cocktail cre-
ates a situation where other enzymes become the nearest substrate
for the protease. While it is acceptable to use all the carbohydrases
together, using protease in a cocktail usually reduces the efficacy of
other enzymes.
Several published and unpublished trials with carps, shrimp and
salmonids showed lower beneficial effects of multi-enzyme compared
to a single protease or a protease-complex.
If intended, it is recommended to use protease either separately or
in a protected form in a multi-enzyme cocktail to prevent hydrolysis
of other enzymes.
Conclusion
Apart from their availability and poor nutrient charac-
terisation, imbalanced amino acid profiles, poor digestibility
of nutrients, presence of various anti-nutritional factors has
been limiting the use of some novel ingredients in aquacul-
ture feed.
Using a protease enzyme would therefore be a useful solution to
address these unknown factors.
It can be assumed that in the near future, similar to phytase,
protease enzymes would become an essential component of feed
as a cost-effective solution to improve the quality of salmonids
feeds.
References:
Chowdhury, M.A.K. 2012. Aquafeed: Advances in Processing & Formulation,
Autumn Issue.
Drew et al. 2005. Animal Feed Science and Technology, 119:117-128
32 | INTERNATIONAL AQUAFEED | March-April 2014
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position of shrimp was detected among the
different treatments (Table 4).
The efficiencies of energy and protein
utilization were calculated from energy and
protein gained in relation to energy and
protein consumed. Here, the superiority
of the ‘mixed’ feeds was more apparent.
Gross energy retention efficiency was 15.1
percent for L. vannamei fed the 60%
polychaete meal, which was significantly
higher than the energy efficiency of shrimp
on the fish meal and polychaete meal only
diets (Figure 1). Similarly the crude pro-
tein retention efficiency was significantly
higher for L. vannamei fed the 60 percent
polychaete at 22.7 percent compared to
shrimp fed the single ingredient diets
(Figure 1). No significant difference was
observed for energy or protein retention
efficiency for shrimp fed the polychaete
only diet compared to the 100 percent
control fishmeal diet.
Conclusion
Polychaete meal inclusion in the diets of
L. vannamei supported equal growth perfor-
mance
and feed efficiency compared to a standard
fish meal diet. Freeze-dried polychaete meal
could thus serve as a total substitution for fish-
meal. The final decision however is dependent
upon availability and price of the product.
Table 4: Proximate composition of juvenile L. vannamei fed diets containing polychaete meal at
increasing levels (per g wet weight).
Dietary
treatment
Initial Fishmeal
30%
Polychaete
60%
Polychaete
100%
Polychaete
Dry matter, g 210 230 ± 6.6 233 ± 8.4 244 ± 9.1 232 ± 11.6
Ash, g 30.0 29.3 ± 1.8 27.1 ± 2.0 28.3 ± 0.7 28.0 ± 0.5
Protein, g 144 162 ± 3.7 162 ± 6.5 170 ± 8.2 161 ± 8.9
Energy, kJ 3.92 4.61 ± 0.1 4.81 ± 0.3 5.13 ± 0.2 4.84 ± 0.3
More InforMatIon:
Ingrid Lupatsch, PhD,
Email: i.lupatsch@swansea.ac.uk
Fig. 1: Protein and energy retention efficiency in shrimp fed the experimental feeds
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Assessing the potential
of polychaete meal in
shrimp feeds
Prospects on dietary trace minerals
– aquafeeds & aquaculture
Volume 17 Issue 3 2014 - mAY | JuNe
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Use of a heat-stable
protease in salmonid feeds
– experiences from Canada and Chile
Fish Farming Technology supplement
Nets and cages
Recirculating aquaculture
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