May | June 2014

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

The International magazine for the aquaculture feed industry

International Aquafeed is published six times a year by Perendale Publishers Ltd of the United Kingdom.All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2014 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

INCORPORAT ING F I SH FARM ING TECHNOLOGY

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.

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 proteaseIn 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 Chileby 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

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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 qualityIn 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 CPThe 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 healthSeveral 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)

FCRVilli 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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Challenges for using a protease enzymeIssues 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.

ConclusionApart from their availabil ity and poor nutrient charac-

terisation, imbalanced amino acid profi les, poor digestibil ity 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.

ConclusionPolychaete 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%

Polychaete60%

Polychaete100%

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

www.indolivestock.com

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Assessing the potential of polychaete meal in

shrimp feeds

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Volume 17 I s sue 3 2 014 - mAY | J uNe

INCORPORAT ING F I SH FARM ING TECHNOLOGY

Use of a heat-stable protease in salmonid feeds

– experiences from Canada and Chile

Fish Farming Technology supplement

Nets and cages Recirculating aquaculture

systems

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