Changes in production technology and marketing and changes in feed ingredients are key structural transformations necessary for the aquaculture sector to grow. Today, with improved genetic techniques novel genetic lines are being bred for maximum efficiency over a shorter production period with lower feed conversions. Thus, the correct amount of micro-nutrients present in their diet is crucial.

1. I N C O R P O R AT I N G
f i s h far m ing t e c h no l og y
November | December 2013
Animal co-product hydrolysates: a source of
key molecules in aquaculture feeds
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3. FEATURE
Animal co-product hydrolysates:
a source of key molecules in aquaculture feeds
by Sergio F Nates, ALAPRE, Costa Rica, Victor Suresh, United Research Pte. Ltd, Singapore, and Kent Swisher, National Renderers
Association, USA
C
hanges in production technology
and marketing and changes in
feed ingredients are key structural transformations necessary for
the aquaculture sector to grow. Today, with
improved genetic techniques, novel genetic
lines are being bred for maximum efficiency
over a shorter production period with lower
feed conversions. Thus, the correct amount
of micro-nutrients present in aqua diets is
crucial.
On the other hand, the rapid growth of
aquaculture worldwide has become increasingly dependent upon the use of external feed
inputs, and in particular upon the use of compound aquafeeds. Pressures to reduce fishmeal consumption for sustainability reasons,
combined with economic reasons, require
intensive research efforts to find candidates
for fishmeal replacement.
However, formulating low fishmeal aquaculture feeds requires the use of combinations
of several ingredients since most feedstuffs
have been shown to have significant nutrient
and functional limitations and cannot be used
table 1: effect of hydrolysis on animal byproducts
effect of hydrolysis
resulting benefit
1. Digestion of protein
Improved digestibility, absorption and
assimilation of peptides
2. Increase in the proportion of low molecular
compounds like short-chain peptides, free
amino acids and nucleotides
enhanced attractability and palatability
3. Production of bioactive peptides
antioxidant and anti-microbial activities
individually at very high levels in the diets of
most aquaculture species.
Fishmeal has always been the main
source and the preferred choice of nutritionists for quality protein, above all in the
formulation and especially in feeds for the
youngest ages. Though, with the market
volatility of fishmeal, the aquaculture feed
industry is looking for cheaper sources of
protein to substitute the fishmeal and this
has become a priority. Additional renewable and sustainable protein alternatives are
needed.
Animal byproducts are well accepted as
aquafeed ingredients these days due to short
supplies and the escalating cost of fishmeal.
Protein content in animal byproducts is higher
and their complement of indispensable amino
acids is superior to those of plant origin.
In
addition,
Animal
Co-Product
Hydrolysates (ACPH) can meet the many
nutritional needs of aquaculture worldwide
as a protein alternative in aquafeeds. ACPHs
can help reduce pressure on natural fisheries
stocks and provide sustainability to the growing demand for aquatic products.
Methods
ACPHs result from controlled enzymatic
digestion of byproducts from the meat
processing industry. Technically, it is feasible
to generate ACPH from most kinds of
10 | INterNatIoNal AquAFeed | November-December 2013

4. FEATURE
Hydrolysis improves the nutritive value
of feed ingredients that are produced from
slaughterhouse waste (Table 1). Enzymatic
digestion of the raw material breaks the
protein chains into peptides that are better
absorbed in the gut. Enzymatic hydrolysis of
poultry meal with endo- and exopeptidases
shows the feasibility of hydrolysing poultry
byproducts so significant amounts of shortchain peptides and free amino acids can be
produced.
High levels of digestible protein characterise poultry protein hydrolysates with a
digestibility index above 95 percent. Feather
hydrolysates produced by bacterial keratinases
have been tested as additives in aquaculture
feeds and several species of bacteria with high
keratinolytic activity has been isolated from
feather meal broth.
In recent studies, it has been established
that pepsin digestibility and amino acid content of fermented feather meal (FFM) can be
far better than those of commercial feather
meal. The microbial cells could also potentially
supply carotenoid pigments to FFM, whereby
the ingredient may be useful in animal feeding
not just as a source of protein but also that
of pigments.
The short-chain peptides and free amino
acids produced as a result of hydrolysis along
with nucleotides that are rich in meats confer
excellent attractability and palatability properties to ACPH.
Poultry liver hydrolysates have been added
to animal feeds at levels as high as 6 percent
and have been found to enhance palatability.
Spray-dried hydrolysates produced from
poultry byproduct meal can contain up to 70
percent protein with compounds that have
molecular weights ranging from 5,800 to
12,000 Da.
We have found that inosine is the dominant nucleoside in poultry meal, a molecule
that is believed to enhance diet attractability in
several fish species, including largemouth bass,
turbot and mackerel.
Among monophosphate forms, adenosine monophosphate (AMP) dominated and
similar trends have been seen in fishmeal
hydrolysates.
It is known that alkali-hydrolysates and
enzyme-hydrolysates from meat and bone
meal, blood meal and feather meal have
similar ash and protein content as the parent
materials, but with concomitant liberation of
bioactive peptides that are encoded within
the protein.
It has been demonstrated that ACPH
prepared under alkaline hydrolysis can be a
source of antioxidants with activities comparable to BHT.
Results from previous studies have also
shown the presence of antioxidant, carnosine,
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slaughterhouse waste such as scrape meat,
offal, feathers, and blood as well as from rendered animal byproducts such as meat and
bone meal, poultry meal, feather, and blood
meal. Protein materials are partially defatted
by hexane extraction prior to hydrolysis.
Hydrolysis is conducted in a thermostat reaction vessel with constant stirring adequate
to prevent the settling of the substrate in
the vessel.
Alkaline hydrolysates are produced
with 0.1 g CaOH/g substrate, at 85
°C. During enzymatic hydrolysis, pH is
monitored continuously and maintained
through the addition of 8M NaOH.
Alkaline hydrolysis reactions are terminated by sparging with CO 2 until the pH
drops to 9, followed by neutralization
with sulphuric acid. Enzymatic reactions
can also be terminated by raising the reaction temperature to 90 °C for 10 minutes.
Residual solid material is removed by
centrifugation followed by filtration.
To test the antioxidant activities of
ACPHs, two hydrolysis methods have been
adopted1, including the alkaline hydrolysis by
a strong base and an enzymatic hydrolysis by
a commercial enzyme protease at a specific
pH condition. Most results have showed that
alkaline hydrolysis produced better antioxidant hydrolysates than the enzymatic
hydrolysis2, 3.
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November-December 2013 | INterNatIoNal AquAFeed | 11

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6. FEATURE
to elicit feeding in satiated animals, and
‘Defensins’ that show antimicrobial activity
against bacteria and fungi, but at this point we
don't know if these molecules are present in
animal by-products.
Compared with fishmeal, dried porcine
blood co-products and bone protein hydrolysates are poor in methionine and lysine6.
However, blood by-products are rich in
microelements, which can improve Ca and
Cu retention in aquaculture species, especially shrimp in which it has been reported
that inadequate supplementary dietary copper
level can result in significant growth depression.
Porcine plasma hydrolysates are also
effective inhibitors of lipid oxidation as
well as metal chelating and reducing
agents.
Conclusions
While we need to keep in mind the ability of bioactive peptides and nutraceuticals
to exert a physiological effect in vivo, these
examples of key molecules found in animal
by-product hydrolysates show the potential
for use as functional ingredients in aquaculture
feeds.
References
a histidine containing dipeptide, in poultry
byproducts4, 5.
Carnosine levels in poultry products
ranged from 0.95-102.3 mg/g (wet basis)
(Table 2). Carnosine levels in meat and bone
meal ranged from 500 to 1,800 ppm, while in
fishmeal they can be as low as 5 ppm. Soy and
higher than that in the control group, and
diets supplemented with carnosine could
increase the levels of GH, IGF-1 and T3 in
their serum indicating that diets supplemented
with carnosine could improve antioxidation
in muscle.
Anti-microbial peptides have also been
Table 2: Carnosine levels in different tissues of stress and non-stress broilers (from Manhiani, 2011)
organ treatment
Calcium
content ppm
Breast non-stress 6.74±0.07a
stress
p-value
Carnosine
p-value
content (wb)2,3
p=0.001
stress
Brain
13.99±0.19b
non-stress 8.48±0.38a
stress
p=0.002
13.78±0.24
1.85±0.241,a p=0.005
7.52±0.97a
P=0.0005
70.89±5.39b
11.10±1.02a
p=0.001 44.47±4.08a
21.25±1.25b
thigh non-stress 10.50±0.37a p=0.006
p-value
17.39±1.33b
11.03±0.13b
Carnosine
content (db)
85.12±5.01b
10.16±1.53a1
p=0.82 45.44±12.37a
10.27±2.77a
55.12±14.88
P=0.002
P=0.54
1. All values are in Mean± S.E.M (N=5)
2. wb= wet basis; db= dry basis
3. Carnosine content is expressed in mg/gm of the original sample
4. Fisher's least significant difference test was used to compare mean values; a-b similar letters
indicate that teh means values are not significantly different (p≥0.05); while different letters
indicate that the mean values are significantly different (p≤0.050
other plant proteins don't contain carnosine.
The results of adding carnosine on growth
performance of Nile tilapia showed that the
body weight and body length of tilapia in the
group supplemented with carnosine were
1. Kalambura S., Kricka T., Jurišic V. and Z. Janjecic
(2008) Alkaline hydrolysis of animal waste as
pretreatment in production of fermented fertilizers.
Journal Cereal Research Communications
36(5):179-182.
2. Bo Li, F. Chen, X. Wang, B. Ji, and Yonnie W.
(2007) Isolation and identification of antioxidative
peptides from porcine collagen hydrolysate by
consecutive chromatography and electrospray
ionization–mass spectrometry. Food Chemistry.
102:1135-1143.
3. Jingbo L., Zhipeng Y., W Zhao, S. Lin, E. Wang,
Y. Zhang, H. Hao, Z. Wang, and Feng C. (2010)
Isolation and identification of angiotensionconverting enzyme inhibitory peptides from
egg white protein hydrolysates. Food Chemistry,
122:1159-1163.
4. Manhiani P. S. (2011) Carnosine content and
antioxidant activity from poultry co-products,
proetin meal and stressed poultry tissues. P.h.D.
Dissertation, Clemson University. 179 pp.
5. Manhiani P.S., Northcult J.K., Bridges W.C.
and Dawson Pl.L. (2013) Antioxidant activity of
carnosine extracted from various poultry tissues.
Poultry Science 92(2): 444-453.
6. Sun, Q. and H. Shen (2011). Antioxidant activity
of hydrolysates and peptide fractions derived from
porcine hemoglobin. Journal of Food Science and
Technology 48(1): 53-60.
identified in PBM and FeM hydrolysates.
These include cysteine-rich antimicrobial peptides. Other potential molecules that can be
found in cattle, chickens and turkeys include
‘Galanin’, which has previously been reported
12 | INterNatIoNal AquAFeed | November-December 2013
More InforMatIon:
Email: sergionates@alapre.org

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