1303

Functional Fe 59. Functional Feeds in Aquaculture

Part J | 59.1
Jorge Olmos Soto, José de Jesús Paniagua-Michel, Lus Lopez, Leonel Ochoa

59.2 Food Formulation Ingredients .............. 1304

The development of functional feed (FF) represents
59.2.1 Fish Meal and Oil....................... 1305
a great opportunity in the aquaculture industry.
59.2.2 Soybean Meal and Oil ................ 1306
FF must promote the growth and health of culti- 59.2.3 Complex
vated organisms, improve their immune systems, and Most Used Carbohydrates..... 1307
and induce physiological benefits beyond tradi- 59.2.4 Probiotic Bacteria ...................... 1308
tional feeds. FF must be economically attractive
and environmentally friendly. In this sense, the 59.3 Conventional Feeds
inclusion of animal products in FF formulations Versus Functional Feeds ....................... 1309
must be partially or totally eliminated, increasing 59.4 Aquaculture Regulations ...................... 1310
the inclusion of alternative economical vegetable 59.4.1 Sanitary Regulations.................. 1310
products. However, the kind and amount of veg- 59.4.2 Environmental Regulations ........ 1311
etable protein, carbohydrates (CHO), and lipids
59.5 Functional Feeds in Aquaculture........... 1311
added to formulations are of great concern for
59.5.1 Advantages and Disadvantages
growth, health, environmental, and economical
of Adhesion .............................. 1311
issues. Therefore, deficiencies in proteases, carbo-
hydrolases, and lipases in shrimp/fish are a major 59.6 Results Obtained in Crustaceans
impediment to the digestion and assimilation of and Fish Using Functional Feeds........... 1312
vegetable sources, limiting its high-level inclusion 59.6.1 Functional and Commercial Feed
in formulations. In this sense, the utilization of Proximal Composition Used
probiotic bacteria has emerged as a solution with in Litopenaeus vannamei
huge applications in the aquaculture industry. and Oreochromis niloticus.......... 1312
Today, Bacillus species are the most investigated 59.6.2 Basal and Functional Feed
bacteria for animal probiotic development due to: Proximal Composition Used
in Atractoscion nobilis ............... 1313
a) The versatility of their growth nutrients 59.6.3 Functional Feed Effects
b) High level of enzyme production on Survival and Growth
c) Secretion of antimicrobial compounds. Performance of L. vannamei,
O. niloticus and A. nobilis .......... 1313
In addition, Bacillus subtilis (Bs) is generally 59.6.4 Functional Feed Effects
recognized as safe (GRAS) by the Food and Drug on L. vannamei
Administration (FDA), meaning that it is harmless and A. nobilis – Health Status .... 1316
to animal and humans. In this work, we present 59.6.5 Functional Feed Effects
the potential benefits of the utilization of FF in the on Environmental Parameters
aquaculture industry. in L. vannamei ......................... 1316
59.6.6 Functional Feed Effects
59.1 Overview ............................................. 1304 on Stress Tolerance
59.1.1 Importance in L. vannamei ......................... 1317
of Aquaculture Development ...... 1304
59.7 Conclusions ......................................... 1317
59.1.2 Development
of Functional Feed .................... 1304 References................................................... 1317
1304 Part J Industrial Applications
59.1 Overview
Part J | 59.2
59.1.1 Importance
of Aquaculture Development
Aquaculture is the fastest growing sector of the world
food economy, increasing by more than 10% per year
and currently accounts for more than 50% of all shrimp/
fish consumed. The United Nations Food and Agri-
culture Organization (FAO) defines aquaculture as the
farming of aquatic organisms including fish, mollusks,
crustaceans, and aquatic plants. Farming implies some
sort of intervention in the rearing process to enhance
production, such as regular stocking, feeding, protec-
tion from predators, etc. Farming also implies that
individual or corporate ownership of the stock being
cultivated [59.1].
The global population’s demand for aquatic food
products is growing in importance; however fisheries’
capture production has leveled off and most of the
main fishing areas have reached their maximum poten-
tial [59.2, 3]. Because of this, fishmeal and oil prices
have increased considerably in recent years (Fig. 59.1).
In addition, concern has also arisen about the nega-
tive impact of fishmeal production on the ecology of
global fisheries and on the environment [59.4, 5]. Aqua-
culture, which is probably the fastest growing food-
producing sector, represents the greatest potential to
meet demands for aquatic food supply. However, in-
stead of helping to ease the crisis in wild fisheries,
unsustainable aquaculture development may exacerbate
the problems and create new ones, damaging our impor-
tant and already-stressed coastal areas [59.6]. In order
to accomplish these goals, the sector will face signif-
icant challenges to increase aquaculture profitability
and, environmental and ecological sustainability.
59.2 Food Formulation Ingredients
Feeds considered traditional are mainly made from
meal and fish oil ingredients and with vegetable flours
as a carbohydrate source. These vegetable flours must
be kept in the formulation of FF and, in fact, must
be increased; besides carbohydrates, FF must con-
tain protein and oil from vegetable origin, preferen-
tially from soy [59.15]. Soy contains a large enough
quantity of proteins and lipids to substitute both an-
imal ingredients in shrimp and fresh-water fish diets.
Additionally, Litopenaeus vannamei and Oreochromis
niloticus require or tolerate protein and carbohydrate
59.1.2 Development
of Functional Feed
Today, feeding represents 4060% of the total pro-
duction costs in shrimp/fish farming, making the aqua-
culture industry a great challenge for future genera-
tions; therefore, new feed formulations must be di-
rected to be well-balanced and less expensive [59.7].
Use of animal protein sources, such as fish meal
(FM) in shrimp/fish feeds, is expected to be con-
siderably reduced or totally eliminated as a conse-
quence of increasing economical, environmental and
sanitary regulations [59.8–11]. Partial or complete FM
substitution by vegetable protein and complex car-
bohydrates sources has been a major concern to the
field for more than 10 years, without promising re-
sults [59.12, 13]. In this sense, functional feeds devel-
opment as foods with dietary ingredients that provide
growth, health, environmental and economical benefits
beyond traditional feeds represents a great opportu-
nity to guarantee the future of Aquaculture [59.14]. FF
must be primarily supplemented with high levels of
vegetable protein, complex carbohydrates and specifi-
cally selected innocuous probiotic bacteria (Fig. 59.2).
A well formulated FF could transform aquaculture in
a sustainable and competitive industry for new gen-
erations [59.15]. The present work evaluated a FF
supplemented with high levels of soy bean meal (SBM),
high levels of complex carbohydrates and a Bacillus
subtilis probiotic strain on; Litopennaeus vannamei,
Oreochromis niloticus and Atractoscion nobilis. Excel-
lent results were obtained opening great opportunities
to new functional feeds development for shrimp and
fish.
(starch) concentrations of around 30% [59.16, 17].
However, carnivorous marine fishes require or toler-
ate carbohydrate concentrations no greater than 10%,
but they need 50% protein concentration for ade-
quate development [59.18]. It is logical to specu-
late that such extreme demands are due to the fact
that this kind of fish does not contain adequate
types and/or amounts of carbohydrolases to digest
starches; therefore their energy requirement must be
obtained from protein, increasing the cost for its
cultivation.

US $ per tonne
1800
1600
Fishmeal
1400
Fish oil
1200
1000
800
600
400
200
0
1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009
Fig. 59.2 Functional feed particle formulated with SBM,
CHO’s, vegetable oil and Bacillus subtilis multifunctional
probiotic strain
On the other hand, it is important to point out that
there are 20 most represented amino acids in the major-
ity of proteins present in nature, coming from animal or
vegetable origin. In this sense, the conformation struc-
ture is the greatest difference between proteins [59.19].
Furthermore, if added probiotics bacteria are capable of
transforming the proteins into amino acids, there will
be no problems for shrimp and/or fish to assimilate and
incorporate them into their biosynthetic pathways.
Similar situations occur with carbohydrates regard-
ing digestion. Although this is more marked in marine
fishes, also fresh-water fishes and shrimp present en-
zymatic deficiencies in their digestion, but in smaller
magnitude [59.20–22]. This is logical if we consider
that aquatic animals did not evolve with starch, the main
component used in formulations, therefore, they did not
have the need to produce carbohydrolases to digest it,
producing severe digestive, health and contamination
problems. Hence, in the last 10 years, many ingredi-
ents of animal and vegetable origin have been used to
Functional Feeds in Aquaculture 59.2 Food Formulation Ingredients 1305
Fig. 59.1 International market price
Part J | 59.2
for fish oil and fish meal
Year
substitute meal and fish oil, without the expected suc-
cess [59.23]. Subsequently, with the increasing knowl-
edge of the digestive physiology, we have tried to
adapt the diets to the enzymatic capability of animals,
which has not been practical or economically viable
for commercial aquaculture. In this sense, our innova-
tion has been focused on understanding the enzymatic
deficiencies in animals and the benefits of vegetable in-
gredients, to general knowledge we will generate FF
that contains enzymatic profiles adequate for diges-
tion and assimilation of proteins, carbohydrates, and
lipids, independently from the source that they come
from.
59.2.1 Fish Meal and Oil
Several decades ago small pelagic fish, were the great-
est economical and nutritional source of protein and
oil that existed. However, the growing aquaculture and
livestock activities in general have provoked an exces-
sive demand, and consequently the over-exploitation of
fish stock used for this purpose, causing an ecological
imbalance and an increase in prices, making it unaf-
fordable to producers. Worse than the elevated prices
is the fact that the capture of fish has been decreasing
since 1996, and human need for consumption of aquatic
products has been increasing [59.3]. Reduced inclusion
of fish meal and oil in the formulations is a tendency
for almost a decade. Furthermore, it has been predicted
that by 2020, the amount of fish meal and oil in diets
used in aquatic cultures will be minimal or null [59.3].
It is evident that in times of abundance, excessive quan-
tities of fish meal and oil were used, without having
a clear idea of the real nutrient necessities for these an-
imals. Now that we have enough knowledge, we know
that if the amount of protein and oil is reduced be-
1306 Part J Industrial Applications

Fig. 59.3 Estimated global use of

Part J | 59.2

Present study IFFO–Jackson (2007)

fish oil within compound aquafeeds
Shrimp
Marine finfish
4% 3% Salmon 2%
5% 27 % 5% 5%
Chinese carps 24%
5% 6%
Trout
6% Eel
Catfish
6% 17 %
Tilapia
Freshwater
11 % 18 % crustaceans 23 %
Misc. freshwater
15 % carnivore fish 17 %
Milkfish

Present study IFFO–Jackson (2007)

Fig. 59.4 Estimated global use of
fish oil within compound aquafeeds
1%
2% 1% Salmon 1%
1%
2% Marine finfish
2% 13 %
4% Trout
Shrimp 3%
12% Catfish
43 % Eel 43 %
Tilapia 10 %
13 % Freshwater
crustaceans
11 %
Misc. freshwater
carnivore fish
20% 14 %
Milkfish

low the nutritional requirements, the animals will not
grow and gain weight properly. From a ton of processed
fish, approximately 25% of meal is obtained, containing
an average of 60% digestible protein. In shrimp and ma-
rine fish feeds the fish meal is added in concentrations
of about 2050%, respectively (Fig. 59.3). Nowadays,
we know that most carnivores require between 2030%
protein and similar amounts of carbohydrates. Regard-
ing this, the aquaculture industry has had to search for
viable alternative digestible and economical ingredi-
ents to satisfy the nutritional requirements of cultivated
animals. Soy is the most probable candidate due to
its high concentration of protein, carbohydrates, and
lipids [59.24].
In addition, the limited capacity of fish and shrimp
to digest starch has induced fish meal to be used by ani-
mals as a source of protein and energy, making feeding
less efficient and more expensive [59.20–22, 25–29].
Regarding fish oil, the problem is even greater than
with fish meal, because the amount generated by a ton
of processed fish is less than 10%, and the require-
ments in shrimp and fish diets are between 10 and
40%, respectively (Fig. 59.4) [59.3, 18]. In fact, all the
produced oil is used in aquaculture feeds, increasing
the price of this ingredient even higher than fish meal
(Fig. 59.1). The possible candidates to replace fish oil
are vegetable oils with profiles of saturated and un-
saturated fatty acids, similar to those present on fish.
In this sense, the oil of some microalgae species ful-
fills all the requirements necessary to replace fish oil.
Analyzing fatty acids, especially the unsaturated ones
contained in fish oil, most come from marine phyto-
plankton, since the animals does not produce them de
novo (Table 59.1). Presently, the technology to pro-
duce fatty acids from microalgae is well established,
due to the interest in the use of these lipids to generate
biodiesel. This technology can facilitate lipid produc-
tion in the required volumes for aquaculture, assure its
availability, and reduce its cost [59.30].
59.2.2 Soybean Meal and Oil
SBM is the most studied ingredient for the substitution
of fish meal in aquatic animal feeds, because it con-
tains high levels of proteins and carbohydrates and may
represent half or less of the FM price. However, un-
 Functional Feeds in Aquaculture 59.2 Food Formulation Ingredients 1307

Table 59.1 Fatty acids comparison between fish oil and algae oil

Part J | 59.2
Fatty acids Formula Fish oil Algae oil SL
Lauric C12:0 0.16; 0.01 0.32; 0.00 ***
Myristic C14:0 7.04; 0.35 9.09; 0.00 ***
Palmitic C16:0 17.33; 0.59 22.86; 0.00 ***
t-Palmitoleic C16:1t 0.57; 0.18 0.07; 0.00 ***
Palmitoleic C16:1 7.96; 0.40 0.21: 0.00 ***
Stearic C18:0 3.50; 0.07 0.57; 0.00 ***
Elaidic C18:1t 1.17; 0.11 0.01; 0.00 ***
Oleic C18:1(!-9) 8.69; 0.29 1.11; 0.02 ***
Vaccenic C18:l(!-7) 3.11; 0.11 0.13; 0.0 1 ***
t-Linoleic C18:2t 0.06; 0.02 0.06; 0.00 sl
Linoleic C18:2(!-6) 1.26; 0.05 0.46; 0.02 ***
Arachidic C20:0 0.20; 0.01 0.00; 0.00 ***
 -Iinolénico C18:3(!-6) 0.22; 0.02 0.22; 0.00 s1
˛-linolenic acid C18:3(!-3) 1.16; 0.07 0.09; 0.00 ***
Behenic C22:0 0.05; 0.00 0.03; 0.00 ***
Brassidic C20:1t 1.84; 0.02 0.41; 0.00 ***
Erucic C22:1 0.05; 0.00 1.71; 0.00 ***
Arachidonic C20:4(!-6) 1.14; 0.10 0.51; 0.01 ***
Eicosapentaenoic C20:5(!-3) 16.92; 2.03 1.25; 0.01 ***
Docosapentaenoic C22:5(!-6) 0.00; 0.00 15.44; 0.00 ***
Nervonic C24:1 0.60; 0.00 0.00; 0.00 ***
Docosapentaenoic C22:5(!-3) 2.23; 0.00 0.22; 0.00 ***
Docosahexaenoic C22:6(!-3) 13.44; 0.58 42.41; 0.02 ***

SL: level of significance; ***: p < 0:001; s1: no significance: p > 0:05

satisfactory results have been obtained because SBM
contains compounds that are toxic and antinutritional
to monogastric animals, and no more than 20% can be
included in shrimp/fish formulations without causing
health problems [59.31, 32].
Furthermore, flour from oleaginous seeds offer an-
nually more than 200 million metric tons (MT), soy
being the most abundant and in expansion. Soybean
meal has a natural resistance to oxidation and in storage
it stays fungus and bacteria free, that can be pathogenic
to shrimp and fish development [59.33, 34]. The protein
fraction of soybean meal is between 4548% for com-
mon meals, while the premium grade exceeds 50%.
Concerning its carbohydrate composition, soybean
meal contains approximately 32% from which 12% are
soluble sugars, distributed as follows; 45% sucrose,
12% raffinose, 3:54:5% stachyose, as well as small
amounts of melibiose and verbascose. 20% the remain-
ing fraction which is difficult to digest are known as
anti-nutritional compounds, due to the toxic effects they
causes in monogastric animals (Table 59.2) [59.34].
Soybean oil is another possible candidate to replace
fish oil, since it also contains enough quantities of es-
sential polyunsaturated fatty acids (omega 3 and 6), to
satisfy aquaculture and consumer needs. The growing
demand of soybean oil in homes around the world is due
to its low cost and the amounts of omega 3 (linolenic
7:5%), omega 6 (linoleic 54%), and omega 9 (oleic
22%). It is important to comment that oleic acid is a mo-
nounsaturated fatty acid, which can be transformed to
polyunsaturated acid by the enzymatic machinery of an-
imals. Furthermore, soybean oil also contains saturated
fatty acids such as palmitic acid (1112%) and stearic
acid (35%).
59.2.3 Complex
and Most Used Carbohydrates
Today starch present in corn and wheat flour is the most
utilized source of energy; for economical and availabil-
ity reasons. Additionally, it has been shown that starch
may replace protein as an energy source without de-
1308 Part J Industrial Applications

Table 59.2 Non-starch polysaccharide (NSP) content from cereals and soybean meal
Part J | 59.3

Dry matter % Carbohydrates Corn Wheat Barley Soybean meal

NSP 9.9 11.9 18.7 21.9
Arabinoxylans Arabinose 2.2 2.9 2.8 2.6
Xylose 3 4.7 5.6 1.9
Uronic acid 0.7 0.4 – 4.8
Pectins Mannose 0.3 0.3 0.4 1.3
Rhamnose – – – 0.3
Galactose 0.5 0.4 0.3 4.1
ˇ-glucans Glucose 0.1 0.8 4.2 –
Cellulose Glucose 2.2 2 4.3 6.2

creasing growth [59.20, 35]. However, starch is one of
the less digestible ingredients in fish and crustaceans,
and high-level inclusions represent an important ob-
stacle to shrimp/fish aquaculture; the carbohydrate di-
gestion capabilities of animals are limited to no more
than 10% in carnivorous fishes and 30% in tilapia and
shrimps [59.20, 21, 36]. Nevertheless, higher carbohy-
drate levels are included in feeds because they are the
most economical and for this reason the most used
ingredient in the aquaculture industry. The identifica-
tion of glucosidases producing probiotic bacteria and
their addition to low-cost diets as a digestive promot-
ing supplement should lead to better assimilation, lower
production cost, faster animals growth, and less pollu-
tion and diseases [59.22].
59.2.4 Probiotic Bacteria
Recently, beneficial microorganisms (probiotics) were
utilized to improve nutrient assimilation and to en-
hance the growth performance and health of mono-
Safe for humans
and animals (FDA)
Natural
antibiotics
Growth in
economical
Bacillus subtilis substrates
Spores
producer
Aerobic
Proteases Anaerobic
Carbohydrolases
Lipases
Fig. 59.5 Bacillus subtilis multifunctional probiotic capacities
gastric animals [59.22, 37, 38]. Additionally, pollution
of shrimp effluent ponds was also treated with mi-
croorganisms [59.39]. In this sense, we have defined
a probiotic as a living microbial supplement that:
a) Positively affects hosts by modifying the host-
associated microbial community and immune
system.
b) Secretes a variety of enzymes to improve feed
degradation, enhancing its assimilation.
c) Improves the quality of environmental parame-
ters [59.14].
Bacillus probiotic strains have been used to im-
prove growth performance, digestive enzyme activity,
and immune response, with good results being ob-
tained in all the parameters measured [59.22, 40–42].
The genus Bacillus constitutes a diverse group of rod-
shaped, Gram-positive bacteria, characterized by their
ability to produce a robust spore. Most Bacillus species
are not harmful to mammals, including humans, and
are commercially important as producers of a high
and diverse amount of secondary metabolites, antibi-
otics, heterologous proteins, fine chemicals, and en-
zymes [59.43–47]. Bacillus enzymes are very efficient
in breaking down a large variety of proteins, carbohy-
drates, and lipids into smaller units. Bacillus species
grow efficiently with very low-cost carbon and nitrogen
sources [59.43]. They also degrade organic accumu-
lated debris in ponds of shrimp cultures [59.14, 48–50].
Taking into account the advantageous characteristics
of Bacillus strains, these bacteria are good candidates
for consideration as probiotics in diets of crustaceans
and fish [59.14, 15]. Additionally, B. subtilis is gener-
ally recognized as safe (GRAS) by the FDA, meaning
that this bacterium is not harmful to animals or humans
(Fig. 59.5).
 Functional Feeds in Aquaculture
59.3 Conventional Feeds Versus Functional Feeds
Currently, the cultivation of shrimp and fish is very
important, socially and economically speaking, being
Litopenaeus vannamei and Oreochromis niloticus the
most produced around the world. However, apart from
the questions about which culture is the most important
or which generates the greatest economical dividends,
the diets utilized for the aquaculture of any animal are
developed with mainly three ingredients:
1) Fish meal, because it contains high concentrations
of easily digestible protein, which promotes optimal
growth and weight gain in cultivated animals.
2) Carbohydrates that come from vegetable flour, spe-
cially those present in corn and wheat starches,
which give the necessary energy for vital function-
ing.
3) Lipids that are obtained as a by-product from the
processing of fish meal and are the structural base
for the production of hormones and other important
bioactive macromolecules.
However the use of fish meal and oil in the pro-
duction of feeds is limited due to costs, principally
since these ingredients come from fish that are at
risk due to unmeasured exploitation [59.3, 51]. In this
sense, the production of feeds that does not depend
on the utilization of fish products has been widely
investigated during the last 10 years, and there has
been experimentation with a wide range of animal
and vegetable ingredients [59.52]. Unfortunately, up
to now even the high cost of fish products and by-
products, feeds are for aquaculture still being formu-
lated with them. This means that alternative formula-
tions that can compete with the benefits of survival,
weight gain, and growth that fish meal and oil ingre-
dients generate has not yet been found. Alternative
feeds produced and sold in a commercial manner as
a consequence of the high prices, are mixtures re-
duced in fish meal and oil content and increased in
the amount of vegetable protein and oils. This has
brought as a consequence low food conversion fac-
tors (FCF), poor growth, intoxication and health sta-
tus detriment. Also, the use of vegetable products
has generated an increase in environmental contami-
nation due to the low digestibility and, consequently,
59.3 Conventional Feeds Versus Functional Feeds 1309
Part J | 59.4
pathogen proliferation and massive death of the cul-
tured animals.
For these reasons, our research group has been try-
ing for more than 10 years to understand the most
important strengths and weaknesses of cultivated an-
imals [59.14, 19]. Additionally, alternative vegetable
ingredients economically viable to substitute fish meal
and oil have been investigated [59.15, 22]. Also, the an-
imal-environment-pathogen relationship had been stud-
ied to generate innovations that can fully satisfy aqua-
culture necessities [59.53, 54]. After all this time, and
after having experimented with crustacean and fish
cultivations, we believe that we have found healthy for-
mulas that are sustainable with the environment and that
are economically viable, that we have named functional
feed.
According to our concept, FF must be formulated
with little or no fish meal and oil, instead vegetable in-
gredients increase is recommended; this will decrease
its price and ammonium pond contamination consider-
ably. Furthermore, it will relax the fish population and
environmental stress. However, vegetable ingredients
are not recommended by most nutritionists, due to their
low digestibility, contents of highly toxic compounds,
and mortality increase. We believe that FF must contain
high levels of vegetable flour and oils; rich in proteins,
carbohydrates, and lipids. Micronutrients must be sup-
plemented for the optimal development of the animals.
In this sense and in our particular point of view, the
most important components of FF are probiotic bacte-
ria, since they produce enzymes with several capacities
to amplify the digestive spectrum and help the animals
to assimilate the ingredients, even if they come from
vegetable origin. Likewise, the enzymes must have the
capacity to degrade the toxic compounds contained in
vegetable flour [59.55]. The enzymatic diversity and ac-
tivity of proteases, carbohydrolases, and lipases must
be maintained, even in adverse physicochemical or
environmental conditions such as temperature, pH, oxy-
genation, etc.; so the enzymes will not be inhibited in
the digestive system of animals or in cultivation ponds
once they are excreted. This also will help with the ef-
fluent bio-remediation. Probiotics must be harmless to
animals and humans, but must have the capacity to se-
lectively inhibit pathogen growth.
1310 Part J Industrial Applications

59.4 Aquaculture Regulations

Part J | 59.4

Recently, the massive development of aquaculture has be the main reason of environmental deterioration
promoted the establishment of sanitary and environ- due to the contamination generated, resulting in the
mental regulations in most countries where this activity spread of diseases and death of cultivated animals
is practiced. This is particularly due to the spread (Fig. 59.7).
of diseases and environmental contamination gener-
ated by poor cultivation management (Fig. 59.6). It 59.4.1 Sanitary Regulations
is important to emphasize that the majority of these
regulations have been generated in little or in great The formulation of feeds with animal origin ingredients
measurement due to the feed; starting with the in- can be a factor in the spread of viral as well as bac-
gredients and concentrations utilized in them, up to terial diseases, that can be contained in animal-based
the amount of feed administered during cultivation. ingredients. The feed utilized in aquaculture is some-
Furthermore, the time period of water exchange in times distributed around the world and can generate
ponds is also a consequence of the feed and the global pandemics and epizooties, like the ones pro-
enzymatic limitation of animals must digest and as- duced by Vibrio genera and the white spot syndrome
similate them. Therefore, poorly formulated feeds can virus [59.56, 57]. Therefore, animal products and by-
products used for the formulation of feed must be certi-
fied by the sanitary authorities of each country to verify
Losses in aquaculture
that it does not contain pathogen microorganisms above
the concentrations permitted by the official norms. This
20.8 % Diseases
will assure its innocuousness but raises the production
30.6 % Transportation costs of these ingredients, and thus, the cost of the for-
Predators mulated feed. Furthermore, cysts and spores are forms
Human error of life that are resistant to extreme conditions presented
13.9 % Water shortage
Pollution
by some pathogenic bacteria [59.58]. This characteris-
6.1 %
Defective design tic allows them to survive the conditions used in the
8.3 % 5.3 % Environment preparation of products and by-products of animal ori-
7.5 % 7.5 % gin, only to germinate once the product is in contact
with water and the cultivated animal. In this sense,
Fig. 59.6 Schematic representation of loss percentages in the prevention of diseases has also generated an un-
aquaculture measured use of antibiotics, which has induced the
development of pathogenic bacteria stock resistant to
a great variety of these compounds. It is important
to keep in mind that human beings can be suscepti-
ble to the antibiotics used in aquaculture, as well as
Environment to pathogens present in cultivated animals, especially
when that person has an immune compromised system.
Vibrio parahemoliticus is an opportunistic pathogen
that can cause death to individuals with problems such
FF as liver cirrhosis, if they consume contaminated oys-
ters. The transmission of the disease known as Mad
Pathogen Host Cow Disease and other diseases that come from poul-
try stock are examples of the importance of the in-
nocuousness and sanitary regulations that have to be
implemented in products from animal origin [59.59].
In this sense, the use of vegetable ingredients in FF
Fig. 59.7 Illustration of the balance involved in the state can eliminate the pathogen transference contained in
of health and disease of a cultivated organism with or with- animal products, as well as the unmeasured use of
out FF antibiotics.
 Functional Feeds in Aquaculture
59.4.2 Environmental Regulations
Environmental regulations with respect to aquaculture,
even when they are stipulated in the official norms and
are very clear regarding the compounds and quanti-
ties that can be thrown into rivers or seas; in most
of the countries where massive production of fishes
and crustaceans is being practiced, are not being com-
plied. Therefore, high concentrations of organic con-
taminant compounds are indiscriminately thrown into
rivers and seas each year, generating constant con-
tamination and deterioration of the environment and
59.5 Functional Feeds in Aquaculture
Autochthonous bacteria of crustaceans and fishes can
be recommended as probiotics, only if they have the
capacity of secreting a wide variety of enzymes that
will allow them to take adequate advantage of differ-
ent sources of protein, carbohydrates, and lipids. Also,
they must be capable of producing antimicrobial com-
pounds to inhibit the growth of different pathogens,
without having any effect on beneficial bacterial flora.
They must also be able of surviving sudden nutritional
and environmental changes, such as temperature, pH,
oxygenation, etc. Furthermore, autochthonous strains
are, in general, bacteria that adhere to the cells of the
animal’s intestine and, therefore, are circumscribed to
scarcely variable nutritional and environmental condi-
tions. For this reason, most of them do not have the
capacities mentioned before. On the other hand, free
life strains like Bacillus subtilis have all these capaci-
ties, hence it can be considered as a potential probiotic.
Also, this bacterium is classified by the FDA as GRAS,
which means that is not a pathogen to animals or hu-
mans [59.22].
59.5.1 Advantages and Disadvantages
of Adhesion
The advantage of adhesion is that probiotics should
only be used a few times until they have colonized
the digestive tract, which would reduce the cost and
would increase the profitability of producers. Other-
wise, the capacity of adhesion means that the strains
are adapted to live within the host, which assures a high
percentage of interaction, as long as the conditions of
the environment are satisfactory. Unfortunately, most
pathogens have a highly developed capacity of adhe-
59.5 Functional Feeds in Aquaculture 1311
ecosystem that surrounds farms. Unfortunately, most
Part J | 59.5
countries where aquaculture is being practiced, even
with the existence of official norms, do not count
with mechanisms to carry out those norms. In addi-
tion, the formulation and usage of FF of easy digestion
and assimilation by cultivated animals will generate
low concentrations of organic waste, and can be a di-
rect mechanism to reduce contamination of ponds and
waste that is thrown into rivers and seas [59.14, 60]. In
this sense, the probiotics used in FF must also have
the capacity to degrade the contaminants generated in
farms.
sion as an invasive mechanism. In this sense, it is almost
a general rule that when cultivation is in its initial
stage, the bacterial flora present is composed by cer-
tain groups. However, once cultivation advances and
conditions deteriorate, more capable bacterial groups
surge, taking advantage of the reigning nutritional and
environmental conditions. Most proliferating bacteria
are opportunistic pathogens with a high capacity of ad-
hesion. Therefore, if we isolate bacterial strains from
aquatic cultivated organisms with the purpose of uti-
lizing them as probiotics, it is recommended to isolate
them from the first stages of the cultivation or from fi-
nalized healthy and high-productivity cultures.
In the late stages of cultivation, the possibility
of the isolation of opportunistic pathogenic bacteria
rises [59.54, 60]. Lately, it has been very complicated
to keep healthy cultures with economically income,
mainly due to food expenses and the great amount of
energy used for water exchange in the required percent-
ages. This is one of the reason for the huge proliferation
of pathogens and the consequent mortality in cultures.
Generally, when an opportunistic pathogenic bacterium
has adequate conditions it takes advantage of it to
proliferate.
If we take probiotics from humans as an example,
most of them are Lactobacillus species with adhesion
capacity to epithelial intestinal cells, which means that
a daily dose must be taken to maintain their active
growth and probiotic capacity. In this sense, we know
for a fact that up to this date there is no commer-
cial strain that is taken once and maintains its activity
during the rest of the host life. For example, daily or
almost daily consumption of Lactobacillus-based prod-
ucts is needed to keep its positive effects; this is due
1312 Part J Industrial Applications

to the feeding conditions and stress factors that restrict This also happens with unauthorized antibiotics and
Part J | 59.6

the proliferation of probiotic strains on cultivated ani- hormones.

mals. Yet, it is more complicated to maintain probiotic
strains that are permanently in contact with a water flow
in the digestive tract, as is the case for fish and crus-
taceans. If we add the fact that most of enzymes and
antimicrobial compounds are produced in a short win-
dow of the stationary phase from most bacteria, the
protection due to the production of these compounds
will also be restricted to when the probiotic remains
inside the animal. Additionally, due to its evolutionary
environment the autochthonous bacteria of crustaceans
and fish have never been in contact with carbohy-
drates of terrestrial origin such as starch, which is the
main ingredient in used formulations. Therefore, the
physiological and economical conditions to utilize an
autochthonous probiotic strain added to the epithelium
cells, do not appear, in theory, to have advantages with
respect to those free living bacteria. Otherwise, if we
have a probiotic that adheres well to epithelium cells,
we need to take into consideration that the elimination
of these bacteria from the digestive tract of the ani-
mals is more complicated. In this sense, if the probiotic
is not recognized as safe and is not approved by the
FDA, it can generate problems when the time comes
to commercialize the animal for human consumption.
Even though the diverse marine and aquatic sys-
tems represent 85% of all forms of life on earth, it
is important to point out that if we are going to use
FF especially formulated with nutrients of land origin,
we should select probiotic strains with the capacity to
digest them without any problem. Therefore, if we de-
velop free living bacteria probiotics strains accustomed
to surviving in extreme conditions, with an abundance
as well as limitations of nutrients, salts, temperature,
and variable pH, they will have a greater adaptive re-
sponse to sudden changes in the culture. Moreover, if
these strains count with the capacity of enzyme se-
cretion to degrade any kind of nutrient, as well as to
produce compounds to solely kill pathogenic microor-
ganisms and do not present toxic effects in animals
and humans, these strains can be considered as a po-
tential probiotics. The only inconvenience is that good
fortune and capacity is needed to isolate and character-
ize them adequately. Additionally, continuous probiotic
supplementation must be carried out to obtain an effec-
tive and therapeutic dose, and consequently, maintain
its probiotic properties. In this sense, FF are the ade-
quate vehicle to introduce probiotics into the animal,
considering stability and price.

59.6 Results Obtained in Crustaceans and Fish Using Functional Feeds

59.6.1 Functional and Commercial Feed is not the case for tilapia because it is an omnivore
Proximal Composition Used with the capacity to tolerate high concentrations of
in Litopenaeus vannamei carbohydrates.
and Oreochromis niloticus The commercial diet for shrimp has 10% more pro-
tein than the basal diet formulated by our research
Table 59.3 shows the proximal composition of the in- group. The origins of the ingredients of the com-
gredients in diets utilized for white shrimp and tilapia mercial diet for shrimp are unknown to us, although
cultivation. The formulated FF (basal diet C probiotic),
was the same for L. vannamei as for tilapia because Table 59.3 Functional and commercial feed proximal
the nutritional requirements are similar for both an- composition used in Litopenaeus vannamei and Ore-
imals. In this sense, analyzing the commercial diet ochromis niloticus
for tilapia we could see its similarity regarding the
Items % Basal C Commercial Commercial
formulated basal diet. The origin of the ingredients P FF feed to L. feed to O.
contained in the commercial diet for tilapia are not vannamei niloticus
known to us, although the basal diet ingredients were Crude protein 27.41 36.39 32
reported previously [59.14]. It is important to point out Total lipid 6.46 3.98 5
that the protein in the basal diet comes from 100%
Carbohydrates 49.50 38.42 50
soybean meal. In both diets, the carbohydrate concen-
Moisture 11.34 9.88 5
tration is around 50%, which could originate health
Ash 5.29 11.33 8
problems in shrimp, since this amount surpasses the nu-
tritional requirements of this ingredient [59.61]. This Probiotic (P) 1 kg t1 – –
 Functional Feeds in Aquaculture 59.6 Crustaceans and Fish Using Functional Feeds 1313

it is a fact that the protein used is of animal ori- with Litopenaeus vannamei (Fig. 59.8), Table 59.5

Part J | 59.6
gin. The commercial diet for shrimp contains 10% shows that the parameters analyzed improved con-
less carbohydrates than the basal diet, which is the siderably in animals fed with the basal diet contain-
closest to what is required and/or tolerated for this ing the probiotic (FF), with respect to the commer-
crustacean [59.61]. cial diet and the basal diet without the probiotic.
The food conversion ratio (FCR) was the parame-
59.6.2 Basal and Functional Feed Proximal ter showing the best result, which confirms that L.
Composition Used vannamei has difficulties digesting vegetable sources
in Atractoscion nobilis of proteins and high levels of CHO. These results
also confirm that the probiotic is the most impor-
Table 59.4 shows the basal diet containing the required tant ingredient in the formulation of FF, since the
ingredients and concentrations for Atractoscion no- inclusions of the bacteria allows the use of alterna-
bilis, a strictly carnivorous fish. From the diets CHO10 tive, economical, healthy and available nutrients [59.14,
to CHO22, the corresponding carbohydrate percentage 22].
was increased and the lipid concentrations reduced,
as indicated in the table. The probiotic was added to
all the CHO diets in concentrations of 1 kg t1 . It has
been widely reported that A. nobilis presents a health
problem when the carbohydrates contained in the diet
surpass 10%. Additionally, the development for marine
fishes is limited when the lipid concentrations in the diet
is reduced. Moreover, it is most important to maintain
the high levels of fish protein in this species, which is
why this ingredient was maintained at 50% in all the
formulated diets.

59.6.3 Functional Feed Effects

on Survival and Growth
Performance of L. vannamei,
O. niloticus and A. nobilis Fig. 59.8 Litopenaeus vannamei Pacific Ocean white
shrimp
Once the animals were fed in controlled conditions,
biometry studies were carried out to determine the di-
etary effects on growth, survival, etc. In experiments

Table 59.4 Basal and functional feed proximal composi-

tion used in Atractoscion nobilis
Items % Basal CHO CHO CHO CHO
feed 10 FF 14 FF 18 FF 22 FF
Crude 56.5 56.1 56.0 54.2 55.9
protein
Total 19.3 18.3 15.0 15.0 14.3
lipid
Carbo- 10.3 10.5 13.9 18.8 21.4
hydrates
(CHO)
Ash 9.8 10.5 10.2 10.8 8.9
Energy 5076 5102 5037 5071 5066 Fig. 59.9 Effect of FF on Oreochromis niloticus develop-
Cal g1 ment. From left to right: animals fed with basal diet C
Probiotic – 1 kg t1 1 kg t1 1 kg t1 1 kg t1 probiotic (FF), commercial diet C probiotic, commercial
(P) diet, and basal diet
1314 Part J Industrial Applications

Table 59.5 FF effects on survival and growth performance in Litopenaeus vanammei

Part J | 59.6

Items Basal feed Basal C P FF Commercial feed

Initial weight (g) 5:98 ˙ 0:22a 5:96 ˙ 0:20a 6:06 ˙ 0:18a
Final weight (g) 9:48 ˙ 0:13c 10:71 ˙ 0:11a 10:38 ˙ 0:16b
Daily weight gain (g d1 ) 0:125 ˙ 0:003c 0:169 ˙ 0:003a 0:154 ˙ 0:001b
Food conversion ratio (FCR) 2:49 ˙ 0:15c 1:54 ˙ 0:07a 2:06 ˙ 0:22b
Survival (%) 96:67 ˙ 3:87a 100a 96:67 ˙ 3:87a
Probiotic (P) 1 kg t1

Means in the same row with different superscripts are significantly different (P < 0:05)

Table 59.6 shows that the values obtained for O. sibilities and hopes for the development of healthy,
niloticus using the basal plus the probiotic bacteria sustainable, and profitable feeds in the immediate
(FF), where almost twofold greater than the results future.
obtained using the basal and the commercial diet, With respect to the results obtained in Atrac-
with the exception of the survival rates, which were toscion nobilis, the CHO22 diet tripled its weight
similar in the three assays. These results mean that and duplicated its size in comparison with the basal
the basal diet formulated for this experiment and the diet (Fig. 59.10). According to studies on this marine
commercial diet are very similar with respect to the
energy content. Moreover, it also means that even
though O. niloticus is an omnivore it shows certain
problems digesting vegetable protein and high con-
centrations of carbohydrates. When the probiotic was
added to the vegetable diet (basal diet), all parame-
ters improved almost 100% (Fig. 59.9). The biomass
and its size doubled in the same period of time and
with the same amount of food, which opens great pos-

Table 59.6 FF effects on survival and growth performance

in Oreochromis niloticus
Items Basal feed Basal C Commercial
P FF feed
Initial weight (g) 3.46a 3.90c 3.63a
Final weight (g) 5.40a 9.40c 6.74b
Absolute growth (g) 1.94a 5.50c 3.11b
Biomass gained (g) 55a 165c 81b
Specific growth rate 3.16a 8.12c 5.40b
(SGR)
Efficiency feed 0.21a 0.49c 0.30b
consumption
Food conversion 4.71a 2.04c 3.37b
ratio (FCR)
Protein efficiency 4.95a 10.78c 6.92b
ratio (PER)
Survival (%) 100a 100a 90b
Probiotic (P) 1 kg t1
Fig. 59.10 Effect of FF on Atractoscion nobilis develop-
Means in the same row with different superscripts are signifi-
ment. From top to bottom: animals fed with basal diet,
cantly different (P < 0:05)
CHO10, CHO14, CHO18, and CHO22 FF
 Functional Feeds in Aquaculture 59.6 Crustaceans and Fish Using Functional Feeds 1315

animals have sources of energy that are easier to as-

Part J | 59.6
a) Ammonium (mgL–1)
similate than protein, they will prefer them because
0.8 c
0.7 Basal diet
the energy wasted to metabolize them is less signif-
0.6 Basal + P icant [59.19]. In this sense, the CHO22 FF diet was
c b
0.5 Commercial very successful in all the parameters evaluated, due to
0.4 b this diet, the animal used protein to grow and carbohy-
0.3 c drates as a source of energy. Because the weight was
b
0.2 tripled using FF, regarding the basal diet, it is probable
0.1 a a that the extra energy from digested-assimilated carbo-
a
0 hydrates was canalized to generate protein, as happened
Day 14 Day 21 Day 36
in assays carried out with L. vannamei [59.14]. The in-
b) Phosphate (mgL–1) terconnection of the biosynthetic pathways gives the
4 c possibility to redirect the carbon flow and send it to
3.5 Basal diet
3 Basal + P where it is required most, as long as the system is
c in balance between the nutrient entry and the output
2.5 Commercial
2 waste discarded from the animal cells. All this shows,
c b
1.5 as demonstrated in this experiment and in the one with
b
1
b
tilapia and shrimp, that it is possible to use complex
0.5 a a a sources of proteins, carbohydrates, and lipids, when
0 probiotic bacteria are included to carry out processes
Day 14 Day 21 Day 36
to control the liberation of nutrients independently of
Fig. 59.11a,b FF environmental parameter reduction. their origin.
(a) Ammonium evaluation and (b) phosphate evaluation According to the results obtained in this study (Ta-
bles 59.5–59.7), the FF effects were more impressive in
and strictly carnivorous species, it tolerates a maxi- fish for the basic and simple reason that fish eat the pel-
mum of 10% carbohydrates in its diet without having let complete and shrimp chew their food. This small but
health problems. However, the results in Table 59.7 great difference induces fish to obtain a complete dose
show that it is possible to increase the carbohydrate of FF and its consequent benefits. This is not the same
levels up to 22% without producing toxic effects in for shrimp, which because of the way they feed, only
the animal. These results also demonstrate that if these take advantage of half or less of the FF.

Table 59.7 Effect of FF on survival and growth performance of Atractoscion nobilis

Items Basal feed CHO 10 FF CHO 14 FF CHO 18 FF CHO 22 FF
Initial weight (g) 9:63 ˙ 0:07 9:52 ˙ 0:04 9:50 ˙ 0:20 9:37 ˙ 0:20 9:51 ˙ 0:11
Final weight (g) 18:26 ˙ 1:04ed 21:37 ˙ 0:15dc 21:67 ˙ 0:59c 27:45 ˙ 1:88b 33:77 ˙ 1:47a
Gained weight (g) 8:63 ˙ 1:02e 11:85 ˙ 0:11dc 12:16 ˙ 0:78c 18:08 ˙ 1:84b 24:26 ˙ 1:38a
Initial length (cm) 9:02 ˙ 0:06 9:03 ˙ 0:04 8:87 ˙ 0:16 8:99 ˙ 0:09 9:04 ˙ 0:05
Final length (cm) 11:66 ˙ 0:05ed 11:90 ˙ 0:02dc 12:12 ˙ 0:10c 12:91 ˙ 0:22b 13:65 ˙ 0:13a
Gained length (cm) 2:64 ˙ 0:07ed 2:88 ˙ 0:02d 3:26 ˙ 0:18c 3:92 ˙ 0:16b 4:62 ˙ 0:08a
Specific Growth Rate 1:06 ˙ 0:09e 1:35 ˙ 0:01dc 1:37 ˙ 0:08c 1:79 ˙ 0:11b 2:11 ˙ 0:06a
(SGR)
Efficiency of feed conver- 0:62 ˙ 0:07ed 0:81 ˙ 0:01c 0:74 ˙ 0:05dc 1:00 ˙ 0:10ba 1:17 ˙ 0:07a
sion (EFC)
Protein efficiency ratio 1:09 ˙ 0:13e 1:44 ˙ 0:01d 2:36 ˙ 0:06a 1:85 ˙ 0:19cb 2:10 ˙ 0:12ba
(PER)
Survival % 88:0 ˙ 0:88 98:0 ˙ 0:00 90:0 ˙ 0:58 93:0 ˙ 0:58 100:0 ˙ 0:00
Probiotic (P) – 1 kg t1 1 kg t1 1 kg t1 1 kg t1

Means in the same row with different superscripts are significantly different (P < 0:05)
1316 Part J Industrial Applications

Table 59.8 Functional feed effects increasing energy sources and immunology parameters in Litopenaeus vannamei and
Part J | 59.6

Atractoscion nobilis
Parameters L. vannamei A. nobilis
Basal feed Basal C P FF Commercial feed Basal feed CHO 22 FF
Glucose (mmol L1 ) 0:470 ˙ 0:02b 0:675 ˙ 0:02a 0:452 ˙ 0:03b 80 ˙ 3:7b 168 ˙ 3:6a
Lactate (mmol L1 ) 0:261 ˙ 0:02b 0:385a ˙ 0:03a 0:249 ˙ 0:03b
Total cholesterol 0:134 ˙ 0:07b 0:323 ˙ 0:08a 0:163 ˙ 0:03b
(mmol L1 )
Haemocytes (cell mL1 ) 9:41106 ˙0:15b 2:02107 ˙0:08a 9:63  106 ˙
0:12b
Red cells (cell mL1 ) 1:5 ˙ 0:3b 2:0 ˙ 0:3a
Hb (G dL1 ) 12:5 ˙ 1:8b 14:3 ˙ 2:3a

Means in the same row with different superscripts are significantly different (P < 0:05)

Table 59.9 FF effects on stress tolerance in Litopenaeus vannamei

Parameters Survival %
Basal feed Basal C P FF Commercial feed
Stress tolerance to high ammonium concentrations 0:0b 83:33 ˙ 0:11b 0:0b
Stress tolerance to low oxygen concentrations 66:67 ˙ 2:22b 91:67 ˙ 3:20a 41:67 ˙ 4:18c

Means in the same row with different superscripts are significantly different (P < 0:05)

59.6.4 Functional Feed Effects
on L. vannamei
and A. nobilis –
Health Status
The capacity of the probiotic used to inhibit the growth
of the pathogens due to the production of antimicro-
bial compounds was demonstrated previously [59.15].
Additionally, the improvement in the health status of
the animals was evidenced with the increase of blood
cells and energy sources like glucose and lactate; these
results were obtained exclusively in treatments where
FF had been used (Table 59.8). This means that an-
imals assimilate nutrients in greater amounts, due to
the increase in the digestibility processes that was
made possible by the probiotic enzymes. Because of
the increase in weight and size, it was evident that
if the animals have a greater nutrient concentration
in the bloodstream, a greater generation of biomass
and energy will be obtained in animals that consume
FF. Conventional nutrition classifies the increase in
glucose, lactose, etc. as stress indicators in animals.
However, if we consider that the animals are in perfect
shape, are bigger, weigh more, and are more vigor-
ous, it is logical to translate these increases in blood
parameters as a direct measurement of health status
improvement, rather than a state of stress in animals
consuming FF. A greater number of blood parameters
evaluated in O. niloticus and A. nobilis will be shown
in studies published in specialized journals in the near
future.
59.6.5 Functional Feed Effects
on Environmental Parameters
in L. vannamei
The environment where cultivated animals develop is as
important as their nutrition and/or state of health, since
the three parameters are in close relationship with each
other (Fig. 59.7). Because the conditions of the environ-
ment are directly related with the quality of food, our
conception of a FF take into account the decrease in wa-
ter contamination; due to the characteristics of the food
with vegetable ingredients, and/or the intrinsic capac-
ity of bioremediation by the probiotic added to the FF.
In this sense, all the contaminants evaluated in the cul-
ture shrimp ponds that used FF showed a considerable
decrease in water concentration, in comparison to the
assays where no FF were used (Fig. 59.11). Regarding
the results obtained with the basal diet without the pro-
biotic, this is due in part by the nature of the food, which
is less pollutant than the commercial diet [59.14, 22].

59.6.6 Functional Feed Effects
on Stress Tolerance
in L. vannamei
Ammonia stress experiments carried out by Olmos
et al. [59.14] showed that shrimp fed with FF tolerated
well high levels of ammonia throughout the 24 h of the
experiment (Table 59.9). Taking into account that basal
and commercial diet do not presented any survival rate
and that glucose and lactate were higher and statisti-
cally different in animals treated with FF (Table 59.8),
we can hypothesize that:
a) Shrimp survived due a higher energy level availabil-
ity on the circulatory system, giving them a greater
response capacity,
b) by a direct ammonia conversion-transformation
reaction accomplished by the probiotic bacte-
ria [59.14].
59.7 Conclusions
The benefits of FF are the following:
1. FF promotes the digestion of ingredients and assim-
ilation of nutrients, improving growth, weight gain,
and FCR.
2. FF increases the health status of animals, elim-
inating pathogens and stimulating the immune
system.
3. FF improves the quality of water in culture ponds,
making them environmentally sustainable.
References
59.1 FAO: The State of World Fisheries and Aquacul-
ture (FAO Fisheries Department, Rome 2006) p.
159
59.2 E.A. Amaya, D.A. Daves, D.B. Rouse: Replacement
of fish meal in practical diets for the Pacific
whiteshrimp (Litopenaeus vannamei) reared un-
der pond conditions, Aquaculture 262, 393–401
(1997)
59.3 A.G.J. Tacon, M. Metian: Global overview on the
use of fish meal and fish oil in industrially com-
pounded aquafeeds: Trends and future prospects,
Aquaculture 285, 146–158 (2008)
59.4 E.A. Amaya, D. Daves, D. Rouse: Alternative diets for
the Pacific white shrimp Litopenaeus vannamei,
Aquaculture 262, 419–425 (1997)
Functional Feeds in Aquaculture References 1317
On the other hand, even when oxygen depletion
Part J | 59
stress-experiments do not presented such drastic mor-
tality results as the one presented by the ammonia
experiments, 35 and 60% shrimp mortality was oc-
curred with the use of basal and commercial diets, with
respect to 10% mortality showed by the FF treated ani-
mals (Table 59.9). These results clearly indicate that:
a) Shrimp fed with FF have the capacity to tolerate
well oxygen depletion.
b) The basal diet without the probiotic bacteria, in
comparison with the commercial diet, presented
less oxygen consumption by itself or by less am-
monia contamination [59.14].
The results obtained in the stress experiments indi-
cate that FF utilization in shrimp/fish aquaculture would
help them to tolerate extreme variation of environmen-
tal parameters in a better way than commercial diets do.
4. FF eliminates ecological stress in seas by using eco-
nomical and abundant vegetable ingredients.
5. FF are cheaper than traditional feeds, which im-
proves the competitiveness and profitability of the
producers.
6. There is no alternative than the development of FF
to assure the future of aquaculture.
7. The movie star is the probiotic, and therefore we
need to isolate a multifunctional strain.
8. Bacillus subtilis is an ideal probiotic bacteria.
59.5 C.F. Arellano, S.J. Olmos: Thermostable ˛-1, 4- and
˛-1-6-glucosidase enzymes from Bacillus sp. iso-
lated from a marine environment, World J. Micro-
biol. Biotechnol. 18, 791–795 (2002)
59.6 K. White, B. O’Neill, Z. Tzankova: At a Crossroads:
Will Aquaculture Fulfill the Promise of the Blue
Revolution? (Sea Web Aquaculture Clearinghouse
Report Providence, Rhode Island 2004)
59.7 M.N. Bautista-Teruel, P. Eusebio, T. Welsh: Utiliza-
tion of feed pea, Pisum sativum, meal as a pro-
tein source in practical diets for juvenile tiger
shrimp, Penaeus monodon, Aquaculture 225, 121–
131 (2003)
59.8 J. Bechard, K. Eastwell, P. Sholberg, G. Mazza,
B. Skura: Isolation and partial chemical character-
1318 Part J Industrial Applications
ization of an antimicrobial Peptide produced by
Part J | 59
a strain of Bacillus subtilis, J. Agric. Food Chem. 46,
5355–5361 (1998)
59.9 A. Bomba, R. Nemcová, S. Gancarcíková, R. Herich,
P. Guba, D. Mudronová: Improvement of the probi-
otic efect of micro-organisms by their combination
with maltodextrins, fructo-oligosaccharides and
polyunsaturated fattyacids, Br. J. Nutr. 88(1), 95–
99 (2002)
59.10 C.E. Boyd, C.S. Tucker: Pond Aquaculture Water
Quality Management (Kluwer, Boston 1998)
59.11 J.C. Chen, P.C. Liu, S.C. Lei: Toxicity of ammonia and
nitrite to Penaeus monodon adolescents, Aquacul-
ture 89, 127–137 (1990)
59.12 J.E. Colt, D. Armstrong: Nitrogen toxicity to crus-
taceans, fish and mollusks, Proc. Bio-eng. Symp.
Fish Culture (1981), pp. 34–47 (FCS Publ. 1)
59.13 C. Rosas, G. Curzon, G. Taboada, C. Pascual, G. Gax-
iola, A. van Wormhoudt: Effect of dietary protein
and energy levels on growth, oxygen consumption,
haemolymph and digestive gland carbohydrates,
nitrogen excretion and osmotic pressure of Litope-
naeus vannamei (Boone) and L. setiferus (Linne)
juveniles (Crustacea, Decapoda; Penaeidae), Aquac.
Res. 32(7), 531–547 (2001)
59.14 S.J. Olmos, S.J.L. Ochoa, J.P. Michel, R. Contr-
eras: Functional feed assessment on Litopenaeus
vannamei using 100% fish meal replacement by
soybean meal, high levels of complex carbohy-
drates and Bacillus probiotic strains, Mar. Drugs 9,
6 (2011)
59.15 J.L. Ochoa: Functional feeds with high soy flour,
starch and Bacillus spp. content, US2012/0128827 A1
(2012)
59.16 S.Y. Shiau: Nutrient requirements of penaeid
shrimps, Aquaculture 164, 241–250 (1998)
59.17 T. Storebakken, S. Refstie, B. Ruyter: Soy products
as fat and protein sources in fish feeds for inten-
sive aquaculture. In: Soy in Animal Nutrition, ed.
by J.K. Drackley (Federation of Animal Science So-
cieties, Savoy 2000) pp. 127–170
59.18 R.P. Wilson: Dicentrarchus labraxs. In: Nutrient Re-
quirements and Feeding of Finfish for Aquacul-
ture, ed. by D.C. Webster, C. Lim (CABI Publishing,
Wallingford 2002) pp. 144–175
59.19 D.L. Nelson, M.M. Cox, A.L. Lehninger: Lehninger
Principles of Biochemistry (W.H. Freeman, New York
2008)
59.20 P. Le Chevalier, A. Van Wormhoudt: Alpha-
glucosidase from the hepatopancreas of the
shrimp, Penaeus vannamei (crustacea-decapoda),
J. Exp. Zool. 280, 384–394 (1998)
59.21 C.F. Arellano, S.J. Olmos: Thermostable ˛-1, 4- and
˛-1-6-glucosidase enzymes from Bacillus sp. iso-
lated from a marine environment, World J. Micro-
biol. Biotechnol. 18, 791–795 (2002)
59.22 S.J.L. Ochoa, S.J. Olmos: The functional property of
Bacillus for shrimp feeds, Food Microbiol. 23, 519–
525 (2006)
59.23 T.M. Samocha, D.A. Davis, I.P. Saoud, K. DeBault:
Substitution of fish meal by co-extruded soy-
bean poultry by-product meal in practical diets for
the pacific white shrimp, Litopenaeus vannamei,
Aquaculture 231, 197–203 (2004)
59.24 D. Sookying, D.A. Davis: Pond production of Pacific
white shrimp (Litopenaeus vannamei) fed high
levels of soybean meal in various combinations,
Aquaculture 319, 141–149 (2011)
59.25 P. Enes, S. Panserat, S.C. Kaushik, A. Oliva-Teles:
Growth performance and metabolic utilization of
diets with native and waxy maize starch by gilt-
head sea bream (Sparus aurata) juveniles, Aqua-
culture 274, 101–108 (2008)
59.26 D. German, M. Horn, A. Gawlicka: Digestive enzyme
activities in herbivorous and carnivorous prick-
leback fish (Teleostei: Stichaeidae): Ontogenetic,
dietary and phylogenetic effects, Physiol. Biochem.
Zool. 77(5), 789–804 (2004)
59.27 G.I. Hemre, S.Y. Shiau, F.D. Deng, G.I. Hemre,
T. Storebakken, S.Y. Shiau, S.S. Hung: Utilization
of hydrolyzed potato starch by juvenile atlantic
salmon when using a restricted feeding regime,
Aquac. Res. 31, 207–212 (2002)
59.28 C. Pascual, E. Zenteno, G. Curzon, A. Sánchez,
G. Gaxiola, G. Taboada, J. Suárez, T. Maldonado,
C. Rosas: Litopenaeus vannamei juveniles ener-
getic balance and immunological response to di-
etary protein, Aquaculture 236, 431–450 (2004)
59.29 C. Rosas, G. Curzon, G. Gaxiola, L. Arena, P. Lemaire,
C. Soyez, A. Van Wormhoudt: Influence of di-
etary carbohydrate on the metabolism of juvenile
Litopenaeus stylirostris, J. Exp. Mar. Biol. Ecol. 249,
181–198 (2000)
59.30 A. Conchillo, I. Valencia, A. Puente, D. Ansorena,
I. Astiasarán: Componentes funcionales en aceites
de pescado y de alga, Nutr. Hospitalaria 21(3), 369–
373 (2006)
59.31 L.E. Cruz-Suarez, M.D. Ricque, J.D. Pinal-Mansilla,
P. Wesche-Ebelling: Effect of different carbohydrate
sources on the growth of P. vannamei, Econ. Impact
Aquac. 123, 349–360 (1994)
59.32 W. Dall , B.J. Hill, P.C. Rothlisberg, D.J. Sharples:
The biology of the Penaeidae. In: Advances in Ma-
rine Biology, Vol. 27, ed. by W. Dall (Academic Press,
London 1990) p. 489
59.33 C. Berger: Aportes de la Bio-Tecnología a la Al-
imentación ya la Inmuno-Estimulación de Ca-
marones peneidos, Av.Nut. Acuícola. Mem. V Simp.
Int. Nut. Acuícola (2000) pp. 19–22
59.34 M. Sørensen, M. Penn, A. El-Mowafi, T. Store-
bakken, C. Chunfang, M. Øverland, A. Krogdahl:
Effect of stachyose, raffinose and soya-saponins
supplementation on nutrient digestibility, diges-
tive enzymes, gut morphology and growth perfor-
mance in Atlantic salmon (Salmo salar), Aquacul-
ture 314(1), 145–152 (2011)
59.35 M.J. Alvarez, C.J. Lopez-Bote, A. Diez, G. Cor-
raze, J. Arzel, J. Dias, S.J. Kaushik, J.M. Bautista:

The partial substitution of digestible protein with
gelatinized starch as an energy source reduces
susceptibility to lipid oxidation in rainbow trout
Oncorhynchus mykiss and sea bass Dicentrarchus
labrax muscle, J. Animal Sci. 77(12), 3322–3329
(1999)
59.36 C. Pascual, L. Arena, G. Curzon, G. Gaxiola,
G. Taboada, M. Valenzuela, C. Rosas: Effect of
a size-based selection program on blood metabo-
lites and immune response of Litopenaeus van-
namei juveniles fed different dietary carbohydrate
levels, Aquaculture 230, 405–416 (2004)
59.37 R. Fuller: History and development of probiotics.
In: The Scientific Basis Probiotics, ed. by R. Fuller
(Chapman Hall, New York 1992) pp. 1–8
59.38 P. Cheeke: Applied Animal Nutrition: Feeds and
Feeding (Macmillan, New York 1991)
59.39 Lezama-Cervantes, Carlos, J. Paniagua-Michel: Ef-
fects of constructed microbial mats on water qual-
ity and performance of Litopenaeus vannamei
post-larvae, Aquac. Eng. 42(2), 75–81 (2010)
59.40 F.J. Gatesoupe: The use of probiotics in aquacul-
ture, Aquaculture 180, 147–165 (1999)
59.41 L. Verschuere, G. Rombaut, P. Sorgeloos, W. Ver-
straete: Probiotic bacteria as biological control
agents in aquaculture, Microbiol. Mol. Biol. Rev.
64, 655–671 (2000)
59.42 A. Farzanfar: The use of probiotics in shrimp aqua-
culture, FEMS Immunol. Med. Microbiol. 48, 149–
158 (2006)
59.43 A.L. Sonnenschein, R. Losick, J.A. Hoch: Bacil-
lus Subtilis and Others Gram-Positive Bacteria:
Biochemistry, Physiology and Molecular Genet-
ics (American Society for Microbiology, Washington
1993)
59.44 T. Godfrey, S. West: Industrial Enzymology
(Macmillan, New York 1996)
59.45 S.J. Olmos, V. Bolaños, S. Causey, E. Ferrari, F. Bo-
livar, F. Valle: A functional SpoOA is required for
maximal aprE expression in Bacillus subtilis, FEBS
Lett. 381, 29–31 (1996)
59.46 S.J. Olmos, R. De Anda, E. Ferrari, F. Bolívar, F. Valle:
Effects of the sinR and degU32 (Hy) mutations on
the regulation of the aprE in Bacillus subtilis, Mol.
Gen. Genet. 253, 562–567 (1997)
59.47 J. Olmos-Soto: Molecular characterization and
phylogenetic identification of marine microorgan-
isms, X Congr. Nac. Biotecnol. Bioing. (Puerto Val-
larta, Jalisco 2003)
59.48 C.K. Lin: Progression of intensive marine shrimp
culture in Thailand, Swimming Through Troubled
Water. Proc. Spec. Sess. Shrimp Farming, Aquac. ’95
(1995) pp. 13–23
59.49 S. Rengpipat, W. Phianphak, S. Piyatiratitivo-
rakul, P. Menasveta: Effects of a probiotic bac-
Functional Feeds in Aquaculture References 1319
terium on black tiger shrimp Penaeus monodon
Part J | 59
survival and growth, Aquaculture 167, 301–313
(1998)
59.50 J.J. Guo, K.F. Liu, S.H. Cheng, C.I. Chang, J.J. Lay,
Y.O. Hsu, J.Y. Yang, T.I. Chen: Selection of probiotic
bacteria for use in shrimp larviculture, Aquac. Res.
40(5), 609–618 (2009)
59.51 R.L. Naylor, R.J. Goldburg, J.H. Primavera,
N. Kautsky, M.C.M. Beveridge, J. Clay, C. Folke,
J. Lubchenco, H. Mooney, M. Troell: Effect of aqua-
culture on world fish supplies, Nature 405(6790),
1017–1024 (2000)
59.52 A.G.J. Tacon: Use of fish meal and fish oil in aqua-
culture: A global perspective, Aquat. Resour. Cult.
Dev. 1(1), 3–14 (2004)
59.53 Z.G. Hernandez, S.J. Olmos: Identification of bac-
terial diversity in the oyster Crassostrea gigas by
fluorescent in situ hybridization and polymerase
chain reaction, J. Appl. Microbiol. 100, 664–672
(2006)
59.54 T.A. García, S.J. Olmos: Quantification of fluorescent
in-situ hybridization of bacteria associated with
Litopenaeus vannamei larvae in Mexican shrimp
hatchery, Aquaculture 26, 211–218 (2007)
59.55 I. Němečková, H. Dragounová, M. Pechačová,
J. Rysová, P. Roubal: Fermentation of vegetable
substrates by lactic acid bacteria as a basis of func-
tional foods, Czechoslov J. Food Sci. 29, S42–S48
(2011), Spec. Issue
59.56 Z.G. Hernandez, S.J. Olmos: Molecular identifica-
tion of pathogenic and nonpathogenic strains of
Vibrio harveyi using PCR and RAPD, Appl. Microbiol.
Biotechnol. 63(6), 722–727 (2004)
59.57 S.R. Martorelli, R.M. Overstreet, J.A. Jovonovich:
First report of viral pathogens WSSV and IHHNV in
Argentine crustaceans, Bull. Mar. Sci. 86(1), 117–131
(2010)
59.58 A.K. Chakrabarty, B.R. Boro: Prevalence of food-
poisoning (enterotoxigenic) Clostridium perfrin-
gens type A in blood and fish meal, Zentralblatt
Bakteriol. Mikrobiol. Hyg. 1. Abt. Orig. B, Hyg. 172(4–
5), 427 (1981)
59.59 C. Pacífico, M.G. Jorge: Enfermedades por pri-
ones, Rev. Cienc. Agrar. Tecnol. Aliment. (2002),
p. 20
59.60 J. Paniagua-Michel, A. Franco-Rivera, J.J.L. Can-
tera, L.Y. Stein: Activity of nitrifying biofilms
constructed on low-density polyester enhances
bioremediation of a coastal wastewater efflu-
ent, World J. Microbiol. Biotechnol. 21(8), 1371–1377
(2005)
59.61 D.M. Akiyama, W.G. Dominy, A.L. Lawrence: Pe-
naeid shrimp nutrition. In: Marine Shrimp Culture:
Principles and Practices, ed. by A.L. Fast, L.J. Lester
(Elsevier, Amsterdam 1992)