Bioencapsulation_Atrayee et al

74 International Journal of Research in Fisheries and Aquaculture 2015; 5(2): 74-83
ISSN 2277-7729
Original Article
An Overview on Bioencapsulation of Live Food Organisms with Probiotics for Better
Growth and Survival of Freshwater Fish Juveniles
Atrayee Dey1
, Koushik Ghosh2
and Niladri Hazra1*
1
Entomology Research Unit, Department of Zoology, The University of Burdwan, WB, 713104
2
Aquaculture Laboratory, Department of Zoology, The University of Burdwan, WB, 713104
*E–mail: hazra.niladri@gmail.com; Cell phone: +919474141470
Received 13 April 2015; accepted 29 April 2015
Abstract
Aquaculture is globally expanding into new directions. A constant goal of global aquaculture is to maximize the
effectiveness of production to optimize profitability. Intensification and commercialization of aquaculture production need
to develop microbial control strategy. Conventional culture methods involve delivery of micronutrients as well as
probiotics as feed supplements or direct use in the aquaculture. Providing probiotics directly to the aquaculture industry
does not guarantee for its intake by fishes. Moreover, these conventional methods may not be conclusive as some of the
bioactive compounds are not able to withstand feed processing temperature and thereby lose its activity. Cultivation of
larval stages of different species involved in aquaculture is still largely dependent on live food. Normal growth and
behaviour of fish juveniles are dependent on the quality of the diet provided to them. Since live feed is rich in proteins,
carbohydrates and fats along with diverse types of vitamins and minerals, it is always preferable to have usual supply of
live feed. Bio–encapsulation and bio–enrichment are the techniques by which nutritional status of the live feed can be
increased so that nutritional status of the fishes feeding on them could be improved. Bio–encapsulation of live feeds with
probiotics and their supply for improving growth and survival rate of fish juveniles is a new advent to aquaculture.
Generally, probiotics offer possible alternatives by providing benefits to the host primarily via the direct or indirect
modulation of the intestinal microbiota, improved immune system and growth, stimulate enzyme activity and enhanced
disease resistance. Probiotics are live microorganisms, which when administered in sufficient amount confer a health
benefit on the host. Therefore, bio–encapsulated probiotic bacteria may have immense promise in the enhancement of
growth and survival of different species of fish juveniles; thus, help to recognize a new avenue in aquaculture.
© 2015 Universal Research Publications. All rights reserved
Key words: Aquaculture, probiotics, bio–encapsulation, bio–enrichment, live food.
1. Introduction
In aquaculture, the increase in fish disease is mainly due to
the augmentation of production and the rise in fish density
in ponds. The widespread application of antibiotics may
cause significant problems such as the spread of drug
resistant pathogens and hazards on the food chain and on
the environment. Modern aquaculture requires alternatives
that can keep healthy environment for best production
practices[1]
(Ai et al., 2011). Prevention of bacterial
infection by avoiding the colonization of pathogenic
bacteria is possible by the use of probiotics. Bio–
encapsulation is the method involved in improving the
nutritional and/or beneficial status of live food organisms
either by feeding or integrating within them various kinds
of nutrients. Live food has been used as vectors for
delivering compounds of diverse nutritional value to larval
stages of aquatic animals[2]
(Cappellaro et al., 1993). Some
common live food organisms like Artemia, Daphnia, and
chironomid larvae can be used as possible vectors for the
delivery of different substances such as probiotics, nutrients
etc. Intensive rearing of fish juveniles suffer from heavy
mortalities, which may be minimized by introducing
beneficial bacteria in the rearing system with live food.
Roles and effects of probiotics in aquaculture as an
alternative to antimicrobial drugs have been carefully
examined by the scientific community. Probiotics have
positive effects on fish survival[3]
(Villamil et al., 2002),
growth[4]
(Burr et al., 2005), stress resistance[5]
(Smith and
Davey, 1993), immune system enhancement[6]
(Erickson
and Hubbard, 2000), and finally general welfare[7]
(Balcázar et al., 2006). In the present review,
we have addressed the issue to provide an overview of the
Available online at http://www.urpjournals.com
International Journal of Research in Fisheries and Aquaculture
Universal Research Publications. All rights reserved

Table 1. Some potent probionts in aquaculture.
Probiotics
Method of
Administration
Mode of action References
Lactobacillus sp. and
Carnobacterium sp.
enrichment of rotifers
antagonism and/or improved
nutritional value of the rotifers
Gatesoupe, 1994[21]
Vibrio alginolyticus bathing in bacterial
suspension
antagonism Austin et al., 1995[22]
Vibrio salmonicida and
Lactobacillus
plantarum
addition to culture water immunostimulation Olafsen, 1998[23]
Pseudomonas
ﬂuorescens
addition to culture water antagonism Gram et al., 1999[24]
Carnobacterium addition to diet immunomodulation Robertson et al., 2000[25]
Bacillus circulans addition to diet immunostimulation Ghosh et al., 2004[26]
Bacillus subtilis and
Bacillus circulans
optimization of cellulase
production by gut
bacteria
sole product of fermentative
metabolism
Ray et al., 2007[27]
Lactobacillus
rhamnosus
in vivo production
antagonism or immunostimulation of
Listeria monocystogenes
Panigrahi et al., 2010[28]
Bacillus subtilis and
Lactococcus lacti
in vivo production
production of enzymes like subtilisin
and catalase, results in a positive
environment for beneficial bacteria
such as Lactobacilli
Mohapatra et al., 2012[29]
Pseudomonas
aeruginosa
in vivo production Immunostimulation Giri et al., 2012[30]
Enterococcus faecium
MC13
in vitro production
biofilm inhibition, especially with
Listeria
Kanmani et al., 2013[31]
information available on the evaluation of the efficacy of
the use of probiotics or beneficial bacteria for bio–
encapsulation of live food organisms and their utilization
for enhancement of growth and survivability in fish
juveniles. The results cited include works published in
well–known as well as minimally circulated journals. This
is performed to indicate that there are numerous interesting
investigations published on this topic.
2. Probiotics in Aquaculture
The term probiotic has been originating from the Greek
words “pro” and “bios”[8]
(Gismondo et al., 1999), which
simply means “for life”. According to[9]
Fuller (1989)
probiotic is “a live microbial feed supplement which
beneficially affects the host animal by improving its
intestinal balance”[10]
. Tannock (1997) defined probiotics as
“living microbial cells administered as dietary supplements
with the aim of improving health”. Although probiotics
have been an area of much attention and research in the
past 30 years, the innovative idea was possibly shaped by
Metchnikoff in the early 1900s. Metchnikoff[11]
(1907)
theorized that human wellbeing could be aided through the
intake of fermented milk products. Aquatic probiotics must
possess certain important criteria in order to aid in correct
establishment of new, helpful and safe products[12]
(Verschuere et al., 2000). A probiotic should: a) be of fish
origin, b) be able to adhere to gut epithelial cells and reduce
colonization of pathogens, c) be non–pathogenic in nature,
d) modulate immune response, e) be resistant to gastric
acid, bile salts etc., f) secrete antibacterial substances
(bacteriocins and organic acids), g) compete for nutrients
essential for pathogen survival, and producing an antitoxin
effect.
The difference in the surrounding environment is a
consequence of the difference in the intestinal flora of
aquatic animals. Increment of bacterial colonization in the
larval gut occurs at the onset of exogenous feeding,
resembling the microflora similar to that of live food as
opposed to that of the surrounding environment. Therefore,
it can be said that the gut microbiota of aquatic animals
mostly resembles the microbiota in the aquatic
environment[13]
(Tuan et al., 2013). To maximize the
competitive advantage of probiotics, early delivery seems
to be greatest[14]
(Ringø et al., 1996). Major indigenous
microbiota of a variety of species of marine fish is
constituted by Gram–negative facultative anaerobic
bacteria such as Vibrio and Pseudomonas[15]
(Onarheim et
al., 1994). In opposition to brackish water fish, the
indigenous microorganisms of freshwater fish species tend
to be dominated by the members of the genera Aeromonas
and Plesiomonas. These are representatives of the family
Enterobacteriaceae, and obligate anaerobic bacteria of the
genera Bacteroides, Fusubacterium, and Eubacterium[16]
(Sakata, 1990). Extracellular enzyme producing gut
bacteria in fresh water teleost fishes have been identified as
Gram–positive aerobic bacteria such as Bacillus[17] [18]
(Khan and Ghosh, 2013; Das and Ghosh, 2014). The
constancy and preservation of microbial flora within
aquatic animals is correlated with external environmental
factors[19]
(Lara–Flores, 2011). This may be contradictory
that anaerobic bacteria are probably the most important
contributors to fish nutrition. More than 50% of
Aeromonas, Bacteroidaceae and Clostridium strains are

able to produce amylase efficiently, while Acinetobacter,
Enterobacteriaceae, Moraxella, Plesiomonas and
Streptococcus strains are not[20]
(Ray et al., 2012). Some
effective probionts used in aquaculture are listed in Table 1.
3. Autochthonous and Allochthonous microbiota
Autochthonous or indigenous microbiota of fish is able to
adhere and inhabit the host’s gut epithelial surface.
Allochthonous microbiota is incidental visitors in the
Gastrointestinal (GI) tract and is discarded after some time
without colonizing[32]
(Merrifield et al., 2011). Ringø and
Birkbeck[33]
(1999) proposed some criteria for testing
indigenous microorganisms in fish on the basis of the
characteristics for testing autochthony of microorganisms
reported in the GI tracts of endothermic animals: a) the
microorganisms should be detected in healthy individuals,
b) demonstrated in both free–living and hatchery–cultured
fish, c) able to grow anaerobically, d) colonize early stages
and persist throughout the life cycles, and e) be detected
associated with the epithelial mucosa in the stomach,
proximal or distal intestine. Apart from these criteria, a
number of factors such as a) gastric acidity, b) bile salts, c)
peristalsis, d) digestive enzymes, e) immune response and
f) native bacteria and the antibacterial compounds that they
produce are suggested to influence adhesion and
colonization of the microbiota within the digestive tract[34]
(Ringø et al., 2003).
4. Importance of Probiotics in Aquaculture
The necessity for sustainable aquaculture has promoted
research into the use of probiotics on aquatic organisms.
The early interest was focused on their use as growth
promoters and to develop the health of animals; though,
new areas have been established, such as their consequence
on reproduction or stress tolerance, even though this
requires a more scientific development.The significant fact
is to note that the population of endogenous microbiota
may depend on genetic, nutritional and environment
factors. In case of aquatic animals, microorganisms present
in the immediate environment have a much larger influence
on the health status than with terrestrial animals or humans.
4.1 Inhibitory compound production
Diverse of chemical compounds are released by probiotic
bacteria which are inhibitory to both Gram–positive and
Gram–negative bacteria. Thus, microbial interaction plays a
principal role in the stability between beneficial and
pathogenic microorganisms. The inhibitory compounds
include bacteriocins, sideropheres, lysozymes, proteases,
hydrogen peroxides etc. Lactic acid bacteria (LAB) are
well–known to produce compounds such as bacteriocins
that are inhibitory to other microbes[35]
(Saurabh et al.,
2005).
4.2 Inhibitory effect on pathogens
Prevention of disease in aquaculture was done by the use of
antibiotics for a long time. Using antibiotics caused a
variety of problems such as the production of bacterial
resistance mechanisms, presence of antibiotic residues in
animal tissues, as well as an inequity in the gastrointestinal
microbiota of aquatic species, which affected their health.
Probiotic bacteria are able to liberate chemical substances
with bactericidal or bacteriostatic effect on harmful
pathogenic bacteria that are in the intestine of the host, thus
constituting an obstacle against the propagation of
opportunistic pathogens. There are some bacteria used as
candidate probiotics show antiviral effects. Several
laboratory tests showed that the inactivation of viruses can
occur by chemical or biological substances[36]
(Denev et al.,
2009). The autochthonous gut bacteria in Catla catla have
probiotic potential and antagonistic activity against fish
pathogens[37]
(Mukherjee and Ghosh, 2014).
4.3 Influence on water quality
Probiotics are also responsible for changing water quality
in aquatic ponds[38]
(Moriarty, 1997). It has been found that,
improved water quality is particularly connected with
Bacillus sp. The justification behind this statement is that
Gram–positive bacteria are better converters of organic
matters back to carbon dioxide than Gram–negative ones.
Fish excrete nitrogen wastes as NH3 or NH4
+
resulting in
rapid increase of ammonium compounds which are very
toxic to them[39]
(Hagopian and Riley, 1998). Few bacteria
like Nitrosomonas, convert ammonia to nitrite and other
bacteria e.g. Nitrobacter, again mineralize nitrite to nitrate.
Nitrate, is considerably less toxic being tolerated in
concentrations of several thousand mg per liter. Some
bacteria oxidize organic carbon using sulfur as a source of
molecular oxygen, known as sulfur–reducing bacteria. The
hydrogen ions released when organic carbon fragments are
oxidized is united with sulfate to form sulfide which is less
toxic to the aquatic species. Methane–reducing bacteria
utilize carbon dioxide as a source of molecular oxygen.
Diffusion of methane into the air improves the water
quality.
4.4 Growth promoting effect
Administration of probiotic microorganisms over a long
period of time may result in colonization in gastrointestinal
tract because they have a higher multiplication rate than the
rate of expulsion[7]
(Balcázar et al., 2006). Addition of
probiotics in fish diets has become prevalent in aquaculture
industry. Reduced feed cost may result in, due to
application of probiotics which plays a significant role in
determining the practices of aquaculture[13]
(Tuan et al.,
2013). Probiotics when constantly added to fish cultures,
adhere to the intestinal mucosa of them, developing and
exercising their multiple benefits. Colonizing probiotics in
the digestive tract of the host affect the process of digestion
through increased production of microbial enzymes,
improving the intestinal microbial balance and in turn help
in digestibility and absorption of feed and feed
utilization[30]
(Mohapatra et al., 2012).
4.5 Competition for adhesion for sites and nutrients
Probiotic microorganisms compete with the pathogens for
the adhesion sites and food in the gut epithelial surface and
finally prevent their colonization[40]
(Vanbelle et al., 1990).
The host–specific adhesion of probiotic bacteria to mucosal
surfaces is significant in the competitive exclusion of
pathogenic microorganisms and draws special attention[41]
(Bengmark 1998). The ability of bacteria to colonize the
intestinal mucosa depends on some factors such as,
a) bacterial factors that help the organisms to persist in the
GI tract, b) host factors that help to resist the colonization
by enteropathogens, c) interaction of the colonizing
enteropathogens and beneficial microflora in the gut that

may inhibit the adherence of the given enteropathogen and
survival in the GI tract. Application of high number of
beneficial bacteria or probiotics results in inhibition of
adherence of harmful or pathogenic bacteria in the GI tract.
Probiotics utilize nutrients otherwise consumed by
pathogenic microbes. Reports on competition for iron have
been found as a crucial factor in marine bacteria for their
growth, but in general is limited in the tissues and body
fluids of animals and in the insoluble ferric form[12]
(Verscuere et al., 2000). Successful application of
probiotics to natural situation is a major task for microbial
ecologists.
4.6 Digestive enzymes
According to some researchers probiotic microorganisms
have positive effect in the digestive processes of aquatic
animals. According to Sakata[16]
(1990), Bacteroides sp.
and Clostridium sp. have provided to the host’s nutrition,
particularly by supplying fatty acids and vitamins. During
the juvenile stages, the digestive capability changes and
develops as the larva grows[42]
(Zambonino–Infante and
Cahu, 2001). Isolated bacteria from fish digestive system
are able to digest chitin[43,44]
(Hamid et al., 1979;
MacDonald et al., 1986), starch[43,44,45]
(Hamid et al., 1979;
MacDonald et al., 1986; Gatesoupe et al., 1997),
protein[43,45]
(Hamid et al., 1979; Gatesoupe et al., 1997),
cellulose[46,47]
(Erasmus et al., 1997; Bairagi et al., 2002)
and lipids[45,48]
(Gatesoupe et al., 1997; Vine, 2004) in vitro.
In a previous study, Ghosh et al.[49]
(2008) examined four
different inclusion levels of B. subtilis isolated from C.
mrigala on existence, proximate composition, feed
conversion ratio, specific growth rate, intestinal amylase
and protease activity. Length, weight, existence, body ash,
protein content and gut enzyme activity were significantly
developed by including Bacilli in the diets. Besides, the
population level of gut bacteria belonging to motile
aeromonads, Pseudomonas and total coliforms was
significantly reduced by probiotic feeding.
5. Importance of Live food Organisms in Aquaculture
Healthy culture stock is required for successful aquaculture.
Disease free healthy stock of fish fingerling is largely
dependent on the availability of suitable live food
organisms for feeding fish larvae, fry and fingerlings. In
terms of preference, nutritional and other factors, live food
organisms are superior to artificial larval feeds. Feeding
habit of fishes in natural water bodies is diverse among the
species but all the fishes necessitate protein rich live food
for their better development, efficient breeding and
survival[50]
(Mandal et al., 2009). In addition to providing
protein and energy, they supply other important nutrients
like vitamins, polyunsaturated fatty acids (PUFA), sterols
and pigments which are transferred through the food
web[51]
(Das et al., 2012). In case of cultured fish, they
require good amount of nutrients especially when fish
larvae are of altricial type. Altricial larvae possess
rudimentary digestive system, where stomach is lacking
and much of the protein digestion takes place in the hindgut
epithelial cells[52]
(Govoni et al., 1986). They remain in a
relatively undeveloped state until the yolk sac is exhausted.
This type of rudimentary digestive system is not capable of
processing artificial supplemented diets in a manner that
allows survival and growth of the larvae equivalent to those
fed live food organisms. In aquaculture industry high
mortality of larval stages can be observed, which is
considered as one of the critical phases of aquaculture.
Jhingran et al.[53]
(1991) observed that the spawn do not
take artificial feed for a short period after hatching during
their ‘critical period’ but survive on natural food and ‘live
feed’ serving as ‘living capsules’ of nutrition. Special
strategies and planning are necessary to overcome the risk
of high mortality during this phase of aquaculture. Feeding
of most species of interest relies on live feeds during the
early life stages.
Besides low digestive capability of altricial larvae, live
prey are also capable to swim and/or undulate in the water
column and are thus constantly available to the larvae. The
movement of live feed in the water is likely to stimulate
larval feeding responses. Live feed having thin
exoskeleton, high water content, lower nutrient
concentration are more palatable than that of hard, dry
formulated diet.
6. Selection of Livefood
The selection of an appropriate and nutritious diet should
be based on a number of criteria. Live food contaminated
by bacteria is not necessarily hazardous but may have
remarkable impact on the microbial populations in the
associated culture medium and ultimately in the fish gut
flora, and as a result it makes an impact on the overall
health status and the digestive capability of the larva.
7. Selective significant Live feeds
7.1 Microalgae
There are many marine and fresh water potential species of
microalgae. The use of microalgae as a possible source of
protein food was recognized by the researchers in mid-20th
century. These are photosynthetic, calcified, and capable of
high lipid production. Microalgae also constitute an
important source of food for live food organisms (rotifers,
copepods, cladocerans, brine shrimp etc.). Considerable
amount of eicosapentanoic acid (EPA; 20:5n–3), high
concentrations of docosahexanoic acid (DHA; 22:6n–3) are
found in microalgae, mostly in Thraustochytriidae (e.g.
Schizochtrium sp.), which can contain over 70% of its
weight as lipids and have a DHA content upto 35% of their
total fatty acids[54]
(Becker, 2004).
7.2 Rotifers
Rotifers are an important group of live food organisms for
use in aquaculture. Brachionus, is the most common form
of all rotifers, serve as an ideal starter diet for juvenile
stages of many fish and prawn species in marine as well as
freshwater. The rotifer, B. plicatilis and B. rotundiformis,
have been indispensable as a live food for mass larval
rearing of many aquatic organisms[55]
(Maruyama et al.,
1997). Rotifer feed is constituted by DHA and EPA can be
nutritious for fish larvae. They are composed of about 52–
59% protein, upto 13% fat and 3.1% n–3 HUFA[56,57]
(Awais, 1992; Oie and Olsen, 1997).
Stock culture of B. plicatilis is started by collecting them
from brackish water with a scoop net. Rotifer is normally
mass cultured in 10 to 15 ppt saline water, where nearly all
reproduction occur. They are fed with yeast @ 200 ppm or
Chlorella at a cell density of 10 × 106
cells per ml. When

B. plicatilis population reaches 100 to 150 individuals per 1
ml, about 25 % of culture is harvested and shifted to the
another tank. This method helps in continuous supply of B.
plicatilis for aqua hatcheries.
7.3 Copepods
Copepods are large group of common zooplankton of
freshwater and brackish water. These are microscopic
crustaceans. In nature, most marine fish larvae feed on
copepod eggs during the first few weeks of life. Because
some species of copepods have very small size larvae (a
necessity for some species of fish larvae) and can have very
high levels of HUFAs. In general copepods have high
protein content (44–52%) and a good amino acid profile,
with the exception of methionine and histidine and other
crucial nutrients, they are an outstanding food source for
first–feeding larvae. Copepods are cylindrical in shape
consisting of head, thorax and abdomen. Larval stages of
copepods consist of six naupliar and six copepodite stages.
They can be administered under different forms, either as
nauplii or copepodites at start feeding and as ongrown
copepods until weaning. They do not reproduce asexually
like rotifers and Artemia. Female can produce about 250–
750 fertilized eggs. Several candidate species belonging to
both the calanoid (very long first antennae 16–26 segments)
and the harpacticoid groups (short first antennae fewer than
10 segments) have been studied for mass production.
Culture of copepods is more difficult than that of rotifers on
the commercial basis. One attractive benefit of copepods is
that under appropriate conditions some species will
produce a resting egg alike Artemia. So once commercial
techniques are developed, copepod eggs could be collected
in large numbers and stored for months, like Artemia (brine
shrimp) and rotifer cysts. According to Evjemo et al.[58]
(2003) copepodite and adult stages of the marine copepods
Temora longicornis and Eurytemora sp. had a total lipid
content varying between 7% and 14% of dry weight (DW)
and the protein content of various copepods varied between
52.4% and 57.6% of dry weight (DW).
7.4 Artemia
The brine shrimp Artemia can be considered as the most
widely used live prey in aquaculture. Artemia remains
necessary in most marine finfish and shellfish hatchery
operations particularly during the earliest life stages[59]
(Kolkovski et al., 2004). The ease and simplicity of
hatching brine shrimp nauplii makes them the most suitable
and least labour–intensive live foods accessible for
aquaculture[60]
(Lavens and Sorgeloos, 2000). According to
Akbar et al.[61]
(2014) although enrichment of Artemia is
broadly used in aquaculture, it is unclear as to what
proportion of the enrichment is integrated into body tissue
and how much remains resident in the gut. The gut loading
and evacuation trial established the gut loading and
retention time of 2 days old Artemia nauplii bio–
encapsulated with fish oils, vegetable oils and probiotics. A
huge disadvantage of brine shrimp is their intrinsic
insufficiency in essential fatty acids. Artemia has high
contents of canthaxanthin pigments that may have
significant roles as antioxidants and sources of Vitamin A
in fish larvae nutrition.
Artemia cysts are hatched into nauplii by following the
steps: hydration of cysts, decapsulation of cysts and
hatching of decapsulated cysts. During hatching of cysts
cylindroconical container is used with saline medium
slightly less than that of sea water. Optimum temperature
for hatching should be maintained at 30º
C. Decapsulation is
a process by which the chorion of the brine shrimp cysts is
removed without affecting the viability of the embryos. The
process of decapsulation includes hydration of the cysts and
exclusion of the chorion in a hypochlorite solution. The
decapsulated cysts are disinfected and can be fed to larval
forms directly.
7.5 Tubifex
Tubifex are worms under the class Oligochaeta of the
phylum Annelida. These worms are found in clusters in
sewage drains. For the cultured fish species this worm is
excellent live food, which can be used alone or in
combination with other food. Tubifex accelerates growth,
stimulates the appetite, makes feeds more attractive, so the
animals come to feed better and waste is avoided. Tubifex
are easily cultured in huge amount with pond mud, some
decaying materials. Constant flow of mild water is
necessary for their survival. They can be cultured in
laboratory condition in a container.
7.6 Chironomid Larvae
Chironomids are insects under the order Diptera. The larval
forms are commonly known as blood worms due to the
presence of haemoglobin in their body fluid[62]
(Madlen,
2005). They can be used as universal food to fishes,
shrimps, and cultured invertebrates. They can also be used
in brackish water. These larvae are low–priced and very
protein rich food. Initially the larvae live in soft tubes made
up of organic matter which can be clearly seen at bottom of
the track. After 2 to 3 days, they come out of the tubes and
freely swim in water vertically. The larvae are collected
with scoop net and washed thoroughly before feeding. It
constitutes one of the staple food items of nearly all
carnivorous young fishes.
Midge larvae are remarkable source of protein and other
nutrients, such as, lipid, vitamins and minerals. They are
able to promote growth of fish, crustaceans etc. due to their
high digestibility (73.6%) value[63]
(Noue and Choubert,
1985). Two fifths of the food of adult freshwater fishes is
insects, the most important of which are bloodworms,
mayfly naiads and caddis fly larvae[64]
(Metcalf et al.,
1962). According to Basu et al.[65]
(2010) a positive
correlation between the increase of planktonic food items in
the midge gut and consequent exoenzyme producing
bacterial load suggest a symbiotic relationship between
these bacteria and the midge larvae in regard to their
feeding habit. These midge larvae are utilized as a potent
source of live feed in aquaculture practice.
8. Methods of Bio–encapsulation of Live food
organisms with Probiotics
Live nauplii of the brine shrimp (Artemia sp.), rotifers
(Brachionus sp.) etc. can be considered as vectors for
delivering compounds of varied nutritional and/or
therapeutic value to larval stages of aquatic animals, a
process known as bio–encapsulation.
 The live food organisms which are to be encapsulated
are collected, reared and sterilized by using antibiotics

Table 2. Probiotic Administration via Fortified Live Food (Bio-encapsulation).
Probiotics Live prey used as vector Observations References
Bacillus toyoi Disinfected rotifers
enhanced growth rate of
turbot and control of Vibrio
alginolyticus density
Gatesoupe, 1990[67]
Lactobacillus plantarum Rotifers
Increased population
density, reduced aerobic
bacterial loads and
increased dietary value of
the rotifers and inhibition of
Aeromonas salmonicida
Gatesoupe, 1991[68]
Flavobacterium sp. Microalgae
Improved growth
characteristics
Suminto and Hirayama,
1997[69]
Lactococcus lactis Brachionus plicatilis
Enhanced growth rate of B.
plicatilis and inhibitory
effect against Vibrio
anguillarum
Shiri Harzevilli et al.,
1998[70]
Vibrio alginolyticus C14 Artemia nauplii Prevention of mortality Gomez-Gil et al., 1998[71]
Selection criteria for food sources of fish juveniles from the viewpoint of the culturist and the cultured fish juveniles can be presented as
follows (According to Léger et al., 1987) [66]
Selection criteria for food sources
For the culturist For the predator
Accessibility
Cost effectively
Simplicity
Versatility
Physical
Factors
Purity
Accessibilitry
Acceptability
Nutritional
Factors
Digestibility
Energetic requirements
Nutrient requirements
or surface disinfectant.
 Specific probiotic strains are isolated and different
concentrations of bacterial suspension (CFU/ml) are
provided.
 The live food organisms are incubated in the bacterial
suspensions for some hours at specific temperature.
 After the incubation period, they accumulate probiotic
strains within them.
The bio–encapsulated live feeds are used as a vector to
carry probiotic strains to digestive system of fish juveniles.
Probiotic bacteria significantly promote final body weight,
body length and specific growth rate (SGR%) in fish.
In modern years, much importance has been given to
improve the nutritional status of live food organisms
through various techniques of enrichment and bio–
encapsulation. The use of ascorbylpalmitate as a supreme
source of vitamin C supplementation in live food can give
an important tool to construct stress and disease resistance
during larval rearing in hatcheries.
Administration of probiotics via bio–encapsulated live food
like microalgae, rotifers, Artemia etc. is an attractive
approach (Table 2). Although, the process of administration
through fortified live food seems to be economically not
viable and basically difficult in large scale aquaculture
practice. The influence of bacteria brought by live food
organism is particularly dramatic during first feeding[72]
(Munro et al., 1993). Delivery of probiotic bacteria to live
prey can not only provide as control agent of opportunistic
or pathogenic bacteria but also be a vehicle for introducing
probiotics to fish juveniles (bio–encapsulation)[73]
(Makridis et al., 2000).

To the authors’ experiment on 2013–14 extracellular
enzyme producing gut bacteria have been isolated from
fresh water air breathing walking catfish (Clarias
batrachus). Out of which three bacterial strains (one of
them identified as Bacillus aryabhattai while remaining
two yet to be submitted to GenBank) capable of producing
enzymes amylase, protease, and lipase were selected and
used for bio–encapsulation of third instar midge larvae of
Chironomus striatipennis. Chironomid larvae were used as
vector to deliver probiotics to the nearly 40 days old
Clarias juveniles; better growth and survival have been
observed in case of experimental juveniles in comparison to
controlled juvenile fishes (Unpublished data).
9. Major Limitations in Live feed culture and
Administration of Probiotics in Aquaculture
The most practical solution for minimizing mortality of fish
juveniles remains the use of live food organisms. Still, it is
not easy to maintain and provide cost effective adequate
quantity of live feed at suitable time during intensive fish
culture. Main restriction of using live feed especially in
smaller hatcheries is high production cost and lack of
infrastructure for controlled laboratory condition for culture
maintenance. Apart from this, it is also difficult to get pure
strains. Dietary status of the live feed should be examined
before feeding different larval stages of fish and shellfish.
New process of encapsulation and enrichment is not
affordable to farmers. There are some constraints in
administration of probiotics in aquaculture industry. The
probiotic strains are difficult to be produced commercially
and resulting manifestation on a large scale. Still it is easier
to administer probiotics in terrestrial animals than aquatic
animals.
10. Future Perception
In aquaculture industry use of probiotics for disease control
is an area of increasing interest, as the use of antibiotics is
causing apprehension of the possible development of
antibiotic resistant bacteria. Probiotics can be delivered
easily to the larval gastrointestinal tract, and this may
suggest that the ability of a probiotic to attach to live food
could be used as selection criteria for probiotics used in
larviculture. The introduction of large numbers of probiotic
bacteria during premature stages could amplify the
percentage contribution of the probiotic in the overall
microflora. More research is required to test the hypothesis.
Apart from this, whether probionts can progress resistance
against infections through conferred resistance should also
be tested. Finfish producers are concerned with improving
the quality, quantity and cost effectiveness of their live feed
production facilities. Many of them now supplement
cultures with omega yeast, vitamins (E, D, C and B12),
marine oils or other HUFA sources, and vitamin B12
producing bacteria to improve feed quality. Nowadays, live
feeds for fish larvae are being superior by modifying their
biochemistry through controlling their diet and
supplementing the cultures with emulsified oils or
microencapsulated feeds. Producers are finding new
species of live food organisms better suited for specific
culture situations.
Further study should be carried out to establish suitable
culture environment for the bio–encapsulated as well as
bio–enriched live food organisms which in turn will act as
an effective source for fish juveniles of commercial value.
Therefore, inoculation of probiotics through live feed may
re–establish a good balance of the gut microflora and
thereby contribute to an optimal growth and health status of
the fish[74]
(Singh et al., 1994). The use of live feeds
discussed in this paper may have a positive impact on
aquaculture industry.
Acknowledgements
Authors are grateful to University Grants Commission,
Govt. of India for providing financial assistance for the
project and to the Head, Dept. of Zoolgy, University of
Burdwan for Laboratory and internet facilities.
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Source of support: Nil; Conflict of interest: None declared

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