March | April 2010

Feature title: Evaluation of probiotic bacteria in prawn production

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The International magazine for the aquaculture feed industry


Evaluation of probiotic
bacteria in prawn production
by Tim O’brine, university of Plymouth, united Kingdom
This article focuses on our current knowledge of the benefits of probiotic application, selecting probiotics
and their modes of actions in combating microbial diseases in prawn production
G
lobally, two million metric and in the live feed and they usually cause
tonnes of prawn valued disease under sub-optimal culture condi-
at US$14.4 billion were tions. Therefore the successful completion
produced in 2004(1), with of the larval cycle in prawns requires a
large industry growth observed in healthy microbial balance in the system (3).
Asian countries. During the larval stages of prawn
production, luminescent vibriosis caused
Intensification of prawn production has by Vibrio spp. is particularly severe and
resulted in an increase of bacterial infec- sporadically causes temporary closure of
tions which frequently cause mass mortali- economic activities in prawn larviculture
ties of prawn larvae due to the proliferation facilities (3).
of opportunistic pathogenic bacteria. Vibriosis elicits signs of lethargy, degener-
Disease is often the result of stress ation of the gills, lymphoid organs, digestive
caused by poor water quality and by reduc- glands and detachment of the epithelium
ing stress, less mortality may occur. lining the mid-gut, all of which lead to even-
The single most critical factor governing tual or rapid mortality.
optimal growth and survival of prawn is Application of disinfectants and antimi-
water quality. Prawns are delicate creatures crobials can complete the larval cycle in
and can be stressed by adverse environ- prawns but has led to the emergence of
mental conditions in culture ponds, result- antibiotic-resistant pathogens with threat
ing in reduced appetite, slow growth, as well of gene transfer to humans as well as waste
as being more susceptible to disease (2). antimicrobials ending up in the environment.
Vibrio spp. are normally present in the These negative consequences of antibiotic
culture facilities, larval gut flora of prawns treatments have led to the development
18 InternatIonal AquAFeed
18 || InternatIonal AquAFeed || March-april
March-april 2010
2010
of probiotics to manipulate the microbial
balance (3).
Probiotics are organisms that create
a favourable environment for the growth
and well-being of the host animals by their
transience through the gastrointestinal tract
or merely by their presence in the water.
The selection of probiotics for specific
marine aquatic species has to be considered
carefully in order to make them spe-
cies specific (4). Probiotic candidates are
most often bacteria with inhibitory activity
against a target pathogen or the dominant
intestinal bacterial flora in larvae from well
performing prawn-rearing groups.
For example, Hill et al. (5) identified
Bacillus pumilis in the mid gut of P. monodon
which had an inhibitory effect against patho-
genic bacteria. The dominant bacterial flora
is possibly capable of excluding invading
bacteria from the adhesion sites of the gut
wall thus supporting the endemic bacterial
flora of prawn - a more natural approach to
protecting prawns from diseases.
F: Probiotic
How should probiotics
be administered?
Probiotics could be used to replace
chemicals and antibiotics and have
already been shown to increase the
immune response, survival and disease
resistance in prawns (6). Probiotics
could either be incorporated into the
feed or administered into the rearing
water. Hai et al. (4) found that both feed
and water administered application was
efficient in the transfer of probiotics
to the prawns. However, survival, spe-
cific growth rates and total haemocyte
counts (THCs) concentrations were
higher when probiotics were supple-
mented through feed (4) .
Thus, feed incorporated probiotics are
more beneficial than probiotics adminis-
tered to the rearing water. THCs play
important roles in the crustacean immune
response as the haemocytes encapsulate
bacteria, thus a high haemocyte count clears
the bacterial load in the haemolymph.
or on the sediments. The spores of Bacillus
sp. can be easily introduced into dry food
Bacillus spp. to inhibiting the growth of
pathogenic bacteria could be one of the
and are unlikely to use genes for antibiotic enzymes produced by Bacillus, including
How do probiotics work? resistance or virulence from the vibrios or protease, amylase, lipase and gelatinase (6).
Proposed mode of actions of probiotics related gram-negative bacteria (6) and have The success of Bacillus spp. has been
includes immunostimulation, inhibition or not been associated with aquatic organism demonstrated by the development and use
competition (antagonistic) with potential pathologies.
pathogens or simply maintenance of healthy For this
and diverse intestinal microbiota. reason, their
ECOBIOL
Non pathogenic strains of Vibrio, use has been ®
Aeromonas, Pseudomonas, Bacillus and promoted and
Alteromonas that are known to have antago- widely accept-
nistic properties to pathogenic strains ed within
can be used as probiotic strains (8). The the industry.
The probiotic for healthy growth
inhibitory compounds produced from the Banerjee et al.
antagonistic organisms can control the (9) observed 90
pathogenic bacteria population in a system percent inhibi-
without affecting the water quality and tion of patho-
therefore could be very beneficial to prawn genic bacteria
production. by Bacillus
Examples of antagonistic bacteria that subtilis along
secrete inhibitory substances include nisin with competi-
(antibacterial peptide) of Lactobacillus lactis, tive exclusion
pediocin (proteinaceous bacteriocin) of of patho-
Pediococcus acidlactici, sidephore-like mol- genic vibrios
ecules and pyocyanin (antibiotic pigment) by Bacillus spp.
of Pseudomonas spp. The primary require- Moriar ty(6)
ment for the demonstration of antagonistic reported that, Improvements in growth and Feed Conversion Ratio
mode of action of probiotics in aquaculture Lower mortality in the ponds
when Bacillus
Increases biomass production
systems is the assurance of the minimum sp. was added Keeps pathogenic bacteria populations under control
inhibitory concentration of the antagonistic to pond water Helps maintain optimal health status
compound in the culture, which is supple- in penaeid
mented to the larval rearing tank (3). prawn culture,
Antagonistic, gram-positive Bacillus spp. there was a
have been used as a probiotic in fish and decrease in
l
shellfish (9) and could be used in prawn luminous Vibrio Natunrautrition
aquaculture. densities.
Bacillus spp. are naturally ingested by The antago- NOREL,S.A. • Jesús Aprendiz, 19, 1º A y B • 28007 Madrid (SPAIN)
Tel. +34 91 501 40 41 • Fax +34 91 501 46 44 • www.norel.es
prawns and other organisms that feed in nistic factor of
March-april
March-april 2010 InternatIonal AquAFeed
2010 || InternatIonal AquAFeed || 19
19
F: Probiotic
of SANOLIFE® MIC, a mixture of strains
of Bacillus spp. Decamp et al. (10) reported
on the performance of SANOLIFE® MIC
on Asian and Latin-American hatcheries,
with P. monodon and L. vannamei and found
that SANOLIFE® MIC performed as well as
prophylactic use of antibiotics and was also
a cost effective alternative.
Pai et al. (3) suggested another mode of
action of Bacillus; immunostimulation and
enhanced food acceptance which results in
improved general health of prawns rather
than the antagonistic mode described above.
In their study they found that the bacteria
that showed the greatest antagonism was
the Pseudomonas bacteria.
The Pseudomonas bacteria suppressed
the vibrio population in a P. monodon larval
rearing system by production of pyocyanin
giving them a competitive advantage over
V. harveyi, mediated by the killing of the
pathogen (3).
The unusual redox properties of pyo-
cyanin and other phenazine compounds
secreted by fluorescent Pseudomonads can
enhance the production of hydroxide and
20 | InternatIonal AquAFeed | March-april 2010
super oxide radicals, resulting in the inhibi-
tion of bacteria due to oxidative damage
and lipid peroxidation.
As a result, prawns fed the probiotics
Pseudomonas synxantha and P. aeruginosa
had higher specific growth rates, survival
and food conversion ratio compared to
prawns not fed probiotics (11). Probiotics
stimulated the prawns to produce a
higher THC and resulted in the lowest
bacterial load in the haemolmpyh, thus
improving the health of the prawns (4).
Nakayama et al. (12) demonstrated
that B. Subtilis could be effective in
the inhibition of Vibrio growth, while
B. Licheniformis and B. Megaterium
could be effective in reducing V. harveyi
haemolytic activities, suggesting that
compounds produced by Bacillus might
be effective against Vibrio growth and
toxin production.
Balcazar and Rohas-Luna (13)reported
that B. subtilis produced antimicrobial
activity against pathogenic Vibrio spp. and
that prawn mortality was reduced by
treatment with B. subtilis. Zhou et al. (14)
also reported that B. coagulans
used as a water additive at a
certain concentration could sig-
nificantly increase the survival
rate and some digestive enzyme
activities of prawn larvae. In
addition, the use of B. subtilis
was improved prawn immune
responses by increases in phe-
noloxidase activity, phagocytic
activity and clearance efficiency
against Vibrio alginolyticus.
Several probiotics
versus one probiotic
Combining probiotics to pro-
duce a range of beneficial effects
on prawn production has been
studied and is often employed
in commercial probiotics, as
previously mentioned by the
use of multiple Bacillus spp. in
SANOLIFE® MIC (10).
The combination of two pro-
biotics in Western king prawn
production was not found to
significantly improve the specific
growth rate of prawns compared
to single use of a probiotic,
but the prawns exposed to the
combined probiotics did elicit a
lower proportion of hyalinocytes
(involved in phagocytosis) as well
as having a lower bacterial load
in the haemolymph, hence were
healthier than those exposed to the
individual probiotics (4).
Far et al. (15) also compared the use of
one probiotic against multiple probiotics
through feed incorporation. Treatment
groups consisted of prawns fed diet
with B. subtilis and prawns fed a diet
mixed with B. subtilis and a commercial
probiotic (BIO-GOLD, Korea) which con-
sisted of L. acidophilus, Bacillus subtilis
and L. plantarum. Interestingly, diets just
consisting of B. subtilis had significantly
Dry with 20-50% less energy
higher survival and yield compared with
the diet incorporating both B. subtilis
and BIO-GOLD. Thus it appears that B.
subtilis alone, out performs BIO-GOLD,
but it should also be noted that a diet just
containing BIO-GOLD was not different
from the diet containing B. Subtilis and
BIO-GOLD.
Therefore, an inhibitory action between
the consortium of probiotics in the BIO-
GOLD and B. subtilis diet can be ruled out.
Overall though, the addition of B. subtilis
was found to improve prawn (L. vannamei) Geelen Counterflow / T +31-475-592315
survival rate and yield (15). Geelen Counterflow USA Inc. / T +1-772-559-4338
Geelen Counterflow América Latina / T +54-9-2362-418899
E info@geelencounterflow.com / I www.geelencounterflow.com
 F: Probiotic
of western king prawns (Penaeus latisulcatus
Wang et al. (16) evaluated a consortium probiotics led to the market being flooded Kishinouye, 1896). Fish & Shellfish Immunology PROFIL
of commercial probiotics (Bacillus sp., pho- with commercial probiotics with little or E: Tech
27(2):100-104. nolo gy
tosynthetic bacteria, yeast, Saccharomyces no scientific basing (10).
Hill JE, Baiano JCF, and Barnes AC. 2009.
cervisiae, Nitrosomonas sp. and Nitrobacter Also, probiotics are only beneficial and COMPA
Isolation of a novel strain of Bacillus pumilus NY LO
sp.) and found that the feed conversion cost-effective when they are properly
from penaeid shrimp that is inhibitory against
TECHNOLOGY PrOFiLE W=110
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ratio and population density of beneficial applied together with suitable farm manage- marine pathogens. Journal of Fish Diseases Coming soon to International Aquafeed Comp
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Tel: +44 nham, GL50
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"Intensification of prawn production,

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nts adva
Hai N, Buller N, and Fotedar R. 2009. Effects of Feature tu pa
s can ha res have been ge versions of ts nces,
prebiotics (Bio-Mos For full
details
ve exist
please
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contact rts replaced or stomers as po re that has ap
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probiotics (Pseudomonas synxantha and Basic Pr James Ta removed int of sa peared
We aim to bring you the latest innovations in the PRODU
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which provide industry and how they can benefit your business.
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selected bacteria Kishinouye, 1896). Aquaculture Research For more information about including your products and
amplifies the need for alternatives to to encourage their 40:590-602. equipment into this section, please contact Caroline Wearn Techni
cal Sp
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proliferation in the for more information: PRODU Interna

tional Aq cation:

prevent disease outbreaks, and the

uafeed
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Page
• Exsistin s feature
Moriatry D. 2003. Probiotics in aquaculture. W=80m
m x H= GE • Full iss
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TECHN
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Microbiology Australia 24:15-17. gloss pa
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survival and spe- Banerjee S, Devaraja TN, Shariff M, and Yusoff For mor
Tel: +44
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fighting the microbial community of

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Email: jam 42 267706 act: Jam
FM. 2007. Comparison of four antibiotics with est@aq es Taylo
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indigenous marine Bacillus spp. in controlling

prawns (11).
farms is developing" Perhaps more
pathogenic bacteria from shrimp and artemia.
Journal of Fish Diseases 30(7):383-390. QUART
importantly though, ER PAG
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W=190
Decamp O, Moriarty D, and Lavens P. 2008. mm x H=
T
as emphasised at
58m m

Probiotics for shrimp larviculture: review of field

the beginning of this
data from Asia and Latin America. Aquaculture
article, maintaining
Research 39(4):334-338.
Tech_
Profile 1 | INTE
_IAF.in RNATIO
dd 1 NAL AQ

the water quality is

UAFEED
|

important, as this Hai N and Fotedar R. 2009. Comparison of

the effects of the prebiotics (Bio-Mos and -1,
factor will dictate
3-D-glucan) and the customised probiotics 23/02/
2010
11:26

the level of stress

(Pseudomonas synxantha and P. aeruginosa) Naturally ahead
on the prawns, and
on the culture of juvenile western king prawns
good water qual- (Penaeus latisulcatus Kishinouye, 1896).
ity will result in Aquaculture 289(3-4):310-316.
healthier prawns,
Nakayama T, Lu H, and Nomura N. 2009.
with no addition of

the use of probiotics significantly increased
dissolved oxygen concentrations and signifi-
cantly reduced dissolved phosphorus and
total inorganic nitrogen concentrations as
well as decreasing the chemical oxygen.
This study highlights the use of probiot-
ics to improve water quality, which in turn
will reduce stress on the prawns, making
them less susceptible to disease.
Conclusion
Intensification of prawn production,
subsequent increases in disease and anti-
biotic resistant strains of bacteria ampli-
fies the need for alternatives to prevent
disease outbreaks, and the concept of
maintaining, rather than fighting the micro-
bial community of farms is developing. This
article has clearly demonstrated numerous
studies that show that probiotics can
antimicrobials, pro-
biotics or prebiotics needed – this is how
a sustainable production system should
attain to be.
References
FAO. 2007. The State of World Fisheries and
Aquaculture 2006. Rome.
Boyd CE. 2001. Soil and water quality
considerations in shrimp farming. In Methods
for improving shrimp farming in Central America.
Managua, Nicaragua: Central American University
Press.
Pai SS, Anas A, Jayaprakash NS, Priyaja P,
Sreelakshmi B, Preetha R, Philip R, Mohandas A,
and Singh ISB. 2009. Penaeus monodon larvae
can be protected from <i>Vibrio harveyi</i>
infection by pre-emptive treatment of a rearing
system with antagonistic or non-antagonistic
bacterial probiotics. Aquaculture Research
9999(9999):
Biomin® P.E.P.
Inhibitory effects of Bacillus probionts on
growth and toxin production of Vibrio harveyi
pathogens of shrimp. Letters in Applied
Microbiology 9999(999A):
Balcazar JL and Rojas-Luna T. 2007. Inhibitory Naturally ahead in phytogenics!
activity of probiotic Bacillus subtilis UTM
126 against vibrio species confers protection
against vibriosis in juvenile shrimp (Litopenaeus
vannamei). Current Microbiology 55(5):409-412. Biomin® P.E.P. is made with a unique
blend of essential oils and prebiotics to provide
Zhou X, Wang Y, and Li W. 2009. Effect of
probiotic on larvae shrimp (Penaeus vannamei) a synergistic formula.
based on water quality, survival rate and It is designed specifically to support digestion
digestive enzyme activities. Aquaculture 287(3-
4):349-353. and improve feed conversion.
Far HZ, Saad CRB, Daud HM, Harmin SA, and
Shakibazadeh S. 2009. Effect of Bacillus subtilis
on the growth and survival rate of shrimp
(Litopenaeus vannamei). African Journal of
Biotechnology 8(14):3369-3376.
Wang YB, Xu ZR, and Xia MS. 2005. The
effectiveness of commercial probiotics in For enquiry, please e-mail

www.aqua.biomin.net
northern white shrimp Penaeus vannamei ponds.
benefit prawn production. It should also Hai NV, Buller N, and Fotedar R. 2009. The Fisheries Science 71(5):1036-1041. aqua@biomin.net
be stressed that the increased demand for use of customised probiotics in the cultivation

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