Shrimp aquaculture, as well as other industries, constantly requires new techniques in order to increase product performance. New technologies and other sciences such as biotechnology and microbiology are important tools that could lead to a higher quality and greater quantity of products.

Probiotics, as ‘bio-friendly agents’ such as lactic acid bacteria and Bacillus spp., can be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms.

Probiotics are nonpathogenic and nontoxic microorganisms without undesirable side-effects when administered to aquatic organisms.

On fishes

Pathogenic bacteria are located in every where of the aquatic environment. The fish egg is the first victims that could be exposed to pathogenic bacteria. Therefore, a barrier nonpathogenic present on eggs is necessary to prevent the formation of pathogenic colonies on egg.

In addition, Larvae may ingest substantial amounts of bacteria. This affects the primary colonization of juveniles in next stage of fish.

Some experiments that used probiotic to larvae fish tanks, from egg to through transformation. They observed an increasing of survival, an uniformity size, and a good growth rate.

Previously, Gildberg et al. (1997) had analysed the effect of a probiotic of lactic acid bacteria in the feed of Atlantic cod fry (Gadus morha) on growth and survival rates. A certain improvement in disease resistance was obtained after 3 weeks of feeding, and at the end of the experiment lactic acid bacteria dominated the intestinal flora in surviving fish given feed supplemented with lactic acid bacteria.

Lara-Flores et al. (2003) used two probiotic bacteria and the yeast, Saccharomyces cerevisiae as growth promoters in Nile tilapia (Oreochromis niloticus) fry. The results of this study indicated that the fry subjected to diets with a probiotic supplement exhibited greater growth than those fed with the control diet. In addition, they suggested that the yeast is an appropriate growth-stimulating additive in tilapia cultivation.


On crustaceans

During the last few decades, aquaculture has become the world's fastest growing food production sector, with cultured shrimp growing at an annual rate of 16.8%. Meanwhile, according to a World Bank report, global losses resulting from shrimp diseases are around 3 billion US dollars. The potential negative consequences of using antibiotics in aquaculture, such as the development of antibio-resistant bacteria and the reduced efficiency of antibiotic for human and animal diseases, have led to suggestions of the use of nonpathogenic bacteria as probiotic control agents (Vaseeharan & Ramasamy, 2003).


Moriarty (1999) reported on his success of using probiotic bacteria instead of antibiotics to control Luminus vibrios in shrimp farms in Negros, Philipine.


The effects of probiotics on the survival of black tiger shrimp (Penaeus monodon) were also recorded by Meunpol et al. (2003) and by Rengpipat et al. (2003). They investigated the effects of the probiotic Bacillus sp on Vibrio harveyi control and, on the growth rate and survival of shrimp.


According to the results of their study, shrimp survival after probiotic treatment increased significantly compared with controls. They also found that the growth and survival rates of shrimps fed on the probiotic supplement were significantly greater than those of the controls.


The antagonistic effect of Bacillus against the pathogenic Vibrios was evaluated in black tiger shrimp, and it was suggested as an alternative treatment factor instead of antibiotics in shrimp aquaculture (Vaseeharan & Ramasamy, 2003).


Selection of probiotics


The principal purpose of the use of probiotics is to produce a proper relationship between useful microorganisms and the pathogenic microflora of digestive organs and their environment. Hence, a successful probiotic is expected to have a few specific properties as follows:

1. Antagonism to pathogens, which is one property of probiotic bacteria. Probiotics should stimulate the immunity of the host by increasing the number of erythrocytes, macrophages and lymphocytes.

One sign of antagonistic properties against bacteria is the production of antimicrobial substances such as organic acids, hydrogen peroxide, sideropheros and lysozyme (Ali, 2000; Verschuere et al., 2000; Irianto & Austin, 2002).


2. Benefits to the host animal in some ways. In order to have a beneficial effect in the form of a growth promoter or to protect fish against bacterial pathogens, the strains should produce important substances, for example vitamins such as biotin and vitamin B12 (Fuller, 1992; Ali, 2000; Irianto & Austin, 2002).


3. The capability of surviving in or colonizing the gut of an aquatic organism by adhesion (Fuller, 1992; Ali, 2000; Verschuere et al., 2000).


4. Adhesion is one of the most important selection criteria for probiotic bacteria because it is considered a prerequisite for colonization (Fuller, 1992; Ali, 2000; Verschuere et al., 2000).


5. Applied microorganisms should be stable for long periods under storage as well as in field conditions (Fuller, 1992).


6. Probiotic microorganisms will, of course, have to be nonpathogenic and nontoxic in order to avoid undesirable side-effects when administered to aquatic organisms (Fuller, 1992).


7. Probiotics should be of animal-species origin. This criterion is based on ecological reasons, and takes into consideration the original habitat of the selected bacteria (in intestinal flora). Many workers believe these bacteria have a better chance of out-competing resident bacteria and establishing themselves at a numerically significant level in their new host (Rengpipat et al., 2003; Riquelme et al., 1997; Alvandi et al., 2004; Jöborn et al., 1997).


Lactic acid bacteria

These are classified in the group of Gram-positive bacteria. They usually have no mobility and are nonsporulating bacteria that produce lactic acid.

Some members of this group of lactic acid bacteria, such as StreptococcusLeuconostocPediococcusAerococcusEnterococcusVagococcusLactobacillusCamilobacterium have adapted to grow under widely different environmental conditions. They are found in the gastrointestinal tract of various endothermic animals, in milk and dairy products, seafood products, and on some plant surfaces (Ringø & Gatesoupe, 1998).

Although lactic acid bacteria are not dominant in the normal intestinal microbiota of larval or growing fish, several trials have been undertaken to induce an artificial dominance of lactic acid bacteria in aquatic animals (Verschuere et al., 2000).

Lactobacilli, as a major group of lactic acid bacteria, have the ability to

1. adhere to cells;

2. exclude or reduce pathogenic adherence;

3. compete for essential nutrients;

4. stimulate the immunity of the host;

5. persist and multiply;

6. produce acids, hydrogen peroxide, and bacteriocins antagonistic to pathogen growth;

7. be safe and therefore noninvasive, noncarcinogenic, and nonpathogenic;

8. coaggregate and form a normal, balanced flora.


Bacillus bacteria

Bacillus subtilis is currently being used for aquaculture, terrestrial livestock and in human consumption as an oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders. Bacillus species are saprophytic Gram-positive, nonpathogenic, spore-forming organisms normally found in air, water, dust, soil and sediments (Gatesoupe, 1999; Green, 1999; Moriarty, 1999).


Bacillus subtilis is a gram-positive, nonpathogenic, spore-forming organism, and the robustness of spores is thought to enable passage across the gastric barrier, and population, albeit briefly, of the intestinal tract. In addition, the clinical effects of B. subtilis as an immunostimulatory agent in a variety of diseases in human and animals, as an in vitro and in vivo stimulant of secretor immunoglobulin A, and as an in vitro mitogenic agent have been documented (Green et al., 1999).


Furthermore, one of the most important advantages of using Bacillus species is that they are unlikely to use genes for antibiotic resistance or virulence from the Vibrios or related Gram-negative bacteria. There are barriers at the transcriptional and translational levels to the expression of genes from plasmid, phages and chromosomal DNA of Escherichia coli in B.subtilis (Moriarty, 1999).


There are many other reports regarding the advantages of using Gram-positive bacteria in aquaculture. For instance, Vaseeharan & Ramasamy (2003) reported on the antagonistic effect of B. sublitis BT23 against the pathogenic Vibrios in shrimp P. monodon, and a 90% reduction in accumulated mortality. The application of Bacillus as a probiotic bacteria can improve the survival rate of larvae, increasing feed absorption by enhancing protease levels, and gave better growth. Moreover, the probiotic decreased the number of unsuspected pathogenic bacteria in the gut (Irianto & Austin, 2002).


The microbial species composition in hatchery tanks or large aquaculture ponds can be changed by adding selected bacterial species to displace the harmful bacteria. This influence is particularly important in the larval stages because larvae may ingest bacteria by grazing on or filtering the suspended particles. It is suggested that probiotics may be most effective when applied to larvae stages, when the larvae have not yet started feeding and the digestive tract is not yet developed completely and the immune system is still incomplete. Therefore, the intestinal microbiota of larvae may change rapidly with the intrusion of microorganisms coming from water and food (Vadstein, 1997; Gatesoupe, 1999; Olafsen, 2001).


When microbial control is desired, single strains of probiotics are less effective than mixed-culture probiotics. The approach should be systemic, i.e. based on a number of strains capable of acting and interacting under a variety of conditions and able to maintain themselves in a dynamic way.

In addition, the microbial community in the gut of aquatic organisms may vary with changes in many factors. It is unlikely that a single bacterial species will be able to remain dominant in a continuously changing environment. Furthermore, the probability that a beneficial bacterium will dominate the associated microbiota is higher when several bacteria are administered than when only one probiotic strain is involved. 


By those reasons, Cenzone Tech Inc. designed YEASTURE, CENMOS and MICROBOND products with mixed-culture probiotic and their performance has been proved in many years and in many countries. These products combined  Bacillus subtilis, Lactobacillus acidophilus, Saccharomyces cerevisiae and their cell wall extracts.