In the modern world probiotics are almost as common as vitamins. They are used in antibiotic therapy, immunosuppression and to treat gastrointestinal diseases. They are available as medical products or various types of milk drinks containing selected strains of bacteria. Not all users know how probiotics work but their effects are pretty visible. So why not try products with probiotics for aquarium fish? Knowing how difficult it is to diagnose and treat this group of animals, wouldn’t be a good idea to take care of their natural immunity instead? After all, probiotics in fish food are becoming more and more popular with every day.
Short history lesson of probiotics
First beneficial effects of fermented milk on the human body were recognized in ancient times. Nowadays it’s a common knowledge that this drink contains strains of bacteria with probiotic properties. But back then only the effects of its consumption were known. Piliny the Elder recommended the use of fermented drinks in gastrointestinal disorders and so did other famous ancient thinkers such as Hippocrates and Galen.
But the one who made a huge contribution to the development of microbiology was Louis Pasteur. Between 1857 and 1868 he conducted research on fermentation processes and he discovered that it was carried out by microorganisms. He also noted that some bacteria definitely did not like the company of others. This discovery was undoubtedly the first step towards the use of other bacteria to fight against pathogenic bacteria.
Ilya Ilyich Mechnikov, a Russian immunologist who was awarded the Nobel Prize in medicine in 1908 for his work on immunity, in his book entitles “Studies in human nature: Essay in optimistic philosophy” claims that: “with various foods subjected to lactic fermentation and eaten raw (sour milk, kefir, cabbage, cucumbers), people have introduced into their gastrointestinal tract huge amounts of microbes, which can also reproduce there”. Mechnikov himself drank sour milk every day and was a promoter of the consumption of fermented products.
In 1907 Serkowski noticed that “sour milk may save milk from rotting”, and that „milk fermentation plays an important role in animal feed because fermented feed does not rot”. So he started wondering if milk fermentation would prevent rotting in human gastrointestinal tract as well.
In 1989, Roy Fuller defined the characteristics that a probiotic organism should have:
- resistance to low stomach pH, enzymes and bile acids in duodenum;
- ability to survive and carry out its metabolic activity in the large intestine environment;
- ability to adhere to intestinal epithelium cells and permanent or transient colonization of the gastrointestinal tract;
- beneficial effects on the host organism;
- persistence and durability during storage and in an unfavorable environment (e.g. in the gastrointestinal tract: low pH, digestive enzymes, factors inhibiting growth or adhesion capacity);
- coming from man/fish/target animal.
In Poland, a number of scientists were interested in this subject, including a pediatrician and neurologist Józef Brudziński who treated some intestinal diseases in infants with lactic bacteria. In the 70’s and 80’s, Zychowicz and Cieplińska demonstrated that Lactobacillus acidophilus has antagonistic effects on Salmonella sp. and Shigella sp. and Shigella sp. Next, they were the first to use fermented milk formula in the treatment of children during the long-term epidemic of dysentery in the State Orphanage in Mrągowo, where all other methods of treatment failed. Their therapy were effective after just 2-3 weeks. Not only was dysentery eliminated, but also changes in children’s behavior were observed. Their appetite and immunity increased and there was a clear leap in their psychomotor development.
So what is a probiotic?
The current definition says that probiotics are living micro-organisms which have beneficial health effects, if administered in appropriate quantities. But…. in the case of fish, this definition should be extended. The micro-organisms living in the digestive tract of fish are the same as those present in their environment or in food. One can say that in an aquatic environment, the host and micro-organisms share one ecosystem. In the case of fish, microorganisms can „choose” whether they want to live in the gastrointestinal tract, on the skin, gills or simply in the surrounding aquatic environment. There is one more aspect to bear in mind. In fish, whose vast majority lay eggs to the environment, the hatching does not come into contact with the parent’s microflora. Eggs are colonized by bacteria from the environment, as well as the digestive tract, gills and skin of fry, which do not have their own microflora after hatching.
Therefore, in the case of aquatic animals, the definition of probiotic should read as follows: Probiotics are living micro-organisms which have beneficial health effects by modifying the bacterial flora of the host and the surrounding environment, when administered in appropriate quantities.
There is a great number of probiotic microorganisms but it’s important to remember that each strain may have different properties. Two different strains, even of the same species, do not have to behave in the same way. The strains have numbers which are written after the species name of the bacteria, e.g. Lactobacillus casei DN 11400. In fish, the following bacteria are most commonly used as probiotics: Bacillus subtilis, Lactobacillus acidophilus,Lactobacillus plantarum, Lactobacillus lactis,Streptococcus termophilus. And apart from them fungi such as Sacharomyces cerevisiae and Sacharomyces exigous and Phaffia rhodozyma algae are used.
How do probiotic organisms work?
We already know what a probiotic is and what qualities it should have. But how does it function in the host’s body? If you take a closer look at the abilities of probiotic organisms, you can come to the conclusion that they are sophisticated killers. In order to eliminate other bacteria, they use both physical and chemical methods.
They restrict access to ‚living’ areas
In order to colonize the gastrointestinal tract the bacteria need to adhere to the intestinal epithelium. Probiotic bacteria often show greater ability to adhere than pathogenic bacteria, thus creating a tight barrier on the epithelium surface, which prevents the settlement of other bacteria.
They shape the environment
A large group of probiotic bacteria produce organic acids such as lactic acid, which lower the pH in the gastrointestinal tract beyond the optimal range for pathogens such as Escherichia coli, Clostridium sp. or Salmonella sp. In addition, organic acids take part in the production of a protective barrier on the surface of the intestinal epithelium, thus inhibiting the adhesion of pathogens.
They poison subtenants
They produce hydrogen peroxide (H2O2), which breaks disulphide bridges in bacterial proteins.
They reach for biological weapons
They can produce proteolytic proteins, which have a destructive effect on the cell walls of bacteria, and also produce bacteriocins – substances that can be described as natural antibiotics. They can destroy gram-positive bacteria.
They restrict access to food
Bacteria living in the gastrointestinal tract are called heterotrophs and they use organic components as sources of carbon and energy. Probiotic organisms, which feel perfect in intestinal conditions and after some time gain an advantage in numbers, use nutrients, limiting access to them for less ‘go-getting’ bacteria.
Immunostimulation – secret weapon
The mechanism of influence on the host’s immune system is extremely complicated. Bacteria stimulate the lymphatic system associated with the gastrointestinal tract to produce lymphotics (immune cells), and these in turn produce IgA immunoglobulins, which are the first line of defence of the organism against harmful factors of the external environment, responsible for agglutination of bacteria, inhibition of bacterial cell adhesion to mucous epithelium, as well as neutralization of viruses, toxins and enzymes produced by microorganisms.
Probiotics in fish food – studies
One of the latest products for aquarium fish in Tropical’s portfolio is Pro Defence, which uses Bacillus subtilis C-3102 bacteria as a probiotic. The research on the use of this strain in ornamental fish nutrition was carried out in 2008 by Shubhadeep Gosh and his team. Four popular species of egg-living fish were selected for the study: guppies, platies, gladiolus and molynesia. In the study, each species was represented by 400 fish. Due to the fact that the effect of 4 different doses of probiotic (plus control group) on each fish species was studied, 5 tanks were prepared in 4 repetitions, which totaled 20 aquariums with a capacity of 150 litres.
The content of Bacillus subtilis in the food used in the experiment:
5 x 1011 CFU Bacillus subtilis / kg of food
5 x 1010 CFU Bacillus subtilis / kg of food
5 x 109 CFU Bacillus subtilis / kg of food
5 x 108 CFU Bacillus subtilis / kg of food
The unit used to administer the number of bacteria is the so-called colony-forming unit (CFU). It determines the number of cells in the test material using a culture that distributes the material sample as evenly as possible on the medium so that all microorganisms are lying alone and at a distance from each other, forming a single colony by propagation. In other words, CHU denotes the number of individual cells from which cell colonies will be formed as a result of the divisions. Under perfect conditions the number of colonies is equal to the number of microorganisms in the sample.
After 90 days of the experiment, one more test was carried out. The fish were infected with gram-negative bacteria Aeromonas hydrophila. It is one of the most common pathogens of tropical fish, which causes furunculosis, causing mass deaths of fish in farms.
The number of fish
Starting weight of fish
The number of tanks
Volume of the aquarium
The number of fish in the aquarium
4% of body weight
4% of body weight
4% of body weight
4% of body weight
Amount of probiotics in the food
Duration of the experiment
As a result of these studies the following results were observed:
- greater growth of all four species of fish getting doses of 5 x 109 – 5 x 1011 CFU/kg of feed compared to the control group
- increase in the number of B.subtilis bacteria in the gastrointestinal tract of fish
- increased resistance to Aeromonas hydrophila
The research team also conducted an analysis of the food used in terms of changes in the content of probiotic bacteria over time. A decrease in the number of bacteria was observed, however, much smallerwhen compared to other probiotic bacteria species. Scientists explained this by the fact that Bacilus subtilis produces endospores, a specific type of spore. The whole process of spoluration is extremely complicated, although we are dealing with a single-cellular organism. Around the nucleotide there is a cytoplasm and the whole is covered with a thick cortex (the thickest layer of spore) and a three-layer wall. The endospore is resistant to temperatures even above 100oC, although for the bacterium itself this feature does not matter much and such a temperature can only be found in humans. However, what is important for bacteria and what is believed to have contributed to the development of their ability to spolurate is endospores’ resistance to drought, UV radiation, unsuitable pH and their ability to survive in the absence of food. The germination of endospor is as complicated as their formation. This process is independent of external sources of energy and food.
The same team of scientists also conducted one-year-lasting research on the effects of probiotics on the reproduction of the above mentioned fish species. For all species higher fertility, higher gonado-somatic index*, higher survival rate of fry, lower number of deformities and faster growth were observed.
The next research on Bacillus subtilis were conducted in 2011 and this time they involved koi. The main objective was to determine the effect of probiotics on fish growth. This effect was proved to be positive. The bacteria were found in the gastrointestinal tract and inhibition of the development of Aeromonas veronii, a pathogen common in fish farms, was observed.
The influence of Bacillus subtilis on immunity was also studied in 2014 by Telli and his team. They used a Nile tilapie for his research. High density of fish (50 fish in a 800l tank) allowed for obtaining stress conditions.
|The number of fish||520 fish|
|Starting weight of fish||32.63 g|
|Volume of the aquarium||800 liters|
|The number of tanks||16|
|The number of fish in the aquarium (low and high density)||15 50|
|Feeding||3 times a day to the full|
|Amount of probiotics in the food||500 mg/kg|
|Duration of the experiment||84 days|
Microbiological analysis showed the presence of live B.subtilis in the gastrointestinal tract of tilapia. However, the most interesting founding was the positive effect of probiotics on non-specific immunity of fish under stress conditions. The scientists found an increase in phagocytic index and lysozyme activity under stress in fish that received probiotics compared to the control group.
Phagocytes play an important role in the innate immune system. They are able to recognize and destroy the cells of pathogens. An increase in phagocytic index in fish fed with probiotic food and kept at high densities is a response to unfavorable living conditions and stress. Lysozyme, on the other hand, is a protein that breaks down the cell walls of bacteria. The probiotic organism may increase its secretion by the host’s immune system, as well as produce it by itself.
Benefits for fish from the use of probiotics in fish food
Probiotics improve nutrition through the synthesis of essential ingredients such as vitamins, hort-chain fatty acids and enzymes (amylases, proteases). Moreover, probiotic bacteria decompose food components that would not be digestible without their participation. They increase resistance to infections. They eliminate unwanted bacteria, including substances produced by the, which inhibit growth. In addition, they have a beneficial effect on reproduction, the quality of the offspring and their survival.
With probiotic food we can influence the composition of the gastrointestinal microflora. The probiotic is given to fish which already have a mature digestive tract microflora. In order for the probiotic share in the total mass of microorganisms to reach a level at which its action is visible, time must elapse and administration of the food should be regular during the first period.
*GSI – gonado-somatic index (gonade mass/body mass * 100). It is a tool to assess the sexual maturity of animals in relation to the degree of development of ovaries and testicles.
Fuller R. (1989) Probiotics in man and animals. J. Appl. Bacteriol., 66, 365–78.
Ghosh S., Sinha A., Sahu C. (2007) Effect of probiotic on reproductive performance in female livebearing ornamental fish. Aquaculture Research, 38, 518-526.
Ghosh S., Sinha A., Sahu C. (2008) Dietary probiotic supplementation in growth and health of live-bearing ornamental fishes. Aquaculture Nutrition, 14, 289-299.
He at al. (2011) Evaluation of probiotic strain Bacillus subtilis C-3102 as a feed supplement for koi carp (Cyprinus carpio), Aquaculture Research & Development, January.
Nowak A., Śliżewska K., Libudzisz Z. (2010) Probiotyki – historia i miechanizmy działania. ŻYWNOŚĆ. Nauka. Technologia. Jakość, 4 (71), 5 – 19
Telli G. i inni. (2014) Dietary administration of Bacillus subtilis on hematology and non-specific immunity of Nile tilapia Oreochromis niloticus raised at different stocking densities. Fish & Shellfish Immunology, 39, 305-311.