Innovation in natural growth promotion

The growth of aquaculture production has been increasing at a fast rate and this trend is expected to continue in the future. This growth is also accompanied by more intensive farming conditions that may lead to a higher incidence of fish diseases. Antibiotic growth promoters (AGPs) have been used in such challenging conditions to keep pathogenic bacteria under control.

There is a growing awareness regarding the misuse of antibiotic growth promoters (AGP’s), which leads to the need to find more sustainable alternatives for responsible aquaculture production. Among these alternatives several feed additives, including organic acids, may be promising substitutes to the AGP’s in aquaculture.

Organic acids are known to have positive effects on growth performance and animal health. For terrestrial animals, mainly pigs and poultry, these effects are already well documented. Also in fish there is mounting evidence that organic acids improve growth performance and health of fish.

Several studies reported the effects of organic acids in different species including carnivorous fish such as Atlantic salmon, rainbow trout, and Artic charr, also herbivorous fish such as tilapia, and omnivorous fish such as carp and catfish. These studies mainly reported an improvement in performance parameters such as lower feed conversion ratios, increased weight gain and improved nutrient digestibility. These effects are mediated by an antimicrobial effect of organic acids. The antimicrobial effect of organic acids is also shown by the reduced mortality of tilapia fed with acids and challenged with Vibrio anguillarum. Interestingly, for arctic charr it was also reported that fish fed diets with organic acids had a slower gastric emptying rate and this could enhance the antimicrobial potential of the acid salts.

The presence of bacteria represents not only a possible threat to the animals’ health resulting in economic loses, it is also resulting in a loss of energy and nutrients. This is, as possibly necessary immune responses require energy and the host is competing with the bacteria for nutrients. Therefore, it needs to be taken care that bacteria are effectively controlled. In recent years, great effort was put into enhancing the antimicrobial effects seen when using organic acids in aquaculture feeds. The successful control of pathogenic bacteria can hereby be enhanced by combining organic acid with other natural derived products, such as a phytochemical and the new Per4izer^®, a permeabilising substance, which increases the permeability of the outer membrane of gram-negative bacteria. This is a totally new strategy as in this way the antimicrobial effect is boosted considerably.

Effects of the presence of pathogenic bacteria on growth performance

On one side, animals live in symbiosis with different bacteria, while some bacteria are also known to cause diseases. The so-called beneficial bacteria can protect the host from pathogenic bacteria, regulate the development of the gut or produce vitamins and hormones for the host.

However, the presence of possibly harmful bacteria in the gut always represents a possible threat to growth performance.  In general, bacteria within the gastro-intestinal tract compete with the host animal for nutrients. Furthermore, bacteria secrete toxic compounds i.e. toxic amino acid catabolites, decrease fat digestibility, stimulate rapid turnover of absorptive epithelial cells, require an increased rate of mucus secretion by intestinal goblet cells and stimulate immune system development and inflammatory responses. All of these effects lead to a loss of net energy to the microflora, resulting in decreased growth performance.

Therefore, it is not only highly important to control possibly harmful bacteria, but also to keep the bacterial population within the gut well balanced.

Naturally combating bacteria

Already a long time ago organic acids were identified to be able to alter the gastro-intestinal microflora by reducing in particular acid-intolerant bacterial species such as E. Coli, Salmonella and Campylobacter resulting in increased growth performance. However, when combating bacteria the structural differences of gram-positive and gram-negative bacteria have to be considered. The cytoplasm of the cell is surrounded by the cytoplasmic membrane. The cytoplasmic membrane is covered by a thick cell wall layer. This cell wall layer is significantly thinner in gram-negative bacteria compared to gram-positive bacteria. However, gram-negative bacteria are surrounded by an additional outer membrane, which provides the bacteria with an inherent resistance to hydrophobic antibiotics and detergents.

Often organic acids were combined with other naturally derived products such as essential oils in an attempt to use possible synergisms to more powerful combat pathogenic bacteria. Essential oils in general serve as antioxidants, stimulate the immune system, suppress harmful microorganisms on one side but stimulate beneficial microbes on the other, they regulate the activity of certain enzymes, are known to protect the gut villi and to interfere with the DNA replication of bacterial cells, therefore having anti-bacterial effects. However, the mode of action of different phytochemicals varies a lot. So for example cinnamaldehyde, which is a phytochemical derived from cinnamon bark oil, has a more complex mode of action as it targets the FtsZ protein. This protein plays a major role in the cell division of potentially harmful bacteria. FtsZ polymerizes into filaments, which assemble at the place within the cell, where the cell division takes place. There they form into a polymeric structure known as the Z-ring on the inner membrane in the mid of the cell, which is responsible for the division of the cell. Cinnamaldehyde inhibits not only the formation of FtsZ into filaments, but also inhibits essential processes involved in the Z-ring formation and its function and thus the cell division. This results in a reduction of the bacterial load, within the gastro-intestinal tract.

Even if the outer membrane of the gram-negative cell is acting as a protective barrier for external agents it is possible to weaken the outer membrane by agents commonly characterised as permeabilizers. However, permeabilizing substances have different modes of action.  Some of them remove stabilizing cations from the outer membrane, while others bind to the outer membrane resulting in the loss of barrier function. When permeabilizing substances weaken the outer membrane of gram-negative bacteria, the activity of other antimicrobials is increased by facilitating external substances, which inhibit or destroy cellular functions, into the cell is facilitated. This leads to synergistic effects when a permeabilizing substance is added to a mixture of organic acids. However, if a synergism can be found highly depends on the right combination of organic acids and permeabilizing substances, as not with each combination a synergism will be found.

Effects on the inhibition of fish pathogens

Synergistic effects on the inhibition of commonly found bacteria in terrestrial animals such as E. Coli or Salmonella were found when adding the Per4izer^® - a permeabilizing substance – to the formula of organic acids and a phytochemical. This is an indication that the permeabilization of the outer membrane of gram-negative bacteria facilitates organic acids and phytochemicals to penetrate the gram-negative cell destroying cellular functions, leading to cellular death. Seen synergistic effects on the inhibition of general pathogenic bacteria draw the attention to the potential effects on the inhibition of pathogenic bacteria specific for fish. Therefore, an in vitro trial was set up, at the BIOMIN Research Center in Tulln in Austria, to test the effects on the inhibition of bacterial growth of known fish pathogens by an acid mixture combined with a phytochemical and the Per4izer^®. Three different pathogens, specific for aquacultured species were tested. This was Yersinia ruckeri, which was shown to cause enteric redmouth disease, a disease which causes significant losses especially in salmonid aquaculture worldwide. The second fish pathogen tested was Vibrio Harveyi, a bacterium responsible for the outbreak of white spot disease in fish. As a third pathogen specific in fish, Aeromonas veronii was chosen. Aeromonas spp. are often isolated from humans suffering from diarrhoea, but also fish with hemorrhagic septicaemia. Also the epizootic ulcerative syndrome in fish, which is causing severe economical losses, is associated with the presence of fish pathogens such as Aeromonas or Vibrio.

Results show that inhibition of Yersinia ruckeri was increased by the addition of the Per4izer^® to the antimicrobial mixture of organic acids and the phytochemical. For Vibiro Harveyi a 100% inhibition was already achieved with the antimicrobial mixtrure. A real synergism between the antimicrobial mixture and the Per4izer^® was found for the inhibition of Aeromonas veronii. While the acid mixture and the antimicrobial mixture had inhibiton rates lower than 20%, the addition of the Per4izer^® enhanced the inhibition to near 100%.

Figure 1: Inhibition of fish pathogens by an antimicrobial mixture (acid mixture, plus a phytochemical) and an antimicrobial mixture and the Per4izer^®

Effects on growth performance

As explained earlier these mixture of acids combined with the phytochemical and the Per4izer^® - permeabilizing substance – can also have important impact as a growth promoter. Therefore, an in vivo trial was set up at the BIOMIN Research Center in Bangkok in Thailand to test this hypothesis

The fish species used in this trial was red tilapia (Oreochromis mossambicus x Oreochromis niloticus). Groups of 60 fish averaging appr. 45g were stocked in 6 experimental tanks (Volume: 500l). Fish were fed to near satiety 3 times per day for a period of 56 days. Fish were fed a diet based on soybean meal, rice bran and cassava and wheat flower containing 30% crude protein. The control group diet contained no feed additives, whereas the diet of the trial group was supplemented with the natural growth promoter Biotronic^® Top3 containing an organic acid mixture, a phytochemical and the Per4izer^® at an inclusion rate of 1.0 kg/t feed.

Growth performance was improved due to the supplementation of the diets with the acidifier Biotronic^® Top3. Feed intake was not affected by the treatment. However, feed conversion ratio was significantly improved by 5% (P>0.05) in the Biotronic^® Top3 group compared to the control group.  This higher efficiency of ingested feed resulted in significantly higher (P<0.05) final body weight by 5% in the Biotronic^® Top3 group compared to the control group. Also daily growth coefficient was significantly (P<0.05) improved by 6% in the Biotronic^® Top3 compared to the control group.

In short, the synergy caused by the inclusion of the permeabilizing substance boosts the effect of the antimicrobial ingredients, resulting in economical benefits for the end user. Therefore, it presents an innovative strategy to act against bacteria. The company has launched the natural growth promoter as Biotronic^® Top3.