Why You Should Use a Combination of Beneficial Bacteria in Aquaculture

In any group of people, some have skills and talents that others do not possess. Each person has his or her own strengths, weaknesses, skills and talents that allow them to perform and successfully accomplish a task. The same is true for beneficial bacteria, or probiotics. Different species have different characteristics that affect their efficacy, and ultimately their probiotic usefulness. This seems like common sense, but then why are the majority of commercial probiotic formulations focused on a single genera, Bacillus? There are a few reasons. Firstly, most common Bacillus spp. are generally not too difficult to grow at moderate cost.

Secondly, they can form bacterial spores, which allows for a longer shelf life and gives greater (but not limitless) heat stability. Thirdly, they have an extensive safety record with only a couple of species known to be pathogenic or toxic to animals (Bacillus anthracis and Bacillus cereus). This safety record makes it relatively easy to register Bacillus spp. products, since they appear on many approved lists (e.g. GRAS - Generally Recognized as Safe in USA, and QPS – Qualified Presumption of Safety in EU). However, these benefits are more useful for probiotic producers than they are for the animal. Although there is scientific evidence to support the use of Bacillus-based probiotics in aquaculture, it is unlikely that a single genera of bacteria can do everything. The intestine is an extremely complex ecosystem, and therefore specific microbial drivers are needed, in the right concentration, for specific tasks within the gut. The probiotic additive should be chosen based on the desired outcome, e.g. growth performance, immunity, disease resistance, survival rate.

Probiotic colonization

It is controversial whether ‘true’ probiotic colonization is possible. A number of studies have reported that once probiotic feeding has stopped, the probiotic can persist in the intestine for a certain period, therefore demonstrating temporal colonization. In aquatic species, the current literature suggests this period can be anywhere between three days to more than three weeks and is dependent on the probiotic species, host species, environmental factors, dosage and duration of probiotic supplementation. The intestinal microbiota can be split into two distinct groups, those that are transient (allochthonous) and those associated with the epithelia (autochthonous). The best probiotic colonizers are the autochthonous bacteria.
By attaching to the intestinal epithelia, they compete with pathogens for adhesion sites, preventing their attachment and subsequent translocation leading to immune response, sub-acute stage of energy loss, or even acute infection. Furthermore, through a complex system of molecular receptors, authochthonous bacteria can also interact with the host immune system, improving immunity and increasing disease resistance.

Figure 1. Schematic diagram of where intestinal cells were isolated from the digestive system of rainbow trout (O. mykiss). The intestine was split by region, opened up longitudinally and epithelial cells isolated for further in vitro culture

Source: Langan et al., 2018
Figure 1. Schematic diagram of where intestinal cells were isolated from the digestive system of rainbow trout (O. mykiss).

Different probiotic species possess different adhesion properties, and this can ultimately affect their ability to colonize the intestine and exert their benefits. As important mucosal surfaces, two aquatic epithelial cell lines from rainbow trout (Oncorhynchus mykiss) were chosen to evaluate probiotic adhesion; commercial gill cell lines (RTgill-W1, ATCC, Virginia, US) and intestinal cell lines, isolated fresh by scientists at the BIOMIN Research Centre, Austria (Figure 1). The probiotic species (Lactobacillus sp., Pediococcus sp., Enterococcus sp. and Bacillus sp.) were chosen based on their well-documented benefits in aquatic animals. Their combination makes up the commercially available probiotics from BIOMIN, AquaStar® Growout and AquaStar® Hatchery. In general, lactic acid bacteria (LAB; e.g. Lactobacillus spp., Pediococcus spp. and Enterococcus spp.) can be colonizing bacteria in the gut. As this is one crucial criterion for an efficacious probiotic, all LAB strains selected for AquaStar® products are able to attach to the gill and gut epithelial cells, with stronger adhesion observed in the intestinal cell lines (Figure 2).
AquaStar® Lactobacillus sp. was extremely good at attaching to epithelial cells, with an average of >100 and >300 probiotic cells attaching to an individual gill and gut epithelial cell, respectively. AquaStar® Pediococcus sp. exhibited the second strongest adhesion properties, with more than 30 probiotic cells attaching to a single enterocyte (gut epithelial cell). Enterococcus sp. also displayed adhesion abilities, albeit at a lower level than Lactobacillus and Pediococcus. Also of interest was the complete inability of Bacillus sp. to attach to either epithelial cell types (less than one Bacillus cell per epithelial cell). In the intestinal environment, this suggests that their main function is in the lumen of the gut. It is hypothesized that, due to their proteolytic nature (discussed below), it would actually be negative and potentially harmful for the animal if a Bacillus probiotic were to attach to epithelial cells. These data suggest that for better colonization of the intestine, an in-feed probiotic should favor LAB, instead of Bacillus spp.

Figure 2. Adhesion properties of selected probiotic bacteria to gill (a) and gut (b) epithelial cell lines. The data indicate the number of probiotic cells attached to a single epithelial cell

Source: BIOMIN
Figure 2. Adhesion properties of selected probiotic bacteria to gill (a) and gut (b) epithelial cell lines.

Enzyme production by Bacillus spp.

Bacillus spp. are well known for their ability to produce enzymes. In the intestine, these enzymes can improve the digestibility of feeds, contributing to better feed conversion and growth performance, whilst in the environment they can help break down organic matter in the water and sediments. Using in vitro techniques, BIOMIN scientists have documented the ability of numerous Bacillus probiotic candidates to produce proteolytic, amylolytic and cellulolytic enzymes. It was clear from the variation that the ability to produce enzymes was not universal within the Bacillus genus (Figure 3) and therefore caution must be applied when choosing a commercial probiotic. Most interesting was the variability within a single species. For example, when looking at protease production, the inhibition zone from B. subtilis 1 was approximately half that of B. subtilis 5, indicating considerably lower protease production. Similarly, amylase production differed between the Bacillus spp. tested. Certain Bacillus candidates were not able to produce amylase at all (e.g. B. licheniformis 1), whereas others (e.g. B. subtilis 5) could produce more amylase. While carbohydrates are not fully utilized in aquatic animals, starches help with pellet binding and expansion in extruded aqua feeds, affecting pellet density. For example, sinking feeds will typically have 6-8% starch, while floating feeds will have over 20%.

Figure 3. Ability of Bacillus spp. to produce extracellular proteolytic, amylolytic and cellulytic enzymes. Higher bars indicate greater production or activity of the respective enzyme. The red box indicates the B. subtilis strain in AquaStar® products

Source: BIOMIN
Figure 3. Ability of Bacillus spp. to produce extracellular proteolytic, amylolytic and cellulytic enzymes.

Conclusion

The intestinal microbiota is infinitely complex, with different groups of bacteria having different roles and bringing different benefits to the host. Therefore, it is extremely unlikely that a single probiotic species, or even genera, can offer a “silver bullet” solution to the diverse challenges in aquaculture. Producers and feed millers can address these complexities by choosing formulations that utilize a multi-genus and multi-benefit concept, such as AquaStar®. Bacillus sp. can produce high volumes and activity of enzymes, contributing to improved digestibility and feed conversion, whilst AquaStar® containing LAB can colonize the intestine, reducing pathogen load, improving immunity and increasing disease resistance.
Clearly, there is a place for simple Bacillus probiotics, but perhaps it is time that that they were substituted, in favor of alternative and maybe more promising, systematic probiotic products.

In Brief

  • The environment within the gut is very complex, with different groups of bacteria having different roles and bringing different benefits to the host.
  • Using a single-strain probiotic is unlikely to provide a solution to the many challenges faced.
  • AquaStar® from BIOMIN contains multiple species and therefore delivers multiple benefits.

Reference

Langan, L.M., Owen, S.F., and Jha, A.N. (2018). Establishment and long-term maintenance of primary intestinal epithelial cells cultured from the rainbow trout, Oncorhynchus mykiss. Biology Open 2018 7. doi: 10.1242/bio.032870

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