Probiotics: Key criteria and role in gut health
Abstract. Searching for alternatives to antibiotic growth promoters (AGP) has become an imperative task in animal industry today. Probiotics appear to be one of the promising solutions. Probiotics refer to various microbial strains, either in single or in combination. Selection criteria are numerous, mainly 1) safe to host, meaning not to cause cyto-toxicity or hemolysis; 2) stable to feed processing and gastro-intestinal conditions; 3) benefit intestinal health through improving gut barrier function and enhancing gut immunity; 4) able to improve birds' performance, especially under challenging conditions; 5) doesn't contain antibiotic resistance genes; and 6) not too difficult for end users to conduct quality check.
According to Food and Agriculture Organization and World Health Organization, the definition of probiotic is "live microorganisms which, when administered in the right amount, confer a health benefit on the host" (FAO, WHO 2001, Bajagai et al., 2016). Among a wide variety of probiotics are bacterial strains belonging to Lactobacillus, Enterococcus, Bifidobacterium, Lactococcus, Streptococcus, Pediococcus, Bacillus genera as well as yeast species. They can be classified as bacterial or non-bacterial (yeasts), spore forming (Bacillus) or non-spore forming (Lactobacillus, Bifidobacterium), multi-species or single-species, allochtonous (not present in the GIT such as yeasts) or autochtonous (such as Lactobacillus).
Ideal probiotics must be non-pathogenic, host-specific, able to survive to gastrointestinal conditions like acidity, bile salts and digestive enzymes, modulate gastrointestinal microbiota and/or gut health parameters such as barrier integrity, immune response, inflammatory status, prevent pathogen bacteria development, and survive processing and storage. Bacillus species are able to form endospores, stable to passage of the stomach, bile salts, chemical and physical stresses, making them very attractive for animal feeding.
Mode of Action
The mode of action of probiotics has not been entirely understood by far but it is clear that it does rely on complementary mechanisms. It is very well accepted that the mode of action depends on the nature of the microorganism. The phylogeny and full identification of the strain used as probiotic is therefore crucial. Hence, it is important not only to know the genus, the species but also the strain. For instance, the degree of homology among Bacillus subtilis strains was calculated from 79% to 97% alike. As a comparison, human and mice share 92% of their genes and their difference is clear.
The mode of action of probiotics can be either direct or indirect, through an action on intestinal microbiota. Clear evidence can be obtained by simple in vitro tests where the growth of bacteria is monitored in presence or not of the probiotics. Probiotics produce compounds and signals that will affect other bacteria, positively or negatively.
Therefore, probiotics can change intestinal microbial population in the gut creating a more favorable microbial population due to a shift in the balance of beneficial and harmful microbes, resulting in better gut health. Scientific publications have shown increases in the populations of Lactobacillus sp, Bifidobacteria sp. and conversely a decrease in E coli and Clostridium perfringens pathogenic species (Mountzouris et al., 2009).
Also, probiotics can have an effect on the host. There is evidence that probiotics increase the villi length and the villus height:crypt ratio thus enhancing the intestinal surface area for better absorption of nutrients. Probiotics and particularly Bacillus based ones have shown a direct effect on the enterocytes and the intestinal barrier. Maintaining an optimal tight junction functioning and reducing inflammation of the gastro-intestinal tract directly improve gut health under challenged conditions. Different in vitro and in vivo models can demonstrate the stimulation of immune response (IL-8 production, T-cell and the relevant CD3+, CD4+, CD8+, IgA… for instance) under either standard conditions or immuno-challenges (after virus, E coli, IL-1… administration).
Different probiotics may have similar mode of action whilst the same species but different strains may have very different mode of action. It is also clear that in order to benefit from probiotics, one must supplement them prior to infectious challenges or exposure to various stress conditions.
In order to select optimal strain of probiotics, more than 800 strains from Novozymes extensive culture collection were tested. They were QPS and American GRAS/AAFCO listed, primarily Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus pumilus, which were independently isolated. The first selection criterion was the absence of beta-hemolysis assessed as recommended by EFSA on Tryptic Soy Agar +5% sheep blood (EFSA, 2011). Second criterion was a radial diffusion assay for a performance relevant activity. The third criterion was compliance to EFSA guideline for antibiotic resistance (EFSA, 2012).
From the 800 strains initially included in the screening, 32% were non-haemolytic and about 10% of the isolates were positive in the radial diffusion assay, only 26 isolates fulfilled the above criteria. After testing for susceptibility to antibiotics according to EFSA's guideline, only six strains met the criteria. In the end, only one strain, Bacillus subtilis DSM29784, was selected for further testing.
To study the phylogeny, a comparative analysis of the gyrB gene sequences was performed. This was done essentially as for Bacillus subtilis and related strains using about 1200 nt partial gyrB gene sequences (Wang et al., 2007). The analysis showed that Bacillus subtilis DSM29784 is different from the type strain of Bacillus subtilis at a level similar to the already known and described subspecies; Bacillus subtilis subsp. spizizenii, Bacillus subtilis subsp. inaquosorum. DSM29784 showed the highest degree of identity to Bacillus subtilis subsp. Inaquosorum, being a novel subspecies of Bacillus subtilis (Figure 1). The competitor product X showed close relationship to the type strain of Bacillus subtilis.
Figure 1 Phylogenetic tree showing the relationship of Bacillus subtilis DSM 29784 to reference strains of Bacillus species and the competitor product
Three performance studies were conducted in three different research facilities in order to test different experimental conditions and broiler strains. The study design was the same for all studies, with three treatments (Control, Control + Product X and Control + B. subtilis DSM29784). The diets were based on corn-soybean meal and similar among all the studies, but differed by the presence or absence of animal by-products and phytase. Diet nutrient levels were calculated to meet the requirements of the animals, according to the relevant breeder recommendations. For each study, at day 0, male broilers were randomly assigned to pens and numbers of broilers per pen and pens per treatment were determined to allow statistical analyses. Animal performance was measured at day 28 and day 35.
The results of the three studies (Table 1) were analysed independently. In all studies, the effects of the different Bacillus subtilis strains were more developed at day 35 than at day 28, and the strains did not show the same degree of efficacy. The Bacillus subtilis DSM29784 significantly improved the performance in all 3 studies, with average improvement 3.8% on BWG and 3.2% on FCR. The competitor product improved significantly BWG and/or FCR in only one study (average improvement in the 3 studies: 1.7% for BWG and -2.2% for FCR).
Moreover, this Bacillus strain was also tested in challenging conditions, using a well-established challenge model, the results showed the challenge was detrimental and increased mortality, and reduced performance. The addition of Bacillus subtilis DSM29784 showed potential to restore animal performance, with no significant difference to the unchallenged animals, and to the level of the antibiotic treatment for BWG and FCR.
Bacillus subtilis belongs to a bacterial species that covers quite extensive diversity, which is acknowledged in a comparative analysis of genome sequences (Earl et al., 2012). The specificity of strains is crucial when comes to the efficacy of probiotic, and selection process should be extremely rigorous.
Our further studies on host-microbe interaction have demonstrated improvements on overall gut health, such as intestinal barrier integrity, immuno-function and reduction of the inflammation in the small intestines of broiler birds. The consistency observed from in vivo trials with Bacillus subtilis DSM29784 suggests that this strain behaves more naturally in the intestinal system of broiler chicken than the already known strains of this species.
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Article made possible through the contribution of Dr. Y G Liu, Adisseo Asia Pacific P/L; Dr. Estelle Devillard, Adisseo France, S.A.S.