Probiotics, belonging to the functional group of gut flora stabilisers within the category of zootechnical feed additives (according to the Regulation EC No 1831/2003) is a fast growing market. In 2004, the global market value of probiotics was €32 million, with a forecasted annual growth of approximately 3%. However, due to the ban of antimicrobial feed additives, the probiotic market in Western Europe showed an annual growth of more than 7%. In 2006, Western Europe produced around 296 tons of probiotics, with a value of €15.5 million. With 1012 CFU (equivalent to about 100g) usually added to a ton of mixed feed, approximately 3 million tons of feed containing probiotics was produced last year.
Authorised probiotics for pigs
Of the 18 authorised probiotics in the EU, 12 are authorised for pig feed (10 are approved for piglets, 6 for sows and 5 for fattening pigs). The micro-organisms for pig feed are of various origins (Table 1 ). Most preparations contain defined strains of bacteria and only three of them contain yeasts. Bacillus strains are spore forming bacteria and are applied as spore preparations while enterococci and pediococci do not form spores and are applied as desiccated vegetative ells. Therefore, Bacillus probiotics are much more stable during feed processing (including pelleting and during in-feed storage). We have found that the recovery of B. cereus var. toyoi was 95% after pelleting (conditioner 80°C, dye 87°C), while the recovery of viable counts of an E. faecium strain decreased with increasing treatment temperature (Figure 1). However, the stability of vegetative cells can be improved by various techniques (soaking on globuli, coating). Although viability losses can be compensated by initial overdosing during feed production if the rate of inactivation is known, storage of the complete feed is still a matter of concern. Bacterial spores, on the other hand, are remarkably stable during storage in pelleted feed (Figure 2 ). Yeasts are the most sensitive to heat treatment. Probably influenced by their stability characteristics, sales volume of used probiotic organisms in pig feeding can be categorised as follows: Bacillus spore probiotics > Enterococcus strains (lactic acid bacteria) > yeast probiotics (viable cells).
Effect on bacterial communities
Considering the fact that bacterial population outnumber the ingested probiotic bacterium by a factor of 10-1000 in the stomach/small intestine and more than 104 in the hind gut, one may doubt that any effect could occur. However, there are a number of studies on the influence of probiotics in piglets that show significant modifications of bacterial composition and hence bacterial activity, as well as influences on intestinal immunology, epithelial physiology and anatomy. Bacillus cereus and Enterococcus faecium, two commonly used probiotics in pig nutrition, are of completely different origin. Both species are able to modify bacterial population in young animals, yet their modes of action may be completely different. In our recent realtime-PCR study (Vahjen et al., 2006), we found that the ratio of probiotic E. faecium cells to total Enterococcus spp. cells in the intestine of suckling and weaned piglets was only in the range of about 0.1 - 12% (Figure 3) and even less for total bacterial cells in the intestine. However, the same study showed that E. faecalis - not E. faecium - cell numbers (excluding the probiotic strain) were significantly reduced due to the presence of the probiotic E. faecium. Thus, one probiotic mode of action of this strain may be the growth inhibition of similar species, which may trigger further changes of the bacterial composition. Similar studies on Bacillus spp . probiotics are not available; however, classical culturing studies show decreased growth of enterobacteria and enterococci, but not for total lactic acid bacteria or other main bacterial groups. As bacilli are alien to the intestine, the local immune system of the piglet will treat probiotic bacilli as a potentially pathogenic threat. Indeed, the contact of suckling piglets with a Bacillus cereus probiotic via mother faeces led to an upregulation of key small intestinal immune parameters.
Interestingly, the same group found a reduced immune response in suckling piglets which had contact with E. faecium probiotic via mother faeces (Scharek et al., 2005). This led to the assumption that the E. faecium probiotic itself or the modified bacterial population in mother faeces (mother sows received the probiotic in feed) were less active in triggering an immune response in the piglet. Thus, different modes of action regarding the modification of intestinal bacterial communities can be expected in different probiotic bacteria.
Effects on pig performance
A summary of 49 peer-reviewed publications on probiotics in pigs between 1973 and 2007, which covered 11 different bacterial species in several different strains and in various preparations, yield a very heterogeneous picture regarding piglet performance and post-weaning diarrhoea.
Concentrating on weaned piglets, for which the reviewed literature is reasonably comparable, the relative change (probiotic vs. control group) in average daily gain (ADG) of post-weaning piglets ranged from -8% to +24% (median +5.5%), but compromised just 14 trials (35%) with significant positive effects out of 40 analysed trials.
The relative change in feed conversion ratio (FCR) in these trials ranged from -12.5 to +5%, had a median change of 0% and was significantly improved compared to the control group in only 10 trials.
Our own studies, which used Enterococcus faecium NCIMB 10415 and Bacillus cereus var. toyoi in feed for sows during gestation/lactation and for piglets pre-/ postweaning, led to an 11% numerical increase in ADG (P>0.05) and a highly significant improvement of about 9% for FCR (P<0.01) in the case of the B. cereus strain (Taras et al ., 2005). E. faecium NCIMB 10415 had no significant affect on overall performance of weaned piglets in any of three supplementation initiation variants; although the overall FCR was improved numerically by about 1%.
Correlation of the ADG in control groups with the relative ADG differences observed in the respective probiotic groups has led to the impression that a significant improvement of ADG by probiotics might become more and more unlikely with increasing ADG in the control group (Figure 4). In addition, it might be suggested that with increasing ADG in the control group, the magnitude of the probiotic effect decreases. This may indicate that probiotics work most efficient in young piglets early on in the development of microbial communities in the gut. Immunological parameters that exhibited the largest difference to control samples in 14 day old probiotic piglets measured in one of our own trial might support this hypothesis (Scharek et al., 2005).
Effects on post weaning diarrhoea
Of the 49 reviewed publications, 19 trials in pre-/postweaning piglets considered diarrhoea incidence and 74% of those trials reported a reduction in diarrhoea incidence. However, a large range from a 78% increase in the number of affected piglets to total elimination of diarrhoea in the probiotic group (median 49% reduction) can be observed, although significant differences were always in favour of the probiotic group (11 trials). Evaluation of the effect of different starting points of probiotic application with E. faecium NCIMB 10415 in our own trials demonstrated that the relative magnitude of the reduction in diarrhoea was frequency largely independent of dietary probiotic concentration or starting time of supplementation (Figure 5 ).
Future developments and potential
Despite the apparent inconsistencies in conclusions from published research, investigations are undertaken to genetically engineer probiotics with improved abilities (acid stability, epithelial adhesion and metabolite profile). The European Union has not yet implemented regulations for the risk assessment of genetically modified microorganisms (GMO) in animal nutrition.
Especially when GMOs are used to deliver drugs or vaccines, they could not be regulated as feed additives but have to be treated as therapeutic agents. For the development of probiotics with an improved potential, such sophisticated genetic tools might be unnecessary.
Several work groups isolate novel bacteria from the habitat of interest as done with the isolation of Bifidobacterium spp. from pigs to test their probiotic potential. The use of Bifidobacterium spp. and Lactobacillus spp. as probiotics in pigs might be a further future development if technological solutions (e.g. drinking water application or non-thermal feed processing) can be developed, which overcome their instability in common feed processing techniques.
This article can be found in Feed Mix magazine Vol. 15 nr. 1