Agriculture Business Week

More and more knowledge and experience is being gained on how probiotics should be used to give reliable results. Through a more exact knowledge of the mode of action of each individual probiotic, it will be possible to make further improvements.

In spite of the unknown data on the physiology of the gut microflora, from the results obtained with  probiotic use, we are beginning to understand more about the variations involved. Indeed, despite their present low efficiency, it can be a starting point to achieve more efficient microbial strains, thanks to today’s biotechnological processes.

The use of foods derived from microbial activity goes back to the dawn of human civilisation and fermented milks were probably the first foods to contain active microorganisms. The beneficial effect of fermented milk was given a scientific basis at the beginning of the twentieth century by Elie Metchnikoff. Interest in the gut microflora revived after the Second World War and it the necessity that the microbial cells be viable. Probiotics are live micro-organisms that, when administered through the digestive route, are favourable to the host’s health.

Micro-organisms for animal feed
The micro-organisms used in animal feed are mainly bacterial strains of Gram positive bacteria belonging to the types Lactobacillus, Enterococcus, Pediococcus and Bacillus. Some other probiotics are microscopic fungi such as strains of yeast belonging to the Saccharomyces cerevisiae species (Table 1).

It should be noted that amongst the bacterial species used as probiotics, the Bacillus and the Lactobacillus differ in many characteristics. Moreover, Lactobacillus and the Enterococcus are bacterial families present in large quantities, 108 and 105-106 per gram respectively, in the digestive microflora of animals. On the other hand, the Bacillus and the yeasts are not usual components of the gut microflora Colonisation ability because the microorganisms classed as probiotics include species that are usual residents of the digestive tract (Lactobacillus and Enterococcus), and others that are not (Bacillus and Saccharomyces), we have studied the ability of some strains of probiotics to colonise the gut of axenic and gnotoxenic chickens.

A probiotic strain of Enterococcus faecium is able to colonise the axenic and gnotoxenic gut after a single administration. The population size of the strain in the intestines of gnotobiotic birds is similar to the population size of the resident Enterococcus. Similar results were obtained with Pediococcus and Lactobacillus.

For Bacillus, spores administrated to axenic and gnotobiotic birds don’t colonise the gut and are referred to as transiets. Continuously administered in feed, the bacterial strain is diluted in the proportion of a decimal logarithm, but present in different parts of the gut.

Dose and mode of administration
A bacterium will have an activity in the gut if the concentration is outstanding, that is to say in great numbers, so that the quantity of substances it produces, such as amino acids, vitamins, antimicrobial molecules, should be sufficient to be active. Many microbiologists consider that unless it has 106-107 per gram in the intestinal contents, the density is not sufficient to obtain a balance between the probiotic and the bacteria of the resident flora, and to have a considerable activity on the host. Although the physiology of the bacteria in the gut is not well known, this close estimate gives an idea of the bacterial population size to be reached to obtain a possible effect.

For many animal species, the mode of administration has mainly been through the feed or drinking water. Feeding by means of a spray in the hatcheries is also recommended in breeding conditions so as to obtain one day old chicks contaminated with probiotics. Feed is the safest way to get the correct proportion and quantity introduced daily.

Continuous feeding over the breeding period is the rule to keep steady and high probiotic populations permanently in the gut. Feeding tests of Lactobacillus every second day in the drinking water don’t show any change in the results obtained. On the other hand, the consumption of pelleted feeds by poultry requires the perfecting of technological methods in order to protect nonsporulated bacteria such as is the case for Lactobacillus, Enterococcus and Pediococcus.

Modes of action
The gut microflora forms a complex ecosystem with its host animal or bird, and microbial interactions ensure the stability of the ecosystem and the health of the host. In some cases, the gut microflora is unbalanced and the biological defences against pathogenic agents less effective. The introduction of a probiotic is an unnatural event  which will act on the natural and complex interactions of the microbial flora. The global positive effects observed are better zootechnical results in the form of weight gain and feed conversion efficiency. Currently, little is known about the way in which probiotics work, however, according to the general knowledge about gut microecology, several ways in which probiotic supplements may be influencing the composition of the gut microflora and affecting the health of the host have been suggested.

The positive effect may be the result of a direct nutritional effect, similar to the effect obtained with antibiotics, or a health or sanitary effect, where the probiotic acts as a bioregulator of the gut microflora and reinforces the host’s natural defences.

The different mechanisms of action suggested are:
1. Nutritional effects – Direct feeding of probiotics (the “direct-fed microbials”) has a number of beneficial effects on the host’s nutritional status. Reducing the ability of pathogens to produce toxins by a competitive exclusion mechanism (i.e.  the probiotics prevent pathogens from using nutrients for toxin formation) is beneficial to the bird’s gut morphology. The presence of probiotics themselves stimulates the production of indigenous enzymes, enhancing the digestive function of the bird. The production of vitamins and antimicrobial substances which probiotic bacteria are thought to secrete will also improve the bird’s health status.
2. Sanitary effect – Increasing the resistance of the bird to colonisation by pathogenic species of bacteria would improve it’s overall health status when challenged by a pathogenic attack. Furthermore, probioticsare thought to stimulate the bird’s immune response, though the mechanism of action has not been clarified.

Most of the mechanisms of action are still only hypotheses and need to be accurately demonstrated. Few of them were demonstrated in vitro or on laboratory animals.

Some experiments have demonstrated in vitro the effects of strains of Saccharomyces cerevisiae on the activity of intestinal microorganisms. The majority of the research has been performed in ruminant animals, but positive effects have also been reported in poultry. The addition of S. cerevisiae live cells to cultures of some species of cellulolytic fungi stimulated zoospore germination and cellulose degradation. The addition of yeasts also stimulate the growth of some anaerobic bacteria, including the lactic acid utilising bacteria. Certain strains of Saccharomyces cerevisiae also appear to have the ability to bind pathogenic bacteria and, since they are unable to bind to the gut  lining, escort them out of the intestinal tract.

Efficacy of probiotics
It is known that probiotics can be useful for animal husbandry, but it is also known that the preparations being used do not always exert reproducible effects. Effective results of field trials with probiotics are frequently divergent in many animal species (Table 2). When there are control groups, quite often the difference observed between them and the treated group is not statistically significant. This is due to the fact that when probiotics have a positive effect on growth, it is usually inferior to that obtained with antibiotics. Therefore efficient measures have to be taken under conditions of controlled breeding in order to look for nutritional or sanitary effects. For example, under similar conditions, we have studied the efficiency of strains of Bacillus and Enterococcus on the growth of chickens. The Bacillus strain was found to improve growth by 1.5%, compared to 2.1% for bacitracin, whereas the strain of Enterococcus reduced it by about 1.7% (Table 3). These husbandry conditions, which exclude the phenomena linked to the environment, show the unquestionable but low efficiency of some probiotics. Several tests carried out on husbandry conditions confirm that in poultry, as well as in other animal species, various types of stress and subclinical pathology reduce the zootechnical performance of animals and therefore probiotics appear to only have superior activity under these conditions.

Under gnobiotic husbandry conditions, we have carried out experiments on a controlled caecal coccidiosis with Eimeria tenella associated with Salmonella carriage and tested the eventual effect linked to the administration of a strain of Bacillus to the chickens. We observed a reduction in the clinical symptoms linked to better growth in the groups receiving Bacillus spores.  The results obtained were reproducible and statistically significant (Table 4).

However, the efficiency is not comparable with the effect of anticoccidial drugs or antibiotics, and no differences in Salmonella carriage were observed between treated  and untreated birds in this experiment.

Guidelines for the use of probiotics in poultry

Since 1970, the use of additives in animal feedingstuffs is regulated in Europe (Directive 70/524/EEC). This directive was adapted in 1994 to include the micro-organisms and enzymes (Directive 94/40/EC). The principal modifications introduced to the guidelines taking out the assessment of micro-organisms and enzymes as additives are related to specifications necessary for the identification and characterisation of the active substance and to studies to be carried out to guarantee the safety of the use of the product. With respect to the product’s safety:
1. Micro-organisms must be free of diffusible antibiotic resistance genes and non-pathogenic and non toxigenic for target species and for man under the expected conditions of use.
2. Some studies on the target species are required concerning the toxicological and microbiological aspects, namely tolerance test and tests to determine the effect on the colonisation of the digestive tract.

Since 1996, the request for authorisation should be accompanied by a dossier drawn up in accordance with the guidelines, and at present the opinion of the Scientific Committee for Animal Nutrition (SCAN) on the safety of the microbiological strains is needed by the European Commission for each probiotic

Table 1 – Microbial species used as probiotics in animals
Gram + bacteria       Lactobacillus acidophilus, farciminis, rhamnosus, reuteri, salivarius
Enterococcus faecium, mundtii
Pedicoccus acidilactici
Bacillus cereus, licheniformis, subtilis
Yeast                 Saccharomyces cerevisiae

Table 2 – Efficiency of some probiotics in poultry (Wolter and Henry, 1987)
Bacteria                     Species        Feeding period      Weight gain   Feedconversion
Lactobacillus  acidophilus   chickens         1-28 days            -5.1           +5.1
chickens         1-21 days            -0.4           -0.3
chickens         1-49 days            +2.31           0
layers           150-210 days         +3.8           +3.3
turkeys          28-112 days          +5.8           +2.2
Lactobacillus mixture        chickens         1-43 days       +2.3
Bacillus toyoi               chickens         1-43 days            +1.6
Streptococcus faecium(SF684) chickens         1-35 days            +1.6           -3.5
chickens         1-43 days            +2.7

Guidelines for the Assessment of micro-organisms in animal nutrition. Directive 94/40/EC
1. Identity and specifications concerning the active substance.
Name and place of culture collection recognised as International Depositary Authority where the strain is deposited.
Physiological and genetic characteristics and modifications. Stability and incompatibility. Conditions of use (cfu/g of product). Methods of detection, enumeration and identification.
2 Efficacy.
Improvements in the characteristics of feedingstuffs. Effects on animal production.
3. Safety
Tolerance studies on target species,Microbiological studies: ability to colonise the digestive tract
antibiotic resistance and genetic basis of the resistance, genetically modified or not. Acute toxicity: not for micro-organisms.

Table 3 – Efficiency of a strain of Bacillus and Enterococcus on the growth of chickens (Guillot and Yvoré, 1990)

Feed                   Weight at 42 days     Feed conversion            Mortality
Bacillus  Enterococcus   Bacillus   Enterococcus    Bacillus  Enterococcus
Pellet    1900        1928         1.85         1.84          2.5          2.1
Flour                 1772         1807         1.88         1.89          1.6          1.2
Flour + bacitracin    1811         1828         1.88         1.87          2.3          1.9
Flour + probiotic    1799         1775         1.88         1.93          2.7          1.6

By Professor J.F. Guillot