Trial 35: Comparative experimentation in relation to the efficacy of probiotics in animal nutrition

The probiotics Turval 6, Turval Swine and Yea-Sac1026 have nonetheless maintained a good overall fermentation potential, which should allow for a better regulating activity of the intestinal microflora than the other products compared.

Prof. Piero Susmel
Università degli Studi di Udine - Dipartimento di Produzione Animale

1 - Introduction: probiotics and antibiotics

The increased sensitivity in regards to natural products and the necessity, imposed by norms and economical needs, to diminish the “medicalization” of breeding farms, make it obligatory, on the part of the research sectors working on the exploitation of feeds for zootechnical use and animal production, to conduct a careful scientific examination of the physiological and biological behavior of the microorganisms commonly called probiotics.
In fact, the appearance and the increase in bacteria strains dangerous for man and animals and resistant to therapies has been widely attributed to a widespread use of antibiotics as dietary additives. This negative secondary effect has pushed the legislator to set more and more restrictive limits on the utilization of these substances and impose research to study experimental protocols that also consider companies’ needs to contain costs, which have often become excessive for a rational economic budget, due precisely to the use of these antibiotic substances.
Probiotics are vital microorganisms able to give benefit to its host through the re-balancing of the intestinal microflora. This definition, given by R. Fuller regarding human medicine, is most certainly extensible to the zootechnical sector. Probiotics are often commonly called “lactic yeasts” and their positive properties in the diet of animals and humans have been known for a long time, even though only recently their physiological action has begun to be scientifically evaluated and analyzed.
In fact, it has been long noted that the positive characteristics of these microorganisms, which favor the well-being and health of animals, are closely connected with the capacity of fermentation of organic substrata, with the production of various metabolites, among which acetic acid and, better yet, lactic acid.

The Department of Animal Production Sciences, having determined the interest of further scientific investigation on the physiological and biological processes of probiotics and of the economical aspects of the market for the use of commercial products which contain these organisms, tested a number of probiotics in commerce, comparing them with one another and with brewer’s yeast, traditionally used for animals because it’s a low cost supplement and derived from the brewing industry.

2 - Objective - evaluation of the efficacy of probiotics

The study of digestive and metabolic processes in vivo (directly on the animals) is not easily interpreted, due to the numerous factors that characterize the gastric and intestinal digestive process including the diversity of species and breeds, the ingredients of the diet, the chemical and nutritional characteristics of the ration and the method of breeding. The same factors interfere with the secretion activity of the enzymes and on the motility of the intestine, and, therefore, influence the fermentation activity of the microorganisms. For these motives, it was estimated to be more useful, for the aim of comparison between products, to test the probiotics in vitro, analyzing the fermentative potential and the capacity of the microorganisms to produce metabolites contained in the products compared, rather than using the in vivo method, based on the “final” results obtained in animals.
In order to reduce the dietary and breeding variables and to be able to describe the phenomena of interest, laboratory methods were developed in vitro, which represent simplified but more exemplary models of what happens in vivo.

3 - List of products utilized for the trials

Product	Company	Microorganism	CFU/g
		declared
Turval Swine	Turval Labs	Kluyveromyces f B0399	>2 x 10⁶
Turval 6 horse	Turval Labs	Kluyveromyces f B0399	>5 x 10⁶
Entero	Stamina	Saccharomyces cerevisiae
Biosprint	Prosol	Saccharomyces cerevisiae
Equiferm	Agrolabo	Kluyveromyces fragilis Saccharomyces cerevisiae Saccharomyces kurlsbergiensis
Yea-Sacc¹⁰²⁶	Alltech	Saccharomyces cerevisiae1026	100 x 10⁶
Lievito di birra		Saccharomyces cerevisiae

4 - Methodology of experimentation

4.1 Fermentation potential

The evaluation of the quality of probiotics was made to examine the fermentative potential, before and after gastric digestion, in terms of gas production and of principal products of fermentation (lactic and acetic acid). To this end the “gas test” technique was employed, which is often utilized as trial of microbial activity in the study of fermentation potential of feeds for ruminants. The test consists in the incubation of a microbial inoculum with a alimentary substratum in special air-tight the
period of incubation was reduced to 24hrs, as this duration of time is closer to that of the average permanence of feed in the intestinal tract. At the end of the fermentations, the production of lactic and acetic acid in the liquid phase was measured.
The first trial was made by weighing in a syringe 1.00g of the feed utilized as a base and 0,5 g of product to be tested and adding 30 ml of phosphate buffer 0.2 M, pH 6.8. The syringe was then sealed by the piston and put in incubation in a thermostatic bath at 39°C. After 2, 6 ,8, 10, 16 and 24 hrs, the production of gas was measured and recorded. Upon termination of incubation, the contents of the syringe was filtered on cellulose filters (porosity 40-60 m) and the liquid was salvaged in 50 ml centrifugation test tubes. Two successive centrifugations (2500 rpm) were then executed, in order to prepare the solutions for the evaluation of the organic acids produced during the fermentations.
The fermentation capacity was also evaluated for the digestive products in the gastric phase. To this end, since it was demonstrated that the gastric digestibility was around an average value of 50%, 1,00g of probiotic were incubated with HCl and pepsin, as previously reported. After the conclusion of the incubation, the entire contents of the Erlenmeyer flasks was decanted in 50 ml centrifugation test tubes and centrifuged at 13500 rpm, in order to recuperate the entire solid phase. The sediment was separated from the surnatante through the aspiration of the latter and was retreated with 30 ml of phosphate buffer 0.2M, pH 6.8; the solution thus obtained was introduced in the syringe containing 1.00g of feed
The evaluation of the fermentation capacity of the products before and after gastric digestion was executed in parallel.
The lactic acid was measured with a commercial kit (ALIFAX, Padova ), which is based on the reaction of hydrogen peroxide, produced by the oxidation of lactic acid, with a chromogen and spectrophotometric reading at 550nm. The acetic acid was measured with the kit made by Boheringer Mannhein (Germany), which utilizes the enzymatic conversion of acetic acid in oxalacetic acid, which in the reaction derives from the dehydrogenation of a sick subject, the spectrophotometric measurement consists in the evaluation of the velocity of production of NADH at 340 nm.

5 - Results

5.1 Products not digested
5.1a. Production of lactic acid
The comparison between the values of lactic acid produced by the 7 probiotics not digested, shows a major concentration for the probiotics Turval 6 and Turval Swine (P<0.01). Progressively decreasing production of lactic acid was measured for Yea-Sacc1026, Biosprint, Brewers Yeast, Entero and Equiferm (see chart. 2 and fig. 2)

Chart 2. Profile of fermentation (lactic acid) of the products examined.

	Product
	Turval Swine	Turval 6	Entero	Biosprint	Equiferm	Yea-Sacc	Brewer’s yeast

lactic acid	2152^A	3534^A	109^C	344^B	94^C	354^B	214^B
(mg/l)
	**	**	ns	*	ns	ns	*

A,B,C= denote statistically significant differences by P<0.01 between the averages
* = significant differences between whole and digested, by P<0.05
**= significant differences between whole and digested, by P<0.01
ns= non-significant differences between whole and digested

Figure 2. Profile of fermentation (lactic acid) of the products examined

susmel 1.jpg

5.1.b - Production of acetate and lactate

The detailed results of the comparison trials of the overall production of organic acids (lactic and acetic), reported in chart 3 and fig. 3. have shown an elevated heterolactic fermentative activity (which starting from glucose produces acetate) for the products not digested of Turval 6, Turval Swine and Entero (P<0.01), while an intermediate activity resulted for Yea-Sacc1026 and low for the others.

Chart 3. Products not digested. Profile of fermentation in vitro (acetic acid +lactic acid) in the products examined.

	Product
	Turval Swine	Turval 6	Entero	Biosprint	Equiferm	Yea-Sacc	Brewer yeast

acetic acid
+	3483	4750	1309	555	115	927	330
lactic acid
(mg/l)

Figure 3. Products not digested. Profile of fermentation in vitro (acetic acid +lactic acid) in the products examined

susmel 3.jpg

5.2. Products digested

The characteristics of the total fermentation activity after gastric digestion showed the major production of organic acids for Turval Swine and Turval 6, followed by Yea-Sacc1026 and Entero, while Brewer’s Yeast had the worst result, confirmed by the scarce microbial activity of this product once it was digested (see chart 4 and fig. 4).

Chart 4. Products digested. Profile of fermentation in vitro (acetic acid +lactic acid) of the products examined

	Product
	Turval Swine	Turval 6	Entero	Biosprint	Equiferm	Yea-Sacc	Brewer’s yeast
Acetic
+	1708	1650	1072	190	867	1103	295
lactic acids
(mg/l)

Figure 4. Products digested. Profile of fermentation in vitro (acetic acid + lactic acid) of the products examined

Fig 4.jpg

6 - Conclusions
6.1 Scientific results
In order to evaluate the mechanisms of action of probiotics, without incurring variables found in experimentation on live animals, a method of examination in vitro was developed , able to simulate, and simplifying, the microenvironmental conditions of the gastro-intestinal apparatus of the animal host in which the microorganisms contained in the commercial products are found.
The parameters measured give an indication of the microbiotic activity before and after gastric digestion, from which scientific hypotheses can be drawn regarding the effects found on animals in campo.
The results of the laboratory tests indicate a considerable variation of probiotic activity in vitro and a substantial modification of their fermentation capacities after gastric digestion.
The probiotics Turval 6, Turval Swine and Yea-Sacc1026 have nonetheless maintained a good overall fermentation potential, which should allow for a better regulating activity of the intestinal microflora than the other products compared.
Lastly, it should be noted that the laboratory method of evaluation in vitro developed, because it can be standardized and repeated, represents an innovative system of evaluation of the microbiological activity of a product, which could also be utilized for analyses on other products on the market with similar characteristics of action.
It should also be pointed out that in eventual other experimentations , it would be opportune to re-calibrate the quantity of product to use for each sample.

6.2 Economic comparisons for the products in relation to livestock

In order to have a more correct comparison, the quantity of organic acids produced per 0,50 US$ of product was calculated, taking into consideration the average Italian market price of probiotics (see Chart.5). In the case of brewer's yeast, the figure refers to a product with 90% of dry substance.
The result of this comparison shows that the Turval probiotic produced, in specific conditions in vitro in which the test was made, 240 mg of organic acids per 0,5 US$ of cost a figure two times superior to that of Biosprint, four times to that of Yea-Sacc1026 and eight times to that of brewer's yeast (see Fig. 5).

Figure 5. Production of organic acids (acetic + lactic acid) per 0,5 US$ of product.

Fig 5.jpg