Microorganisms in the bovine rumen represent an extremely diversified
and important component for the nutrition of ruminants. Their capacity
to act on ingested food and transform it in assimilation compounds is
their main function (Fraser et al., 2007a). Another role extremely
aimed at by the researches is represented by the protection effect they
may have against some pathogens. Thus, together with food, the type and
number of micro-organisms which reach the rumen is very high. Due to the
conditions in here and to the role of these strains, a favourable balance
for the farm animal is kept (Cerrato-Sánchez et al., 2007).
The majority of bacteria strains in the rumen are Gram- negative. The
Gram-positive ones reach the rumen at the same time with food (Jalc et
al., 2002). An important input is produced when food is supplemented
by such products based on viable probiotic biomass. This type of bacteria
is strictly anaerobe and since a great part tolerate oxygen to a small
extent, it creates a series of advantages from a trade and practical point
of view (Cerrato-Sánchez et al., 2007). A very important
aspect is constituted by resistance to pH, temperature and different acids
or enzymes. Marketed products use Gram positive strains which resist at
a pH between 5 and 7 and have maximum productivity at temperatures of
37-39 °C. When choosing such strains, their resistance to different
acids and to the enzymatic action in the digestive tube of the animal
has to be considered. The strains used are carefully selected so as to
use those strains with a high resistance, because, generally this is not
the strongest characteristic of the lactic bacteria (Colombatto et
Products which might improve zootechnic performances and health of the
animals are mainly based on yeasts and acidolactic bacteria. Strains are
carefully selected so as to offer maximum protection and a quick effect
by improving the health of the digestive system. (Wadhwa et al.,
2001a,b) Thus, one uses strains which can adhere and are resistant to
a low pH, digestive enzymes or various other factors, such as temperature
(Vallimont et al., 2004). The resistance and persistency in the
animal`s digestive system represent some of the basic criteria because
probiotic strains have to prevail over pathogen strains which can survive
for a long time in unfavourable conditions. Thus, an important modality
for prevailing is their number and their increased capacity of multiplying
in the conditions of the animal`s digestive tube. This is why one doesn`t
use only one strain or only one type of micro-organims but a combination
of several micro-organisms, different genus, in general. For example,
the combination between yeast and bacteria is an adequate one taking into
account the improvement of the animal`s health and production increase.
In this sense also, the use of different genus of each type of micro-organisms
is considered (Greenfield et al., 2001). Due to the restrictions
imposed by the latest regulations of the European Union in this domain,
the use of such natural products is stimulated in the detriment of antibiotics
or other synthesis substances.
The purpose of this study consists in testing some yeast and bacteria
selected strains so as to formulate a probiotic product used in animal
nutrition. Tests consisted in establishing viability under the influence
of some physical and chemical factors. Their action is exerted either
in the handling and conditioning period of the product of after it is
ingested by the animal. Testing the effectiveness was done by inoculating
the rumen content of a calf with the probiotic product, based on lyophylizing
biomass (Silveira et al., 2007).
MATERIALS AND METHODS
Experimental studies were done in the laboratory of Fermenting Bio-technologies
under the Biotechnological Centre in Bucharest, from January 2007-July
Biological material: Microbial strains are kept in the freezer
at -82 °C, in protecting environment with glycerol 20%. We use 6 yeast
strains and 2 lactic bacteria strains: T1: Saccharomyces
cerevisiae 2-15; T2: Saccharomyces cerevisiae 1-29;
T3: Saccharomyces cerevisiae R-BF; T4: Kluyveromyces
marxianus R-CS; T5: Issatchenkia orientalis R-BC;
T6: Trichosporon beigelii R-LF; T7: Lactobacillus
paracasei CMGB16; T8: Enterococcus faecium GM8.
Cultivation mediums and fermentations conditions: Revitalization
of the yeast cells is done by using YM modified medium: yeast extract
0.3%, glucose 2%, peptone 0.5%, malt extract 0.3%, pH 6. Sowing is done
with cryotube bow and the development takes places at 30 °C, 48 h.
For Lactobacillus paracasei CMGB16, revitalization is done through
cultivation on MRS medium, la 37 °C, for 24 h (Novik et al.,
For Enterococcus faecium GM8, the revitalization of the strain
is done through the cultivation on a medium specific to the strain of
Enterococcus, MEI noted, which contains (g L-1): yeast
extract 10 g, peptone 8 g, glucose 10 g. The sowing medium is introduced
in the thermostat at 31 °C, 24 h.
pH effect and of the temperature on the viability of the probiotic
strain: The role of these tests consists in the fact that when fodder
is produced, it may undergo some thermal treatments (e.g., granulation)
or keeping them for a longer period of time may also imply a pH variation,
mainly its decrease. Thus, viability tests at extreme values were done
acid pH 1, 2, 3 and basic pH 8, 10 and 12. For temperature, viability
was tested at values of 50, 70 and 90 °C.
So, as to perform the tests, 2 mL of fresh culture from an Epperdorf
sterile tube is put. For each strain, viability was tested at every pH
interval by using NaOH 20% or concentrated HCl. After 30, the value of
the pH was brought to 7 and viability was determined. So, as to test viability
in the 3 intervals of temperature, the culture was introduced in a thermostat
bath for 30. After this interval, the tube was put on ice bath in a special
ice box and after the liquid cooled viability was determined.
Enzyme effect on probiotic strains: So, as to accomplish this
experiment, the following enzymes are used: pepsin (Sigma-Aldrich), pancreatin
(Fluka Biochemika) and biliary salts. For testing the protective effect
of some substances on strains, the same set of tests was done but NaCl
0.5% was supplemented with casein and mucin with a concentration of 1
g L-1, the procedure is the same for the rest (Perea Vélez
et al., 2007).
After performing the tests with the two enzymes viability/mortality was
determined according to Blaenka et al. (2000).
The effect of the probiotic biomass on the microbiota in the rumen:
So, as to test the formula of probiotic product, the inoculation of the
rumen recently extracted from the stomach of the calf with the mixture
of strains was used. So, the following mixture was used: rumen content
500 g, 200 mL saliva, 100 mL sterile distilled water (Boguhn et al.,
2006). The experiment was done for a period of maximum 12 days, at 37-40
°C, with samples taken at 6 and 12 days. From these samples, pH, lactic
acid and viability were determined. For the majority of the yeast, the
following medium formula was used, YEPD (g L-1): yeast extract
10 g; peptone 20 g; agar 20 g. So, as to determine the number of lactic
bacteria, Rogosa medium was uses; for the determination of Enterobacteriaceae,
the Istrate-Meittert medium was used (Abel et al., 2006; Kamra,
All the tests done were based on the consideration of a viability of
105 and minimum 106, because according to the data
in the literature in the domain with respect to probiotic, these are the
minimum viabilities necessary for taking into consideration a microbial
strain as a possible probiotic.
RESULTS AND DISCUSSION
Upon performing experimental studies, two strains were used, in parallel,
as close as possible from a phylogenetic point of view so as to better
observe their evolution. Another aspect of this choice is represented
by the use of one of the strains for the formula of the final product.
From Table 1 a very good viability of the 2 lactic
strains at the temperature of 50 °C can be observed. However, at higher
temperatures, the viability of the 2 strains is 0. What needs to be observed
is the resistance of the strain Lactobacillus paracasei CMGB16
at pH between values 2 and 12, the strain doesn`t resist at pH1.
The strain Enterococcus faecium GM8 is not viable at a pH higher
than 8. A very important fact is that both strains keep the viability
of 106 irrespectively of the value of the pH from where the
measurement was done.
In Table 2, one can observe that the strain Issatchenkia
orientalis R-BC is viable at 50 °C. For the other two intervals
of temperature, viability doesn`t exist in the 2 yeast strains. Although
it is an important strain from a probiotic and point of view and more
others, Kluyveromyces marxianus R-CS has a maximum viability only
in the pH interval 2-3. After this interval viability decreases from 106
to 105, after which the strain is no longer viable. Moreover,
Issatchenkia orientalis R-BC has a maximum viability in the pH
interval 1-8. At a pH higher than 8, viability is 0.
Trichosporon beigelii R-LF and Saccharomyces cerevisiae R-BF
are not viable in the 3 tested intervals of temperature (Table
3). What needs to be observed is that the strain Saccharomyces
cerevisiae R-BF has a maximum viability in the pH interval 1-12, with
a smaller number of colonies in the case of a basic pH than in the case
of an acid one. Trichosporon beigelii R-LF is not viable at a pH
higher than 8, in other cases viability is high at any tested interval.
The strain tolerates very well the low values of the pH, the number of
colonies exceeding the one considered to be necessary so as to validate
In Table 4, only the strain Saccharomyces cerevisiae
1-29 is viable at the temperature of 50 °C. The fact that the strain
Saccharomyces cerevisiae 2-15 is not viable in the conditions of
a basic pH, but is viable in the case of the acid one is made obvious.
The number of colonies at maximum dilution decreases proportionally to
the decrease of the pH. The strain Saccharomyces cerevisiae 1-29
has maximum viability in the pH interval 1-8, after which at pH 10, viability
decreases up to 105 and becomes 0 at pH 12.
In Table 1-4 one can observe that
none of the strains has a maximum viability at temperatures higher than
50 °C. However, the strains Kluyveromyces marxianus R-CS,
Trichosporon beigelii R-LF, Saccharomyces cerevisiae R-BF
and Saccharomyces cerevisiae 2-15 are not viable at 50 °C.
From the point of view of the pH, only one yeast strain is viable at
all the intervals of the pH. Except for Kluyveromyces marxianus
R-CS and Saccharomyces cerevisiae 1-29, for which the maximum value
of the pH determines a decrease of the viability at 105, all
the other strains have a maximum viability. Thus, we can generally consider
that strains are resistant at extreme values of pH and the number of colonies
at the maximum value of the viability is mostly high, more than 5. The
majority of the strains with a maximum viability have more than 10 colonies
at the pH tested values.
In case they are exposed to the 2 enzymatic solutions, it can be observed
from Table 5 that, once the concentration of the biliary
salts increases, viability decreases, but not in a uniform way. It needs
to be observed that the two strains of lactic bacteria are not viable
when such a treatment is applied. However, the use of protective solutions
(mucin, casein) determines the preservation of a significant part of the
viability of the probiotic strains. This fact is not important for the
strain of Enterococcus faecium which is not viable in the presence
of such substances. The essential conclusion is that the yeast strains
are much more resistant than the lactic ones to the enzymatic action,
but the use of support mediums or of some protections given to the probiotic
cells confer additional resistance and increase viability.
|| Viability of the Enterococcus faecium GM8 strain
and Lactobacillus paracasei CMGB16 at different intervals of
pH and temperature
|| Viability of the strain Kluyveromyces marxianus
R-CS and Issatchenkia orientalis R-BC at different intervals
of pH and temperature
|| Viability of the strain Trichosporon beigelii
R-LF and Saccharomyces cerevisiae R-BF at different intervals
of pH and temperature
|| The number of viable cells in case of exposure to solutions
1, 2 and 3
||The variation of the viability (%) after exposure to
the solutions 1 and 2 function of the concentration of the biliary
||The viability rate for each strain in case of exposure
to the protective solution
||Mortality variation (%) after exposure to solutions
1 and 2 function of the concentration of the biliary salts
Thus, from the diagrams below one can observe that viability decreases
when the concentration of biliary salts increases (Fig.
1). Some strains (T2 or T4) have a constant
viability at a certain concentration. From all the analysed strains, the
worst results were observed in the case of the strain T7. From
Fig. 2 one can observe that yeasts are much more resistant,
in case protective solutions are used. Good results are also obtained
in the case of the strain T6, the presence of the protectors
||Mortality rate for each strain in case of exposure to
the protective solution
In the case of mortality, the increase of the concentration of the biliary
salts determines an increase of the mortality rate also (Fig.
3). This tendency can be mainly observed for concentrations of 1.5
and 2 mg mL-1. The mortality rate (Fig. 4)
is very high for the strain T7, however, for the strain T6
it is relatively low. Strains T1, T2 and T5
have the highest mortality values in the presence of the protector.
In the formula Fp1 only the strains for which experimental
results were the best are kept and those intestinal ecosystem of the animal.
In the structure of the formula Fp2 all the strains used are
distributed in variable percent. The relation between them expresses the
experimental results obtained in the performed tests, related to the data
which exist in the literature in the domain.
Taking into account the tests performed, 2 formula of probiotic product
Thus, so as to verify viability, the number of micro-organisms developed
on the designated mediums was determined: YEPD, Rogosa and Istrate-Meittert,
rumen sowed with probiotic strains. What can be observed is that the
number of yeast (41%) is identical to the one of anaerobic or strictly
anaerobic micro-organisms (42%), in the case of the Rogosa medium. We
conclude strains are of major importance (lactic bacteria) in that the
animal from which the sample was taken had an equilibrated metabolism.
Microbial balance was also equilibrated with a slight advantage in favour
of the strains which tolerate air. The number of entero-bacteria is low
(17%), significantly under the values of the other two categories. This
small disequilibrium can be explained by the fact that the animal was
fed close to the moment when the sample was taken.
In first case, one can observe that 6 days after the sowing with Fp1,
the majority of the strains is anaerobic and especially those of lactic
bacteria. The number of entero-bacteria is very low, only 2%. After more
6 days from the incubation, the balance starts to equilibrate, the strains
of the entero-bacteria are found in a percent of 25%. The most important
aspect is that anaerobic strains are still of a majority, being those
strains which ensure the decrease of the pH and inhibition of the pathogen
In second case, one can observe that Fp2 is not a viable formula
because it leads to an important disequilibrium between the aerobic and
the anaerobic species in the animal`s rumen. Thus, after 6 days aerobic
strains are preponderant (74%). The anaerobic ones are of maximum 1%.
After 12 days the disequilibrium is manifested through the inexistence
of the anaerobic strains, no lactic bacteria strain can be isolated. However,
the strains which appear are intensely coloured and give off a heavy smell,
specific to the strains of aerobic bacilli.
Selective pressures exerted by antibiotics on the digestive micro-flora
lead to a disequilibrium of the intestinal microbial ecosystem. This microbial
ecological equilibrium established between hundred of different bacterial
populations is maintained due to a subtle game of interactions between
different biotic and abiotic constituents of the ecosystem. The association
of some non pathogen micro-organisms with the antibiotic therapy, known
for a long period of time leads to the recovery of the microbic ecological
The quality of the product obtained from yeast and bacteria probiotic
strains is determined first of all by the content of viable micro-organisms
and by their genus. Another aspect of novelty and originality of the study
is represented by the formula of the product, the mixture of micro-organisms
and their association. The most important parameter is represented by
the microbiological determination of the number of micro-organisms (successive
dilutions and spreading on a specific agar medium). The results of the
determination of the viability titre of the probiotic obtained from yeast
and bacteria strains are presented in Table 6.
|| Determination of the viability titre for the selected
It results from the table that all the studied strains have a very good
viability, at cultivation in the given conditions. In the case of lactic
bacteria strains, low viability is not determined by the low productivity
of the strains but by limitative factors (pH) which manifest their presence
during the lactic fermentation.
The 6 strains were tested with different physical and chemical agents.
The most resistant strains were chosen and 2 formula of probiotic product
were established. Tests were done in fresh rumen content in conditions
similar to those in the animal rumen. Pursuant to the performed tests
Fp1 determined a positive result. In this case anaerobic strains
were preponderant (lactic bacteria) and the aerobic ones had a maximum
of 25%. For Fp2 the results were negative, in this case aerobic
strains which cause a heavy smell in the tested system were preponderant.