Probiotics are non-pathogenic
microorganisms and when they are ingested they exercise a positive effect
on the host`s health and physiology. They can influence the physiology
of the gut both directly and indirectly by modulating the endogenous ecosystem
or the immune system (Reid et al., 2003). There are many references
regarding probiotics, particularly to their potential in preventing or
healing gut problems; however, only several strains of probiotics have
proven to be efficient in the controlled clinical cycles (Fernandes and
Among the microbial strains considered to be probiotics,
the following can be itemized: Saccharomyces cerevisiae, Saccharomyces
boulardii, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus
casei, Streptococcus thermophilus, Bifidobacterium bifidum (Lee et
al., 1999). A growing emphasis has been recently laid on the use of
the Lactobacillus plantarum strain, as a human use symbiotic product.
Originally used as an animal use probiotic strain, it is now proven that,
in humans, it has a more and more important effect in cholesterol assimilation.
Because of this reason, the Lactobacillus plantarum strains are
more and more often used in the attempt to support the elimination of
the cholesterol in hypercholesterolemia patients (Maria et al.,
Another issue recently approached in the study of symbiotic
products is that of gut problems. Although there are some studies and
even probiotic products against colitis, lately the emphasis has been
laid on preventing the colon cancer formation by using symbiotic products
(Meydani and Woel, 2000). If by now the emphasis used to be laid on just
one probiotic strain (e.g., Lactobacillus acidophilus or Streptococcus
thermophilus), the last studies show the use of a product that contains
two or several probiotic strains and a probiotic product i.e., a symbiotic
product. The role of the probiotic is to stimulate, at the gut level,
the development of probiotic strains of the Lactobacillus and Bifidobacterium
type (Overton et al., 1994).
Special emphasis is laid on these two issues, because they
appear because of environment pollution, sedentariness, consumption of
food additives, high consumption of fats and low-fiber content food (Reid,
1999). This way, it is considered that the change of the food diet leads
to the decrease of the risk of colon cancer formation, by ingesting fibers,
prebiotic and symbiotic products. The factors that lead to the formation
of colon cancer are inhibited by the consumption of symbiotic products,
as these products inhibit the precancerous lesions and even tumors (Roberfroid
and Delzenne, 1998).
Honey has lately been very often quoted as a prebiotic product.
In its composition there are some fructo-oligosaccharides that are considered
to be prebiotic. The combination between pollen and honey is an ideal
medium, full of nutritive substances, for the multiplication of these
types of bacteria. The final product offers, in exchange, a very good
protection of the probiotic strains, due to its composition. This finding
is important because probiotics are inhibited during the intestinal transit.
Thus, the mixture of pollen with honey provides an important protection
to microbial probiotic strains (Salminen et al., 2004).
The objectives of this study were the following: (1) Determination
of the pollen and bee honey on growth and development and the effect of
the bee honey on growth, development and activity of the Lactobacillus
plantarum and Lactobacillus acidophilus strains, (2) The bio-productive
effect on the tested lab animals and (3) Influence of the symbiotic product
on the cholesterol level of the tested lab animals.
The first objective was studied in the microbial bio-technology
labs of the Bio-technologies Faculty and Biotehnol Center and the 2nd
and 3rd objectives were studied in the pharmacy-bio-technology labs of
the National Institute for Chemical Pharmaceutical Researches-ICCF Bucharest.
Biological material: In the research, the Lactobacillus plantarum
2s and Lactobacillus acidophilus 1a strains were used, existing
in the collection of the Biotehnol Center.
Culture media and fermentation conditions: The strains used were
kept at the temperature of 4°C on a medium containing 9% milk powder and
0.2% yeast extract (called LE by the authors). To revitalize the microorganism,
a volume of sterile MRS liquid of 25 mL is sowed with 10% culture on LE.
It takes place at 37°C, under static conditions, for 24 h (Vamanu et
To perform the research, 4 pollen- and honey-based media
were used: P1: 20 g non-milled pollen, 3 g honey, 5 mL distilled water;
P2: 20 g milled pollen, 3 g honey, 5 mL distilled water; P3: 20% non-milled
pollen, 3% honey; P4: 20% milled pollen, 3% honey. The media P1 and P2
are semisolid media obtained by mixing the components and adding a minimum
quantity of water, but sufficient to smash the pollen grains and obtain
a relatively homogenous medium. The 4 culture media were inoculated in
a ratio of 1% with the Lactobacillus plantarum 2s and Lactobacillus
acidophilus 1a strains, developed on LE, 48 h, at 37°C (Vamanu et
The medium was prepared in tightly closed plastic recipients
and after inoculation they were placed at the temperature of 37°C. The
tubes were statically maintained.
Determination of the sugar quantity by using the o-toluidine method:
It was performed with the o-toluidine test, made by the National Institute
of Chemical-Pharmaceutical Research-Development-ICCF Bucharest (Rosoiu
et al., 2005).
Determination of the lactic acid quantity: The lactic acid accumulation
was determined by titration with HCl 0.1N. For determination, the fact
that 1 mL HCl 0.1N corresponds to 0.009008 g lactic acid is taken into
account Rosoiu et al., 2005).
Determination of the viability: To establish the number of Colony
Forming Units (CFU) the successive dilutions method was used. For determination,
the Rogosa medium was used (Haddadin et al., 2004).
Testing of the nutritive value of the symbiotic product: The experiment
was made on Wistar rats, males and females, divided into lots of 10 animals
each. The animals were kept in the acclimatization vivarium conditions
for one week, with standard food and ad libitum water. For the
experiment, the animals that showed no clinical signs of disease and whose
body weights were somewhere between 140-180 g were chosen.
Four lots were formed: three experimental lots, which were
administered symbiotic product every day, in their food, in intakes of
2 mg kg-1 (lot I), 20 mg kg-1 (lot II) and 200 mg
kg-1 (lot III) and a control. The animals were monitored every
day and weighed every week.
In the beginning of the experiment and then every week,
the animals were weighed, calculating the weight gain index. Blood samples
were taken to determine the cholesterol level.
The weight gain indexes in the 3 batches were calculated
by the following formula:
Development of the Lactobacillus
plantarum 2s and Lactobacillus acidophilus 1a strains on pollen
and honey-based media: The development of the
strains was analyzed comparatively on the two groups of culture media.
The first group (P1 and P2 media) has a higher consistency due to the
low water content. The second group (P3 and P4 media) contains the media
similar to the classic ones.
The determination of the lactic acid accumulation, the viability
and the sugar consumption were made in parallel. The development period
is of 72 h, also monitoring the aroma and savor of the symbiotic product.
First of all, the quantity of lactic acid was determined,
because this is an important feature in such a product. The existence
of a quantity as large as possible is an additional guarantee for the
preservation of the product sterility. The largest quantity is found in
the P2 medium (Fig. 1). For the P1 medium, the non-milled
pollen determines a weaker synthesis of the lactic acid, which is due
to a large quantity of nutrients located in the pollen grain, which are
gradually released due to the fermentative action of the 2 strains.
For the classic media, the largest accumulation is on the
P4 medium, representing 50% of the quantity synthesized on the P2 medium.
The low quantity of nutrients, due to the large quantity of water, determines
low values of the lactic acid.
The largest sugar consumption is on the milled pollen media,
P2 and P4. From the viewpoint of the quantity remained after 72 h of fermentation,
the strains consume most of the sugar existing on the P3 and P4 media.
But, from the viewpoint of the consumed quantity, the P2 medium is optimal
because, when using it, the strains use the largest quantity of sugar
Figure 3 shows that the P2 medium is optimal
for the concomitant cultivation of the Lactobacillus plantarum 2s
and Lactobacillus acidophilus 1a strains, with a view to obtain
a symbiotic product. Even if a large viability is also obtained on the
P3 and P4 media, the large quantity of the water existing in them needs
Sugar consumption on the P1,
P2, P3, P4 media
Viability of the Lactobacillus
plantarum 2s and Lactobacillus acidophilus 1a strains
on the P1, P2, P3, P4 media
From the three graphs it results that the milling of the pollen has an essential
role in the quick multiplication of the Lactobacillus plantarum
and Lactobacillus acidophilus
Several organoleptic features
of the symbiotic product on the 4 culture media
Average values of cholesterol
found in the lots of female and male rats treated with symbiotic
Other features monitored in obtaining the symbiotic product
are homogeneity and taste. The results are shown in Table
Table 1 shows that homogeneity is kept
during the entire fermentation period for the P1 and P2 media. On the
other two media, homogeneity is not kept.
The taste at the inoculation time is sweet, due to honey
and pollen. Once the lactic acid is formed, the taste becomes an appropriate
sweet and sour mixture. Besides these characteristics, the aroma of the
product was also monitored, which, after 72 h of fermentation, is characteristic
to pollen and lactic acid.
Testing of the nutritive value of the symbiotic product: The evolution
of the body weight of the rats treated with symbiotic product, obtained
on the P1 medium, in intakes of 2, 20 and 200 mg kg-1, respectively,
is shown in Fig. 4.
Figure 4 shows that, in all the lots treated
with symbiotic product, the weight gain was higher than that of control.
This increase of the weight gain is noticed both in females and in males.
The average gain of the female lots was of 105% and the average gain of
the male lots was of only 103%. For females, the maximum average gain
was reached during the third week, being 107%. For males, the maximum
average gain was reached during the fourth week, being 105%. The maximum
gain was obtained after the 20 mg kg-1 intake. It can be noticed
that the weight gain was maximum during the third and fourth week of treatment.
Another monitored parameter was the cholesterol level. The
determined experimental data is shown in Table 2.
The lowest cholesterol level is found in the lots treated with 20 mg
of symbiotic product. Thus, we can conclude that the controlled used has
a positive influence on the decrease of the blood cholesterol level. This
way, using this product determines a balancing of the intestinal microflora
and can determine the decrease of the cholesterol level.
Body weight of the rats treated
with symbiotic product
Lot 1-treated with 2 mg of product
Lot 2-treated with 20 mg of product
Lot 3-treated with 200 mg of product
The highest viability of the 2 Lactobacillus strains
is seen when using the 2 culture environments with ground pollen and honey
3% (P2 and P4), as they contain the highest quantity of glucides. Many
glucides from pollen and honey are oligosaccharides that can be considered
prebiotic, positively influencing the development, the activity and the
viability of Lactobacillus plantarum 2s and Lactobacillus acidophilus
1a strains. And so, the most important result is that the high number
of micro-organisms determined by the composition of P2 environment can
lower the cholesterol level. So, the composition of the culture environment
has an indirect positive effect on the level of cholesterol. If the direct
level is exercised on the growth of the milk bacillus number, the indirect
one is important as well, as it influences the number of lactic micro-organisms.
In the specialty literature, studies are published regarding
the growth and the development of different lactic bacteria strains in
milk products, which contain honey in different percentages. Most of the
studies are limited to supplementing a culture environment with a certain
quantity of honey. But they are not studies on the growth and development
of milk bacillus on pollen and honey. The results obtained on this type
of combination of the 2 Lactobacillus strains are original,
sustained by the composition of the culture environments, the new type
of product which can be obtained and by the indirect effect. So, cholesterol
lowering can be considered as the first attribute resulting from such
a bio-technology process.
The benefic effects of pollen on health are combined
with the positive effect exercised by the microbial biomass. Hence, the
synergic effect of the components ensures the functionality of the symbiotic
product. The taste, aroma and homogeneity of the product are important
In the case of male and female rats treated for four
weeks with symbiotic product, the cholesterol level generally decreases
in the treated lots, compared to the control, which proves the lack of
toxicity of the product. Practically, the values are within the physiological
limits in all the treated lots.