The effect of prebiotic and probiotic products used in feed to stimulate the bee colony (Apis mellifera) on intestines of working bees

Abstract

Results are presented of a study of the effects of prebiotic products (lactic and acetic acids) and of probiotic products (Enterobiotic: Lactobacillus acidophilus LA-14 and Bifidobacterium lactis BI-04); Enterolactis Plus: (Lactobacillus casei). Total of 110 colonies of Apis mellifera were allocated to 11 equal treatment groups each of 10 colonies of equal strength. Experimental groups were fed sugar syrup incorporating, at different dosages, the additives mentioned. Histological studies of worker bee intestines were made after three weeks. It was shown that intestinal development was closely correlated with absorption of nutrients and also with a good development of the bee colonies during the active season. Introduction Beekeepers, along with researchers in apiculture and related areas are necessarily concerned with the maintenance of colony health in view of the need to better exploit the bees' potential. This includes their importance as pollinators

Full text

Journal of Food, Agriculture & Environment, Vol.11 (3&4), July-October 2013 2461
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Journal of Food, Agriculture & Environment Vol.11 (3&4): 2461-2464. 2013
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Received 6 July 2013, accepted 10 September 2013.
The effect of prebiotic and probiotic products used in feed to stimulate the bee colony
(Apis mellifera) on intestines of working bees
Silvia Pătruică 1, 2*, Gabi Dumitrescu 3, Roxana Popescu 4 and Nicoleta Măriora Filimon 5
1 Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and Biotechnologies,
Department of Beekeeping, Calea Aradului, 119, 300645, Timisoara, Romania. 2 Vasile Goldiş West University Arad, Bulevardul
Revoluţiei, 94. 3 Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and
Biotechnologies, Department of Histology, Calea Aradului, 119, 300645, Timisoara, Romania.4 University of Medicine and
Pharmacy V. Babes, 300041 Timisoara, Piata E. Murgu 2, Romania. 5West University, Faculty of Biology, Chemistry, Geography,
300315, Pestalozzi, 16, Timisoara, Romania. *e-mail: patruica_silvia@yahoo.com, gdumitrescu@animalsci-tm.ro
Abstract
Results are presented of a study of the effects of prebiotic products (lactic and acetic acids) and of probiotic products (Enterobiotic: Lactobacillus
acidophilus LA-14 and Bifidobacterium lactis BI-04); Enterolactis Plus: (Lactobacillus casei). Total of 110 colonies of Apis mellifera were allocated
to 11 equal treatment groups each of 10 colonies of equal strength. Experimental groups were fed sugar syrup incorporating, at different dosages, the
additives mentioned. Histological studies of worker bee intestines were made after three weeks. It was shown that intestinal development was closely
correlated with absorption of nutrients and also with a good development of the bee colonies during the active season.
Key words: Bee feeding, acidifying substances, lactic acid bacteria and intestinal villi.
Introduction
Beekeepers, along with researchers in apiculture and related
areas are necessarily concerned with the maintenance of colony
health in view of the need to better exploit the bees’ potential.
This includes their importance as pollinators 1, 5, 21, the health
benefits available from use of bee products 2, 6, 15 and, not least,
the role they can play in monitoring environmental pollution 8,
12, 28, 29.
One means of improving colony health involves the addition of
prebiotic and probiotic products to supplementary feeds 11, 17, 22, 26.
In addition to their direct effect of pH lowering, acidficants also
exert an antimicrobial effect, inhibiting the growth of potentially
pathogenic bacteria and creating a favorable environment for the
development of beneficial species 4, 19. Feeding colonies with sugar
syrups having pH between 3 and 6.5 has been shown to result in
significant reductions in the count of pathogenic bacteria, with
the lowest counts being observed at pH 3 26. Both improvement of
health and better colony development were observed by Ceksteryte
and Racys 7 when bees were given acidified feeds.
Probiotics are microorganisms which may exert a favorable
influence on the health of the digestive tract by contributing to
the maintenance or re-establishment of the intestinal microfloral
equilbrium 13, 14, 16, 18, 20. Numerous studies have shown, in addition
to the fact that prebiotics may prevent or restore digestive
dysfunction, that they may also have beneficial effects on
organism immunity 9, 10, 32, inhibit colonisation of the intestine with
pathogenic spores which are in evidence in particular situations
(stress, illness)31. For this reason probiotic bacteria can be used
both prophylactically and in the treatment of a number of bee
diseases 22.
Acidifying substances, as used in the dosages employed by us,
do not impact negatively either on the quality of apicultural
products or on health, given that the reduction in pH of the sugar
feed syrup gives it a pH similar to that of honey. Probiotic products
(Enterobiotics and Enterolactic Plus) are often used in the treatment
of humans to restore a beneficial balance of intestinal microflora
following various digestive disturbances or following antibiotic
treatment.
The use of prebiotic and probiotic products in the spring
stimulation feeding of bee colonies has been shown to lead to
increased production of rape and acacia honies and thus to a
growth in profits resulting from their sale 27.
The aim of this investigation was to observe the intestinal
histological modifications produced by the use of prebiotics and
probiotics as additives to the supplementary spring feed of bees
and to follow the progress of the treated colonies over the active
season.
Materials and Methods
Experimental work was carried out using 110 bee (Apis mellifera)
colonies divided into 11 treatment groups each made up of 10
colonies, each of comparable vigor and with queens of the same
age. It was conducted between March 25th and September 15th,
2011 in Berini, Romania (Europe) with the bee colonies being
maintained in multisection hives under equal conditions of care.
Colony feeding was done using sugar syrup (1:1 sugar:water)
into which pre- and probiotic products were incorporated in the

2462 Journal of Food, Agriculture & Environment, Vol.11 (3&4), July-October 2013
range of doses shown in Table 1.
Three successive weekly sugar syrup feeds were delivered
directly to the combs. After a further three weeks 10 sample
worker bees from each experimental treatment group were
collected for preparation of permanent histological sections. In
the laboratory mid-sections of bee abdomen were fixed in 80%
ethanol and double stained using haemotoxilin-eosin 3. The
sections obtained were viewed under an IOR binocular
microscope.
With a view to establishing the effect of the prebiotic
substances studied on colony development over the course of
the season the proportion of comb occupied by brood was
estimated at the start of the experimental period (March 25th),
three weeks after the administration of pre- and probiotically
doped feed, and again on August 15th. On September 15th the
number of worker bees ready to overwinter was estimated from
the extent to which they covered the combs.
Measurement of intestinal villus length was achieved with the
aid of the QUIKFOTO MICRO 2.2 software package and
statistical analysis (Dunnett’s test) was carried out using IBM
SPSS Statistics, version 19, ANOVA to compare the performance
of experimental treatment groups with the control which had
been fed on pure (undoped) sugar syrup.
Results
Transverse sections from intestines of bees fed with sugar syrup
showed oblong intestinal villi covered with simple columnar
epithelium, with a visible peritrophic membrane. In the
perinuclear cytoplasm small vacuoles are to be
observed while in the conjunctival corion a
lymphatic network can be discerned (Fig. 1).
Bees fed either lactic or acetic acid doped
sugar syrup, with a pH between 3 and 4, had larger
intestinal lumens, while the mucosa showed villi
which were both longer and more numerous than
those found in bees fed undoped syrup. Villi were
slightly heteromorphic, some being oblong,
others appearing triangular (Fig. 2) with mean
dimensions falling between 225 and 248
microns, significantly different in size (P<0.01)
from the control group fed the unacidified syrup
(Table 2).
The intestinal epithelium of bees fed on acidified sugar syrup
presented a slightly disorganised appearance in that among the
parenchyma cells. One feature which developed in bees fed with
lactic acid supplements was the presence of lymphatic networks,
both in the corion of the villi and in the basal corion (Fig. 2).
Numerous cells with spongiform cytoplasm were present in the
intestinal epithelial structure of bees fed acidified syrup, while
the cell apices showed many peritrophic membranes (well-
developed microvilli), – indicative of their enhanced absorption
function.
The intestinal mucosa of bees fed with probiotically doped
sugar syrup (Enterobiotics or Enterolactis Plus) showed more
numerous villi than that of the other treatment groups. Mean
villus dimensions fell between 160 and 183 µm (Table 2). These
Experimental groups
Feed
composition M EG
1
EG
2
EG
3
EG
4
EG
5
EG
6
EG
7
EG
8
EG
9
EG
10
Sugar
syrup (l) 1 1 1 1 1 1 1 1 1 1 1
98% Lactic
acid (ml) - 2 2.5 - - - - - - 2.5 2.5
Acetic
acid (ml) - - - 30 20 - - - - - -
Enterobiotics
(g) - - - - - 1.25 2.5 - - 2.5 -
Enterolactis
Plus (g) - - - - - - - 1.2 2.4 - 2.4
Table 1. Experiment organization scheme.
M = control group; EG1 = Experimental group 1; EG2 = Experimental group 2; EG3 = Experimental group 3; EG4 = Experimental
group 4; EG5 = Experimental group 5; EG6 = Experimental group 6; EG7 = Experimental group 7; EG8 = Experimental group 8;
EG9 = Experimental group 9; EG10 = Experimental group 10.
Figure 1. Control group. Intestinal villi (1000x).
Table 2. Mean and standard deviation values for intestinal villus
dimension (microns) of bees fed with prebiotic and
probiotic products (N=120).
**P <0.01, M = control group; EG1 = lactic acid (2 ml) treatment group; EG2 = lactic acid (2.5 ml)
treatment group; EG3 = acetic acid (20 ml) treatment group; EG4 = acetic acid (30 ml) treatment group;
EG5 = Enterobiotics (1.25 g) treatment group; EG6 = Enterobiotics (2.5 g) treatment group; EG7 =
Enterolactis Plus (1.2 g) treatment group; EG8 = Enterolactis Plus (2.4 g) treatment group; EG9 = lactic
acid (2.5) and Enterobiotics (2.5 g) treatment group; EG10 = lactic acid (2.5) and Enterolactis Plus (2.4
g) treatment group.
Prebiotic
products
Probiotic
products
Prebiotic and
probiotic
products
No prebioti
c
or probiotic
products
EG
1
240±23
**
EG
5
176±23
**
EG
9
201±15
**
M 106±12
EG
2
248±21
**
EG
6
183±19
**
EG
10
198±26
**
EG
3
238±26
**
EG
7
160±14
**
EG
4
225±18
**
EG
8
168±17
**
Figure 2. Experimental treatment group 2. Intestinal villi showing
lymphatic network (1000x).

Journal of Food, Agriculture & Environment, Vol.11 (3&4), July-October 2013 2463
villi were covered with simple epithelia of columnar cells with
peritrophic membranes arranged in a thick layer. A thickening of
the peritrophic membrane in bees from colonies fed with
probiotic supplements has also been reported by Szymaś et al. 30.
A number of spherical cells were found among the parenchyma
cells underlying the epithelium. These had basophilic cytoplasm
and large spherical nuclei. These cells showed a high level of
mitotic activity with very many being found in one of the various
stages of mitotic division (Fig. 3). Loose connective tissue
presence in the axes of the villi was much reduced.
In experimental treatment 6 (fed with a double strength dose
of Enterobiotics product) the muscularis externa was formed of
two thick superimposed bands of smooth muscle. A small number
of cells with spongiform cytoplasm and spherical, intensely
staining, nuclei were present in the parenchyma. The morphology
of these cells suggests a high level of secretory activity (Fig. 4).
In the treatment groups fed with lactic acid in conjunction with
a probiotic product (Enterobiotics or Enterolactis Plus) the
intestinal wall showed villi that were long (198-201 microns) and
branching. The covering epithelium of these villi was simple,
with a thick layer of peritrophic membrane (Fig. 5). Within the
loose connective tissue in the interior axis of the villus a number
of large spherical or oval shaped cells with central or eccentric
nuclei and vacuolated cytoplasm were found.
Figure 3. Experimental treatment group 5. Intestinal villi (1000x).
Figure 4. Experimental treatment group 6. Intestinal villi (1000x).
Discussion
The addition of acidificants (lactic or acetic acids) to the spring
sugar syrup feeds given to bees after the cleaning flight leads,
through reduction of intestinal pH levels, to an inhibition of the
growth of pathogenic bacteria and to the improvement of digestion
through an enhanced development of intestinal villi (statistically
significant, P<0.01), compared with that found in the control group.
We can therefore say that, in addition to the effect of acidifiers in
significantly reducing the number of germ 26 they also led to
important modifications of the tissue structure of the intestinal
wall, with direct implications for an increase in the efficiency of
assimilation of nutrients from pollen and nectar. This shows a
clear correlation with the significant increase in the area of brood
comb found three weeks after the acidified syrup feeding was
completed 23.
Addition of probiotic products to feed improves colony health
through intestinal colonisation with beneficial bacterial
(Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus
casei) 26 while also favouring better digestion through increasing
the surface area for absorption, due to the promotion of greater
development of intestinal villi. Experimental results show mean
villus lengths of between 160 and 183 microns in treated groups
compared with a mean of 106 microns in the untreated control.
The probiotic bacteria reaching the intestine with the sugar
syrup have been shown to cause a reduction in the number of
germs and their replacement with Lactobacillus acidophilus LA-
14, Bifidobacterium lactis BI-04 and Lactobacillus casei, present
in the administered preparations 26. The increased intestinal
secretory activity associated with the observed histological
changes could explain the better development of the bee colonies
studied, as seen in the enhanced number of brood cells found
three weeks after the administration of the probiotic products 24.
In the treatment groups fed with lactic acid in conjunction with
a probiotic product (Enterobiotics or Enterolactis Plus), reduction
of intestinal pH has been shown to lead to a reduction in the
number of potentially pathogenic spores and the colonisation of
the intestine with beneficial bacteria present in the products
administered 26 as well as to an increase in the area of brood
comb three weeks subsequent to the adminstration 25.
In apicultural practice the opportunity to encourage intestinal
villus development could be particularly valuable, given that such
Figure 5. Experimental treatment group 9. Peritrophic membrane
(1000x).

2464 Journal of Food, Agriculture & Environment, Vol.11 (3&4), July-October 2013
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a development leads to an increase in the surface area available
for the absorption of nutrients and thus to better digestive
assimilation. This might also increase the efficiency of
absorption of other pharmacological treatments used as additives
in feeding syrup.
Although colonies fed with pre- and probiotically modified
feeds showed enhanced development during the first part of the
season, in the autumn the number of combs covered with bees
and the reserves of food for winter were comparable with those
found in the control group.
Acknowledgments
This work was co-financed by the European Social Fund through
Sectoral Operational Programme Human Resources Development
2007-2013, project number POSDRU/89/1.5/S/63258 “Postdoctoral
school for zootechnical biodiversity and food biotechnology
based on the eco-economy and the bio-economy required by eco-
san-genesys”. The authors declare that they have no conflicts of
interest.