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Prebiotic and probiotic properties of honey

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Abstract

Laboratory studies have shown that honey contains olygosaccharides and low molecular weight polysaccharides exhibiting prebiotic properties. Like the well-known commercial prebiotics the honey oligosaccharides are not digested in the upper part of gastrointestinal tract but are fermented by beneficial microflora in large intestine of human and animals and stimulate its growth and vital activity. It is emphasized that prebiotic properties of honey depend on its plant origin. It is shown that fresh honey also contains probiotics - the microorganisms beneficial for human and animals that inhibit the growth and development of pathogenic and conditionally pathogenic microflora, and can also be a source of biologically active substances with antimicrobial activity. Bifidobacteria and lactobacilli inhabiting honey stomach can survive in honey within 2-3 months after its harvest. The microflora composition of honey stomach and fresh honey may depend on the botanical origin of honey, as well as habitat and subspecies of honey bees. The probiotic microorganisms are involved in the development of honey bee resistance to adverse environmental factors directly inhibiting the growth ofpathogens, and stimulating components of the immune system. Antagonistic activity of probiotic bacteria against a broad spectrum of pathogenic microorganisms enable their application for prophylaxis and treatment of honey bee diseases, and in human and veterinary medicine.
Chapter
PREBIOTIC AND PROBIOTIC
PROPERTIES OF HONEY
Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.*
Institute of Biochemistry and Genetics of Ufa Scientific Center of Russian Academy of
Science, Ufa, Russia
ABSTRACT
Laboratory studies have shown that honey contains olygosaccharides and low molecular
weight polysaccharides exhibiting prebiotic properties. Like the well-known commercial
prebiotics the honey oligosaccharides are not digested in the upper part of gastrointestinal
tract but are fermented by beneficial microflora in large intestine of human and animals
and stimulate its growth and vital activity. It is emphasized that prebiotic properties of
honey depend on its plant origin. It is shown that fresh honey also contains probiotics -
the microorganisms beneficial for human and animals that inhibit the growth and
development of pathogenic and conditionally pathogenic microflora, and can also be a
source of biologically active substances with antimicrobial activity. Bifidobacteria and
lactobacilli inhabiting honey stomach can survive in honey within 2-3 months after its
harvest. The microflora composition of honey stomach and fresh honey may depend on
the botanical origin of honey, as well as habitat and subspecies of honey bees. The
probiotic microorganisms are involved in the development of honey bee resistance to
adverse environmental factors directly inhibiting the growth of pathogens, and
stimulating components of the immune system. Antagonistic activity of probiotic bacteria
against a broad spectrum of pathogenic microorganisms enable their application for
prophylaxis and treatment of honey bee diseases, and in human and veterinary medicine.
Keywords: honey, prebiotics, probiotics, honey carbohydrates, lactic acid
bacteria
** Corresponding author: Email: lurim78@mail.ru.
2Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
INTRODUCTION
In the past 20 years a surge of interest in medicinal honey properties is
observed. The antimicrobial activity of honey against human pathogenic
microorganisms is shown (Conway et al., 2010; Angela, 2012, Gomashe et al.,
2014). This activity is caused by osmolality, acidity, hydrogen peroxide (White et
al., 1963), flavonoids, phenolic acids (Taormina et al., 2001) and other
antimicrobial components depending on honey type (Olofsson and Vásquez,
2008). Honey is also used for treatment of diseases of the respiratory system,
gastrointestinal tract, cardiovascular system, etc. (Yaniv and Rudich, 1996). The
scientific mechanism of honey therapeutic effect is currently not fully understood.
However, the vast majority of scientific findings suggest that some of the honey
beneficial properties are due to their probiotic and prebiotic components.
Probiotics are beneficial to human living microorganisms, which are normal
inhabitants of healthy human intestine, inhibiting the growth and development of
pathogenic and conditionally pathogenic flora. The most famous probiotic
microorganismes are Lactobacillus acidophilus, L.casei, L. delbrueckii subsp.
bulgaricus, L. reuteri, L. brevis, L. cellobiosus, L. curvatus, L. fermentum, L.
plantarum, gram-positive cocci Lactococcus lactis subsp. cremoris, Streptococcus
salivarius subsp. thermophilus, Enterococcus faecium, S. diaacetylactis, S.
intermedius, Bifidobacterium bifidum, B. adolescentis, B. animalis, B. infantis, B.
longum, and B. thermophilum (Kashirskaya, 2000). The mechanism of probiotic
effect may be due to competition for adhesion receptors on the intestinal
epithelium (Johnsson and Conway, 1992) and nutrients (Freter, 1992), the
production of antibacterial substances (Sarkar and Banerjee, 1996) and the
stimulation of the immune system (Nousiainen and Setala, 1993). Lactic acid
bacteria in the intestine produce metabolites that inhibit the growth of pathogenic
microorganisms and increase resistance of the host organism. Produced organic
acids also decrease the intestinal pH, which can inhibit other bacterial pathogens.
Prebiotics are nondigestible food ingredients which promote the improvement
of health by selectively stimulating the growth and/or metabolic activity of one or
more groups of probiotic bacteria inhabiting the colon. Prebiotics are not
hydrolyzed by the human digestive enzymes, not absorbed in the upper digestive
tract and are selective substrates for growth and/or metabolic activation of one
species or group of probiotic microorganisms, resulting in their ratio
normalization (Van Loo et al., 1999). Most often prebiotics are different oligo-
and polysaccharides in which molecule residues are connected by -glycosidicβ
linkages (Buchakhchyan et al., 2011). Human enzyme systems do not contain -β
3
Prebiotic and Probiotic Properties of Honey
glycosidases, therefore prebiotics are hydrolyzed only by the normal intestinal
microflora. The best-known prebiotics are inulin, fructo-oligosaccharides (FOS),
galacto-oligosaccharides (GOS), soya-oligosaccharides, xylo-oligosaccharides,
pyrodextrins, isomalto-oligosaccharides and lactulose (Conway et al., 2010).
A large number of oligosaccharides and low molecular weight polysaccharides
in honey attract a research interest to the honey as a source of nutrients for colon
microflora.
PREBIOTICS IN HONEY
[1] The Carbohydrate Composition of Honey
Qualitative and quantitative carbohydrate composition of honey is variable
and depends on the floral source of honey. In fact, the honey is a supersaturated
sugar solution with about 17-20% water. 90% honey carbohydrates are glucose
and fructose monosaccharides, and other carbohydrates include more than 30
different oligosaccharides (Anklam, 1998; Morales et al., 2007; Ruiz-Matute et
al., 2010). Fructose is the predominant sugar with concentration range of 36-50%,
glucose amounts to 28-36%. Disaccharides, trisaccharides, tetrasaccharides,
hexasaccharides and other oligosaccharides are present in much smaller quantities
than glucose and fructose (The National Honey Board, 2008). Honey
disaccharides include sucrose, maltose, isomaltose, nigerose, turanose, maltulose,
leucrose, kojibiose, neotrehalase, gentiobiose, laminaribiose isomaltulose,
melibiose, palatinose, trehalose, and trehalulose (DArcy et al., 1999; Sanz et al.,
2004; de la Fuente et al., 2007). 25 trisaccharides including planteose and a-30-
glucosyl-isomaltose, which have been reported in honey for the first time, and 10
tetrasaccharides were identified in Spanish and New Zealand honeys (Ruiz-
Matute et al., 2010). Honeydew honey is characterized by a high concentration of
oligosaccharides, mainly the trisaccharides melezitose and raffinose, which
usually are not found in blossom honeys (Bogdanov et al., 2004). New Zealand
honeydew honeys contain tetrasaccharides maltotetraose, alpha-panasyl-D-
fructofuranoside and alpha-maltosyl-D-fructofuranoside, two pentasaccharides
and one hexasaccharide (Morales et al., 2007). Shin H.S. and Ustunol Z. (2005)
have reported that bee alpha-D-glucosidase catalyzes the transfer of alpha-D-
glucopyranosyl groups from sucrose to an acceptor carbohydrate resulting in the
formation of FOS and various other oligosaccharides in different amounts.
Different grades of honey are found to contain specific oligosaccharides. For
example, the New Zealand honey contains isomaltose and melezitose (Weston and
4Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
Brocklebank, 1999), and the Italian honey contains raffinose (Oddo et al., 1995).
Sugar composition and the ratio of particular carbohydrates are fairly reliable
indicators for honey classification and authentication in the case of unifloral
honeys with very high amount of dominating plant (Kaˇskoniene and
Venskutonis, 2010). Morales and others (2007) found differences in the higher
oligosaccharide compositions of ten different honeys by extraction of
oligosaccharides with activated charcoal. It is also shown that sage, alfalfa and
oxydendrum honey contains 3.8, 5.5 and 10.9% oligosaccharides respectively
(Popa and Ustunol, 2011). The total content of kojibiose, maltose, nigerose, and
turanose was highest in Spanish honey samples of rosemary, lavender, sunflower,
eucalyptus, heather, and honeydew origin (Mateo and Bosch-Reig, 1997). Cotte
and others (2004) determined the predominant disaccharides in some France
honeys: maltose and turanose in acacia; maltulose and turanose in chestnut and
linden; turanose and trehalose in fir; and sucrose and maltose in lavender origin
honey. Maltose was the major disaccharide in 80 Brazilian honey samples
(Eucalyptus spp., extra-floral, and multifloral honeys) (Da Costa Leite et al.,
2000). Heather honey were characterized by the presence of erlose and nigerose;
forest honey contained higher amounts of trehalose and
melezitose; spike lavender honey was specified by isomaltose; while French
lavender and thyme honeys were noted for the panose presence (Nozal et al.,
2005).
The variability of qualitative and quantitative carbohydrates composition of
honey causes the differences in the glycemic index (GI) of different honeys and
consequently in their dietary properties. Australian honeys Yellow Box,
Stringybark, Red Gum, Ironbark, and Yapunyah have low values of GI (Arcot and
Brand-Miller, 2005). In contrast, the average GI of four American honeys was
72.6 with no significant differences between the tested varieties (The National
Honey Board, 2008). Obviously a low content of glucose and increase in share of
oligo- and polysaccharides contributes to decrease in honey GI.
[2] The Effect of Honey on the Probiotic Microorganisms
In vitro and in vivo investigations demonstrate a stimulating effect of honey
and its carbohydrate components on the beneficial microorganisms inhabiting the
lower sections of human and animal digestive tract (Table 1).
Bifidobacteria are quite fastidious organisms. Many researchers reported that
bifidobacteria grow poorly in the milk and therefore require the addition of
5
Prebiotic and Probiotic Properties of Honey
specific growth factors (Rybka and Fleet, 1997; Dave and Shah, 1998; Chick et
al., 2001). It is assumed that the more preferred substrates for bifidobacteria are
polysaccharides with a low degree of polymerization that are present in honey
(Chick et al., 2001; Kajiwara et al., 2000).
Thus, sourwood, alfalfa, and sage honeys are shown to have stimulating action
on the growth and activity of B. longum, B. adolescentis, B. breve, B. Bifidum,
and B. infanti inhabiting the human gut and are used in the production of
fermented milk products (Shin and Ustunol, 2005). Presented honey effect was
similar to that of the commercial oligosaccharides FOS, GOS, and inulin
(Kajiwara et al., 2002; Ustunol, 2007). Moreover the native honey was more
effective than the combination of its purified basic saccharide components. Based
on these results, a synergistic effect of honey carbohydrate components to
enhance the growth and activity of bifidobacteria has been proposed. However, it
is not impossible that tested honey could contain additional unexplored
saccharides that are more effective in enhancing the growth of bifidobacteria. Two
types of monofloral honey, dark chestnut honey and light acacia honey, increased
the enzyme activity and the number of viable cells of B. lactis (Bb-12) and B.
longum (Bb-46) in soy milk (Slacanac et al., 2012). Furthermore, the addition of
honey, especially chestnut honey, increased the inhibitory potential of the
fermented soymilk against Listeria monocytogenes. Jordanian honeys enhanced
the growth and increased short
chain fatty acids production of the two intestinal bacteria, B. infantis and L.
acidophilus (Haddadin et al., 2007). In addition, different bifidobacteria strains
specifically responded to the addition of this honey type in the medium.
Australian honeys contributed to the growing of B. lactis and L. plantarum more
than sucrose and inulin (Conway et al., 2010).
Differences in carbohydrate composition of various honeys suggest their
diverse prebiotic effect. Thus, glucose-rich yellow box honey stimulated the
growth of coliform bacteria, while fructose-rich banksia honey contributed to the
growth of lactic acid bacteria (Conway et al., 2010). Comparison of sage, alfalfa,
and oxydendrum honeys with sucrose, corn syrup with a high fructose content,
and inulin was carried out by their ability to support the growth, activity and
viability of bifidobacteria and lactic acid bacteria commonly used in the
production of yogurt (Popa and Ustunol, 2011). Alfalfa honey was more effective
in enhancing the growth of Streptococcus thermophilus (St-133), sourwood honey
- L. delbrueckii subsp. bulgaricus (Lr-78), and all three types of honey contributed
to the growing of B. bifidum (Bf-1). Alfalfa and sourwood honeys were the most
effective in stimulation of the lactic acid production of L. delbrueckii subsp.
bulgaricus (Lr-78) and L. acidophilus (La-7). An important characteristic of
bifidobacteria is the production of lactic and acetic acids as the final products of
6Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
sugar fermentation. In an ideal synthetic medium fermentation of 2 moles of
glucose by bifidobacteria leads to the formation of 3 moles of acetic acid and 2
moles of lactic acid (Scardovi and Trovatelli, 1965). In medium containing other
substrates, including prebiotic oligosaccharides, this ratio is not supported. High
acetate levels create a "vinegar" taste of foods. Therefore, an increase in the
proportion of lactate and acetate production decline would be useful for dairy
products manufacturing technology, since it would improve the organoleptic
characteristics of the products. In this study, alfalfa honey increased in B. bifidum
(Bf-1) production of lactic acid, and sourwood honey - acetic acid. Chic et al.
(2001) also reported increases in lactic acid production to the level of acetic acid
in the fermentation of milk by B. bifidum (Bf-1) in the presence of clover honey.
Thus, applications as a sweetener of the types of honey, which alter lactate/acetate
ratio in favor of lactate, can be recommended for the dairy production technology.
Based on the results of these studies, such honeys as alfalfa and clover honeys
stimulate the growth of bifidobacteria and lactic acid production.
Table 1. Monofloral honeys, stimulating the growth and
activity of probiotic microorganisms
Microorganism The botanical origin of honey Reference
Lactobacillus acidophilus
L. delbrueckii subsp.bulgaricus
L. plantarum
L. ramnosus
L. paracasei
Streptococcus thermophilus
Bifidobacterium bifidum
B. adolescentis
B. infantis
B. longum
B. breve
B. lactis
Eucalyptus sideroxylon,
Eucryphia lucida
Oxydendrum arboreum,
Medicago sp.
E. sideroxylon, Banksia sp.,
E. melliodora, E. paniculata,
E. longifolia, E. triantha
E. sideroxylon, E.lucida
E.lucida
Medicago sp.
O. arboreum, Medicago sp.
Salvia sp., Trifolium sp.
O. arboreum, Medicago sp.
Salvia sp.
O. arboreum, Medicago sp.
Salvia sp.
O. arboreum, Medicago sp.
Salvia sp., Castanea sp., Acacia
sp.,
O. arboreum, Medicago sp.
Salvia sp.
Castanea sp., Acacia sp., E.
sideroxylon, E. melliodora,
E. paniculata, E. longifolia, E.
triantha
Conway et al., 2010
Popa and Ustunol, 2011
Conway et al., 2010
Conway et al., 2010
Conway et al., 2010
Popa and Ustunol, 2011
Popa and Ustunol, 2011;
Chick et al., 2001
Popa and Ustunol, 2011
Popa and Ustunol, 2011
Popa and Ustunol, 2011;
Slacanac et al., 2012
Popa and Ustunol, 2011
Popa and Ustunol, 2011;
Slacanac et al., 2012
7
Prebiotic and Probiotic Properties of Honey
In a detailed study of Australian honeys carried out by Conway P.L. et al.
(2010), mugga honey showed prebiotic effect for four probiotic cultures: B. lactis,
L. rhamnosus, L. plantarum, and L. acidophilus. Tasmanian leatherwood honey
demonstrated good prebiotic effects on 3 probiotic cultures: L. rhamnosus, L.
acidophilus, and L. paracasei. Banksia honey, woolly butt honey, grey ironbark
honey and yellow stringybark honey had a prebiotic effect on at least one
probiotic culture. In comparison with inulin and sucrose all tested honey showed
higher values of prebiotic index (the PI), measured by changes in the number of
four bacterial groups (bifidobacteria, lactobacilli, clostridia and bacteroides): from
about 130 in creek woolybutt honey up to 420 in mugga ironbark honey against
46 in inulin. The authors assumed that this result reflects the synergistic effect of
simple and complex sugars that are present in honey, and assessed the prebiotic
potential of oligosaccharides isolated from the investigated honeys. These
oligosaccharides are also largely contributed to the growth of L. acidophilus. At
the same time, PI values of honey oligosaccharides were much lower than in
honeys, but almost the same as in inulin.
The situation in which honey monosaccharides are digested in the upper
human intestinal tract and oligosaccharides pass into the colon, where indigenous
bacteria are living, was modeled (Sanz et al., 2005). For this, monosaccharides
have been removed from honey and oligosaccharide fraction was studied in
comparison with FOS on prebiotic activity in relation to fecal bifidobacteria,
lactobacilli, and eubacteria. According to the study, honey oligosaccharides have a
potential probiotic activity (PI values between 3.38 and 4.24) increasing the
population of bifidobacteria and lactobacilli, although not to the level of FOS (PI
6, 89). In another study the prebiotic potential of honey was evaluated in
comparison with inulin and gum acacia towards lactobacilli isolated from human
feces (Tejpal, Goyal, 2009). The highest specific rate of growth of lactobacilli was
observed in the presence of honey.
According to the studies conducted by Shamala et al. (2000) in vitro, the
amount of L. acidophilus and L. plantarum increased by 10 - 100 times in the
presence of honey compared with sucrose. In vivo study found that the number of
viable lactic acid bacteria from the intestine of rats was significantly higher when
feeding with honey than with sucrose. From these results the authors concluded
that a known curative effect of honey on the liver, cardiovascular system and
gastrointestinal tract can be associated with changes in the microbial profile, with
a significant increase in the amount of lactic acid bacteria, which in turn affect the
physiology and health of the host. In experiments carried out in vivo by El-Arab et
al. (2006) the addition of honey in mice diet has been shown to increase the
amount of bifidobacteria and lactobacilli in the mice intestine too, and also reduce
the histopathological and genotoxic effects of mycotoxins.
8Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
PREBIOTICS IN HONEY
[3] Probiotic Lactic Acid Bacteria in Honey
Species Content of Lactic Acid Bacteria in Honey
At present we know that fresh honey contains a large number of lactic acid
bacteria (LAB), derived from honey bee stomachs, that possess a wide spectrum
of antimicrobial activity against various microorganisms, pathogenic to bees and
humans (Olofsson et al., 2014).
LAB are a clade of Gram-positive microaerophilic microorganisms
functionally associated by their ability to ferment carbohydrates in homo- or
heterofermentative metabolism with lactic acid formation (Salminen et al., 2004).
Traditionally LAB include immobile, catalase negative, asporous, either rod- or
cocci-shaped representatives of Lactobacillales. Bifidobacterium are distant
relatives of Lactobacillales. It is genus of Gram-positive, anaerobic, catalase
negative, asporogenous, rod-shaped bacteria, by definition, is not a “true”'
member of the LAB. However Bifidobacterium are generally regarded to LAB
group due to their lactic acid production, use in the manufacture of dairy products
and well-known beneficial effects on the flora of the gastrointestinal tract of
humans and animals (Coenye and Vandamme, 2003).
Recently for the first time the symbiotic flora from honey stomachs and fresh
honey of Swedish honey bees has been detected and associated with many healing
honey properties (Olofsson and Vásquez, 2008). Approximately 40 LAB strains
with 13 taxonomically identified species of Lactobacillus (9 spp.) and
Bifidobacterium (4 spp) has been found in the new microbiota: L. kunkeei Fhon2,
L. apinorum Fhon13, L. mellis Hon2, L. mellifer Bin4, L. kullabergensis Biut2, L.
kimbladii Hma2, L. helsingborgensis Bma5, L. melliventris Hma8, L. apis
Hma11, B. coryneforme Bma6, B. asteroides Bin2, B. sp Bin7, and B. sp Hma3
(Butler et al., 2014). Most of these LAB symbionts are the species described for
the first time. It is shown that the discovered LAB symbionts present in honey
stomachs of honey bees of all species, as well as stingless bees, and in the
corresponding fresh honey on all continents of the world (Vasquez et al., 2009;
2012; Olofsson et al., 2011; Tajabadi et al., 2011; 2013). In addition to the unique
honey bee LAB flora honey may also contain other LAB strains, for example, L.
acidophilus contributing to its antibacterial activity (Angela, 2012; Aween et al.,
2012.). The maximum registered number of viable LAB in fresh honey is 108
LAB/g of honey (Vasquez et al., 2012). In process of honey dehydration number
9
Prebiotic and Probiotic Properties of Honey
of viable LAB decreases and becomes zero at a water content of less than 20%
(Olofsson et al., 2014).
These LAB are not collected by bees from flowers, but are symbiotic
organisms that inhabit honey bee stomachs. The number and species composition
of honey stomach LAB microflora depends on the season, the source and amount
of nectar, the health of bees, and the presence of other microorganisms in the
collected nectar (Olofsson and Vásquez, 2008; Vasquez et al., 2009; 2012;
Tajabadi et al., 2011; Butler et al., 2013; Forsgren et al., 2010). Thus, the number
of LAB flora is lowest in early spring, when bees start collecting pollen and
nectar after winter, and increases with foraging activity. Transient microbes
collected from flowers, provoke the growth of LAB microbiota in honey bees and
the production of anti-microbial proteins (Butler et al., 2013).
Value of LAB in the Honeybee Organism
Each member of the discovered honey bee LAB microbiota ferments nectar,
excretes strain-specific spectrum of metabolites and thus participates in the
process of conversion of nectar to honey (Olofsson et al., 2014). The substances
produced by LAB are present in fresh honey and stored in the mature honey. In
addition, these LAB are assumed to play a key role in the production of bee bread
from a pollen (Vasquez and Olofsson, 2009).
Environment of honey stomach is characterized by the micro aerobic
condition, the presence of nectar sugars and sufficiently optimal temperature
(35°C), independent from the outside air temperature (Jones et al., 2004), and is
the optimal niche for LAB. Based on the received data, Olofsson T. and Vásquez
A. (2008) suggested that bees and LAB flora developed in mutual dependence on
each other: LAB received a niche in which nutrients were available, and the bees
obtained a protection from harmful. Thus, it is known that certain types of LAB
can produce bioactive compounds, such as organic acids, free fatty acid, ethanol,
benzoate, enzymes, hydrogen peroxide, bacteriocins, and antimicrobial peptides
(De Vuyst and Leroy, 2008). At the same time, there is a distinct difference in the
production of antimicrobial substances and other useful properties between
various species and genera in LAB (Pfeiler and Klaenhammer, 2007). Different
extracellular LAB metabolites possess bactericidal or bacteriostatic properties,
and have different mechanisms of action, such as violation of cell membrane
permeability and the DNA synthesis or changing of the growth conditions, for
example by reducing the pH (Butler et al., 2014). These qualities together result in
a broad inhibiting spectrum against pathogens: 55 species of bacteria and 5
species of yeast which are found in flowers (; Forsgren et al., 2010; Vasquez et al.,
2012).
10 Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
Genomic analysis of LAB flora in honey stomach has shown that the genome
sizes of these microorganisms vary within 1,5 - 2,2 mega-base pairs (Mbps),
which indicates a clear distinction in produced proteins and the specialized
functions of bee microbiota and its adaptation to the host (Butler et al., 2013).
L. kunkeei is a dominant species in honey bee stomach microbiota (Olofsson
and Vásquez, 2008, Vasquez et al., 2012). It is remarkable that this microorganism
was first isolated during spoilage of wine, as a strain strongly inhibiting alcoholic
fermentation of yeasts Saccharomyces bayanus and S. cerevisiae (Huang et al.,
1996; Edwards et al., 1998). It was also shown that namely the bees distribute L.
kunker among the damaged grapes with which this microorganism gets into the
wine raw (Bae et al., 2006). Therefore, the value of L. kunkeei for bees may be to
inhibit the process of fermentation of immature honey by yeast Saccharomyces
causing honey spoilage (Snowdon and Cliver, 1996; Madras-Majewska et al.,
2016).
Paenibacillus larvae infection of bee colonies was associated with the
presence in bee organisms of bacterial phylotypes closely associated with genera
Actinobacillus and Phocoenobacter of the family Pasteurellaceae (Olofsson and
Vásquez, 2008). The authors have suggested that the interaction of P. larvae,
Pasteurellaceae phylotypes and LAB flora of honey stomach affects the number
of pathogens. In another study, in all bee colonies Bifidobacterium activity
negatively correlated with the activity of pathogenic microbes (Mattila et al.,
2012). Forsgren et al. (2010) have demonstrated strong inhibitory effects of
combined LAB flora of honey stomach on the growth of P. larvae in vitro. LAB
addition to honey bee young larvaes contaminated with P. larvae spores reduced
the proportion of larvae infected with the causative agent of American foulbrood.
It is remarkable that individual phylotypes of LAB inhibited P. larvae strains
differently. Thus, the dominant strain L. kunkeei Fhon2 showed only partial
inhibition of P. larvae, whereas L. apis Hma11 and L. kullabergensis Biut2
possessed strong inhibitory activity on the growth of P. larvae. Based on these
data, it was concluded by the authors that the whole LAB flora may act
synergistically against P. larvae and possibly other harmful microorganisms.
Field observations indicate that P. larvae infection may be present in bee
colonies without causing clinical symptoms and can be naturally reduced to
undetectable levels (Fries et al., 2006). This phenomenon may be due to the action
of LAB flora, the activity of which can be changed with the switching of bees to
another nectar source. For example, in colonies infected with P. larvae, the
number of the pathogen grown when collecting of oil-seed rape and wild
raspberry nectars, but subsequently decreased until the disappearance of American
foulbrood pathogen when collecting of linden nectar (Olofsson, Vásquez, 2008).
11
Prebiotic and Probiotic Properties of Honey
Organic acids such as formic acid, which is produced by bifidobacteria (Van
der Meulen et al., 2006), lactic and acetic acids, which are produced by LAB, are
antimicrobial agents and may be important in the protecting of bees against
pathogens. Thus in vitro experiments have shown that lactic acid produced by L.
johnsonii inhibit P. larvae (Audisio et al., 2011), and metabolites produced by
bifidobacteria exhibit antagonistic effects on the growth of the pathogen of
European foulbrood Melissococcus plutonius (Wu et al., 2013). It is indicative
that these acids are widely used by beekeepers for protection of bees from Varroa
destructor and Nosema apis. In conditions of microbial stress (under the action of
the representatives of Pseudomonas, Enterobacteriaceae, Bacillus and Candida
from the flowers and the environment) LAB symbionts of honey bees produce
extracellular proteins: enzymes, DNA-chaperones, S-layer proteins, bacteriocins,
lysozyme and a number of new proteins with presumably antimicrobial function
(Vasquez et al., 2012; Butler et al., 2013).
Currently, immunotropic and immune stimulatory mechanisms of probiotics
action on human oragnizm are clinically and experimentally proven (De Vrese
and Schrezenmeir, 2008; Ng et al., 2009). Indirectly LAB action on pathogens by
activating of the immune system is also shown in the organism of honey bee. So,
the addition of Lactobacillus and Bifidobacterium to the bee feed caused increase
of the gene expression level of abaecin (Evans, Lopez, 2004) which is specific to
Hymenoptera antibacterial peptide having bactericidal activity against Gram-
positive and Gram-negative bacteria (Rahnamaeian et al., 2015). Per os treatment
of larvae and adult bees with LAB also caused increasing of gene transcription
levels of antimicrobial peptides abaecin, defensin and hymenoptecin (Yoshiyama
et al., 2013).
These results show that LAB stimulate the innate immune response in honey
bees, and may be useful for the prevention of bee infectious diseases. Preventive
methods which enhance the bee LAB flora or additional food with LAB, can
promote sustainability of honey bee, which is especially relevant in light of the
current problems of the colony collapse disorder (Vasquez et al., 2012). Thus, it is
shown that the stimulating fertilizers containing probiotic additives based on
LAB, improve intestinal microbiocenosis of bees, increase their strength, winter
hardiness, productivity of bee colonies, and reproductive performance of queens
(Mishukovskaya, 2015).
The Antibacterial Activity of Honey LAB against Human and Animal Pathogens
The antagonistic effect of LAB isolated from honey stomach and honey
against a broad spectrum of microorganisms creates a perspective of their
application in the fight against human and animal pathogens, including those
resistant to modern antibiotics. Thus, it is shown that LAB symbionts individually
12 Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
and together have a strong antimicrobial activity against a wide range of human
pathogens, including antibiotic resistant strains such as methicillin-resistant
Staphylococcus aureus (MRSA) (Olofsson et al., 2014). Cells and metabolites of
L. acidophilus strains isolated from Malaysian honeys inhibited the growth of
human pathogens with multiple antibiotic resistance: S. aureus, S. epidermis, and
B. subtilis (Aween et al., 2012).
In vivo experiments proved the effectiveness of dressings based on honey
enriched with the bee LAB for the treatment of wounds infected with various
pathogens (Butler et al., 2014;. Olofsson et al., 2014). In these studies, the strain-
dependence of the LAB properties and substances produced by them were taken
into account. So, L. mellifer Bin4 was shown to inhibit all investigated wound
pathogens and produce benzene, which is a toxic volatile compound that increases
the rate of wound closure and epithelialization. L. kunkeei Fhon2 produced a wide
variety of extracellular proteins in response to microbial stress and three different
3-OH fatty acid as well as had the most potent activity against human wound
pathogens, particularly against members of the Pseudomonas spp. (Butler et al.,
2013). Pseudomonas is widely distributed on plants and at the same time is one of
the therapeutically-resistant pathogens in human chronic wounds due to biofilm
formation, drug resistance, and interactions with other microbes in the wound
environment (Scales and Huffnagle, 2013). Honey in combination with LAB also
inhibited the growth of bovine mastitis pathogens Staphylococcus spp.,
Streptococcus spp., Escherichia coli, and Klebsiella pneumoniae including those
exhibiting the antimicrobial resistance to one or more antibacterial compounds
(Piccart et al., 2016).
[4] Spore-Forming Probiotic Bacteria in Honey
In addition to the LAB flora honey also contains spores of aerobic spore-
forming bacteria of the genus Bacillus spp. which are collected by bees from
plants during foraging (Madras-Majewska et al., 2016). Physico-chemical
properties of honey do not allow these bacteria spores to pass into the vegetative
form. However, the intestine of honey bee and human represents a suitable habitat
for their germination and reproduction, where some strains of Bacillus spp.
manifest themselves as probiotics. Thus, strains isolated from honey B. cereus
(m363, mv86, mv81, mv75), B. circulans (Fr231, m448b), B. megaterium (m435),
B. pumilus (m354), B. subtilis (m329) и Paenibacillus alvei (m321) showed
antagonistic effect against honey bee fungal pathogen Ascosphaera apis (Reynaldi
et al., 2004), and the strains B. subtilis (m351), В. pumilus (M350), B.
licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B.
13
Prebiotic and Probiotic Properties of Honey
megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus
(BLAT170) и B. laterosporus (BLAT171) had the antagonistic activity to P.
larvae (Alippi and Reynaldi, 2006). Evans J.D. and Armstrong T.N. (2006) also
demonstrated antagonistic effects of bee symbionts Bacillus spp. against P.
larvae.
We have shown the enhancement of honey bee humoral defense as a result of
the probiotic impact based on B. subtilis (Gaifullina et al., 2016). We have
registered the activity increase of phenoloxidase, which is an integral link of the
insect immune reactions involved in almost all cellular and humoral immune
responses (Theopold et al., 2004), and antioxidant enzymes that are functionally
associated with phenoloxidase and reduce the oxidative damage of cells and
tissues (Dubovskii et al., 2010). Furthermore, in our experiments B. subtilis has
caused an increase of abaecin and vitellogenin gene expression levels of honey
bees. We have mentioned above about the immune functions of abaecin. Levels of
this peptide increase with the penetration of different pathogens into bee organism
and can serve as an immunocompetence criterion of separate honey bee colonies
(Evans and Lopes, 2004). Vitellogenin is a protein carrying out many functions in
honey bee organism, among which the antioxidant (Havukainen et al., 2013) and
the immune (Zhang et al., 2011) properties are especially remarkable in this
context. Vitellogenin participates in the immunological recognition of Gram-
negative bacterium E. coli and a Gram-positive bacterium P. larvae, as well as
pathogen-associated molecular patterns (Salmela et al., 2015).
Like LAB, various strains of Bacillus spp. exhibit antagonistic activity against
pathogens of human and vertebrates, and also stimulate the immune system of
mammals, in connection with which find application as probiotics in medicine
and veterinary (Lazovskaya et al., 2013).
CONCLUSION
Taking into account all the facts and conclusions presented here, it can be
summarized that honey is a fermented food product which is a nectar, partially
digested by enzymes of bees and their LAB symbionts. The presence of prebiotic
substances and probiotic microorganisms in fresh honey defines it as a synbiotic,
the physiologically functional food ingredient, which is a combination of
probiotics and prebiotics in which probiotics and prebiotics have a synergistic
effect on the host organism (Figure 1).
14 Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
Figure 1. Honey as a synbiotic food product.
Using as a substrate honey polysaccharides and oligosaccharides, honey bee
LAB symbionts during their vital activity produce metabolites that are involved in
the formation of honey organoleptic characteristics (taste, flavor, texture) and the
spectrum of its therapeutic properties. Qualitative and quantitative composition of
honey prebiotic sugars and LAB flora, and consequently bacterial metabolites
depends on many factors, such as geographical and botanical origin of honey,
taxonomic affiliation and health state of bees, which explains the various
medicinal properties of different honey types. Synergistic direct antimicrobial
effect of all microorganisms of beneficial bee microbiota and indirect
immunostimulation effect determines the important role of probiotic bacteria in
honeybee pathology and, consequently, in the manufacture of high-quality
apiculture products. This fact defines the opportunity for development of
adaptogenic supplements for bees based on probiotic bacteria, that is particularly
relevant taking into consideration the massive loss of bee colonies around the
world. The high level of prebiotic activity of honey oligo- and polysaccharides in
relation to human beneficial intestinal flora, and antagonistic action of bacteria in
fresh honey on human pathogens, makes the honey an attractive source of
15
Prebiotic and Probiotic Properties of Honey
components for new prebiotic, probiotic and synbiotic supplements for human. In
honey and honey stomach microbiota the detection of bacterial strains with high
levels of antimicrobial activity against pathogens, that are resistant to antibiotics,
opens up the possibility for development of new alternative tools to overcome the
therapeutically resistant infectious agents.
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BIOGRAPHICAL SKETCH
Name: Gaifullina Louisa Rimovna
Affiliation: Institute of biochemistry and genetics of Ufa scientific center of
RAS
Education: Bashkir State University
Address: Russian Federation, 450054, Ufa, Prospect of October Street, 71
building
22 Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
Research and Professional Experience: 16 years
Professional Appointments: PhD, Researcher of the Laboratory of
biochemistry of insect adaptability
Honors:
Publications Last 3 Years:
1. Ilyasov R.A., Gaifullina L.R., Saltykova E.S., Poskryakov A.V.,
Nikolenko A.G. Role of antimicrobial peptide defensin in the immunity of
the bee colonies. Russian Journal of Beekeeping. 2014. No 1. P. 26-28.
2. Saltykova, E.S., Gaifullna, L.R., Nikolenko, A.G. Differences in induced
defense reactions of honeybee subspecies. Sixth European Conference of
Apidology, 9-11 September, 2014. Murcia: Pilar De la Rua. 2014. P. 168.
3. Ilyasov R. A., Gaifullina L. R., Saltykova E. S., Nikolenko A. G. Biology,
distribution and prevention of the microsporidia Nosema parasited in
honey bees. Biomics. 2014. V. 6. No. 3. P. 145-154.
4. Saltykova E.S., Gaifullina L.R., Poskryakov A.V., Nikolenko A.G.
Implementation of protective response to a bacterial drug in a Bashkir
population of dark forest bees. In: R.A. Ilyasov, A.G. Nikolenko,
N.M. Saifullina editors. Dark forest bee Apis mellifera mellifera L. of the
Republic of Bashkortostan. Ufa: Gilem, 2015; pp. 201-208.
5. Gaifullina L.R., Saltykova E.S., Nikolenko A.G. Differences in cellular
immune response in bee subspecies of the Bashkortostan Republic. In:
R.A. Ilyasov, A.G. Nikolenko, N.M. Saifullina editors. Dark forest bee
Apis mellifera mellifera L. of the Republic of Bashkortostan. Ufa:
Gilem, 2015; pp. 209-221.
6. Saltykova E.S., Gaifullina L.R., Nikolenko A.G. The role of vitellogenin
gene expression level in Apis mellifera mellifera L. longevity. 44th
APIMONDIA International Apicultural Congress. 2015, P. 216-217.
7. Nikolenko A.G., Gaifullina L.R., Saltykova E.S. Background
concentrations of imidacloprid cause degradation of drone sperm in field
studies // 44th APIMONDIA International Apicultural Congress. 2015, P.
208.
8. Gaifullina L.R., Saltykova E.S., Matniyazov R.T., Nikolenko A.G.
Optimal conditions for applying of probiotics as adaptogens based on the
analysis of the honey bee immune status. Biomics. 2016. V. 8. No 2. P. 76-
78.
9. Karimova A.A., Saltykova E.S., Gaifullina L.R., Matniyazov R.T.,
Poskryakov A.V. Nikolenko A.G. Vitellogenin gene expression and
regulating lifespan of working individuals dark forest bee. Biomics. 2016.
V. 8. No 2. P. 110-112.
Name: Saltykova Elena Stanislavovna
23
Prebiotic and Probiotic Properties of Honey
Affiliation: Institute of biochemistry and genetics of Ufa scientific center of
RAS
Education: Bashkir State University
Address: Russian Federation, 450054, Ufa, Prospect of October Street, 71
building
Research and Professional Experience: 29 years
Professional Appointments: PhD, Senior Researcher of the Laboratory of
biochemistry of insect adaptability
Honors:
Publications Last 3 Years:
1. Ilyasov R.A., Gaifullina L.R., Saltykova E.S., Poskryakov A.V.,
Nikolenko A.G. Role of antimicrobial peptide defensin in the immunity of
the bee colonies. Russian Journal of Beekeeping. 2014. No 1. P. 26-28.
2. Saltykova, E.S., Gaifullna, L.R., Nikolenko, A.G. Differences in induced
defense reactions of honeybee subspecies. Sixth European Conference of
Apidology, 9-11 September, 2014. Murcia: Pilar De la Rua. 2014. P. 168.
3. Ilyasov R. A., Gaifullina L. R., Saltykova E. S., Nikolenko A. G. Biology,
distribution and prevention of the microsporidia Nosema parasited in
honey bees. Biomics. 2014. V. 6. No. 3. P. 145-154.
4. Saltykova E.S., Gaifullina L.R., Poskryakov A.V., Nikolenko A.G.
Implementation of protective response to a bacterial drug in a Bashkir
population of dark forest bees. In: R.A. Ilyasov, A.G. Nikolenko,
N.M. Saifullina editors. Dark forest bee Apis mellifera mellifera L. of the
Republic of Bashkortostan. Ufa: Gilem, 2015; pp. 201-208.
5. Gaifullina L.R., Saltykova E.S., Nikolenko A.G. Differences in cellular
immune response in bee subspecies of the Bashkortostan Republic. In:
R.A. Ilyasov, A.G. Nikolenko, N.M. Saifullina editors. Dark forest bee
Apis mellifera mellifera L. of the Republic of Bashkortostan. Ufa:
Gilem, 2015; pp. 209-221.
6. Saltykova E.S., Gaifullina L.R., Nikolenko A.G. The role of vitellogenin
gene expression level in Apis mellifera mellifera L. longevity. 44th
APIMONDIA International Apicultural Congress. 2015, P. 216-217.
7. Nikolenko A.G., Gaifullina L.R., Saltykova E.S. Background
concentrations of imidacloprid cause degradation of drone sperm in field
studies. 44th APIMONDIA International Apicultural Congress. 2015, P.
208.
8. Gaifullina L.R., Saltykova E.S., Matniyazov R.T., Nikolenko A.G.
Optimal conditions for applying of probiotics as adaptogens based on the
analysis of the honey bee immune status. Biomics. 2016. V. 8. No 2. P. 76-
78.
24 Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
9. Karimova A.A., Saltykova E.S., Gaifullina L.R., Matniyazov R.T.,
Poskryakov A.V. Nikolenko A.G. Vitellogenin gene expression and
regulating lifespan of working individuals dark forest bee. Biomics. 2016.
V. 8. No 2. P. 110-112.
Name: Nikolenko Alexey Gennadyevich
Affiliation: Institute of biochemistry and genetics of Ufa scientific center of
RAS
Education: Bashkir State University
Address: Russian Federation, 450054, Ufa, Prospect of October Street, 71
building
Research and Professional Experience: 28 years
Professional Appointments: Prof., Head of the Laboratory of biochemistry
of insect adaptability
Publications Last 3 Years:
1. Ilyasov R.A., Gaifullina L.R., Saltykova E.S., Poskryakov A.V.,
Nikolenko A.G. Role of antimicrobial peptide defensin in the immunity of
the bee colonies. Russian Journal of Beekeeping. 2014. No 1. P. 26-28.
2. Saltykova, E.S., Gaifullna, L.R., Nikolenko, A.G. Differences in induced
defense reactions of honeybee subspecies. Sixth European Conference of
Apidology, 9-11 September, 2014. Murcia: Pilar De la Rua. 2014. P. 168.
3. Ilyasov R. A., Gaifullina L. R., Saltykova E. S., Nikolenko A. G. Biology,
distribution and prevention of the microsporidia Nosema parasited in
honey bees. Biomics. 2014. V. 6. No. 3. P. 145-154.
4. Saltykova E.S., Gaifullina L.R., Poskryakov A.V., Nikolenko A.G.
Implementation of protective response to a bacterial drug in a Bashkir
population of dark forest bees. In: R.A. Ilyasov, A.G. Nikolenko,
N.M. Saifullina editors. Dark forest bee Apis mellifera mellifera L. of the
Republic of Bashkortostan. Ufa: Gilem, 2015; pp. 201-208.
5. Gaifullina L.R., Saltykova E.S., Nikolenko A.G. Differences in cellular
immune response in bee subspecies of the Bashkortostan Republic. In:
R.A. Ilyasov, A.G. Nikolenko, N.M. Saifullina editors. Dark forest bee
Apis mellifera mellifera L. of the Republic of Bashkortostan. Ufa:
Gilem, 2015; pp. 209-221.
6. Saltykova E.S., Gaifullina L.R., Nikolenko A.G. The role of vitellogenin
gene expression level in Apis mellifera mellifera L. longevity. 44th
APIMONDIA International Apicultural Congress. 2015, P. 216-217.
7. Nikolenko A.G., Gaifullina L.R., Saltykova E.S. Background
concentrations of imidacloprid cause degradation of drone sperm in field
25
Prebiotic and Probiotic Properties of Honey
studies. 44th APIMONDIA International Apicultural Congress. 2015, P.
208.
8. Ilyasov R. A., Poskryakov A. V., Nikolenko A. G. Nucleotide
polymorphism of the gene VG of honey bees. Biomics. 2015. V. 7. No. 1.
P. 54-61.
9. Ilyasov R. A., Poskryakov A. V., Nikolenko A. G. Current status and
preservation of the dark European bees Apis mellifera mellifera in Russia
and Europe. Biomics. 2015. V. 7. No. 2. P. 121-127.
10. Ilyasov R. A., Poskryakov A. V., Petukhov A. V., Nikolenko A. G. The
gene pool analisys of the current population of the dark European honey
bee A. m. mellifera in the Ural and Volga region (translation). Biomics.
2015. V. 7. No. 3. P. 167-191.
11. Ilyasov R. A., Poskryakov A. V., Nikolenko A. G. New SNP markers of
the honeybee vitellogenin gene (Vg) used for diagnostics of subspecies
Apis mellifera mellifera L. in Russia. Russian Journal of Genetics. 2015.
V. 51. No. 2. P. 163-168.
12. Ilyasov R. A., Poskryakov A. V., Petukhov A. V., Nikolenko A. G. Genetic
differentiation of local populations of the dark european bee Apis mellifera
mellifera L. in the Urals. Russian Journal of Genetics. 2015. V. 51. No. 7.
P. 677-682.
13. Kaskinova M. D., Ilyasov R. A., Poskryakov A. V., Nikolenko A. G.
Analysis of the genetic structure of honeybee (Apis mellifera L.)
populations. Russian Journal of Genetics. 2015. V. 51. No. 10. P. 1033-
1035.
14. Gaifullina L.R., Saltykova E.S., Matniyazov R.T., Nikolenko A.G.
Optimal conditions for applying of probiotics as adaptogens based on the
analysis of the honey bee immune status. Biomics. 2016. V. 8. No 2. P. 76-
78.
15. Karimova A.A., Saltykova E.S., Gaifullina L.R., Matniyazov R.T.,
Poskryakov A.V. Nikolenko A.G. Vitellogenin gene expression and
regulating lifespan of working individuals dark forest bee. Biomics. 2016.
V. 8. No 2. P. 110-112.
16. Ilyasov R. A., Poskryakov A. V., Nikolenko A. G. Chapter 1. The Genetic
structure of dark european honey bee population in the Ural. In
Honeybees: biology, behavior and benefits. Hauppauge, New York: Nova
Science Publishers. 2016. pp. 1-13.
17. Ilyasov R. A., Poskryakov A. V., Petukhov A. V., Nikolenko A. G. New
approach to the mitotype classification in black honeybee Apis mellifera
mellifera and Iberian honeybee Apis mellifera iberiensis. Russian Journal
of Genetics. 2016. V. 52. No. 3. P. 281-291.
26 Gaifullina L.R., Saltykova E.S. and Nikolenko A.G.
Page layout by Anvi Composers.
Article
Full-text available
Bee bread is the only fermented product of the beehive. It constitutes the main source of proteins, lipids, vitamins, and macro- and microelements in honeybee nutrition and it exerts antioxidant and antimicrobial properties, though research on these aspects has been limited so far. In this study 18 samples of Greek bee bread, two of which were monofloral, were collected during different seasons from diverse locations such as Crete and Mount Athos and were tested for their bioactivity. Samples were analyzed for their antibacterial properties, antioxidant activity, total phenolic content (TPC), and total flavonoid content (TFC). The antimicrobial activity of each sample was tested against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella typhimurium. Our data demonstrate that all samples exert inhibitory and most of them bactericidal activity against at least two pathogens. Furthermore, all samples exert significant antioxidant activity, where the monofloral Castanea Sativa sample demonstrated superior antioxidant activity. Nevertheless, the antioxidant and antimicrobial activity were not strongly correlated. Furthermore, machine learning methods demonstrated that the palynological composition of the samples is a good predictor of their TPC and ABTS activity. This is the first study that focuses on the biological properties of Greek bee bread and demonstrates that bee bread can be considered a functional food and a possible source of novel antimicrobial compounds.
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