Food Chemistry 332 (2020) 127229

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

A current perspective on hydrogen peroxide production in honey. A review T

Katrina Brudzynski
Bee-Biomedicals Inc., St. Catharines, ON L2T 3T4, Canada

ARTICLE INFO ABSTRACT

Keywords: Hydrogen peroxide plays a key role in honey antibacterial activity. The production of H2O2 in honey requires
Hydrogen peroxide glucose oxidase (GOx) that oxidizes glucose to gluconolactone and reduces molecular oxygen to hydrogen
Honey peroxide. The content of GOx of honeybee origin was believed to be the main predictor of H2O2 concentration in
Glucose oxidase honey. The observed variations in H2O2 levels among honeys questioned however the direct GOx-H2O2 re-
Non-enzymatic production of H2O2
lationship and left its absence opened for exploration. Here, we evaluated principal causes underlying the im-
Colloidal structure of honey
Nectar Redox Cycle
balance in the quantitative enzyme-product relationship with respect to: (a) enzyme and the product inactivation
Fungal and bacterial glucose oxidases or destruction by honey compounds; (b) non-enzymatic pathway of H2O2 formation, and (c) a potential con-
tribution of enzymes with GOx activity originating from nectars and microorganisms inhabiting honey. We also
bring new facts on the relationship between honey colloidal structure and H2O2 production that change our
traditional understanding of honey function as antimicrobial agent.

1. Introduction
Generation of hydrogen peroxide (H2O2) is a dominant mechanism
by which honey exerts bacteriostatic and bactericidal activity. The
amount of H2O2 produced in honey significantly correlates with the
honey Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC) (White, Subers, & Schepartz, 1963;
Allen, Molan, & Reid, 1991; Molan, 1992; Brudzynski, 2006;
Brudzynski, Abubaker, Martin, & Castle (2011). Despite the fact that
several other honey compounds (polyphenolic acids, flavonoids, anti-
microbial peptides) and factors (osmolarity, acidity, pH) contribute to
its antibacterial activity (Molan, 1992; Kwakman et al., 2010), the only
two honey compounds, namely hydrogen peroxide and methylglyoxal
produce minimum inhibitory concentrations (MIC) in the range
10–1000 µg/ml that classify them as true antibacterials (Brudzynski
et al., 2011; Strelec et al., 2018; Mavric, Wittmann, Barth, & Henle,
2008; Hayashi, Fukushima, Hayashi-Nishino, & Nishino, 2014). Fur-
thermore, a significant reduction of antibacterial activity after treat-
ment of honeys with catalase, that hydrolyzes H2O2 to water and
oxygen, confirms that H2O2 is the main antibacterial agent (White et al.,
1963; Taormina, Niemira, & Beuchat, 2001; Brudzynski, 2006;
Brudzynski, Abubaker, & Wang, 2012a). Therefore, the pathways of
H2O2 production and destruction are highly relevant to honey anti-
bacterial activity.
Hydrogen peroxide is produced by honeybee GOx in the process of
glucose oxidation. The concentration of the end product depends on the
enzyme concentration and its activity (Schepartz & Subers, 1964).
However, repeated observations have been made that suggest a lack of
correlation between levels of H2O2 and GOx activity in honey
(Bucekova et al., 2014; Strelec et al., 2018). In a quest to find ex-
planation for this apparent contradiction, we reviewed the current
knowledge on GOx reaction in honey along with the physical/chemical
factors that affect the enzyme efficacy and might hold a key to these
differences. We also bring new facts on the relationship between honey
colloidal structure and H2O2 production that can transform our tradi-
tional understanding of honey function as antimicrobial agent.
Historically, studies on antibacterial activity of honey began with
the discovery made by Dold, Du, Dziao (1937) of the substance that
inhibited bacterial growth in diluted honey and thus appropriately
called inhibine. Subsequently, inhibine was identified as hydrogen
peroxide produced by a honey GOx (White et al., 1963). Gauhe, 1941
was first to report GOx synthesis in hypopharyngeal glands (HPs) of
honeybees. Later, several proteomic studies supported ubiquitous ex-
pression of GOx in hypopharyngeal glands of insects. In relation to
honeybees, the levels of GOx in HPs were comparatively quantified in
different bee species, at different stage of the bee development and
according to tasks performed (nursing vs foraging bees) (Kubo et al.,
1996; Feng, Fang, & Li, 2009; Bucekova et al., 2014). The results
highlighted quantitative differences in GOx production by HPs de-
pending on the age and cast of the honeybee. GOx represented the
second largest group of proteins synthesized in the HPs of Africanized
and European honeybees, (the first, most abundant group of HP pro-
teins comprises a family of Major Royal Jelly Proteins, MRJP) (Sano
et al., 2004; Santos, Santos, Mendes, de Souza, Malaspina & Palma,

E-mail address: beebio@sympatico.ca.

https://doi.org/10.1016/j.foodchem.2020.127229
Received 17 December 2019; Received in revised form 28 May 2020; Accepted 1 June 2020
Available online 05 June 2020
0308-8146/ © 2020 Published by Elsevier Ltd.
K. Brudzynski
2005).
Further studies of Burgett, 1974, concluded that honeys produced
by all social Hymenoptera contain GOx, including honeys produced by
Apis species (A. mellifera, A. cerana, A dorsat, A. indica, A. florae) and
Trigona species (stringless bees). The common occurrence of GOx in
honeys found support in recent proteomic studies (DiGromolo,
D’Amato, & Righetti, 2012; Erban, Shcherbachenko, Talacko & Harant,
2019).
Until now, the long-standing view was that all H2O2 in honey is
produced by GOx of honeybee origin, introduced to honey with the
secretion of hypopharyngeal glands during nectar harvesting (White
et al., 1963; Schepartz & Subers, 1964; Molan, 1992). This presumption
has been recently questioned in light of considerable variations in the
H2O2 concentration in different honeys that can reach up to 200-fold
difference (Bucekova et al., 2014, Strelec et al., 2018). The observed
differences could not be attributed to the botanical origin of honey and
hence variations in the plant species-dependent catalase levels, since
such variability was observed even between samples of the same bo-
tanical origin (Strelec et al., 2018). Neither they result from the action
of endogenous enzyme since the recent proteomic analysis failed to
detect catalase of honeybee origin that could be a factor in H2O2 de-
struction in honey (Erban et al., 2019). The apparent contradiction has
prompted a reassessment of our understanding on the regulation of
H2O2 levels in honey.
We began the review with a detailed description of the kinetic
pathway of H2O2 production by GOx under experimental, in in vitro
conditions to show some critical dissimilarity in its formation compared
to the conditions existing in honey (high sugar concentration, low water
activity). Subsequently, the additional, plausible pathways of H2O2
synthesis (beyond GOx of honeybee origin) and its destruction (beyond
plant catalase) are considered in the following parts of the review.
These include the role of plant secondary metabolites, polyphenols and
flavonoids, in non-enzymatic generation of H2O2 and its destruction.
Next, we present new facts on the possible contribution of GOx of plant
and microbial origin that are transferred with nectar to honey as well as
the important, causal relationship between colloidal structure of honey
and H2O2 production. Together, this review represents an advance in
our understanding on how H2O2 is produced and destroyed in honey
2. Enzymatic production of hydrogen peroxide by glucose oxidase
in in vitro system
Hydrogen peroxide is produced enzymatically by glucose oxidase
(β-D-glucose oxidoreductase, EC 1.1.3.4) in two-step redox reaction.
Each of these reactions contains its own substrate, glucose and oxygen
(Wohlfart et al., 1999). Paramount to GOx activity is a flavin adenine
dinucleotide (FAD) cofactor that is responsible for the enzyme redox
activity. In the first, reductive half reaction, GOx catalyzes oxidation of
β-D-glucose by transferring two electrons and a proton from the first
hydroxyl group of β-D-glucose to the FAD cofactor with concomitant
Fig. 1. Glucose oxidase reaction and formation of hydrogen peroxide.
2
Food Chemistry 332 (2020) 127229
oxidation of glucose to D-glucono-δ-lactone (Fig. 1). D-glucono-δ-lactone
is then removed from GOx by a spontaneous hydrolysis to gluconic acid
in aqueous solutions. In the second, oxidative half reaction, the reduced
GOx-FADH2 is re-oxidized by molecular oxygen (a second substrate).
Oxygen accepts two electrons transferred from FADH2 regenerating the
catalytic system with the concomitant formation of hydrogen peroxide
(Fig. 1). The reaction follows ping pong kinetics where substrates of
GOx (glucose and oxygen) appear to bounce on (ping) and off (pong)
the enzyme (Bright & Porter, 1975; Leskovac, Trivic, Wohlfahrt,
Kandrac, & Pericin, 2005). The significance of the ping pong me-
chanism is such that GOx can bind the second substrate (oxygen) only
after the first substrate, glucose, bounce off the enzyme and GOx-
FADH2 is oxidized.
The H2O2 formation depends on the successful completion of the
redox cycling by a FAD cofactor. Each of the half-reactions is in-
dependently regulated by competitive and non-competitive inhibitors
acting on the enzyme and/or on its FAD cofactor (Gibson, Swoboda, &
Massey, 1964). The overall rate of glucose oxidation by GOx depends
on kinetics of two half reactions: the GOx/glucose kinetics and the
oxygen-dependent FAD re-oxidation kinetics.
The generated H2O2 is subsequently hydrolyzed by catalase to water
and oxygen. The efficient removal of H2O2 and the intermediate, glu-
cocnolactone, drives the reaction in a unidirectional manner and also
protects GOx from oxidation.
3. Principal causes of GOx inactivity and the lack of H2O2
production in undiluted honey.
The rate of hydrogen peroxide formation in the GOx reaction de-
pends on the concentration of three reactants; GOx, glucose and oxygen
as substrates. In vitro, the GOx reaction complies with the Michaelis-
Menten kinetic model (Schepartz & Subers, 1964). At the constant
concentration of the enzyme, the initial rate of glucose oxidation is
linear to the glucose concentration per unit time and represents the first
order kinetics. With increasing glucose concentrations and increasing
saturation of GOx binding sites, the rate of the reaction slows down and
reach plateau at maximum velocity (V max). At high glucose con-
centrations, the enzyme is fully saturated and inactive. No hydrogen
peroxide is produced. The kinetics of GOx reaction under in vitro
conditions is significantly different from that existing in undiluted
honey.
3.1. Low water activity
In undiluted honey however, the kinetic of the GOx reaction is
complicated by the honey physical state, mostly by high glucose con-
centration, low water activity and high viscosity that inhibits the dif-
fusion of reactants and the end product within the matrix.
Availability of water is a fundamental factor for GOx–dependent
catalysis. The water activity in natural, undiluted honey is low and
K. Brudzynski
Table 1
Factors affecting GOx activity in honey.
Factors affecting glucose oxidase activity
Enzyme concentration
Concentration of substrate
Concentration of product
Activator Cofactor FAD
Inhibitors Competitive
Substrate inhibition
Product inhibition
Non-competitive
High sugar concentration (viscosity)
Low water activity
amounts to about 0.5762 (Abramovic, Jamnik, Burkan, & Kac, 2008;
Brudzynski et al., 2017). At this stage, most of water molecules are
chemically bound to sugars and honey macromolecules and only low
levels of free water is available for chemical reactions. GOx is a gly-
coprotein containing about 11–13% carbohydrate moiety of the high-
mannose type (Kalisz, Hendle, & Schmid, 1997). This carbohydrate
component helps to bind water molecules, allowing GOx to maintain its
native conformation required for the enzymatic activity (Lewin, 1971).
However, in addition to solvation of molecules, water is needed as a
solvent in several reactions associated with H2O2 production: the GOx-
catalyzed glucose oxidation, in H2O2 hydrolysis by catalase and in the
spontaneous break down of a D-glucono-δ-lactone to gluconic acid.
Under low water activity in undiluted honeys, these reactions are se-
verely impaired and the production of H2O2 is negligible (Dold et al.,
1937; White et al., 1963; Molan, 1992; Bang, Buntting, & Molan, 2004;
Brudzynski, 2006; Bucekova et al., 2014; Strelec et al., 2018).
Secondly, lack of water reduces diffusion rates of reactants and
H2O2 in a viscous honey matrix. Reduced mobility of molecules causes
accumulation of substrates, intermediates and the end products in close
proximity to each other. Considering ping pong mechanism of GOx
reaction, a D-glucono-δ-lactone intermediate must be removed from the
GOx substrate binding site to allow oxygen binding and re-oxidation of
FADH2. With reduced mobility of reactants, D-glucono-δ-lactone be-
comes a competitive inhibitor to oxygen, fighting for the access to the
GOx binding site and non-competitive inhibitor to glucose (Nakamura
& Ogura, 1968). Moreover, its break down to gluconic acid is impeded
at low levels of free water in undiluted honey.
Reduced mobility of molecules also affects diffusion rates of H2O2. If
H2O2 is not removed from the reaction, it may initiate the feedback
reaction and accumulation of gluconolactone. Under these circum-
stances, H2O2 becomes a competitive inhibitor for glucose and a non-
competitive inhibitor for oxygen.
3.2. Substrate inhibition; effects of glucose and oxygen concentrations
β-deoxy-D-glucose is the first substrate in GOx reaction toward
production of H2O2 and it is at high supply in honey. On average,
honeys contain 31% of glucose that together with fructose forms a su-
persaturated honey solution. In in vitro studies conducted by several
groups indicated that the high concentration of glucose saturates GOx
binding sites and blocks binding of the second substrate, oxygen
(Nakamura & Ogura, 1962; Gibson et al., 1964; Schepartz & Subers,
1964; Tomotani, das Neves & Vitolo, 2005). At the concentration higher
then 100 mM in in vitro model, glucose became competitive inhibitor
for oxygen, disallowing regeneration of reduced FADH2. The substrate
inhibition might be one of the reasons for the lack of GOx activity and
H2O2 production in undiluted honey.
GOx requires oxygen as a second substrate in glucose oxidation re-
action and water as a solvent. Oxygen for the GOx reaction might be
initially provided with the air-saturated water present in nectars.
However, water evaporation during honey ripening reduces oxygen
3
Food Chemistry 332 (2020) 127229
concentrations to the levels that might prevent re-oxidation of FADH2-
GOx. Regeneration of FADH2 to FAD must take place for the con-
tinuous, uninterrupted GOx activity (Schepartz & Subers, 1964). In
diluted honey, oxygen is provided by decomposition of H2O2 by cata-
lase to water and molecular oxygen. However, H2O2 production is
negligible in undiluted honey, leaving levels of oxygen as a substrate in
a short supply.
3.3. Product inhibition; effect of H2O2 on the GOx activity
In addition to the substrate inhibition, GOx activity could suffer
from the product inhibition if H2O2 is not removed from the reaction
pathway. The removal of this end product is necessary to prevent its
accumulation to the levels that can reverse the direction of the
reaction·H2O2 competes with O2 for binding to FADH2-GOx and thereby
prevents regeneration of reduced FAD to its oxidized form (Bao,
Furumoto, Yoshimoto, Fukunaga, & Nakao, 2003). The reduced form of
FAD, FADH2 is 100 times more sensitive to H2O2 than its oxidized form
(Kleppe, 1966).
In addition to blocking FAD redox cycling, H2O2 is involved in a
direct oxidation of methionine located in the GOx active site. The for-
mation of methionine sulfoxide reduces the substrate recognition by the
modified enzyme, impacting the enzyme specificity to glucose
(Malikkides & Weiland, 1982).
Table1summarizes the effects of factors described in this section.
The GOx reaction and its kinetics in in vitro system are dissimilar to
that in honey. In undiluted honey, the rate of catalysis by the enzyme is
affected by the reduced diffusion rates and mobility of reactants in
supersaturated sugar solution, by the substrate inhibition at high glu-
cose concentration, and by potential product inhibition. Low water
activity in honey also affects enzymatic hydrolysis of H2O2 by catalase
and non-enzymatic hydrolysis of gluconolactone to gluconic acid. It is
evident that H2O2 production can be influenced by many factors.
4. Activation of GOx system upon honey dilution; the inverted U-
shaped relationship between H2O2 production and dilution
The low water activity is a fundamental factor for the inhibition of
GOx –dependent production of H2O2 in undiluted honey. The system is
activated immediately after addition of water (White et al., 1963,
Molan, 1992, Bang et al., 2003, Brudzynski et al., 2017, Strelec et al.,
2018). Initially, the concentration of H2O2 increases with the degree of
honey dilution, reaching the maximum point, after which it declines
rapidly with further dilutions (White et al., 1963; Bang et al. 2003;
Strelec et al., 2018, Brudzynski et al., 2017). The maximum H2O2
concentration appears at different dilutions in different honeys but in
most of honeys tested it occurred at honey concentration ranging from
15% to 50% (Bang et al. 2003; Strelec et al., 2018, Brudzynski et al.,
2017). Dark honeys such as buckwheat, heather, Manuka often produce
higher amounts of H2O2 than lighter honeys, and the decline in its
production is observed beyond 12.5% to 6.25% of honey concentration
(w/v) (Brudzynski, 2006, Brudzynski et al., 2017, Strelec et al., 2018;
Bang et al., 2004). Further honey dilution beyond this maximum point
did not support a higher H2O2 production. This type of relationship
between H2O2 concentration and honey dilution takes an inverted U-
shape, where a turning point occurs at maximum point of H2O2 pro-
duction (Brudzynski et al., 2017; Strelec et al., 2018). At the present
time, the detailed mechanisms underlying the inverted U-shaped re-
lationship is not clear although the honey colloidal structure plays
important role (as discussed below, paragraph 6.1).
From practical point of view, it became apparent that measuring
peroxide concentrations should be conducted at the ascending part of
the inverted U curve, preferable at the maximum point of H2O2 pro-
duction upon honey dilution. Measurements at a descending part of the
inverted U curve, where there is a lack of relationship between these
two variables, would erroneously present the concentration of H2O2 in
K. Brudzynski
honey. Due to the individual differences in curve patterns between
honeys, it is highly recommended to establish first the linear and
nonlinear parts of the inverted U curve for each honey before de-
termining H2O2 concentration. This is of particular importance in
comparative studies on H2O2 production in different honeys. Since the
maximum point of production is different for different honeys, the ar-
bitrarily chosen honey dilution for the measurements of H2O2 could
contribute to observed variability in reported H2O2 concentrations.
5. Principal causes of variability in H2O2 concentrations in diluted
honeys
In in vitro model system using isolated, purified GOx, there was a
simple, linear relationship between the concentration of GOx, its ac-
tivity and the rate of H2O2 formation (Schepartz & Subers, 1964). Such
correlation is lost in a complex milieu of honey. Although the con-
centration of H2O2 depended on the GOx content, the final product
concentration was negatively affected by “various minor components
(from sources such as nectar, pollen, yeast) in the honey that interacted
with the enzyme or with the product” (White & Subers, 1963). Cor-
recting for the modulating action of “various small molecules” the au-
thors presented a modified view that the final concentration of H2O2 in
honey results from the difference between levels of its production by
GOx and levels of its destruction by catalase and other honey con-
stituents. Many of small molecules and factors affecting the final H2O2
concentration can vary with floral sources, therefore it was expected
that the final concentrations of H2O2 in different honeys would differ.
White (1966) also brought attention to a high sensitivity of honey H2O2
to destruction by light and heat (White, 1966, White & Subers, 1964).
On the other hand, there is scarcity of quantitative data on the
content of GOx in different honey that would allow establishing an
accurate GOx – H2O2 relationship. In bees, the GOx production by
hypopharyngeal glands fluctuates with the age and task performed by
the bee (Kubo et al., 1996; Feng et al., 2009, Bucekova et al., 2014).
These differences may be reflected by varying amount of GOx in-
troduced to honey with the secretion of hypopharyngeal glands during
nectar collection. It is therefore plausible that the differences in the
level of expression of GOx in HPs might be the first source of the
variability in the content of GOx in different honeys. In turn, variability
in the levels of GOx in honey would translate to variability in the
content of the final product.
Some recent studies that analyzed both the GOx content in honey
and the H2O2 production seemed to agree on the lack of a direct re-
lationship between these variables (Bucekova et al., 2014, Strelec et al.,
2018). In many cases, the concentration of H2O2 in diluted honey ex-
ceeded the GOx capacity to produce such amounts of H2O2 alone.
Summarizing reviewed data, one could conclude that the produc-
tion and destruction of hydrogen peroxide in honey is a multifactorial
phenomenon. Some mechanisms of enzymatic and non-enzymatic
pathways of H2O2 destruction and production are well known in bio-
chemistry and are briefly presented in the next paragraphs.
5.1. Hydrogen peroxide decomposition by catalase
The main pathway of H2O2 decomposition involves its hydrolysis to
water and oxygen (2H2O + O2) by catalase of plant origin. It is gen-
erally assumed that the content and activity of catalase in different
honeys determines the final levels of H2O2 (Schepartz, 1966). However,
the issue of a direct, quantitative relationship between catalase and
H2O2 concentrations remains a controversial one. Some studies sup-
ported such relationship (Dustman, 1971, Weston, 2000) while others
did not (Huidobro, Sánchez, Muniatequi, & Sancho, 2005; Schepartz &
Subers, 1966). These contradictory results indicated that there are ad-
ditional mechanisms in honey influencing H2O2 degradation.
4
Food Chemistry 332 (2020) 127229
5.2. Hydrogen peroxide decomposition by metal-containing enzymes
Indeed, besides catalase, H2O2 decomposition could be achieved by
other metal-containing enzyme. Metalloenzymes that contain iron or
other transition metal share similar mechanism of metal-induced redox
reaction to catalase. The catalase catalytic cycle includes 2 molecules of
hydrogen peroxide and Fe (III). In the first step, H2O2 serves as oxi-
dizing agent, oxidizing Fe (III) to Fe (IV) O oxyferryl intermediate. The
one-electron transfer from Fe (III) to the first H2O2 molecule breaks the
OeO bond in the molecules and reduces it to water and oxygen. In the
second part of reaction, second H2O2 molecule acts as reducing agent. A
two-electron reduction of Fe (IV) O oxyferryl intermediate by hydrogen
peroxide restores the original ferricatalase, forming oxygen and another
molecule of water in the same time (Jones & Dunford, 1977).
This mechanism of H2O2 decomposition is common for other metal-
containing enzymes such as peroxidases or thiol-containing peroxir-
edoxins where metal ions serve as the source of electrons (Zámocký,
Gasselhuber, Furtmüller, & Obinger, 2012). Therefore, metalloenzymes
other than catalase may participate directly or indirectly (superoxide
dismutase for example) in H2O2 destruction, adding to variability in its
concentration (see below).
5.3. Hydrogen peroxide decomposition by ascorbic acid
H2O2 might be decomposed also by non-enzymatic mechanism.
Under aerobic conditions, some antioxidants such as ascorbic acid can
be oxidized by H2O2 (instead of O2) to dehydroascorbic acid by trans-
ferring two electrons and two protons to hydrogen peroxide with re-
duction of the latter to water and oxygen (Fig. 2). Ascorbic acid is
present in flower nectars and is often found in honeys (Schepartz, 1966;
Kerkvliet, 1996). It has been observed, that the addition of ascorbic acid
to honey increased by five-fold the rate of H2O2 removal (White, 1975).
In plants, ascorbic acid is the most important antioxidant responsible
for the degradation of hydrogen peroxide. Its removal eliminates pos-
sibility of H2O2 to act as a substrate in the ROS generation (Foyer,
1993). However, depending on its concentrations, ascorbic acid may
exhibit anti-oxidant or pro-oxidant activity, the latter characterized by
the production of H2O2 instead of its degradation (see below, paragraph
5.4).
5.4. Production of hydrogen peroxide by polyphenols autoxidation
In addition to enzymatic generation of hydrogen peroxide by GOx,
this compound could be produced as a result of pro-oxidant activity of
polyphenols and via oxidation of antioxidants such as ascorbic acid.
Polyphenols are known as powerful antioxidants, but in the presence of
transition metals ions, specifically Fe (II) and Cu (I) and under aerobic
conditions, they can be easily oxidized to their quinones, producing
H2O2 as by-product of autoxidation. Production of H2O2 upon poly-
phenol autoxidation is well known phenomenon observed in tea, coffee,
red wine and fruit juices (Long, Lan, Hsuan, & Halliwell, 1999;
Akagawa, Shigemitsu, & Suyama, 2003; Grzesik et al., 2019;
Waterhouse & Laurie, 2006; Oliveira, Ferreira, De Freitas, & Silva,
2011).
Fig. 2. Destruction of hydrogen peroxide by ascorbic acid.
K. Brudzynski
The yield of H2O2 depends on polyphenol structure, polyphenol
concentration, presence of transition metal ions and O2.
The presence of the orto 3′, 4′-dihydroxy catechol structure and a 1,
2, 3-trihydroxybenzene structure (a galloyl group) determines the
maximal electron donating effect and autoxidation to corresponding
quinones (Cao, Sofic, & Prior, 1997; Fukumoto & Mazza, 2000;
Sakihama, Cohen, Grace, & Yamasaki, 2002). The autoxidation begins
with the transfer of one electron from polyphenol hydroxyl group to O2
leading to formation of ortho-semiquinone anion radical (Q̇¯). Reaction
of o-semiquinone radicals with O2 generates a superoxide anion radical
(O2̇¯) that is subsequently dismutated to H2O2. Overall reaction invol-
ving the sequential transfer of two electrons to O2 to form O22−
(O2 + e− → O2·− + e− → O22−) and the protonation of O22− will lead
to H2O2 formation (O22− + 2H+ → H2O2).
Auto-oxidation of catechins of green and black teas is the best
known example of foods that produce copious amounts of hydrogen
peroxide (Agakawa et al., 2003, Long et al., 1999; Grzesik et al., 2019).
In teas, concentrations of H2O2 were dose-dependently correlated with
the total catechin content. The addition of catalase abolished catechin
(epigallocatechin gallate) oxidation confirming H2O2 involvement in
this reaction (Akagawa et al., 2003; Long et al., 1999). Likewise, phe-
nolics of apple juice possessing the catechol or galloyl structure showed
propensity to autoxidation with the formation of H2O2. Chlorogenic,
caffeic acids and flavonoids, such as rutin and quercetin, strongly
contributed to H2O2 production while flavanols and chalcons were in-
effective (Bellion et al., 2009).
Presence of transition metal is essential for auto-oxidation of poly-
phenols because oxygen does not react directly with catechol or gallolyl
groups (Oliveira et al., 2011). Differences in the concentrations of Fe
(II), Cu(I) and Mn (II) transition metals were shown to be the main
factors determining the yield of H2O2 formation in tea, red wine and
honey. Removal of the transition metal by chelators (phenantroline,
desferoxamine) decreased H2O2 formation with tea catechins (Grzesik
et al., 2019).
Autoxidation of polyphenols is pH-dependent and is accelerated by
alkaline pH. No production of this compound was observed below
neutral pH (pH 7) (Grzesik et al., 2019).
In regard to honey, many of honey polyphenolic compounds possess
the structural elements required for both anti- and pro-oxidant activ-
ities (for example, flavonoids, quercetin, apigenin, rutin or cafeic,
ferulic acids). In general, ability to auto-oxidize or serve as antioxidant
with radical scavenging or metal chelating activities concerns the in-
volvement of the same chemical groups in both activities, mainly the
catechol group and a number of hydroxyl groups (Rice-Evans, Miller, &
Paganga, 1996; Bors, Heller, Michel, & Saran, 1990). There is a growing
proof that a switch from antioxidant to pro-oxidant activities depends
on the polyphenols concentration, presence of metal ions and pH (Cao
et al., 1997; Fukumoto & Mazza, 2000). In honey, there is a strong
correlation between the antioxidant activity and polyphenol con-
centrations (Fig. 1; Gheldof, Xiao-Hong, & Engeseth, 2002; Gheldof &
Engeseth, 2002; Blasa et al., 2006; Bertoncelj, Doberšek, Jamnik, &
Golob, 2007; Estevinho, Pereira, Moreira, Dias, & Pereira, 2008).
Higher antioxidant capacity was found for darker honeys that contain
higher concentration of phenolic compounds than light color honeys
(Frankel, Robinson, & Berenbaum, 1998; Gheldof et al., 2002; Nagai,
Sakai, Inoue, Inoue, & Suzuki, 2001; Blasa et al., 2006; Bertoncelj et al.,
2007), and usually the darker honeys had higher total polyphenol
content (Gheldof & Engeseth, 2002; Al-Mamary, Al-Meeri, & Al-Habori,
2002; Vela, de Lorenzo, & Perez, 2007). Therefore, strong antioxidant
properties of polyphenols, such as that of buckwheat honey, can predict
also the pro-oxidant activity. Indeed, pro-oxidant activity of poly-
phenols has been demonstrated in honey (Brudzynski, Abubaker, &
Miotto, 2012c) and honeydew (Bucekova, Buriova, Pekarik, Majtan, &
Majtan, 2018).
Honey polyphenols auto-oxidation comprises the non-enzymatic
pathway of H2O2 production that might significantly contribute to the
5
Food Chemistry 332 (2020) 127229
pool of hydrogen peroxide produced by GOx. Importantly, increased
concentration of H2O2 and increased generation of hydroxyl radicals by
H2O2 degradation (see below) potentiated honey antibacterial activity
in substantial way (Brudzynski, Abubaker, & Wang, 2012a; Brudzynski
& Lannigan, 2012b; Bucekova et al., 2018; Brudzynski et al., 2012c).
These studies illustrate two different mechanisms of H2O2 production in
honey, enzymatic and non-enzymatic, both highly desirable for honey
antibacterial activity.
5.5. Degradation of H2O2 via Fenton reaction
H2O2 generated enzymatically or by autoxidation of polyphenols is
usually immediately converted to ROS via the Fenton reaction in the
presence of Fe (II) or other transition metals. The Fenton reaction is
preceded by the oxygen reduction to superoxide radical (O2 •−) in the
presence of oxidized ferric iron Fe (III). As shown above, superoxide
radicals are formed during polyphenols or ascorbic acid auto-oxidation.
The superoxide radical (O2 •−) enters the Fenton reaction with Fe (III)
reducing it to ferrous ion, Fe (II), with formation of molecular oxygen.
Fe (II) reacts with H2O2 producing Fe (III), hydroxyl radical (HO•) and
hydroxyl anion (OH–). Then superoxide radicals (O2 •−) reacts again
with H2O2 forming hydroxyl radical (HO•), hydroxyl anion (–OH) and
oxygen (O2).
Natural honeys have all necessary substrates for the Fenton reac-
tion; H2O2, polyphenols as well as transition metal ions such as iron and
therefore they have ability to produce superoxide radicals (O2 •−) and
hydroxyl radicals (HO•). These two radicals are most damaging to
bacterial cells, causing growth inhibition and DNA degradation be-
tween other negative effects. Brudzynski and Lannigan (2012b),
showed that supplementing buckwheat honey, containing a high con-
centration of redox-active polyphenols, with either Cu (II) or H2O2,
resulted in a 30-fold increase in bacteriostatic activity against MRSA
and VRE due to formation of hydroxyl radicals. Using p-aminophenyl
fluorescein, a compound that trap (HO•), it was possible to demonstrate
a direct relationship between hydroxyl radical generation and bacterial
growth inhibition. Pre-treatment of honeys with catalase prior to their
supplementation with Cu ions fully restored bacterial growth indicating
that hydroxyl radicals were produced from H2O2 via the Fenton-type
reaction (Brudzynski & Lannigan, 2012b).
Pro-oxidant activity of honey polyphenols and the ROS generated in
the presence of Cu ions has shown to be damaging to bacterial DNA.
The degree of DNA degradation increased with increased concentration
of high redox-active polyphenols. The damage was prevented when
either H2O2 or Cu ions were omitted in the reaction mixture
(Brudzynski et al., 2012a; Brudzynski et al., 2012c).
More evidence that H2O2 production might lead to the formation of
free radicals in honey was provided by Henriques, Jackson, Cooper, and
Burton (2006). The authors compared propensity to the formation of
hydroxyl radicals by two types of honeys, “pasture” honey (clover
honey) that produced H2O2 and manuka honey that did not. The only
honey able to generate radicals in the presence of ferrous ion was
“pasture” honey, while no radical production was seen from manuka
honey (Henriques et al., 2006).
Thus, honey polyphenols can both promote and inhibit ROS gen-
eration. The radical scavenging activity of honey polyphenols as well as
their chelation activity is well described in literature (Bertoncelj et al.,
2007; Gheldof et al., 2002; Kaškonienė, Venskutonis, & Čeksteryte,
2007), however there is also growing evidence of their pro-oxidant
activity and ROS generations. Understanding the conditions under
which either antioxidant or pro-oxidant pathways become dominant in
honeys is important for honey antibacterial activity.
Table 2 summarizes the factors that affect the levels of H2O2 pro-
duction in honey described in this section.
K. Brudzynski Food Chemistry 332 (2020) 127229

Table 2 The existence of colloids in honey was reported for the first time
Factors contributing to variability in H2O2 levels in honey. over 80 years ago (Lothrop & Paine, 1931; Paine, Gertleh, and Lothrop
Factors affecting hydrogen peroxide production in honey (1934). Honey colloids were treated as nuisance disturbing honey
clarity and quality (Lothrop & Paine, 1931; Paine et al. (1934). This
Increase Decrease negative view became radically challenged by recent findings that
colloidal particles are composed of active honey molecules such as
Glucose oxidase activity Inhibitors of glucose oxidase
Glucose oxidase activity Enzymatic destruction: Catalase polyphenols, protein–polyphenol complexes and melanoidins that are
Glucose oxidase activity of plant origin responsible for honey antioxidant and antibacterial activities
Glucose oxidase of microbial origin (Brudzynski et al., 2017). In similarity to phenomenon of molecular
Non-enzymatic synthesis Non-enzymatic destruction by: crowding in living cells, a supersaturated concentration of fructose and
Polyphenol oxidation Ascorbic acid
glucose serve as crowding molecules that confine and immobilize honey
Metalloenzymes
Fenton reaction macromolecules to limited spaces. Close proximity of molecules en-
Colloidal structure (phase separation) Colloidal structure (phase transition) forces their intermolecular interactions, mostly via attractive van der
Waals forces, leading to the formation of larger, colloidal structures
(Minton, 2001). Likewise, molecular crowding compels honey con-
6. Colloidal structure of honey and its role in H2O2 production stituents to form clusters and assemblies. Therefore, there are no free-
floating molecules in honey solution, including enzymes or anti-
A widely held misconception of honey structure is that it comprises microbial compounds. Importantly, the properties and activities of
a mixture of different constituents of plant, honeybee and microbial compounds bound in clusters are different than their free, unbound
origins that are homogenously distributed and freely moving/diffusing forms (Sweetlove & Fernie, 2018). Using scanning electron microscopy,
in sugar solution. However, more evidence has accumulated recently to the colloidal structure of honey could be observed as phase-separated
indicate that honey macromolecules coalesce to form colloidal particles particles of colloidal dimensions dispersed in sugar solution
under conditions of low water activity and high sugar concentration (Brudzynski et al., 2017).
(Brudzynski et al., 2017). Accumulating evidence from our laboratory revealed a key role of

Fig. 3. A relationship between the concentrations of UV absorbing compounds, expressed as the AUC 240–250 nm, and the phase transition point (arrow) upon
honey dilution in dark, medium and light color honeys characterized by high- and low concentrations of UV-absorbing macromolecules. (Reproduced from
Brudzynski et al., 2017, Scientific Reports, 7, 7637. This work is licensed under the Creative Commons Attribution 4.0 International License. Color has been modified).

6
K. Brudzynski Food Chemistry 332 (2020) 127229

colloidal structure of honey in H2O2 production (Brudzynski et al.,

2017). We will briefly discuss two most important facts associated with
colloid formation in honey; phase separation and phase transition.
These two events have opposite effects on synthesis of H2O2; colloidal
structure of honey supported H2O2 production while disintegration of
colloids reduced it.
Formation of colloidal assemblies and phase separation critically
depends on the concentration of macromolecules. High concentration
of macromolecules favours formation of colloidal particles. To measure
concentration of macromolecules in our studies, we took advantage of
the fact that most of honey macromolecules involved in complexation,
such as proteins, polyphenols and early Maillard reaction products,
absorb UV light in the 200 nm to 360 nm. The measurements were
based on the UV scans in the 240–250 nm and 200–400 nm ranges and
the concentration was semi-quantitatively determined from the area
under the curve (AUC) and expressed in the AUC units. Statistical
analyses showed that the UV absorbing compounds were the highest in
dark > medium > light honeys and the differences among honeys were
highly significant (p < 0.0001). Using this approach, and dynamic light
scattering to monitor formation of colloidal particles, it was possible to
show that honeys with the high concentration UV absorbing com-
pounds, usually darker honeys such as buckwheat or Manuka, were able
to form large micron-size colloidal particles. Formation of colloidal
assembly was associated with the efficient H2O2 production and in turn,
with a significant antibacterial effect.
The colloidal structure of honey remained stable as long as the
concentration of UV absorbing molecules was above the critical
threshold, that is, above a phase transition point. We define the phase
transition point as the minimum concentration of the UV absorbing
compounds (expressed in AUC units), at which colloidal particle would
still form. Upon honey dilutions, the reduction of the concentration of
crowding molecules (glucose and fructose) caused the relaxation of
intramolecular interactions within colloidal particles and their disin-
tegration from micro- to nanoparticles, as observed by dynamic light
scattering. Under these conditions, the system changed the conforma-
tion from the two-phase colloidal state to one-phase state of mono-
dispersion at dilute solution conditions. The phase transition point was
observed as the sudden drop in the AUC values upon honey dilution
(Fig. 3). Depending on the concentration of UV absorbing compounds in
honeys, the phase transition point occurred at different dilutions. Mi-
cron-size colloidal assemblies of dark and medium honeys were more
resistant to degradation by water dilution and kept their colloidal
structure up to 16-fold dilution (6.25% (w/v) (Fig. 3). Since the phase
transition point had a significant consequence for H2O2 production, a
mathematical model has been developed to predict the phase transition Fig. 4. The relationship between colloidal structure, antibacterial activity and
point in different honeys upon dilution (Fig. 3) (Brudzynski et al., hydrogen peroxide production upon honey two-fold serial dilution in different
honeys. (A) A schematic presentation of colloidal particles and their dis-
2017). The model showed clearly that in contrast to dark honeys, col-
sociating beyond phase transition point, (B) The relationship between bacter-
loidal assemblies of light color honeys (such as clover) were sensitive to
iostatic activity and honey dilution in different honeys of different botanical
disintegration upon dilution. The degradation was associated with loss
origin and (C) The relationship between H2O2 peroxide production and a phase
of the ability to produce high quantities of H2O2. The level of H2O2 in transition point by dark, medium and light honeys. (Reproduced from
these honeys was not sufficient to inhibit bacterial growth. Brudzynski et al., 2017, Scientific Reports, 7, 7637. This work is licensed under
Thus, there was a causal relationship between the concentration of the Creative Commons Attribution 4.0 International License).
macromolecules, propensity to form colloidal assemblies, H2O2 pro-
duction and antibacterial effect (Fig. 4).
bound within assemblies possess activity many orders of magnitude
higher than the same molecule tested alone in dilute solution in in vitro
6.1. Relationship between colloidal structure of honey and generation of
conditions (Sweetlove & Fernie, 2018).
H2 O 2
Likewise, the production of H2O2 was found highly efficient in
honeys that were able to form colloidal particles. In contrast, the phase
Literature data indicate that macromolecules in colloidal assemblies
transition from the large colloidal to monodispersed state significantly
behave in markedly different way than single molecules in in vitro as-
reduced its synthesis. The H2O2 production under these two states was
says. Specifically, activities of enzymes bound into complexes are more
extremely different (R = 0.7124, p < 0.0009, n = 18). When changes
efficient in the product generation (Johnson, Algar, Malanoski, Ancona,
in conformations of honey upon dilution were investigated in parallel
& Medintz, 2014). The enzymes immobilized on a surface of particle
with the production of hydrogen peroxide, (using dynamic light scat-
usually possessed much higher enzyme activity compared to the single
tering and Amplex Red method, respectively), it was realized un-
isolated, purified enzyme (Sweetlove & Fernie, 2018). For example,
expectedly that the phase transition point coincided with the maximum
enzymes involved in glycolysis or TCA cycle in living cells that are

7
K. Brudzynski
peak of H2O2 production. Both, the phase transition point and the
maximum peak of H2O2 in dark- and medium-color honeys took place
exactly at 16-fold and 8-fold dilutions (corresponding to honey con-
centration of 6.25% and 12.5% v/w), respectively. In contrast, in light
color honeys, a decrease in H2O2 production was observed throughout
the entire dilution process (Fig. 3 and Fig. 4).
Thus, the active generation of H2O2 correlated with honey colloidal
conformation and it ended at a phase transition point. Based on lit-
erature indicating altered kinetics of enzymes bound to complexes, one
can consider possibility that GOx bound into colloidal particles might
have increased activity and produce higher amounts of H2O2. In con-
trast, phase-transition and conformational changes of honey upon di-
lution, that disintegrates colloidal assemblies, would reduce kinetics of
enzymatic reactions and effectiveness of H2O2 production. Such plau-
sible scenario could now explain the U-shaped curve of H2O2 produc-
tion upon honey successive dilutions.
6.2. Significance of honey colloidal structure on antibacterial activity
The colloidal structure and phase separation affected physico-
chemical and functional properties of honey. The colloidal structure
strongly promoted H2O2 production and with that, antibacterial activity
of honey (Brudzynski et al., 2017). However, several other aspects of
colloidal structure of honey also have relevance to its antibacterial
activity. For example, colloidal particles are formed and kept due to
combination of the high sugar concentration, low water activity, and
acidic pH that promote colloid condensation. The same factors (osmo-
larity, pH, water activity) are recognized as inhibitors of bacterial
growth. With respect to water activity, colloidal complexes of macro-
molecules exert the excluded volume effect that reduces the amount of
water that is available for other molecules, including microorganisms,
thus creating antibacterial environment.
From the perspective of honey function, the colloidal assembly
consists of multiple active molecules including polyphenols, proteins,
enzymes, peptides, large proteins complexes (such as MRJPs), Maillard
reaction products etc. which are known to be involved in antibacterial
activity of honey.
In conclusion, the organization of honey constituents into colloidal
particles combines all the factors and compounds associated with an-
tibacterial activity of honey. Thus, colloidal structure of honey provides
new insights into the structure–function relationship with regard to
antibacterial activity.
6.3. Stability of H2O2 molecules in diluted honeys
To what extent the conformational state of honey affects H2O2
stability requires further studies. However, based on the available data
from dynamic light scattering, the phase transition changed the particle
size, increased particle polydisperity and mobility in diluted, less vis-
cous medium (Brudzynski et al., 2017). These changes might affect the
particle–particle interactions, increase the repulsive rather than at-
tractive forces and change distances between particles. It is plausible
that in two-phase conformation, honeys’ active macromolecules
forming micron-size colloidal particles might act in concert to produce
H2O2. The close proximity of particles might influence a spatial dis-
tribution of H2O2. Since diffusion range is short for H2O2, its local ac-
cumulation would increase in areas directly surrounding the particle.
Passing the phase transition point, the diluted state conformation would
affect the H2O2 molecule integrity by exposing it to degradation by
catalase and other honey small molecules. Moreover, the change in pH
upon dilution from acidic toward neutral in diluted honey could also be
destabilizing factor. For commercial H2O2 solution, optimum stability
occurs in the pH below 4.5. Above pH 5, the spontaneous H2O2 de-
composition sharply increases.
Thus, the diluted state conformation would be disadvantageous to
integrity of H2O2.
8
Food Chemistry 332 (2020) 127229
7. Additional sources of H2O2 production in honey
7.1. Nectar redox Cycle of flowering plants
H2O2 production in honey is usually considered in the contexts of
components and enzymes restricted to honey. Currently, the bee GOx
–dependent production of H2O2 is considered to be the main me-
chanism. However, recent evidence indicates that enzymes present in
flower nectar from which honey is produced might also contribute to
the H2O2 levels. Nectars from some flowers possess GOx a part of the
system known as the Nectar Redox Cycle. This is a multiprotein system
originally discovered in the nectar of ornamental tobacco (Carter &
Thornburg, 2000; Carter & Thornburg, 2004). The Nectar Redox Cycle
is composed of 5 enzymes. The subunit known as NEC1 is a manganese
superoxide dismutase that catalyzes the dismutation of the superox-
ide anion into H2O2 and molecular oxygen. The same multimeric
complex possesses also a flavin-containing enzyme (NEC5) with GOx
activity. The redox actions of NEC1 and NEC5 enzymes have been
shown to generate up to 4 mM of H2O2. Such concentration of H2O2 is
significantly higher than required for bactericidal effect (Carter &
Thornburg, 2000; Carter & Thornburg, 2004).
Not all plant nectars possessed Nectar Redox Cycle proteins, but
those nectars that contain the system could potentially operates in
concert with the glucose of oxidase of the bee origin. Thus, the dis-
covery of plant Nectar Redox Cycle enzymes offers novel insights into
the potentially additional source of H2O2 production in honey.
7.2. Hydrogen peroxide production by microbes
Although most studies have focused the attention on the
H2O2 produced by the bee GOx, an often overlooked fact is that H2O2 is
also produced by several microbes that inhabit honey. Fungi such as
Aspergillus and Penicillium spp and yeasts such as Saccharomyces,
Schizosaccharomyces, Ascomycetes, Metschnikowia, Candida,
Zygosacchromyces are commonly found in honey and are known to ex-
press high levels of GOx (Kačániová, Kňazovická, Felšöciová, & Rovná,
2012; Silva et al., 2017; Snowdon & Cliver, 1996). These microorgan-
isms grow well in sugar environment and tolerate high osmotic stress of
honey. Their potential contribution to honey H2O2 levels has not been
investigated yet.
Bacteria also produce H2O2 as a means to compete and defend
themselves against fungal and other bacterial species. Almost all ob-
ligate aerobic and facultative anaerobic bacteria produce H2O2 via their
respiratory electron transfer chain. Among well-documented bacterial
species inhabiting honey, bee bread and the bee crop are lactic acid
bacteria that produce significant amounts of H2O2 (Endo & Salminen,
2013). About 13 different strains of Lactobacillus species were identified
in the crop of honeybee Apis mellifera (Olofsson & Vásquez , 2008) and
shown to produce H2O2 (Olofsson, Butler, Markowicz, Lindholm,
Larsson &Vásquez, 2016). The bee crop is a specialized honeybee sto-
mach in which nectar is carried from flowers to hives. Lactic acid
bacteria are probably introduced to honey from the bee crop during
nectar regurgitation to the honey comb cell.
In addition to lactic acid bacteria, a dominant group of bacteria in
honeys comprise Bacillus genera (Grecka, Kus, Worobo, & Szweda,
2018; Pajor, Worobo, Milewski, & Szweda, 2018; Snowdon & Cliver,
1996). Bacillus species and lactic acid bacteria of genera Enterococcus,
Lactobacillus, Lactococcus, Streptococcus, employ NADH2 oxidases in-
stead of GOx to produce H2O2 as an end product. In the latter cases,
H2O2 is formed by oxidizing NADH2 in the presence of oxygen (Collins
& Aramaki, 1980; Saeki, Nozaki, & Matsumoto, 1985). Escherichia coli, a
common contaminant of honey, could also contribute to the production
of H2O2. It has been shown that the bacterium is able to generate about
14 μM H2O2 per s when grown aerobically in liquid culture containing
glucose (Seaver & Imlay, 2001).
Although, it is well known that the physicochemical conditions of
K. Brudzynski
undiluted honey are prohibitive for growth of microorganisms, it could
be argue however, that growth of microorganism might be still possible
during honey ripening·H2O2 produced by microorganisms could then be
preserved in undiluted honey.
Together, the high variability in the concentration of hydrogen
peroxide in different honeys might be linked to the quantitative var-
iations in microbial contaminations and the presence of the Nectar
Redox Cycle system.
8. Conclusions and future perspective
It is apparent from the work reviewed that H2O2 concentrations in
honey are regulated at several levels, and there is no one simple com-
pound or factor that would allow control and prediction of H2O2 final
quantities. The assumption of the early research that H2O2 levels could
be predicted from the rate of its synthesis by the bee GOx and de-
composition by catalase is now contested. Growing evidence indicates a
multifactorial process influencing the H2O2 levels both at the stage of its
production and decomposition. Firstly, the enzymatic production of
H2O2 might be still a major pathway that depends on the level of GOx of
the bee origin but the contribution of nectar and microbial GOx cannot
be excluded. Secondly, the rate of any enzymatic reaction in honey is
strongly inhibited by low water activity in undiluted honeys. Upon
honey dilution with water, several enzymatic and non-enzymatic re-
actions are initiated and they contribute to H2O2 levels in positive or
negative way (such as synthesis of H2O2 via polyphenol autoxidation
and its destruction in the Fenton reaction).
However, the important stride forward taken so far was discovery of
the close association between colloidal structure of honey and H2O2
production that now could explain the inverted U-shaped relationship
between its production and honey dilution observed by many re-
searchers. The inverted U shape reflected the optimum point in honey
dilution that produced the maximum levels of H2O2. Before or after the
optimal dilutions the levels of H2O2 are markedly decreased. Now, the
process underlying this phenomenon could be explained by the tran-
sition between two conformational states of honey: two-phase colloidal
conformation determined by high concentration of honey UV absorbing
compounds that supported H2O2 production and one-phase colloidal
states that did not. The phase transition point represented the critical,
threshold concentration of UV absorbing compounds needed to uphold
honey two-phase colloidal conformation. This phase transition point
coincided (was the same) with the maximum production of H2O2 upon
honey dilution. In contrast, one-phase diluted state conformation de-
stabilized H2O2 molecules and decreased their concentration. By de-
termining the phase transition point in a given honey, it would be
possible to predict whether the concentrations of H2O2 will reach
bacteriostatic and/or bactericidal quantity.
The effect of honey conformation provides new insight into process
of H2O2 production in honey. With future perspective in mind, we ex-
pect that honey colloidal conformation would continue to be a major
source of discoveries. We envision the colloidal particle and its sur-
rounding area as a reaction chamber allowing the diffusion and ex-
change of molecules with other particles and with the surrounding
sugar solution. Maintaining such conformation would keep H2O2 levels
high, ensuring its stability and in turn, preserving antibacterial activity
of honey.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
9
Food Chemistry 332 (2020) 127229
Acknowledgment
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
References
Abramovic, H., Jamnik, M., Burkan, L., & Kac, M. (2008). Water activity and water
content in Slovenian honeys. Food Control Food Control, 19, 1086–1090.
Akagawa, M., Shigemitsu, T., & Suyama, K. (2003). Production of hydrogen peroxide by
polyphenols and polyphenol-rich beverages under quasi-physiological conditions.
Bioscience Biotechnology and Biochemistry, 67(12), 2632–2640.
Allen, K. L., Molan, P. C., & Reid, G. M. (1991). A survey of the antibacterial activity of
some New Zealand honeys. Journal of Pharmacy and Pharmacology, 43, 817–822.
Al-Mamary., Al-Meeri, A., & Al-Habori, M. (2002). Antioxidant activities and total phe-
nolics of different types of honey. Nutrition Research, 22, 1041–1047.
Bang, L. M., Buntting, C., & Molan, P. (2004). The effect of dilution on the rate of hy-
drogen peroxide production in honey and its implications for wound healing. Journal
of Alternativeand Complementary Medicine, 9(2), 267–273.
Bao, J., Furumoto, K., Yoshimoto, M., Fukunaga, K., & Nakao, K. (2003). Competitive
inhibition by hydrogen peroxide produced in glucose oxidation catalyzed by glucose
oxidase. The Biochemical Engineering Journal. 13, 69–72.
Bellion, P., Olk, M., Will, F., Dietrich, H., Baum, M., Eisenbrand, G., & Janzowski, C.
(2009). Formation of hydrogen peroxide in cell culture media by apple polyphenols
and its effect on antioxidant biomarkers in the colon cell line HT-29. Molecular
Nutrition & Food Research, 53(10), 1226–1236.
Bertoncelj, J., Doberšek, U., Jamnik, M., & Golob, T. (2007). Evaluation of the phenolic
content, antioxidant activity and colour of Slovenian honey. Food Chemistry, 105(2),
822–828.
Blasa, M., Candiracci, A., Accorsi, M. P., Piacentini, M. C., Albertini, M., & Piatti, E.
(2006). Raw Millefiori honey is packed full of antioxidants. Food Chemistry, 97(2),
217–222.
Bors, W., Heller, W., Michel, C., & Saran, M. (1990). Flavonoids as antioxidants:
Determination of radical scavenging efficiencies. Methods in Enzymology, 186,
343–355.
Bright, H. J., & Porter, D. J. T. (1975). Flavoprotein oxidases. Enzymes, 12, 421–505.
Brudzynski, K. (2006). Effect of hydrogen peroxide on antibacterial activities of Canadian
honeys. Canadian Journal of Microbiology, 52, 1228–1237.
Brudzynski, K., Abubaker, K., Martin, L., & Castle, A. (2011). Re-examining the role of
hydrogen peroxide in bacteriostatic and bactericidal activities of honey. Frontiers in
Microbiology, 2, 213.
Brudzynski, K., Abubaker, K., & Wang, T. (2012a). Powerful bacterial killing by buck-
wheat honey is concentration –dependent, involves complete DNA degradation and
requires hydrogen peroxide. Frontiers in Microbiology, 3, 242.
Brudzynski, K., Abubaker, K & Miotto, D. (2012c). Unraveling a mechanism of honey
antibacterial action: polyphenol/H2O2-induced oxidative effect on bacterial cell
growth and on DNA degradation. Food Chemistry, 133, 329-336.
Brudzynski, K., & Lannigan, R. (2012b). Mechanism of honey bacteriostatic action against
MRSA and VRE involves hydroxyl radicals generated from honey’s hydrogen per-
oxide. Frontiers in Microbiology, 3, 36.
Brudzynski, K., Miotto, D., Kim, L., Sjaarda, C., Maldonado-Alvarez, L., & Fukś, H. (2017).
Active macromolecules of honey form colloidal particles essential for honey anti-
bacterial activity and hydrogen peroxide production. Scientific Reports, 7, 7637.
Bucekova, M., Valachova, I., Kohutova, L., Prochazka, E., Klaudiny, J., & Majtan, J.
(2014). Honeybee glucose oxidase-its expression in honeybee workers and com-
parative analyses of its content and H2O2-mediated antibacterial activity in natural
honeys. Naturwissenschaften, 101(8), 661–670.
Bucekova, M., Buriova, M., Pekarik, L., Majtan, V., & Majtan, J. (2018). Phytochemicals-
mediated production of hydrogen peroxide is crucial for high antibacterial activity of
honeydew honey. Scientific Reports, 8(1), 9061.
Burgett, M. D. (1974). Glucose Oxidase: A Food Protective Mechanism in Social
Hymenoptera. Annals of the Entomological Society of America, 67, 545–546.
Cao, G., Sofic, E., & Prior, R. L. (1997). Antioxidant and prooxidant behavior of flavo-
noids: Structure-activity relationships. Free Radical Biology and Medicine, 22(5),
749–760.
Carter, C., & Thornburg, R. W. (2000). Tobacco Nectarin I: Purification and character-
ization as a germin-like, manganese superoxide dismutase implicated in the defense
of floral reproductive tissues. The Journal of Biological Chemistry, 275, 36726–36733.
Carter, C., & Thornburg, R. W. (2004). Tobacco Nectarin V Is a Flavin-Containing
Berberine Bridge Enzyme-Like Protein with Glucose Oxidase Activity. Plant
Physiology, 134(1), 460–469.
Collins, E. B., & Aramaki, K. (1980). Production of Hydrogen Peroxide by Lactobacillus
acidophilus. Journal of Dairy Sciences, 63, 353–357.
DiGromolo, F., D’Amato, A., & Righetti, P. G. (2012). Assessment of the floral origin of
honey via proteomic tools. Journal of Proteomics, 75, 3688–3693.
Dold, H., Du, D. H., & Dziao, S. T. (1937). Nachweis antibakterieller, hitze- und lich-
tempfindlicher Hemmungsstoffe (Inhibine) im Naturhonig Blutenhonig. Zeitschrift für
Hygiene und Infektionskrankheiten, 120, 155–167.
Dustman, J. H. (1971). Über die Katalaseaktivität in Bienenhonig aus der Tracht der
Heidekrautgewächse (Ericaceae). Zeitschrift für Lebensmittel-Untersuchung und
Forschung, 145, 292–295.
Endo, A., & Salminen, S. (2013). Honeybees and beehives are rich sources for fructophilic
lactic acid bacteria. Systematic and Applied Microbiology, 36, 444–448.
Erban, T., Shcherbachenko, E., Talacko, P., & Harant, K. (2019). The Unique Protein
Composition of Honey Revealed by Comprehensive Proteomic Analysis: Allergens,
Venom-like Proteins, Antibacterial Properties, Royal Jelly Proteins, Serine Proteases,
and Their Inhibitors. Journal of Natural Products, 82(5), 1217–1226.
Estevinho, L., Pereira, A., Moreira, L., Dias, L., & Pereira, E. (2008). Antioxidant and
K. Brudzynski
antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey.
Food Chemistry and Toxicology, 46, 3774–3779.
Feng, M., Fang, Y., & Li, J. (2009). Proteomic analysis of honeybee worker (Apis melli-
fera) hypopharyngeal gland development. BMC Genomics, 10, 645.
Foyer, C. H., in Antioxidants in Higher Plants (eds Alscher, R. G. and Hess, J. L.), CRC
Press, Boca Raton, FL, 1993, pp. 31–58.
Frankel, S., Robinson, G. E., & Berenbaum, M. R. (1998). Antioxidant content and cor-
related characteristics of 14 monofloral honeys. Journal of Apiculture Research, 37,
27–31.
Fukumoto, L. R., & Mazza, G. (2000). Assessing antioxidant and prooxidant activities of
phenolic compounds. Journal of Agricultural and Food Chemistry, 48, 3597–3604.
Gauhe, A. (1941). Uber em glukoseoxydierendes Enzym in der Pharynxdruse der
Honigbiene. Zeitschrift für Vergleichende Physiologie, 28, 211–253.
Gheldof, N., & Engeseth, N. J. (2002). Antioxidant capacity of honeys from various floral
sources based on the determination of oxygen radical absorbance capacity and in-
hibition of in vitro lipoprotein oxidation in human serum samples. Journal of
Agricultural and Food Chemistry, 20, 3050–3055.
Gheldof, N., Xiao-Hong, W., & Engeseth, N. (2002). Identification and quantification of
antioxidant components of honeys from various floral sources. Journal of Agricultural
and Food Chemistry, 50, 5870–5877.
Gibson, Q. H., Swoboda, B. E. P., & Massey, V. (1964). Kinetics and mechanism of action
of glucose oxidase. Journal of Biological Chemistry, 239, 3927–3934.
Grecka, K., Kus, P., Worobo, R. W., & Szweda, P. (2018). Study on the anti-staphylococcal
potential of honeys produced in Northern Poland. Molecules, 23, 260.
Grzesik, M., Bartosz, G., Stefaniuk, I., Pichla, M., Namieśnik, J., & Sadowska-Bartosz, I.
(2019). Dietary antioxidants as a source of hydrogen peroxide. Food Chemistry, 25,
692–699.
Hayashi, K., Fukushima, A., Hayashi-Nishino, M., & Nishino, K. (2014). Effect of me-
thylglyoxal on multidrug- resistant Pseudomonas aeruginosa. Frontiers in
Microbiology, 5, article 180.
Henriques, A., Jackson, S., Cooper, R., & Burton, N. (2006). Free radical production and
quenching in honeys with wound healing potential. Journal of Antimicrobial
Chemotherapy, 58(4), 773–777.
Huidobro, J. F., Sánchez, M. P., Muniatequi, S., & Sancho, M. T. (2005). Precise method
for the measurement of catalase activity in honey. Journal of AOAC International,
88(3), 800–804.
Jones, P., & Dunford, H. B. (1977). On the mechanism of compound I formation from
peroxidases and catalases. Journal of Theoretical Biology, 69, 457–470.
Johnson, B. J., Algar, W. R., Malanoski, A. P., Ancona, M. G., & Medintz, I. L. (2014).
Understanding enzymatic acceleration at nanoparticle interfaces: Approaches and
challenges. Nano Today, 9, 102–131.
Kačániová, M., Kňazovická, V., Felšöciová, S., & Rovná, K. (2012). Microscopic fungi
recovered from honey and their toxinogenity. Journal of Environmental Science and
Health, Part A, 47(11), 1659–1664.
Kalisz, H. M., Hendle, J., & Schmid, R. D. (1997). Structural and biochemical properties of
glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense.
Applied Microbiology and Biotechnology, 47, 502–507.
Kaškonienė, V., Venskutonis, R., & Čeksteryte, V. (2007). Radical scavenging activity of
different floral origin honey and beebread phenolic extracts. Food Chemistry, 101(2),
502–514.
Kerkvliet, J. D. (1996). Screening method for the determination of peroxide accumulation
in honey and relation with HMF content. Journal of Apicultural Research, 35(3–4),
110–117.
Kleppe, K. 1966. The effect of hydrogen peroxide on glucose oxidase from Aspergillus
niger. Biochemistry 1966, 5, 1, 139-143.
Kubo, T., Sasaki, M., Namura, J., Sasagawa, H., Ohashi, K., Takeuchi, H., & Natori, S.
(1996). Change in the expression of hypopharingeal-gland proteins of the worker
honeybees (Apis mellifera L.) with the age and/or role. Journal of Biochemistry, 119,
291–295.
Kwakman, P. H. S., te Velde, A. A., de Boer, L., Speijer, D., Vandenbroucke-Grauls, C. M.
J. E., & Zaat, S. A. J. (2010). How honey kills bacteria. FASEB J. 24, 2576–2582.
Leskovac, V., Trivic, S., Wohlfahrt, G., Kandrac, J., & Pericin, D. (2005). Glucose oxidase
from Aspergillus niger: The mechanism of action with molecular oxygen, quinones,
and one-electron acceptors. International Journal of Biochemistry and Cell Biology, 37,
731–750.
Lewin, S. (1971). Water surface energy contribution to adherence of hydrophobic groups
in relation to stability of protein conformations. Nature New Biology. 231, 80–81.
Long, L. H., Lan, A. N., Hsuan, F. T., & Halliwell, B. (1999). Generation of hydrogen
peroxide by “antioxidant” beverages and the effect of milk addition. Is cocoa the best
beverage?. Free Radical Research, 31(1), 67–71.
Lothrop, R. E., & Paine, H. S. (1931). Some properties of honey colloids and the removal
of colloids from honey with bentonite. Industrial & Engineering Chemistry, 23,
328–332.
Malikkides, C. O., & Weiland, R. H. (1982). On the mechanism of immobilized glucose
oxidase deactivation by hydrogen peroxide. Biotechnology and Bioengineering, 24(11),
2419–2439.
Mavric, E., Wittmann, S., Barth, G., & Henle, T. (2008). Identification and quantification
of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum
scoparium) honeys from New Zealand. Molecular Nutrition & Food Research, 52,
483–489.
Minton, A. P. (2001). The influence of macromolecular crowding and macromolecular
confinement on biochemical reactions in physiological media. Journal of Biological
Chemistry, 276(14), 10577–10580.
Molan, P. C. (1992). The antibacterial activity of honey 2. Variation in the potency of the
antibacterial activity. Bee World. 73(1), 59–76.
Nagai, T., Sakai, M., Inoue, R., Inoue, H., & Suzuki, N. (2001). Antioxidative activities of
10
Food Chemistry 332 (2020) 127229
some commercially honeys, royal jelly, and propolis. Food Chemistry, 75, 237–240.
Nakamura, S., & Ogura, Y. (1968). Mode of inhibition of glucose oxidase by metal ions.
The Journal of Biochemistry, 64(4), 439–447.
Nakamura, T., & Ogura, Y. (1962). Kinetic studies on the action of glucose oxidase.
Journal of Biochemistry, 52, 214–220.
Oliveira, C. M., Ferreira, A. C. S., De Freitas, V., & Silva, A. M. M. (2011). Oxidation
mechanisms occurring in wines. Food Research International, 44, 1115–1126.
Olofsson, T. C., & Vásquez, A. (2008). Detection and identification of a novel lactic acid
bacterial flora within the honey stomach of the honeybee Apis mellifera. Current
Microbiololy, 57(4), 356–363.
Olofsson, T. C., Èile Butler, E., Markowicz, P., Christina Lindholm, C., Larsson, L., &
Vásquez, A. (2016). Lactic acid bacterial symbionts in honeybees – an unknown key
to honey’s antimicrobial and therapeutic activities. International Wound Journal,
13(5), 668–679.
Paine, H. S., Gertleh, S. I., & Lothrop, R. E. (1934). Colloidal constituents in honey.
Industrial & Engineering Chemistry, 26, 73–81.
Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure-antioxidant activity re-
lationships of flavonoids and phenolic acids. Free Radical Biology & Medicine, 20,
933–956.
Pajor, M., Worobo, R. W., Milewski, S., & Szweda, P. (2018). The Antimicrobial Potential
of Bacteria Isolated from Honey Samples Produced in the Apiaries Located in
Pomeranian Voivodeship in Northern Poland. International Journal of Environmental
Research and Public Health, 15, 2002.
Saeki, Y., Nozaki, M., & Matsumoto, K. (1985). Purification and Properties of NADH
Oxidase from Bacillus megaterium. Journal of Biochemistry, 98, 1433–1440.
Sakihama, Y., Cohen, M. C., Grace, S. C., & Yamasaki, H. (2002). Plant phenolic anti-
oxidant and pro-oxidant activities: Phenolics-induced oxidative damage mediated by
metals in plants. Toxicology, 177, 67–80.
Sano, O., Kunikata, T., Kohno, K., Iwaki, K., Ikeda, M., & Kurimoto, M. (2004).
Characterizaton of royal jelly proteins in both Africanized and European honeybees
(Apis mellifera) by two-dimensional gel electrophoresis. Journal of Agricultural and
Food Chemistry, 52, 15–20.
Santos, K. S., Santos, L. D., Mendes, M. A., Souza, B. M., Malaspina, O., & Palma, M. S.
(2005). Profiling the proteome complement of the secretion from hypopharyngeal
gland of Africanized nurse honeybees (Apis mellifera L.). Insect Biochemistry and
Molecular Biology, 35, 85–91.
Seaver, L. C., & Imlay, J. A. (2001). Hydrogen Peroxide Fluxes and Compartmentalization
inside Growing Escherichia coli. Journal of Bacteriology, 183(24), 7182–7189.
Schepartz, A. I. & Subers M. H. (1964): The glucose oxidase of honey I. Purification and
some general properties of the enzyme. Biochim. Biophys. Acta 85(2), 228-237.
Schepartz, A. I. (1966). The glucose-oxidase of honey. IV. Some addition observations.
Biochimica et Biophysica Acta, 118, 637–640.
Schepartz, A. I., & Subers, M. H. (1966). Catalase in Honey. Journal of Apicultural
Research, 5(1), 37–43.
Silva, M. S., Rabadzhiev, Y., Eller, M.R., Iliev, I., Ivanova, I & Santana, W.C. (2017).
Microorganisms in Honey. https://www.intechopen.com/books/honey- analysis/
microorganisms-in-honey.
Snowdon, J. A., & Cliver, D. O. (1996). Microorganisms in honey. International Journal of
Food Microbiology, 31, 1–26.
Strelec, I., Crevar, B., Kovač, T., Bilič Rajs, B., Primorac, L & Flanjak, I. (2018). Glucose
oxidase activity and hydrogen peroxide accumulation in Croatian honeys. Croatian
Journal of Food Science and Technology 10, 33-41.
Sweetlove, L. J., & Fernie, A. R. (2018). The role of dynamic enzyme assemblies and
substrate channelling in metabolic regulation. Nature Communications, 9, 2136.
Taormina, P. I., Niemira, B. A., & Beuchat, L. R. (2001). Inhibitory activity of honey
against foodborne pathogens as influenced by the presence of hydrogen peroxide and
level of antioxidant power. International Journal of Food Microbiology, 69, 217–225.
Tomotani, E. J., das Neves, L. C. M., & Vitolo, M. (2005). Oxidation of glucose to gluconic
acid by glucose oxidase in a membrane bioreactor. Applied Biochemistry and
Biotechnology, 121, 149–162.
Weston, R. J. (2000). The contribution of catalase and other natural products to the
antibacterial activity of honey: A review. Food Chemistry, 71(2), 235–323.
Waterhouse, A. L., & Laurie, V. F. (2006). Oxidation of wine phenolics: A critical eva-
luation and hypotheses. American Journal of Enology and Viticulture, 57, 306–313.
White, J. W., Subers, M. H., & Schepartz, A. I. (1963). The identification of inhibine, the
antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-
oxidase system. Biochimica et Biophysica Acta, 73, 57–70.
White, J. W., & Subers, M. H. (1963). Studies on honey inhibine. 2. A chemical assay.
Journal of Apicultural Research, 2(2), 93–100.
White, J. W., & Subers, M. H. (1964). Studies on honey inhibine. 4. Destruction of the
peroxide accumulation by light. Journal of Food Sciences, 29(6), 819–828.
White, J.W. Physical Characteristics of honey. In: Honey, a comprehensive survey. Crane
(ed), Heineman London; UK 1975, 157-206.
White, J. W. (1966). Inhibine and glucose oxidase in honey-A review. American Bee
Journal, 106(6), 80–82.
Wohlfart, G., Witt, S., Hendle, J., Schomburg, D., Kalisz, H. M., & Hecht, H. (1999). 1.8
and 1.9 Å resolution structure of the Penicillium amagasakiense and Aspergillus niger
glucose oxidase as a basis for modelling substrate complexes. Acta Crystallography D,
55, 969–977.
Vela, L., de Lorenzo, C., & Perez, R. A. (2007). Antioxidant capacity of Spanish honeys
and its correlation with polyphenol content and other physicochemical properties.
Journal of the Science of Food and Agriculture, 87, 1069–1075.
Zámocký, M., Gasselhuber, B., Furtmüller, P. G., & Obinger, C. (2012). Molecular evo-
lution of hydrogen peroxide degrading enzymes. Archives of Biochemistry and
Biophysics, 525(2), 131–144.