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Prediction of honey shelf life

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DOI: 10.1111/j.1745-4557.2009.00253.x

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 PREDICTION OF HONEY SHELF LIFE

BIAGIO FALLICO1,3, ELENA ARENA1 and MARIO ZAPPALA2

1
DOFATA sez. Tecnologie Agroalimentari
Universit di Catania
Via S.Sofia 98, 95123 Catania, Italy
2
Assicurazione Qualit Gruppo Zappal
Via Ardichetto sn, 95019 Zafferana Etnea, Italy

Received for Publication July 3, 2007

Accepted for Publication May 22, 2008

ABSTRACT

Fourteen commercial honey samples of different botanical origin

(acacia, chestnut, citrus, eucalyptus, multifloral) were stored for up to 18
months at room temperature. Both 5-hydroxymethylfurfural (HMF) and dia-
stase were evaluated and kinetics carried out. The highest HMF increase was
in citrus and eucalyptus honeys at 3 mg/kg/month; the lowest in chestnut at
0.256 mg/kg/month. The highest diastase deactivation was in eucalyptus
honey at 0.485 DU/kg/month; the lowest was in chestnut at 0.258 DU/kg/
month. Honey shelf life was estimated for both indices, HMF and diastase,
using a Bayesian approach. The results show that commercial honey shelf life
depends on botanical origin as well as processing. Except chestnut, all other
honeys showed shorter shelf lives than the declared one (usually 36 months).
The shortest values, 15 months, were for citrus and eucalyptus honeys. The
longest, 20 months, was for acacia and multifloral honeys.

PRACTICAL APPLICATIONS

Both kinetic equations and results of simulations can be used to estimate,

for quality control purposes as well as regulation requirements, the most
probable value of shelf life for a specific honey.

INTRODUCTION

Honey has always been considered a healthy and natural food. Both
consumer expectations and legislation have addressed the highest guarantees
3
Corresponding author. TEL: 0039957580214; FAX: 0039957141960; EMAIL: bfallico@unict.it

Journal of Food Quality (2009) .

DOI: 10.1111/j.1745-4557.2009.00253.x 1
2009 The Author(s)
Journal compilation 2009 Wiley Periodicals, Inc.
2 B. FALLICO, E. ARENA and M. ZAPPALA

for safety, authenticity and quality. The most commonly used quality index
for honey ageing and/or overheating, is 5-hydroxymethylfurfural (HMF)
(Bogadonov et al. 1999). HMF, as well as diastase, has been used for almost
80 years, although the latter has been strongly criticized (White 1992, 1994).
HMF is the most important end product of the early stages of acid-
catalyzed reactions, e.g., hexose dehydration and Maillard reaction (Belitz and
Grosch 1999). Its use to evaluate honey quality is based on the following
assumptions: (1) easily measurable; (2) absent in fresh honey; (3) responsive
in a predictable way to heating and storage; and (4) independent of honey
composition (White 1994).
The highest allowed amount of HMF in honey has been fixed both by
International Bodies (Codex Alimentarius Commission [CAC]; CODEX
STAN 12-1981 [Rev. 2] 2001) and at national level, but with deep differences.
In fact, the CAC includes HMF levels in the additional composition and
quality criteria, suggesting its use in trading transactions. The proposed limit
is 40 ppm, with the exception of honeys of tropical origin (80 ppm).
At the national level, two different approaches have been used. In the first
case, the HMF level has not been included, while in the second there are
meaningful differences between the different areas.
In the U.S.A., honey has no standard of identity, and, even though the
codex standard (Docket #2006P-0101 2006) has been proposed, it is unlikely
the HMF level will be adopted as a mandatory regulation. For the Australian
and New Zealand honey standard (Australian and New Zealand Food Standard
Code 2007), the HMF level has also not been included.
In those nations where HMF has been included in the standards, a third
limit of 15 ppm for low natural enzyme honeys (Diastase < 8 DU) has been
added to the CAC limits.
Argentina (Codigo Alimentario Argentino 1985) and the MERCOSUR
area (Msys As. N. 003, 11.01.95 1995), as well as Canada (Canada Agricul-
tural Product Act 2007), have adopted these limits, but determined postblend-
ing or processing. The most restrictive HMF standards have been adopted by
the European Union (EU) (Directive 2001/110 EC 2001), guaranteeing the
limits up to the Sell by date, usually 36 months.
In the last few years, it has been highlighted that chemical composition
plays a role in the final level of HMF in honey (Anon and Dart 1995; Singh and
Bath 1997, 1998; Bath and Singh 1999; Horn and Hammes 2002; Tosi et al.
2002). The kinetics of HMF development in unifloral honeys, and its depen-
dence from pH of samples, has been investigated (Fallico et al. 2004). The
negative effects of too restrictive an HMF standard in trading some unifloral
honeys have been highlighted (Fallico et al. 2006). Diastase and HMF evolu-
tion were determined in Spanish honeys after their extraction up to 28 months
of storage (Sancho et al. 1992). The evolution of invertase, in honeys of
 HONEY SHELF LIFE 3

different origin, has been studied (Persano Oddo et al. 1999; Sanchez et al.
2001). The effect of heating and filtration on antioxidant activity during
storage of unifloral honeys has been studied (Wang et al. 2004).
Some researchers have proposed the use of furosine and other
2-furoylmethyl amino acids to assess honey heating and/or storage, analogous
to other foods, and a comparison with HMF levels was investigated (Villamiel
et al. 2001; Sanz et al. 2003). Moreover, an interesting approach which mea-
sures dicarbonyls in honey has been proposed by Weigel et al. (2004).
The purpose of this investigation was to determine HMF and diastase
levels in commercial unifloral honeys during storage for up to 18 months
to obtain models in order to estimate the most probable shelf life for each
honey.

MATERIALS AND METHODS

In storage trials, 14 samples in duplicate (1 kg each) of commercial

unifloral honey were obtained in March 2004. Two samples of acacia (Robinia
pseudoacacia L.), four of citrus (Citrus aurantum L.), two of eucalyptus
(Eucalyptus camaldulensis L.), two of chestnut (Castanea sativa L.) and four
of multifloral honey were stored at room temperature (25 2C, 5560% R.H.)
for up to 18 months. A small amount of each sample was regularly analyzed to
determine HMF content and diastase value. Sampling was conducted monthly,
and in Tables 2 and 3 are reported only data that have shown differences from
previous sampling. Sample descriptions (botanical origin, expiry date and
months to expiry) are reported in Table 1.

Chemical Analyses
The following tests were carried out on each sample:
Moisture was determined by measuring refractive indices at 20C by
Carl Zeiss 16531 refractometer (Carl Zeiss, Gottingen, Germany); the corre-
sponding moisture content (%) was calculated by AOAC method (AOAC
1980).
Electrical conductivity was measured at 20C in a 20% (w/v) solution (dry
matter basis) in deionized water by a Delta Ohm HD 8706 conductivity meter
(Louveaux et al. 1970).
Ash was indirectly determined using electrical conductivity and applying
the following equation: X1 = (X2 - 0.143)/1.743, where: X1 = ash value;
X2 = electrical conductivity in mS/cm at 20C (Piazza et al. 1991).
Free acids, lactones, total acidity and pH were measured using a Mettler
Toledo MP 220 pH Meter (Mettler Toledo, Schwerzenbach, Switzerland)
according to the Official Method of the Italian Republic.
4 B. FALLICO, E. ARENA and M. ZAPPALA

Diastase was determined by enzymatic-spectrophotometer, using a

Phadebas Amylase Test Kit (Pharmacia & Upjohn Spa, Milano, Italy).
For each honey, three samples were analyzed in duplicate.

HMF Determination. Five grams of honey was diluted to 50 mL with

distilled water, 0.45 mm filtered and immediately injected into a high pressure
liquid chromatography (HPLC) (Varian 9012Q, Varian, Palo Alto, CA) with a
diode array detector (Varian, Star 330, Varian). The HPLC column was a
Merck Lichrospher, RP-18, 5 mm, 125 4 mm (Merck, Milan, Italy), fitted
with a guard cartridge packed with the same stationary phase. The HPLC
conditions were the following: isocratic mobile phase, 90% water at 1% acetic
acid and 10% methanol; flow rate, 0.7 mL/min; injection volume, 20 mL. All
the solvents were HPLC grade (Merck). The wavelength range was 220
660 nm and the chromatograms were monitored at 285 nm. The compound
was identified by splitting the honey peak with an HMF standard (Sigma-
Aldrich, Milan, Italy), and by comparing the HMF standards spectrum with
those of the honey samples. HMF was determined from an external calibration
curve, with the signal at l = 285 nm. Five grams of honey proved to be the
optimal minimum, using the Ingamells e Switzer equation (Mannino 2001).
Three samples were analyzed per honey in duplicate: each honey HMF is
therefore the average of six values.

Kinetics and Shelf Life Estimation

Average kinetic equation, using data from Tables 2 and 3, was carried out.
In order to verify if the obtained equation was similar to the equation calcu-
lated for each sample, t-tests were carried out. In only the case of citrus was the
average equation different (t > tcr) than that of a single sample (citrus 3),
notwithstanding it was not excluded.
Honey shelf life estimates were carried out according to Singh (1994).
Zero-order kinetics were applied to both HMF and diastase evolution. In
order to verify if K values for HMF and diastase, respectively, were signifi-
cantly different, a Duncan test was carried out (P < 0.05). Moreover, in order
to keep in account the uncertainty in shelf life estimation, for instance as
a result of the variability in honey composition, a probabilistic approach
(Monte Carlo) was used. It uses random sampling of each probability dis-
tribution to produce a very high number of possible scenarios. This method
is based on Bayesian inference and has three fundamental steps: (1) deter-
mining a prior estimate (Input) of the parameter in the form of a confidence
distribution; (2) finding an appropriate likelihood function of the observed
data; and (3) calculating the posterior estimate of the parameter by multi-
plying prior distribution and likelihood function, then normalizing to Output
 HONEY SHELF LIFE 5

a distribution of confidence (Vose 2001). Simulations were run using the

@Risk 4.5 software (Palisade Inc., Palisade Europe, England). This method,
because of the high number of iterations (e.g., 5,000), allows obtaining: a
very confident value of the best estimate shelf life and all the possible
values, each with a probability, between a confidence level.
Two possible scenarios were hypothesized, first, by a honey producer
who can measure HMF and diastase levels, and can therefore fix the sell by
date each product. As Inputs were used, the HMF and Diastase distributions.
For each simulation were used a minimum of 50 up to 150 Inputs. Each
distribution was obtained by fitting six analyses using data from Tables 2 and
3, respectively. Simulation settings are reported in Box 1 and results are in
Tables 57.
BOX 1.
SIMULATION SETTINGS

Software: @RISK 4.5 (Palisade Inc.)

Number of simulations for each honey: 1
Number of iterations in each simulation: 5,000
Sampling: Latin Hypercube
Inputs: [HMF]T or [Diastase]T
Output: months (shelf life)
Models
Acacia
(1a) Diastase (limit 8 DU): (RiskNormal [{Diastase}T - 8; 0.4] + 0.64)/0.48
(2a) HMF (limit 15 ppm): (RiskNormal [15 - {HMF}T; 0.6] + 1.66 )/1.96
(3a) HMF (limit 40 ppm): (RiskNormal [40 - {HMF}T; 0.6] + 1.66)/1.96
Chestnut
HMF (limit 40 ppm): (RiskNormal [40 - {HMF}T; 0.1] + 0.72)/0.756
Citrus
(1c) Diastase (limit 8 DU): (RiskNormal [{Diastase}T - 8; 0.5] + 0.64)/0.30
(2c) HMF (limit 15 ppm): (RiskNormal [15 - {HMF}T; 0.7] - 0.41)/2.83
(3c) HMF (limit 40 ppm): (RiskNormal [40 - {HMF}T; 0.7] - 0.41)/2.83
(4c) Diastase (8 DU) and HMF (15 ppm): Minimum value coming from Equation 1 or 2
Eucalyptus
HMF (limit 40 ppm): (RiskNormal [40 - {HMF}T; 1.0] - 2.19)/3.31
Multifloral
HMF (limit 40 ppm): (RiskNormal [40 - {HMF}T; 0.3] + 0.26)/1.75

In the second scenario, the producer cannot measure HMF and diastase
levels, but still has to fix the honey shelf life, at least noting the floral origin.
In this case, the Input distributions were obtained from the shelf-life values (in
months) from the formulas in Table 4. Multiflower sample 4, being already
illegal (HMF0 > 40 ppm), was not included in the Inputs of any simulation,
while citrus 2 (HMF0 = 29 ppm) was not considered as an Input of the second
simulation. All Inputs were normally distributed.
6 B. FALLICO, E. ARENA and M. ZAPPALA

RESULTS AND DISCUSSION

Table 1 reports data on the producer, scheduled (total and residual) shelf
life at the beginning of storage and chemical parameters (moisture, ash, con-
ductivity, pH, free acidity, lactones and total acidity) of the honeys. The data
show that independent of botanical origin, producers assign 36 months as
product shelf life and one even assigning 48 months to a multifloral honey
(Multifloral 2). The average moisture value is 17%, with the highest being
19.4% in citrus 4, close to the 20% limit. Ash, conductivity, pH, free and total
acidity, as well as lactones, agree with values reported in the literature for each
honey (Persano Oddo et al. 1995).
HMF levels, both initial and evolution values during 18 months of
storage, are reported in Table 2. During the first 4 months of storage, no
variation of HMF concentration was observed (data not shown). The two
acacias have 13.3 and 5.9 ppm of HMF. The HMF level in citrus honeys ranges
between 5.6 (citrus 4) and 28.7 ppm (citrus 2). The chestnut honeys, as well as
eucalyptus, show the lowest initial HMF (0.1 ppm), while the multifloral show
an HMF ranging from 10.4 to 17.4 ppm, excepting multiflower 4 which
showed the highest value (74.8 ppm). Even though multiflower 4 had been
packaged no longer than 4 months, it could be declared illegal.
In all but the chestnuts and acacia 2, HMF was higher after 18 months
than 40 ppm (Table 2). Data confirm that HMF does not depend exclusively on
storage time and temperature, but also on the honeys chemical parameters and
botanical origin (Fallico et al. 2004). Moreover, the observed HMF increases
during storage at room temperature, is not regular and is quite different from
that during heating (>50C), where HMF in successive samplings was always
higher than the previous one (Fallico et al. 2004). Final HMF in many food
products is considered as the equilibrium between formation and degradation
pathways, depending on pH, temperature and the presence and concentration
of analytes (Belitz and Grosch 1999). Usually, in those foods where HMF is
used as a thermal or ageing index, HMF degradation is not considered.
Data in Table 2 show that, at the studied temperatures, HMF in honey is not
merely an accumulation as a result of formation reactions, but the macroscopic
result of formation and degradation reactions. From a practical point of view,
this has at least two consequences: first, in estimating product shelf life; second,
in establishing when a product is illegal or not. In fact, in many cases, after 10
months of storage, HMF levels were near or higher than 40 ppm. But, the same
samples analyzed a few months later conformed to regulations (Table 2).
Evolution of Diastase
Table 3 reports diastase values for each honey up to 18 months of storage.
In all samples, the initial diastase agrees with the average values reported by
 TABLE 1.
CHEMICAL PARAMETERS OF HONEY SAMPLES

Sample Producer Scheduled Months Moisture Ash Conductivity pH Free acidity Lactones Total
shelf life to expire acidity

% % ms/cm meq/kg meq/kg meq/kg

Acacia 1 E 36 32 17.40 0.01 0.003 0.001 0.148 0.001 3.38 0.02 19.9 0.48 4.2 0.38 24.1 0.51
Acacia 2 D 36 32 17.00 0.01 n.d. 0.130 0.002 3.55 0.03 13.3 0.29 4.3 0.82 17.6 0.93
Chestnut 1 D 36 32 18.00 0.12 0.688 0.002 1.343 0.004 4.98 0.02 17.3 0.29 6.5 0.53 23.7 0.60
Chestnut 2 E 36 32 17.90 0.12 0.929 0.006 1.587 0.002 5.84 0.04 11.4 0.25 5.2 0.53 16.5 0.36
Citrus 1 D 36 32 16.60 0.01 0.046 0.002 0.224 0.003 3.46 0.01 26.3 0.35 4.2 0.46 30.4 0.11
Citrus 2 D 36 32 17.20 0.01 0.120 0.001 0.352 0.001 3.46 0.01 29.8 0.35 3.9 0.92 33.6 1.27
Citrus 3 E 36 32 19.10 0.14 0.047 0.001 0.226 0.002 3.49 0.03 26.0 0.01 5.0 0.32 31.0 0.91
Citrus 4 E 36 32
HONEY SHELF LIFE

19.40 0.01 0.050 0.004 0.230 0.008 3.43 0.01 27.0 0.01 4.7 0.82 31.7 0.82
Eucalyptus 1 D 36 32 15.45 0.17 0.209 0.002 0.507 0.004 3.68 0.01 29.3 0.50 4.8 0.84 34.1 1.08
Eucalyptus 2 E 36 32 16.50 0.35 0.235 0.001 0.553 0.002 3.66 0.01 34.1 0.25 4.5 0.53 38.6 0.36
Multifloral 1 A 36 27 17.05 0.10 0.129 0.001 0.367 0.001 3.92 0.08 24.1 0.85 5.5 0.84 29.6 0.94
Multifloral 2 B 48 44 15.80 0.01 0.061 0.01 0.250 0.001 3.70 0.03 22.9 0.25 6.0 2.08 28.8 2.16
Multifloral 3 C 36 32 16.85 0.06 0.550 0.004 1.101 0.008 4.38 0.03 32.5 0.41 6.1 0.84 38.6 1.04
Multifloral 4 D 36 32 18.03 0.10 0.178 0.002 0.453 0.004 3.76 0.04 38.3 0.29 5.2 0.53 43.4 0.76

n.d., not detected.

7
 8

TABLE 2.
EVOLUTION OF HYDROXYMETHYLFURFURAL (mg/kg) IN HONEYS DURING STORAGE AT ROOM TEMPERATURE

Months Honey
of
storage Acacia Citrus Chestnut Eucalyptus Multifloral

1 2 1 2 3 4 1 2 1 2 1 2 3 4

0 13.3 0.0 5.9 0.0 12.1 0.1 28.7 0.0 6.5 0.3 5.6 0.2 0.1 0.01 n.d. 0.1 0.0 0.1 0.0 11.7 0.3 10.4 0.3 17.4 0.3 74.8 0.7
5 24.6 0.5 15.9 0.1 24.3 0.1 60.4 0.6 9.7 0.3 9.9 0.8 4.9 0.5 0.1 0.01 23.9 2.5 4.7 0.9 15.0 0.8 13.1 2.1 13.9 1.5 103.2 4.2
6 26.6 1.1 18.0 0.3 25.0 0.0 60.2 2.0 22 0.4 21.3 0.9 10.2 0.1 0.1 0.01 53.0 1.8 21.0 0.5 24.7 1.6 28.6 1.4 30.7 0.9 107.6 2.5
8 25.7 1.0 18.2 0.6 25.5 0.5 59.7 1.0 23.9 0.8 22.8 0.8 10.3 0.3 0.1 0.01 47.7 2.8 9.0 1.2 27.0 0.6 27.3 0.7 32.9 0.7 119.8 0.9
10 38.1 0.7 21.2 3.7 33.7 1.0 89.5 1.0 32.8 0.9 32.9 1.9 9.0 0.8 0.1 0.01 73.0 2.4 26.3 0.6 33.5 1.3 35.2 1.5 39.0 2.6 174.7 6.2
12 32.2 1.4 31.9 0.6 29.7 0.8 79.8 3.9 27.4 2.8 28.1 2.9 9.1 0.0 0.1 0.01 68.7 1.0 27.9 0.4 31.7 1.1 31.5 2.0 27.0 1.7 177.1 3.5
14 55.1 3.5 33.9 2.9 59.7 1.2 92.7 5.7 49.1 4.6 49.5 4.2 10.0 0.3 0.1 0.01 64.6 12.1 33.4 1.3 51.2 2.5 46.7 3.0 51.0 3.0 209.7 7.1
15 34.2 0.2 26.0 0.1 39.4 0.6 63.2 2.6 34.0 0.4 33.6 1.3 12.1 0.7 0.1 0.01 44.5 0.4 24.0 0.6 30.9 1.1 36.7 0.8 36.9 1.6 127.7 5.0
17 46.3 0.6 27.9 1.7 47.0 1.4 89.4 1.1 37.6 2.0 45.5 0.5 15.4 0.5 2.1 1.33 68.5 1.4 41.4 0.6 33.2 1.7 41.7 1.4 44.5 0.8 142.8 3.4
18 66.7 4.0 36.8 3.8 61.7 1.4 108.7 0.8 57.4 2.0 58.7 2.6 20.1 0.8 5.8 0.14 83.8 2.4 49.7 2.6 43.9 1.2 45.0 2.0 54.3 2.3 179.2 3.6
B. FALLICO, E. ARENA and M. ZAPPALA

n.d., not detected.

 TABLE 3.
EVOLUTION OF DIASTASE (DU/kg) IN HONEYS DURING STORAGE AT ROOM TEMPERATURE

Months Honey
of
storage Acacia Citrus Chestnut Eucalyptus Multifloral

1 2 1 2 3 4 1 2 1 2 1 2 3 4

0 7.7 0.6 14.8 0.7 13.3 1.2 10.7 1.0 10.0 0.2 13.3 0.1 18.7 1.0 23.0 1.0 22.6 0.2 33.0 1.1 20.2 2.1 20.0 1.4 22.0 0.8 13.7 1.0
8 7.9 0.8 14 0.8 12 0.9 9.5 0.0 10.1 0.3 12.0 0.3 15.7 1.0 21.1 0.9 18.9 0.7 28.0 1.2 20.0 1.6 18.6 1.3 18.9 1.1 13.6 0.5
HONEY SHELF LIFE

12 8.1 0.8 10.2 0.8 8 0.6 7.8 0.6 9.7 0.9 12.3 0.5 15 1.1 20.0 1.2 18.2 0.5 23.8 0.8 16.3 0.8 15.2 0.6 18.3 0.5 13.0 1.1
17 7.1 0.6 7.5 0.6 5.8 0.6 6.4 0.5 8.7 0.3 8.7 0.7 14.3 1.0 18.7 0.9 17.5 0.5 20.2 0.8 13.4 0.7 12.4 0.6 17.5 0.5 12.5 0.6
18 6.7 0.6 6.4 0.6 5.4 0.5 5.9 0.4 7.9 0.7 7.7 0.3 13.8 0.9 18.3 1.1 16.8 0.7 18.7 0.6 12.8 0.9 11.6 0.4 16.7 0.4 11.9 0.3
9
10 B. FALLICO, E. ARENA and M. ZAPPALA

Persano Oddo et al. (1995). During the first 8 months of storage, no variation
of diastase value was found (data not shown).
In acacia honeys, although initial diastase varies, the final value is very
similar after 18 months. Initial diastase in citrus honeys is on average
11.8 D.U, yet, while remaining constant during the first 812 months of
storage (Table 3), it then decreases to lower values (6.7 DU). The high levels
of diastase activity in some honeys (chestnut, eucalyptus and multifloral)
decrease linearly, yet, not one of the samples is lower than 11 D.U. (Table 3)
after 18 months of storage.

Kinetics
Tables 2 and 3 data (HMF and diastase) were used for kinetics. In both
cases, zero-order models were chosen, and, for each sample, the linear equa-
tion and K values were calculated. Table 4 reports the kinetic constants of
HMF accumulation and diastase deactivation and the results of Duncan test.
The HMF kinetic at room temperature, the highest value, at about 3 ppm of
HMF per month, must be ascribed to the citrus and eucalyptus samples. Acacia
and multifloral follow with 2 and 1.8 ppm per month, respectively. The chest-
nut samples showed the lowest values with 0.76 and 0.26 ppm of HMF per
month. As they were very different from each other, both the correspondent

TABLE 4.
KINETICS OF HYDROXYMETHYLFURFURAL (HMF) FORMATION AND DIASTASE
DEACTIVATION AT ROOM TEMPERATURE IN HONEYS

HMF

Samples K (mg/kg/months) Linear equation R2

Acacia (bc) 1.960 0.563 Months = (D[HMF]) + 0.1)/1.96 0.8333

Citrus (cd) 2.830 0.710 Months = (D[HMF]) - 0.41)/2.83 0.8121
Chestnut (a) 0.756 Months = (D[HMF] + 0.72)/0.756 0.8332
0.256* Months = (D[HMF] + 0.082)/0.256 0.7576
Eucalyptus (d) 3.310 1.020 Months = (D[HMF] - 2.19)/3.31 0.8916
Multifloral (b) 1.752 0.227 Months = (D[HMF] + 0.2558)/1.752 0.8557
DIASTASE
K (DU/kg/months)
Acacia (bc) 0.485 0.142 Months = (D[Diastase] + 1.117)/0.485 0.8982
Citrus (ab) 0.299 0.152 Months = (D[Diastase] + 0.640)/0.299 0.9141
Chestnut (a) 0.258 0.001 Months = (D[Diastase] - 0.154)/0.258 0.9915
Eucalyptus (c) 0.515 0.202 Months = (D[Diastase] - 0.162)/0.515 0.9918
Multifloral (abc) 0.324 0.179 Months = (D[Diastase] + 0.515)/0.324 0.9492

* K value calculated using time zero and the last three samplings (0, 15, 17, 18 months).
Different superscripted letters in parentheses mean a significant difference (Duncan test P < 0.05).
D[HMF] = (HMF - HMF0); D[Diastase] = (DU0 - DU).
 HONEY SHELF LIFE 11

kinetic equations are reported in Table 4. The Duncan test shows significant
differences among honeys of different botanical origin, as concerns KHMF
values, with exception of acacia from citrus and acacia from multifloral.
The kinetics of diastase deactivation show K values ranging from 0.258 in
chestnut up to 0.515 in eucalyptus honey. The Duncan test did not show
significant difference among honeys with the exception of chestnut from
eucalyptus.

Prediction of Honey Shelf Life

The development of models and simulation conditions was described in
materials and methods. Tables 5 and 6 report months of honey shelf life using
40 ppm HMF as both the high and low natural diastase level. Each table
reports the most likely, the lowest acceptable (5% P) and highest acceptable
(95% P) values, the scheduled shelf life and the difference between the
scheduled and estimated one.
The data in Table 5 show that the most likely shelf life for the two
chestnut samples is 57.7 and 164 months. It also shows that at worst, the two
samples will break the law after 55 and 156 months. Although the shelf life
estimates are quite far from time zero and do not take into account self-
accelerating effects (Fallico et al. 2004), it is evident that the measured shelf
life does not agree with the scheduled one. In fact, in the first sample, the HMF
limit of 40 ppm will be achieved 21 months later than the scheduled one, while
in the second sample, the limit is achieved almost 10 years later. As the
dehydratation of hexose is an acid-catalyzed reaction (Belitz and Grosch
1999), the higher the honey pH (Table 1), the longer the time to achieve the
HMF limit. Thus, 40 ppm of HMF, as a quality index of chestnut honey, does
not seem appropriate.

TABLE 5.
COMPARISON BETWEEN MEASURED AND SCHEDULED SHELF LIFE OF HONEYS AT
HIGH DIASTASE LEVEL

Sample Estimated shelf life (months) Scheduled shelf Difference

life (months) (months)
Most likely Minimum Maximum
value

Chestnut 1 57.7 54.9 60.6 36 +21.7

Chestnut 2 164 155.8 172.9 36 +128
Eucalyptus 1 15.4 14.7 16.1 36 -20.6
Eucalyptus 2 15.4 14.7 16.1 36 -20.6
Multifloral 1 25.3 24.8 25.7 36 -19.5
Multifloral 2 19.5 18.3 20.6 36 -28.5
Multifloral 3 17.2 16.0 18.4 36 -18.8
12 B. FALLICO, E. ARENA and M. ZAPPALA

TABLE 6.
COMPARISON BETWEEN MEASURED AND SCHEDULED SHELF LIFE OF HONEYS AT
LOW DIASTASE LEVEL

Sample Estimated shelf life (months) Scheduled shelf Difference

life (months) (months)
Most likely Minimum Maximum
value

Acacia 1 17.7 16.9 18.4 36 -18.3

Acacia 2 21.4 20.7 22.2 36 -14.6
Citrus 1 13.7 13 14.5 36 -22.3
Citrus 2 7.8 7.1 8.6 36 -28.2
Citrus 3 15.7 14.9 16.5 36 -20.3
Citrus 4 15.9 15.1 16.8 36 -20.0

As for eucalyptus, although from two different producers (Table 1), they
show identical shelf lives and very narrow distributions. The most likely value
was 15.4 months (Table 5), but values between 14.7 and 16.1 months should
be acceptable.
The multifloral shelf life is the following: the longest shelf life is multi-
floral 1 with 25.3 months, together with a minimum of 24.8 and a maximum
of 25.7 months. Multifloral 2 is 19.5 months, with acceptable ranges of 18.3
and 20.6 months. Multifloral 3 shows the lowest shelf life with 17 months as
the most likely value, and 16 and 18.4 months as the minimum and maximum
values. As expected, the lower the initial HMF (Table 2), the longer the shelf
life (Multifloral 1 and 2).
Data in Tables 2 and 3 highlight that, with the exception of chestnut
honeys, 15 ppm of HMF were reached after only a few months of storage,
when the average diastase level was still higher than 8 DU. Moreover, data in
Tables 6 and 7 confirm this assumption. Table 6 shows that the most likely
acacia shelf life is 17.7 and 21.4 months, assuming 40 ppm limit of HMF. The
lowest acceptable values are almost 17 and 21 months, while about 18 and 22
months are the longest acceptable shelf lives for acacia 1 and 2, respectively.
Citrus shelf life, assuming the same HMF limit, does not exceed
16 months (citrus 3 and 4), with the highest acceptable value being almost
17 months for the same samples. This data show that shelf life declines
very quickly, up to less than 8 months (citrus 2), when the starting HMF level
is high (Table 2). Also for citrus honeys, there is a marked difference
between experimental and scheduled shelf life. The difference is always higher
than 20 months, up to 28 months for citrus 2 (Table 4).
In citrus honeys, a linear correlation (months = -0.3554 [HMF]0 + 18,
R2 1) has been highlighted between the initial HMF value and the estimated
shelf life. This correlation can extrapolate the shelf life of citrus honeys simply
 HONEY SHELF LIFE 13

TABLE 7.
ACACIA AND CITRUS SHELF LIFE USING 15 ppm OF HYDROXYMETHYLFURFURAL
(HMF) OR 8 DU OF DIASTASE AS LIMITS

Sample Estimated shelf life (months) Estimated shelf life (months)

15 ppm of HMF 8 DU of diastase

Most likely value Minimum Maximum Most likely value Minimum Maximum

Acacia 1 4.9 4.8 5.1 0 0 0

Acacia 2 8.7 8.5 8.9 20.3 17.6 23.0
Citrus 1 4.9 4.7 5.1 23.8 17.0 30.7
Citrus 2 7.8 7.1 8.6 15.1 9.2 21.0
Citrus 3 6.9 6.6 7.1 12.8 10.3 15.3
Citrus 4 5.2 7.0 7.4 23.8 21.6 26.1

by measuring the HMF at time zero (e.g., after processing). Moreover, it is

clear that the longest shelf life in citrus honeys does not exceed 18 months,
even in the best case (HMF0 = 0).
Table 7 reports the estimated shelf life of low diastase honeys using
15 ppm of HMF or 8 DU of diastase as limits. This can be the case of such a
honey in the EU area (Directive 2001/110/EC 2001). Data show that both
acacia and citrus reach 15 ppm of HMF very quickly after 58 months, with
the highest values at 9 months only for acacia 2.
Concerning honey shelf life with a diastase index (8 DU as limit), the
models show broad ranges, meaning great uncertainty in shelf life prediction.
For instance, citrus 1 and 2 have 24 and 15 months as the most likely values
of shelf life with acceptable ranges of 1731 and 921 months, respectively.
The shortest shelf life, excepting acacia 1, which has diastase lower than 8
from the start, was citrus 3 (12.8 months).
Citrus honeys, as well as for HMF, revealed a linear correlation
(months = 3.3375 [Diastase]0 - 20.591, R2 1) between the initial level of
diastase and shelf life. For instance, a honey having an 11 DU [Diastase]0 will
not last 16 months of storage (DU < 8); so, in order to guarantee diastase
higher than 8 DU for up to 24 months, its initial value cannot be lower than
1314 DU.
In practice, the data on low diastase honeys previously presented, high-
light that the link between diastase and HMF, to assess honey shelf life,
amounts to doubling the costs without improving the knowledge about the
quality of honeys. This becomes particularly important in the EU area where
both limits have to be maintained up to the last sell-by date.
Simulation results to estimate, not the shelf life of single samples, but of
honeys of different floral origin, e.g., acacia, citrus, eucalyptus and multifloral,
14 B. FALLICO, E. ARENA and M. ZAPPALA

TABLE 8.
ESTIMATION OF HONEYS SHELF LIFE USING 40 ppm OF
HMF AS LIMIT

Honey Estimated shelf life (months)

Most likely value Minimum Maximum

Acacia 19.5 16.0 23.0

Citrus 15.0 13.0 17.0
Eucalyptus 15.4 14.7 16.2
Multifloral 20.3 14.7 26.0

FIG. 1. DISTRIBUTION OF CITRUS HONEY SHELF LIFE USING

HYDROXYMETHYLFURFURAL (HMF) AND DIASTASE AS INDICES

are reported in Table 8 and in Fig. 1, respectively. The most likely shelf life of
acacia is 19.5 months, ranging between 16 and 23. The most probable shelf life
of citrus is 15 months, ranging between 13 and 17. For eucalyptus, it is
between 1516 months, while the most likely for multiflower is 20.3 months,
with a range between 14.7 and 26. Comparing these data with the shelf life for
each sample, Tables 5 and 6 show shorter values and larger ranges.
Lastly, for citrus honey, to take into account the diastase contribution to
honey shelf life, a simulation was run at the same time, using: 40 ppm of HMF
or 8 DU of diastase. The software was required to the output of the lowest shelf
life (derived from HMF or diastase, respectively) for each iteration (5,000).
 HONEY SHELF LIFE 15

The regression sensitivity analysis showed that both indices contribute to shelf
life, with the coefficients for HMF and diastase being 0.618 and 0.537. The
results are summarized in Fig. 1.
The most likely value moves to shorter value (14.4 months), as well as the
lowest and the highest acceptable values, 11.8 and 16.4 months, respectively.
Moreover, the output distribution is negatively skewed (-0.763) which means
that some samples could be outlaw after a few months of storage (Fig. 1).
The data have shown that when honey producers label shelf life, they do
not usually take into account product characteristics, but instead label them
according to habits and customer requirements. With the exception of chestnut
honeys, all the other samples were out of law before the end of scheduled shelf
life. The 36 months of honey shelf life are usually requested or imposed on
producers by shopping house chains. But, as a consequence, this means to not
have conformed products up to the end of shelf life. Moreover, data on low
diastase honeys show that when only HMF is the quality index, it is a good
model to estimate shelf life. The addition to the models of diastase simply
increases the uncertainty associated to the shelf life estimation.

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