food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 299–306

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Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Effect of high hydrostatic pressure applied to a

Mexican honey to increase its microbiological and
functional quality

Diana E. Leyva-Daniel a , Zamantha Escobedo-Avellaneda b ,

Fidel Villalobos-Castillejos a , Liliana Alamilla-Beltrán a ,
Jorge Welti-Chanes b,∗
a Departamento de Graduados e Investigación en Alimentos, Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional, Departamento de Bioquímica, Wilfrido Massieu s/n, Col., Unidad Profesional Adolfo López
Mateos, CP 07738, Gustavo A. Madero, México
b Tecnológico de Monterrey, Escuela de Ingeniería y Ciencias, Centro de Biotecnología FEMSA, Eugenio Garza Sada

Ave. 2501 Sur, CP 64849 Monterrey, NL, México

a r t i c l e i n f o a b s t r a c t

Article history: High hydrostatic pressure (HHP) applied at 600 MPa (0, 2, 5, 8, 12 and 15 min) was used as an
Received 13 March 2016 alternative method to reduce the microbial population, preserving bioactivity (antioxidant
Received in revised form 22 activity (AOA), vitamin C, total phenolic and carotenoid contents) and other quality factors
December 2016 (diastase activity, 5-hydroxymethylfurfural, fructose and glucose contents and rheological
Accepted 4 January 2017 behavior) of a Mexican honey. HHP processing reduces viable microorganisms in honey and
Available online 31 January 2017 the treatment for 15 min reduced the total microbial count below detection limit. AOA and
total phenolic content were increased by 30% and 6%, respectively, after 2 min processing.
Keywords: The vitamin C, fructose, glucose and maltose contents in HHP treated honey were retained
High hydrostatic pressure at all processing times. The total carotenoid content was maintained at processing times up
Mexican honey to 8 min, while violaxanthin content was reduced by 56% after 15 min. HHP did not change
Microbiological flora the contents of 5-hydroxymethylfurfural, while the diastase activity decreased by 10% after
Functional properties 12 min treatment. Honey exhibited a Newtonian behavior and its viscosity was decreased
Quality parameters at all HHP processing conditions. HHP treatment at 600 MPa for 2 min offers an opportunity
Rheological behavior to enhance the antioxidant properties of honey while reducing its microbiological load and
preserving its the general quality parameters of honey.
© 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction tal factors. It has been used with different purposes and has a great
potential to serve as a natural food antioxidant in which phenolic acids
Honey is a natural product produced from the nectar and exudation and flavonoids, certain enzymes (glucose oxidase, catalase), ascorbic
of plants by honeybees (Apis mellifera). The natural honey contains acid, Maillard reaction products, amino acids and proteins contribute
about 200 substances, mainly fructose and glucose, with phenolic com- to this activity (Alvarez-Suarez et al., 2010a,b; Gheldof and Engeseth,
pounds, minerals, proteins, free amino acids and vitamins as minor 2002).
components (Küçük et al., 2007). The composition of honey varies Honey is usually heated to 60 ◦ C or above to inactivate microorgan-
depending on the floral source, processing, seasonal and environmen- isms, facilitate packing and delay crystallization (Kaur Bath and Singh,
1999; Tosi et al., 2004). Heating has a negative effect on honey due to the
loss of compounds, which give its specific aroma, flavor and some of its
∗
Corresponding author.
E-mail addresses: jorge.welti@yahoo.com, jwelti@itesm.mx (J. Welti-Chanes).
http://dx.doi.org/10.1016/j.fbp.2017.01.001
0960-3085/© 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
300 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 299–306
biological activity; the degree of losses are proportional to the temper-
ature and duration of the thermal treatment applied (Tosi et al., 2004).
The damage caused by heating can be evidenced by measuring dias-
tase activity and hydroxymethylfurfural content (HMF) (Bogdanov et al.,
1999). Diastase, one of the most important honey enzymes, can break
down glycosidic linkages in oligo- and polysaccharides. The activity of
this enzyme decreases with the time of storage and heating, being an
important parameter to determine quality of honey. HMF is a cyclic
aldehyde formed by fructose and glucose dehydration in an acid media
as an intermediate in the Maillard reaction (Rizelio et al., 2012; Tosi
et al., 2002). Several factors influence HMF level, such as temperature
and time of heating, storage conditions, pH and floral sources, so it
indicates overheating and inadequate storage (Fallico et al., 2006).
The consumer awareness for healthier foods, has stimulated the
food industry to explore novel potential alternative technologies to
inactivating microorganisms avoiding the negative effects of thermal
processing on organoleptic, nutritional and, functional properties of
foods. The extensive research on HHP processing has originated new
opportunities to improve the balance between the safety and quality of
food products (Vervoort et al., 2012). Due to the non-thermal nature of
HPP processing, it potentially produces high quality food with ‘fresh-
like’ characteristics and improved functionalities (Prasad et al., 2010;
Fauzi et al., 2013).
It has been reported that HHP can increase the total phenolic com-
pounds and AOA of Manuka honey (Akhmazillah et al., 2013; Al-Habsi
and Niranjan, 2012; Fauzi et al., 2013). Quality changes in terms of color
and viscosity when HPP is combined with thermal treatment, have also
been reported (Fauzi et al., 2013). Likewise, changes in minor com-
ponents in Manuka honey such as methylglyoxal and non-peroxide
antibacterial activity (MGO) have been studied after HHP application
(Al-Al-Habsi and Niranjan, 2012; Grainger et al., 2014).
Honey production in Mexico has very long traditions going back to
ancient times; however, its composition and functional properties have
not been studied comprehensively. Mexico is the ninth largest producer
in the world with an annual production of 56 907 t of honey, and it is
also the world’s third leading exporter of honey (FAO, 2013).
The aim of this study was to investigate the effect of HHP at 600 MPa
after different processing times (0, 2, 5, 8, 12, and 15 min) on the
microbial population and contents of total phenolic (TPC), carotenoids,
vitamin C, and AOA of a Mexican honey. Quality parameters such as
HMF, diastase number, sugar content and rheological behavior were
also evaluated.
2. Material and methods
2.1. Raw material
Honey (multifloral) obtained from Guerrero State (25 kg),
Mexico (D’Zarzas S.A. de C.V.) was used in this study (pH of
3.80 ± 0.06, 80.0 ± 0.1◦ Brix, and aw 0.589 ± 0.070). Honey was
placed in plastic bottles (1 kg) and stored at room temperature
(25 ◦ C) while processed. The Harmonized Methods of the Inter-
national Honey Commission (Bogdanov et al., 2009) were used
to evaluate pH, free acidity (meq acid/kg honey) and mois-
ture (% on wet basis, wb). Determinations were carried out in
triplicate.
The color parameters (L*, a* and b*) were determined in raw
sample filled into a clear glass Petri dish and using a Minolta
colorimeter (CR-10, JAN). The results were expressed in accor-
dance with the CIELAB system with reference to illuminant
D65 and with a visual angle of 2◦ . Three replicate samples were
analysed.
2.2. High hydrostatic pressure treatments
About 40 g of honey was vacuum packed (model EVD 4, Tor-
rey, Monterrey, Nuevo León, Mexico) in 15 × 8 cm polyethylene
bags (Filmpack SA de CV, Guadalupe, Nuevo León, Mexico)
and processed in a 2 L capacity high-pressure food processor
(Avure Technologies, Middletown, OH, USA) employing water
as pressurizing medium and operated at 600 MPa, for different
holding times (0, 2, 5, 8, 12 and 15 min), where 0 min indi-
cates the time required to reach the pressure level (1.8 min), in
where once the pressure level was reached, the samples were
immediately depressurized (decompression time was almost
instantaneous).
Once pressure level was reached, final average tempera-
tures during processing time were 37 ± 1, 37 ± 1, 35 ± 2, 31 ± 1,
30 ± 2 and 28 ± 1 ◦ C at 0, 2, 5, 8, 12 and 15 min, respectively.
Duplicate runs of each treatment were performed.
2.3. Microbial analysis
Microbiological determinations were performed according to
the Mexican normativity (NOM-092-SSA1-1994, 1994; NOM-
111-SSA1-1994, 1994). Ten grams of each honey sample was
homogenized into 90 mL of peptone aqueous solution (10−1 ).
Decimal dilutions were made into the same solution (10−2
and 10−3 ), and 1.0 g (100 ) of honey sample or 1.0 mL of each
dilution was plated into triplicate plates of appropriate agar.
Aerobic mesophilic bacteria were counted on standard plate
count agar (PCA) and incubated at 35 ± 2 ◦ C for 48 h, and plates
with 25–250 colonies were counted. The detection limit of the
method used for aerobic mesophilic bacteria was 25 CFU/g.
The molds and yeast were estimated using potato dextrose
agar medium (pH 3.5 ± 0.1) and incubated at 25 ± 2 ◦ C for 5
days, and plates with 15–150 colonies were counted. The
detection limit of the method used for molds and yeast as
stablished for the normativity was 15 CFU/g. Microbial counts
were expressed as colony-forming units per gram of honey
(CFU/g). Log N was calculated to determine the inactivation
effect, where N is the number of viable microorganisms.
2.4. Quality parameters
2.4.1. Hydroxymethylfurfural (HMF) content and diastase
activity
The HMF and diastase activity or diastase number (DN) was
carried out according to the Harmonized Methods of the Inter-
national Honey Commission (Bogdanov et al., 2009). HMF
content was determined after clarifying samples with Car-
rez reagents (I and II) and addition of 0.2% sodium bisulphate.
Sample solutions were placed in 10 mm quartz cells and the
absorbance was measured using UV/vis spectrophotometer
(Genesys 10S UV-vis, Thermo Scientific, China) at 284 and
336 nm. The readings were expressed as mg HMF/kg honey.
The DN is equivalent to Gothe scale number, to determine it
the honey sample was dissolved in acetate buffer (0.1 M, pH
5.3) and sodium chloride solution (0.5 M). The samples were
placed in test tubes with starch solution (2%) in a water bath
at 40 ◦ C. Changes in color were measured at defined inter-
vals of time in the UV/vis spectrophotometer. The results were
expressed in Gothe units. Determinations were performed in
triplicate.
2.4.2. Quantification of glucose and fructose
An HPLC with IR detection (Waters 2114, Milford, MA, USA)
integrated with an auto sampler including temperature con-
trol for the column (Shodex SH1011 CA, Waters, USA) at 60 ◦ C
was used to determine glucose and fructose. The mobile
phase consisted of 5 mM sulfuric acid at a rate of 0.6 mL/min
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 299–306 301

(Bogdanov et al., 2009). All samples were filtered through a trum was used for identification purposes (Chen et al., 1995;
0.2 ␮m filter. Quantification was performed in triplicate. Escobedo-Avellaneda et al., 2014).

2.4.3. Rheological behavior 2.5.4. Antioxidant activity

Rheological behavior of unprocessed and HHP processed The antioxidant activity was measured using the Oxygen
honey samples was determined by measuring viscosity using Radical Absorbance Capacity (ORAC) technique described by
a Rheometer (Physica MCR 101, Anton Paar, USA, Anton PaarTM Escobedo-Avellaneda et al. (2014). Twenty-five microliters
software). The measuring system consists of a Smart Swap of diluted 1:100 sample in sodium phosphate buffer solu-
geometry with 50 mm, 1◦ steel cone. Honey samples of 2 g tion (75 mM PBS, pH 7.4) was placed in a Cotar polystyrene
were poured onto the sample plate. The rotational speed was black plate with 96 round bottom wells. A microplate reader
increased to provide a shear rate in the range of 0.1–100 s−1 . (Synergy HT, BioTek Instruments, Inc., Bad Friedrichshall,
Shear stress and viscosity were measured and recorded at dif- Germany) was used to automatically dispense 150 ␮L of 1 ␮M
ferent shear rates (Fauzi et al., 2013). Triplicate analysis was fluorescein followed by 25 ␮L of 153 ␮M AAPH (2,2 -azobis
performed at 25 ◦ C. (2-methylpropionamidine) dihydrochloride) after 30 min of
incubation at 37 ◦ C. Fluorescence (485/20 nm excitation wave-
2.5. Functional properties length and 528/20 nm emission wavelength) was measured at
37 ◦ C for 1 h at 2 min intervals. The final ORAC values were
2.5.1. Total phenolic content calculated using the net area under the decay curves. The
Total phenolic content (TPC) was measured using the antioxidant activity was expressed in ␮mol Trolox Equivalent
Folin–Ciocalteau methodology (Meda et al., 2005). Honey (TE) per 100 g of sample on a fresh weight. The experiment was
samples were prepared at concentration of 0.2 g/mL with dis- carried out in triplicate.
tilled water and filtered through filter paper (Whatman No.
1). Results were expressed as mg Gallic Acid Equivalents 2.6. Statistical analysis
(GAE)/100 g of honey sample on a fresh weight. Measurements
were done in triplicate. Results were presented as mean and standard deviation of
at least duplicate samples. One-way analysis of variance
2.5.2. Vitamin C (ANOVA) was used to compare means. Differences were con-
Total (l-ascorbic acid + dehydroascorbic acid) and reduced sidered significant at p < 0.05. The separation of treatment
vitamin C (l-ascorbic acid) were measured as described by means was carried out with Tukey’s honestly significant differ-
Escobedo-Avellaneda et al. (2014) with some modifications. ence (HSD) test. All statistical analyses were performed with
Briefly, 250 mg of honey was mixed with 1.75 mL of 6% ® ®
Statistica, version 11 (Statsoft ) and Microsoft Excel 2013.
trichloroacetic acid (TCA) and then centrifuged. Supernatant
aliquots (50 ␮L) were mixed with 25 ␮L potassium phosphate
3. Results and discussion
buffer (75 ␮M, pH 7.0). For the analysis of reduced vitamin C,
50 ␮L water were added to the mix while for the determina-
3.1. Characteristics of unprocessed honey
tion of total vitamin C, 25 ␮L dl-dithiothreitol (DTT) (10 mM)
were added followed by 25 ␮L N-ethyl-maleimide (NEM) (0.5%)
The physicochemical, microbiological and functional char-
after 10 min incubation at room temperature. For all assays,
acteristics of unprocessed honey are presented in Table 1.
335 ␮L of 33.3/26.7/26.7/13.3% (v/v) mixture of 10% TCA: 43%
The HMF content is widely recognized as a parameter of
H3 PO4 :4% ␣-␣-bipyridyl: 3% FeCl3 were added. After 1 h incuba-
honey freshness and tends to increase due to processing
tion at 37 ◦ C, sample was read at 525 nm in a microplate reader
(Synergy HT, Biotek Instruments, Inc., Bad Friedrichshall,
Germany). A five points calibration curve (R2 = 0.998) in the Table 1 – Functional and quality parameters of
0.1–1 mM l-ascorbic acid (AA) range was used to estimate unprocessed honey.
vitamin C concentrations as mg AA/kg sample fresh weight. Parameter Value
Oxidized vitamin C values were calculated by subtracting
reduced vitamin C from total vitamin C concentration. Physicochemical parameters
HMF (mg HMF/kg honey) 6.9 ± 0.1
Diastase activity (Gothe units) 22.3 ± 0.4
2.5.3. Carotenoids Fructose (g/100 g honey) 40.2 ± 0.7
Honey samples (5 g) were weighed under subdued light, Glucose (g/100 g honey) 34.4 ± 0.6
and then homogenized (Ultraturrax T-25 DS1, IKA, Staufen, Viscosity (Pa s) 4.9 ± 0.1
Germany) at room temperature for 1 min at 15,000 rpm with pH 3.8 ± 0.06
10 mL hexane containing 0.1% butylated hydroxytoluene Free acidity (meq acid/kg honey) 32.5 ± 1.8
Moisture (wb, %) 18.4 ± 0.1
(BHT). The final extract was diluted with isopropanol, passed
L* 28.3 ± 0.12
through a 0.45 ␮m membrane (PTEF), and analyzed (50 ␮L) in a
a* 8.8 ± 0.10
HPLC (1200 series, Agilent Technologies, Inc., Santa Clara, CA, b* 0.59 ± 0.0
USA) operated in reversed phase with photodiode array detec-
Functional properties
tion (PDA) using a 5 ␮m 4.6 × 250 mm YMC carotenoid column.
Total phenol content (mg GAE/100 g 29.89 ± 1.0
Methanol and water in a 96:4 (% v/v) ration was used as phase honey)
A, and 100% methyl tert-butyl ether (MTBE) as phase B. Flow Total vitamin C content (mg AA/kg honey) 36.1 ± 1.4
rate was 1 mL/min using the following gradient: 0 min-5% B, Reduced vitamin C (mg AA/kg honey) 21.7 ± 1.0
5 min-20% B, 20 min-30% B, 30 min-47.5% B, 40 min-47.5% B, Total carotenoids content 0.406 ± 0.0
44 min-100% B, 47 min-100% B. The wavelength of maximum (mg ␤-carotene/100 g honey)
Antioxidant activity (␮g TE/100 g honey) 471.7 ± 14.6
absorption max , and the shape of the visible absorption spec-
302 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 299–306
Table 2 – Effect of HHP (600 MPa) on the reduction levels (Log10 (N0 /N)) of microbial native flora of honey.
Processing time (min) Aerobic mesophiles
Log10 N
Unprocessed 1.2 ± 0.06a
0 0.6 ± 0.03b
2 0.6 ± 0.03bc
5 0.5 ± 0.06bc
8 0.5 ± 0.00bc
12 0.4 ± 0.01c
15 NDd
Data are means ± standard deviation. Means values within a column sharing the same letter are not significantly different (p < 0.05), N: CFU/g,
ND: Not detected.
and/or aging of the product (Fallico et al., 2006). The HMF
content of honey according to international trade should be
under 40 mg HMF/kg, some European bee federations propose
a limit of 15 mg/kg (Bogdanov et al., 1999). The HMF con-
centration in Mexican honey was low in unprocessed honey
(6.9 ± 0.1 mg HMF/kg).
According to Honey Quality and International Regulatory
Standards, from the International Honey Commission, the
diastase activity must not be higher than 8, expressed as
Diastase Number (DN) (Bogdanov et al., 2009). The DN in
unprocessed honey was 22.3 ± 0.4 Gothe units. The values are
higher compared with Manuka honey (13.8 Gothe units) (Al-
Habsi and Niranjan, 2012) and it is in the range reported for
Argentinian multifloral honeys (11.2–25.8 Gothe units) (Tosi
et al., 2008). These differences could be attributed to the botan-
ical origin and geographic region of honeys (Fallico et al., 2006).
Fructose was the predominant sugar (∼40%), followed by
glucose (∼34%) in unprocessed Mexican honey. The sugar
composition of honey depends highly on botanical and
geographical origins, and other factors such as weather, pro-
cessing and storage conditions (Dobre et al., 2012).
The TPC was 29.9 ± 0.1 mg GAE/100 g honey; this content
is the in range of TPC reported by Serbian polyfloral honeys
(3.0–139.0 mg GAE/100 g) (Gašić et al., 2014), Cuban monofloral
honeys (21.4–59.6 mg GAE/100 g honey) (Alvarez-Suarez et al.,
2010b) and it is higher than values reported for Italian honeys
(11.1–14.3 mg GAE/100 g honey) (Perna et al., 2013).
The total vitamin C content in unprocessed honey was
36.1 ± 1.4 mg AA/kg and the reduced vitamin C content was
21.7 ± 1.0 mg AA/kg (Table 1). Higher levels of vitamin C con-
tent were found in unprocessed honey compared to the values
reported for Sardinian honey samples (5.8–0.84 mg l-ascorbic
acid/kg honey) (Ciulu et al., 2011). Perna et al. (2013) reported a
vitamin C content of 5.38 ± 0.5 mg l-ascorbic acid/kg for Italian
multifloral honeys.
Honey used in this study exhibited a Newtonian behavior
with a viscosity of 4.9 ± 0.1 Pa s; this value is within the range
found for Chinese (8.3 Pa s) and Brazilian honey (3.13 Pa s) (Al-
Habsi et al., 2013).
Total carotenoid content was 3.6 ± 0.3 mg ␤-carotene/kg of
honey, this value was similar to the values reported for
monofloral Cuban honeys (1.2–5.6 mg ␤C/kg) (Alvarez-Suarez
et al., 2010a).
The AOA in unprocessed honey was 471.7 ± 14.6 ␮mol
TE/100 g. Gheldof and Engeseth (2002) studied different floral
sources of honey, and reported a range from 300 to 1700 ␮mol
TE/100 g. The AOA of honey in this study is in the range of
AOA reported for different fruits (150–3100 ␮mol TE/100 g fresh
weight) and vegetables (50–1940 ␮mol TE/100 g fresh weight),
Molds and yeasts
Log10 (N0 /N) Log10 N Log10 (N0 /N)
2.4 ± 0.06a
0.61 ± 0.01c 0.6 ± 0.03b 1.7 ± 0.06c
0.64 ± 0.01c 0.4 ± 0.01bc 2.0 ± 0.06bc
0.70 ± 0.03bc 0.3 ± 0.02cd 2.1 ± 0.02ab
0.74 ± 0.04bc 0.1 ± 0.01cd 2.3 ± 0.02ab
0.83 ± 0.01b NDd 2.4 ± 0.00a
1.20 ± 0.0a NDd 2.4 ± 0.00a
(Isabelle et al., 2010; Wang et al., 1996). The comparable antiox-
idant activity between honey, fruits and vegetables indicates
that honey, in addition to be a healthy alternative to replace
sucrose in many products, represents a supplementary source
of dietary antioxidants (Gheldof and Engeseth, 2002).
3.2. Effect of HHP on microbial population
The mean initial populations of total aerobic mesophiles in
unprocessed Mexican honey were 1.7 × 101 ± 0.5 CFU/g and
2.3 × 102 ± 0.9 CFU/g of molds and yeast. Levels of aerobic
mesophiles were lower than those reported by other authors:
Gomes et al. (2010) found similar levels of contamination for
aerobic mesophiles (average 2.1 × 102 CFU/g), while the molds
and yeasts (average 2.2 × 102 CFU/g) counts were similar to
the ones found in the Mexican honey. The primary sources
of microbial contamination are likely to include pollen, the
digestive tracts of honeybees, dirt, dust, air and flowers, and
the microorganisms that survive in honey are those that with-
stand the concentrated sugar, acidity and the antimicrobial
compounds of honey (Olaitan et al., 2007).
Immediately after HHP treatment, the counts of aero-
bic mesophiles and molds and yeast were reduced below
the detection limit stablished by the analysis method used
(<15 CFU/g). The microbial inactivation in Mexican honey pro-
cessed with HHP is presented in Table 2. It can be seen that
the processing of the samples only during the time required
to reach 600 MPa (time 0) generated reductions of 0.6 log10 of
total mesophiles and 1.7 log10 of molds and yeasts. HHP treat-
ment at 600 MPa/12 min reduced 0.8 log10 of total mesophiles
and 2.4 log10 of molds and yeasts. Akhmazillah et al. (2012)
reported that more than 350 MPa for 20 min and temperature
above 40 ◦ C is needed to inactivate microorganisms present
in Manuka honey. The same author, reported that the thermal
treatment (60, 70 and 80 ◦ C for 30 min) is not efficient to reduce
the total microbial count in Manuka honey. The yeast and
molds count was below the standard established by Mexican
normativity (<100 CFU/g) (NMX-F-036-1997, 1997).
The results obtained in this study indicate that all evalu-
ated HHP treatment conditions, without using heat, generate
products with suitable microbiological quality.
3.3. Effect of HHP on quality parameters
3.3.1. HMF content and diastase activity
The HMF content after HHP treatments ranged between
6.9 ± 0.1 to 7.2 ± 0.2 mg HMF/kg. The HMF content in Mex-
ican honey was lower than the allowed maximum limit
of 40 mg HMF/kg as suggested by Codex Alimentarius
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 299–306 303

Table 3 – Effect of HHP (600 MPa) on HMF content, diastase activity, viscosity and sugar content of honey.
Processing time (min) HMF (mg/kg Diastase Viscosity Fructose (F) Glucose (G) Ratio F/G
honey) activity (Pa × s) (g/100 g (g/100 g
(Gothe units) honey) honey)

Unprocessed 6.9 ± 0.1a 22.3 ± 0.4a 4.9 ± 0.1a 40.2 ± 0.7a 34.4 ± 0.6a 1.17
0 7.0 ± 0.1a 22.2 ± 0.1ab 4.9 ± 0.1ab 40.4 ± 0.4a 34.2 ± 0.4a 1.18
2 6.9 ± 0.2a 21.3 ± 0.3b 4.3 ± 0.2d 39.7 ± 0.5a 33.4 ± 0.4a 1.19
5 6.9 ± 0.1a 20.0 ± 0.7d 4.5 ± 0.1cd 40.0 ± 0.6a 33.8 ± 0.5a 1.18
8 7.2 ± 0.2a 20.1 ± 0.6d 4.6 ± 0.1bc 39.9 ± 0.8a 33.7 ± 0.7a 1.18
12 7.2 ± 0.1a 20.8 ± 0.6cd 4.6 ± 0.1c 40.2 ± 0.2a 33.9 ± 0.1a 1.19
15 7.2 ± 0.1a 22.3 ± 0.4a 4.6 ± 0.1bc 40.9 ± 1.1a 34.5 ± 1.0a 1.19

Data are means ± standard deviation. Means values within a column sharing the same letter are not significantly different (p < 0.05).

(CODEX STAND, 1999) and Council European Union Directive
(15 mg HMF/kg) (COUNCIL DIRECTIVE, 2002). According to the
statistical analysis, no significant differences were observed
after HHP processing of honey (Table 3). This would indi-
cate that treatment with HHP did not change the content
of HMF, maintaining a high quality product in accordance
with international limits. Likewise, for Manuka honey has
been reported a range from 23.8 to 24.1 mg/kg when HHP is
applied from 100 to 800 MPa during 15 and 160 min (Al-Habsi
and Niranjan, 2012). On the contrary, when honey is treated
with microwave (1.3 W/g, 6 min) or heating (90 ◦ C/60 min) the
HMF concentration of honey increased around 25 and 75%,
respectively (Kowalski, 2013). Fauzi and Farid (2014) found
that when HHP (600 MPa for 10 min) is combined with ther-
mal treatment (70 ◦ C) the HMF content in Manuka honey
increases.
HHP processing caused a slight reduction of the DN. It
was observed that after 5, 8 and 12 min of treatment dias-
tase activity decreased by about 10.4% of its original value
(22.3 ± 0.4 Gothe units). This result coincide with Al-Habsi and
Niranjan (2012) that found that DN decreases by about 4.6%
after HHP. Despite of the reduction of DN observed at some
HHP conditions, DN of HHP processed honey was higher than
the minimum permissible value (8) established by the Inter-
national Honey Commission International (Bogdanov et al.,
2009).
3.3.2. Fructose and glucose content
Honey is a concentrated water solution of two main sugars:
fructose and glucose, with small amounts of various com-
plex sugars. Generally, fructose is the dominant component
(Escuredo et al., 2014). No significant changes in the sugar
content of HHP treated honey were observed (Table 3). To our
knowledge, there are not studies reporting the effect of HHP
on sugars composition of honey.
Crystallization is a natural tendency in honey, which can
potentially cause major problems during handling and pro-
cessing. Crystallization of honey is a matter of interest for
many beekeepers and processors because each variety of
honey crystallizes differently (Venir et al., 2010; Yao et al.,
2003). The ratio fructose/glucose (F/G) is related to the time
required for honey to crystallize (Gleiter et al., 2006; Laos et al.,
2011). Honey samples with F/G values greater than 1.33 have
slow crystallization rates, and honey with F/G values lower
than 1.11 has faster crystallization rates (Smanalieva and
Senge, 2009). The F/G ratio in this study was around 1.17–1.19,
indicating that this honey has an intermediate tendency to
crystallize and HHP treatment did not change that relationship
(Table 3).
3.3.3. Rheological behavior
Both unprocessed and HHP processed honey exhibited a
Newtonian behavior showing a constant viscosity (␩) inde-
pendently of the shear rate (data not shown) as has been
reported previously (Abdul Ghani and Farid, 2007; Juszczak
and Fortuna, 2006; Zhou et al., 2013). There are some litera-
ture reports showing a non-Newtonian behavior (Fauzi et al.,
2013).
The viscosity of all treated samples was found significantly
different to unprocessed honey (Table 3); HHP decreased it
by 6.1–12.2%. The lowest viscosity values were observed after
2 and 5 min of processing (4.3 ± 0.2 and 4.5 ± 0.1 Pa s, respec-
tively).
Fauzi et al. (2013) reported an increase of viscosity with
HHP at ambient temperature in Manuka honey; they sug-
gested changes in oligosaccharides and residual wax present
in honey, induced by HHP. In this study, the reduction of vis-
cosity of the honey treated with HHP may be due to the partial
solubilization of some crystals of not visible sugars, more stud-
ies are needed to confirm this assumption.
3.4. Effect of HHP on functional properties
3.4.1. Total phenolic content
After the 0 and 15 min treatment, honey exhibited significantly
higher (p < 0.05) TPC of 31.5 ± 1.5 and 31.73 ± 0.1 mg GAE/100 g,
respectively, compared to unprocessed honey (29.9 ± 0.1 mg
GAE/100 g). Thus, pressurization of samples only during the
time required to reach the pressure of 600 MPa (treatment indi-
cated as 0 min) caused a significant increase of about 5.4%
while after 15 min TPC increased by 6.2%. In the range of 2
and 5 min a slight increment was observed too. Akhmazillah
et al. (2013) reported an initial TPC of 63.9 ± 0.9 mg GAE/100 g
in Manuka honey; they observed an increment of 47% in the
TPC after treated at 600 MPa for 10 min at ambient tempera-
ture. Differences may be due to different phenolic profile in
both honey samples.
The honey, in contrast with other food matrices, does not
have cell membranes to lead extract the antioxidant com-
ponents; therefore, the increased in TPC with HHP might be
attributed to the possible extraction of phenolic compound
present in honey pollen (Fauzi et al., 2013). Usually, it contains
derivatives of benzoic acid, hydroxycinnamic acids, flavonols
and flavones (Serra Bonvehi et al., 2001). The increase in TPC
could also be attributed to the HHP effects on non-covalent
bonds (hydrophobic and hydrogen bridges) of polyphenols and
proteins. It has been reported that polyphenols have the ability
to bind with proteins through non-covalent bonds form-
ing soluble and insoluble complexes (Brudzynski and Kim,
304 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 299–306

Fig. 1 – Effect of HHP (600 MPa applied for 0–15 min) on total and reduced vitamin C content of honey. Columns with
different letters are significantly different (p < 0.05).

Table 4 – Effect of HHP (600 MPa) on carotenoids (mg ␤-carotene/100 g) of honey.

Processing ␤-Carotene trans-␤- Violaxanthin 9-cis- 13-cis- All-␤-trans- Total
time (min) Carotene Violaxanthin Violaxanthin cryptoxanthin carotenoid
content

Unprocessed 0.076 ± 0.0 0.066 ± 0.0 0.101 ± 0.0 0.043 ± 0.0 0.034 ± 0.0 0.086 ± 0.0 0.406 ± 0.0a
0 0.067 ± 0.0 0.067 ± 0.0 ND 0.074 ± 0.0 0.081 ± 0.0 0.087 ± 0.0 0.377 ± 0.0a
2 0.070 ± 0.0 0.081 ± 0.0 ND 0.074 ± 0.0 0.084 ± 0.0 0.082 ± 0.0 0.393 ± 0.0a
5 0.084 ± 0.0 0.078 ± 0.0 ND 0.086 ± 0.0 0.079 ± 0.0 0.074 ± 0.0 0.403 ± 0.0a
8 0.085 ± 0.0 0.078 ± 0.0 ND 0.076 ± 0.0 0.078 ± 0.0 0.074 ± 0.0 0.391 ± 0.0a
12 0.089 ± 0.0 0.076 ± 0.0 ND 0.076 ± 0.0 ND ND 0.237 ± 0.0bc
15 0.077 ± 0.0 ND ND 0.075 ± 0.0 0.070 ± 0.0 ND 0.227 ± 0.0b

Data are means ± standard deviation. Means values within a column sharing the same letter are not significantly different (p < 0.05), ND: not
detected.

2011; Brudzynski et al., 2013). Brudzynski and Maldonado-
Alvarez (2015) reported a new hypothesis about the potential
association between honey flavonoids and polyphenols such
as association of caffeic acid with flavones, chrysin and
acecatin, and flavanone-, pinocembrin. This kind of asso-
ciation may be affected by HHP applied in honey and,
consequently be released some polyphenols. On the other
hand, the dissociation of protein-polyphenols prevents a
decrease of antibacterial activity and a change of the balance
between antioxidant-pro-oxidant capacities (Brudzynski and
Maldonado-Alvarez, 2015).
3.4.2. Vitamin C
Total and reduced vitamin C contents for unprocessed and
processed honey are shown in Fig. 1. The total vitamin C con-
tent in honey processed with HHP ranged from 34.5 ± 2.3 to
38.3 ± 1.6 mg AA/kg, and the dehydroascorbic acid (oxidized
form) represents around 40% in all treatments. The process
of HHP did not induce significant changes of this form of vita-
min C compared with the unprocessed honey. The effect of
HHP on vitamin C content of honey is reported for the first
time in this study.
3.4.3. Carotenoids
Three epoxycarotenoids (violaxanthin, 9-cis-violaxanthin,
and 13-cis-violaxanthin), one monohydroxycarotenoid (all-
␤-trans-cryptoxanthin), and two carotenes (trans-␤-carotene
and ␤-carotene) were identified in unprocessed honey. Vio-
laxanthin, 9-cis-violaxanthin, 13-cis-violaxanthin, all-␤-trans-
crytoxanthin, trans-␤-carotene and ␤-carotene, represented
about 24.9, 10.6, 8.4, 21.2, 16.3 and 18.7%, respectively, of the
total carotenoids in unprocessed honey.
The impact of HHP on the total carotenoids of honey was
largely dependent on processing time. No significant (p < 0.05)
changes occurred between 0, 2, 5 and 8 min of processing,
while after 12 and 15 min, significant changes were detected
in the content of trans-␤-carotene, violaxanthin and all- ␤-
cryptoxanthin (Table 4).
Violaxanthin was the most affected carotenoid by HHP that
causes isomerization of violaxanthin in 9-cis-violaxanthin
and 13-cis-violaxanthin. After 15 min of processing, the total
carotenoid content decreased by 56%. Honey lacks any of pro-
tective matrix could explain the carotenoid decrease after 12
and 15 min of processing.
3.4.4. Antioxidant activity
The level of AOA in unprocessed honey and after HHP treat-
ments are presented in Fig. 2. AOA ranged from 539.3 ± 30.2
to 611.9 ± 37.0 ␮mol TE/100 g. In general, HHP processed honey
has a higher level of AOA compared to unprocessed honey.
After 2 min treatment, honey showed a significant increase
in AOA (30%, p < 0.05) as has been previously reported for
honey treated at 600 MPa/10 min/room temperature. (Fauzi
et al., 2013). They attributed the increase to the possible pres-
ence of pollen and their components with antioxidant activity
such as enzymes, proteins, B complex vitamins, carotenoids,
flavonoids, anthocyanins, phospholipids and proteins, that
can contribute to the antioxidant activity when HHP is applied
(de Arruda et al., 2013; Fauzi et al., 2013).
 food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 299–306
Fig. 2 – Effect of HHP (600 MPa applied for 0–15 min) on antioxidant activity of honey. Columns with different letters are
significantly different (p < 0.05).
It is worth to mention that in the complex composi-
tion of honey, the synergistic interactions of a wide range
of compounds, including phenolics, peptides, organic acids,
enzymes, and possibly other minor components are the result
of total AOA of honey (Alvarez-Suarez et al., 2010b; Gheldof
and Engeseth, 2002). Higher AOA values could be attributed not
only to the increase of TPC but also to other minor components
present in honey.
4. Conclusion
Multifloral Mexican honey studied in this work contain impor-
tant antioxidant compounds, that after processing with HHP
at 600 MPa (ambient temperature) are retained and in some
cases, were improved. HPP processing may be released some
polyphenols from the association of protein-polyphenols and,
consequently increase the TPC content and antioxidant activ-
ity in honey. The vitamin C and sugar content were not
significantly modified by processing. Total carotenoid content
in honey, reported for the first time in this study, was the
most affected compound after 15 min of processing. In the
same way, it is possible to use HHP to reduce native viable
microorganisms in honey without compromising its general
quality and freshness such as HMF and DN. HHP processing
after 2 min is an excellent alternative to improve the antiox-
idant activity and preserve their original attributes. However,
more studies are needed to determinate the effect of HHP on
TPC in honey.
Acknowledgments
The authors acknowledge the support from Tecnológico de
Monterrey (Research chair funds GEE 1A01001 and CDB081),
IPN-SIP-Project (20160284) and CONACYT Project (216044) and
CONACYT Scholarship Program.
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