The toxic aldehyde, 4-hydroxy-2-trans-nonenal (HNE) formation in natural and imitation mozzarella cheeses: Heat treatment effects

Article abstract of DOI:10.1007/s11746-012-2084-0

The formation of 4-hydroxy-2-trans-nonenal (HNE), a toxic aldehyde formation, was investigated in heat treated imitation Mozzarella cheeses which are made with vegetable oils and in natural Mozzarella cheeses which contain dairy fats. The cheeses were hea

Full text of DOI:10.1007/s11746-012-2084-0

J Am Oil Chem Soc (2012) 89:1801–1805
DOI 10.1007/s11746-012-2084-0
ORIGINAL PAPER
The Toxic Aldehyde, 4-Hydroxy-2-trans-nonenal (HNE)
Formation in Natural and Imitation Mozzarella Cheeses:
Heat Treatment Effects
•
In Hwa Han A. Saari Csallany
Received: 20 October 2011 / Revised: 19 April 2012 / Accepted: 10 May 2012 / Published online: 3 June 2012
Ó AOCS 2012
Abstract The formation of 4-hydroxy-2-trans-nonenal
(HNE), a toxic aldehyde formation, was investigated in heat
treated imitation Mozzarella cheeses which are made with
vegetable oils and in natural Mozzarella cheeses which
contain dairy fats. The cheeses were heat treated at 204 °C
for 30 and 60 min, and at 232 °C for 15 and 30 min. The
HNE formations were much higher in imitation cheeses
than in natural cheeses due to both heat treatments. Average
HNE concentrations in imitation cheeses, after 30 min of
heat treatment at 204 °C, were 110.3 ng HNE/g cheese and
it increased to 877.1 ng HNE/g cheese when the tempera-
ture was raised to 232 °C. In natural cheeses, the average
HNE concentration was much lower only 13.4 ng HNE/g
cheese after 204 °C heat treatment for 30 min and it
increased only to 182.8 ng HNE/g cheese using 232 °C.
Since imitation cheeses are made with vegetable oils which
contain much higher levels of linoleic acid, a precursor for
HNE, than dairy fat, it is not surprising that heat-induced
lipid peroxidation results in increased HNE formation in
imitations cheeses compared to dairy fat containing
cheeses, which are low in linoleic acid.
Introduction
4-Hydroxy-2-trans-nonenal (HNE) is known as a second-
ary lipid peroxidative product of n6 fatty acids, including
linoleic acids [1, 2]. It is a toxic compound and has been
reported to be related to atherosclerosis, LDL oxidation
[3, 4], stroke, Parkinson’s, Alzheimer’s and Huntington’s
diseases [5–10], disease of the liver [11] and other diseases.
Because the toxicity of HNE, (it may induce various
health problems in humans), it is important to investigate
its formation in various foods due to heat treatment.
HNE concentrations ranging from 3.75 to 95.20 lmol/
kg were reported in ham, bacon and sausages after smoking
[12] and this laboratory reported 42.5 lg HNE/g oil in
soybean oils after heating at 185 °C for 6 h in the presence
of air bubbling [14].
HNE is derived from the peroxidation of n6 fatty acids
mostly from linoleic acid [13, 14]; this implies that oils
containing higher concentrations of n6 fatty acids produce
more HNE due to heat treatment than oils low in n6 fatty
acids. Vegetable oils, including corn and soybean oils,
contain high percentages of n6 fatty acids while animal
fats, including milk fat, contain much lower quantities of
n6 fatty acids compared to vegetable oils. Soybean oil
contains about 54 %, corn oil about 60 % and sunflower oil
about 68 % linoleic acid.
Keywords 4-Hydroxy-2-trans-nonenal (HNE) Á
Toxic aldehyde formation Á Lipid peroxidation Á Cheeses
Animal fats such as lard and beef tallow and milkfat
contain less than 10 % of linoleic acid [15]. Therefore
HNE formation, due to heat treatment, is expected to be
higher in vegetable oils than in animal fats. Imitation
Mozzarella cheese is replacing natural Mozzarella cheese
in many instances to lower the cost. Imitation cheeses are
prepared with vegetable oils, therefore HNE formation due
to heat treatment is expected to be higher than in natural
cheeses. In the present study, HNE formation was
Present Address:
I. H. Han
Department of Food and Nutrition,
Gwangju Women’s University, 165 Sanjeong-dong,
Gwangsan-gu, Kwangju 506-713, South Korea
A. S. Csallany (&)
Department of Food Science and Nutrition,
University of Minnesota, 1334 Eckles Ave, St. Paul,
MN 55108, USA
e-mail: ascsalla@umn.edu
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J Am Oil Chem Soc (2012) 89:1801–1805
examined in two imitation Mozzarella cheeses and in two
natural Mozzarella cheeses exposed to various tempera-
tures and heating times.
in a preheated oven and heated for 0, 30, and 60 min at
204 °C for 0, 15 and 30 min at 232 °C. Each heated cheese
sample was mixed with 200 mL hexane and blended in a
laboratory blender for about 10 min, filtered through a
Whatman No. 1 filter paper and the hexane filtrate was
collected. The residue was mixed with 100 mL hexane and
blended for 5 min and after filtration the hexane was col-
lected. The combined hexane extracts were used for HNE
analysis.
Materials and Methods
Chemicals and Materials
Two different low-moisture part-skim Mozzarella cheeses
were purchased from a local store (Roseville, MN) and two
different imitation cheeses were provided by Dr. Metzger
(South Dakota State University). Hexanal and 2,4-dinitro-
phenylhydrazine (DNPH) were purchased from Sigma (ST.
Louis, MO). HNE standard was obtained from Cayman
Chemical Co. (Ann Arbor, MI). Hydrochloric acid was
purchased from J.T. Bakers Inc. (Phillipsburg, NJ), HPLC-
grade methanol from Fisher Scientific (Fair Lawn, NJ), and
HPLC-grade water, n-hexane and dichloromethane from
EMD Chemicals, Inc. (Gibbstown, NJ). Thin layer silica
gel plates (20 9 20 cm, 250 lm layer, Al SIL G) for
chromatography (TLC) and No. 1 filter paper were pur-
chased from Whatman Ltd. (Kent, England).
Preparation of DNPH Derivatives of HNE
from Heat-Treated Cheese Samples
The preparation of DNPH derivatives of HNE from heat-
treated cheese samples was conducted as described by the
detailed method of Seppanen and Csallany [16]. Briefly,
10 ml of hexane extracts, from the heat treated or the
control unheated cheese samples, were reacted overnight
with 5 mL freshly prepared DNPH reagent at room tem-
perature. The DNPH derivatives were extracted with
10 mL of 75:25 (v/v) methanol: water, from the hexane
extract and the DNPH reagent reaction mixture. This
extraction procedure was repeated two more times and the
75:25 (v/v) methanol:water extracts were combined. From
the combined methanol:water extracts, the DNPH deriva-
tives were extracted four times with 10 mL of dichloro-
methane and the combined dichloromethane extracts were
evaporated under N2 gas to about 1 mL.
Preparation of DNPH Reagent
The reagent was prepared daily, according to the method of
Seppanen and Csallany [16], by combining 10 mg DNPH,
recrystallized three times from methanol, with 20 mL 1N
HCl at 50 °C for about 1 h. After cooling, the mixture was
extracted four times with HPLC-grade hexane in a separ-
atory funnel to remove impurities. The aqueous purified
DNPH reagent was used immediately.
Thin Layer Chromatography (Pre-Separation
of DNP Derivatives)
The whole concentrated DNPH dichloromethane extract,
about 1 mL, was applied to two TLC plates and developed
with dichloromethane. On the TLC plates the polar and
nonpolar aldehydes, and osazone regions (Rf = 0.25, 0.75
and 0.50, respectively) were separated. The polar aldehyde
fraction region which included the HNE DNPH derivative
was cut from the TLC plate into small pieces and extracted
three times with 10 mL of methanol. The combined
methanol extracts were centrifuged at 13609g for 15 min
to remove residual silica. The clarified supernatant frac-
tions were combined and concentrated under N2 gas to the
exact volume of 1 mL. Aliquots of 50 lL were injected
into the HPLC column in duplicate for the HNE analysis.
Preparation of Hexanal-DNPH Standard
Pure hexanal-DNPH was prepared by the method devel-
oped in this laboratory [17]. The mixture of 800 mg of
recrystallized DNPH, 80 mL methanol, 2 mL 6N hydro-
chloric acid and 1 mL reagent grade hexanal, was heated
for 10 min at 60 °C, and placed into an ice bath for crys-
tallization. The hexanal-DNPH crystals were filtered
through Whatman No. 1 filter paper and recrystallized two
more times from *50 mL of methanol. After the final
crystallization, the hexanal-DNPH was dried in a desicca-
tor for 3 days and the product was kept at -20 °C in an air
tight container under N2 gas.
HPLC Analysis
Heat Treatment and Solvent Extraction of Natural
and Imitation Cheeses
The HPLC system consisted of a solvent delivery system
(9050, Varian, Walnut Greek, CA), a sample injector (712
WISP, Waters, Milford, MA), Ultrasphere ODS HPLC
column (5 9 4.6 mm, 25 cm), (Beckman, Fullerton, CA)
and a UV–VIS detector (9010, Varian). The integration of
One hundred grams of each cheese sample, in duplicate,
was placed on the surface of aluminum foil on a petri dish
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peaks shown on chromatograms was conducted by Star
Workstation software (Varian, Walnut Greek, CA) instal-
led on the computer connected to the UV–VIS detector. For
polar aldehyde analysis, an isocratic elution with 55:45
(v/v) methanol: water was used for 10 min, a gradient
elution increased to 100 % of methanol over 20 min and
held at 100 % of methanol for an additional 10 min. The
absorbance of the polar aldehydes was monitored at
378 nm. Rejection of peaks was set to 2,000 area counts.
The detection limit of the HPLC system was previously
determined to be 12 ng hexanal (measured as its DNPH
derivatives) per 50 lL injection.
from the hexanal equivalents based on the molecular
weight differences between hexanal and HNE. The HNE
concentrations were expressed as ng HNE/g cheese. In
total, two different natural and two different imitation
cheeses, in duplicate, were analyzed. The HPLC injections
were in duplicate for each duplicate cheese sample.
Statistical Analysis
All measurements were conducted in duplicate. The aver-
age and standard error of the mean (SEM) of HNE con-
centration were calculated for each heating time period,
and for each cheese sample.
The peaks of the HNE from the HPLC chromatograms
were identified by comparing the retention time of the
isolated HNE with the retention time of the peak of pure
standard. Identification of the isolated HNE was also car-
ried out by co-chromatography with pure standard in three
different polarity solvent systems as described in the
method developed in this laboratory [17]. Solvent systems
used for co-chromatography for DNPH derivatives of HNE
were methanol: water, 50:50, 55:45 and 60:40 (v/v). The
percent recovery of added pure aldehyde-DNPH to the
isolated sample from the co-chromatography was calcu-
lated from the increase in the peak area due to the added
pure HNE-DNPH to the original peak area of the isolated
HNE-DNPH from the heat treated cheese samples. HNE-
DNPH derivative was quantified by comparing their peak
area with the area of known amount of pure hexanal-DNPH
standard and expressed as lg hexanal equivalent/g cheese.
One ng of pure hexanal was measured to be equivalent of
4,500 area counts by the HPLC system used in the exper-
iment. The isolated HNE concentrations were calculated
Results and Discussion
The formation of HNE in natural low-moisture part-skim
Mozzarella and imitation Mozzarella cheeses were inves-
tigated due to heat treatment at 204 °C up to 60 min and at
232 °C up to 30 min. These temperatures are commonly
used in home and commercial applications. Typical HPLC
chromatograms of polar aldehydes, including HNE, from
heat treated natural cheese A and imitation cheese B are
shown in Fig. 1. For detection of HNE peak of samples, the
retention time (Rt) on the HPLC column was compared
with pure HNE standard and they were both shown at
28.9 0.4 min Rt. Co-chromatography was conducted for
identification in three different polarity solvent systems.
Recoveries from co-chromatography using three different
polarity solvent systems were the following: 88.3 % using
methanol:water 50:50 (v/v), 91.9 % using 55:45 (v/v) and
Fig. 1 Typical HPLC
chromatograms of polar
aldehydes, including 4-hydroxy-
2-trans-nonenal (HNE), from
natural low-moisture part-skim
Mozzarella cheese heat treated
at 232 °C for 30 min (a) and
imitation Mozzarella cheese
heat treated at 204 °C for
60 min (b)
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Fig. 2 HNE concentration in natural low-moisture part-skim Moz-
zarella and imitation Mozzarella cheese samples heat treated at
204 °C for 0, 30 and 60 min. Imitation cheeses IA, IB and (from two
different sources) and natural low-moisture part-skim Mozzarella
cheeses NA and NB
Fig. 3 HNE concentration in natural low-moisture part-skim Moz-
zarella and imitation Mozzarella cheese samples heat treated at
232 °C for 0, 15 and 30 min. Imitation cheeses IA and IB and natural
low-moisture part-skim Mozzarella cheeses NA and NB
94.6 % using 60:40 (v/v). As shown in Fig. 1 the relative
area of HNE peak was much larger in imitation cheese
(b) than on natural cheese (a).
Figure 2 illustrates the HNE concentrations in imitation
and natural cheeses treated at 204 °C for 0, 30 and 60 min.
HNE concentration increased in all cheeses with the length
of heating time. The imitation cheeses reached higher HNE
concentration than natural cheeses already after 30 min and
much higher levels after 60 min of heat treatment. Cheese
samples A and B are indicating different sources for cheese
samples and duplicate or triplicate HNE analyses. For
imitation cheese samples IA and IB, HNE concentration
after 30 min heating time were 86.5 and 134.0 ng HNE/g
cheese, respectively. For natural cheeses NA and NB, HNE
concentrations were 14.2 and 12.6 ng HNE/g cheese,
respectively. After 60 min of heat treatment the HNE
concentration increased 8–10 times higher in imitation
cheeses compared to natural cheeses. Imitation cheese IA
was 1,120 and IB was 780 ng HNE/g cheese. Natural
cheese NA and NB average was 116 ng HNE/g cheese.
The high increase of HNE concentration in imitation
cheeses was expected since these cheeses are made with
vegetable oils which are high in linoleic acid, a precursor
of HNE [14]. This laboratory has previously reported
higher HNE formation in high linoleic vegetable oils than
in butter oil due to heat treatment [18]. Figure 3 shows the
HNE concentration in imitation cheeses and natural
cheeses treated at higher temperature at 232 °C up to
30 min. HNE concentrations at this temperature rapidly
increased after only 15 min heating time indicating the
accelerated rate of heat induced peroxidation and decom-
position of polyunsaturated fatty acids (PUFA), including
linoleic acid which is responsible for the increase of HNE
in the samples. HNE concentrations increased much more
in the imitation cheeses than the natural cheeses at this
Fig. 4 Average HNE concentrations, lg HNE/g cheese, in natural
low-moisture part-skim Mozzarella and imitation Mozzarella cheeses
heat treated at 204 and 232 °C for 30 min
temperature resulting in a similar pattern as found before at
204 °C. Imitation cheese IA and IB showed very large
increases of HNE concentration, at 30 min compared to
concentration at 15 min of heat treatment. HNE concen-
trations in natural cheeses NA and NB increased to much
lower levels at 30 min, compared to concentrations at
15 min heat treatment. The difference in HNE concentra-
tion between IA and IB samples at 232 °C, due to 30 min
heat treatment, is possibly due to the different types of
vegetable oils used for the manufacture of these imitation
cheeses. It seems that IB sample fatty acid composition
was different from IA sample, most probably the linoleic
acid concentration was in higher level in IB sample since
this N6 fatty acid is a precursor of HNE. Figure 4 shows
the average HNE concentrations of the two imitation and
the two natural cheeses at 204 or 232 °C after heat treat-
ment for 30 min and illustrates the effect of temperature
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References
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cheeses treated at the higher temperature of 232 °C than at
204 °C. Average HNE concentrations in imitation cheeses
were after 30 min heat treatment at 204 °C, 110.3 ng HNE/g
cheese and it increased to 877.1 ng HNE/g cheese when the
temperature was raised to 232 °C. In natural cheeses, the
average HNE concentration was much lower, only 13.4 ng
HNE/g cheese after 204 °C heat treatment for 30 min and
it increased to only 182.8 ng HNE/g cheese using 232 °C.
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HNE, than dairy fat it is not surprising that heat induced
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significant temperature dependence in the oxidative
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Conclusions
The formations of HNE, a toxic aldehyde, were found to be
in much lower concentrations in natural low-moisture part-
skim Mozzarella cheeses which contain dairy fats than in
imitation Mozzarella cheeses containing vegetable oils due
to heat treatments for 15, 30 and 60 min at both 204 and
232 °C. The higher linoleic acid concentration in heat
treated imitation cheeses compared to heat treated natural
cheeses seems to be responsible for the increase in HNE
formations in imitation cheeses. Based on the present
results of this study it is suggested that all cheeses, and
especially imitation cheeses and imitation cheese contain-
ing foods, should be heated at the lowest possible tem-
peratures and the shortest heating time, to lower HNE a
toxic aldehyde formation. Since this aldehyde has been
shown to be readily absorbed from the diet into the gut and
implicated in various pathological conditions, the chronic
consumption of this toxic compound could be negatively
related to human health. However, because to our knowl-
edge no study has yet published on the health related
effects of dietary long term continuous HNE ingestion
from food, more research is needed to establish its toxic
effect.
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