IDT_049.fm Page 171 Thursday, October 17, 2002 6:17 PM
Blackwell Science, Ltd
OR IGINAL
RE S EAR C H
Chemical changes in powdered infant formulas
during storage
E GUER RA-HE R NÁNDE Z ,1 * C L E ON,2 N C OR Z O,3
B GAR CÍA-VIL L ANOVA 1 and J M R OM E R A 2
1
Department of Nutrition and Bromatology, Faculty of Pharmacy, University of Granada, Campus University of Cartuja
18012, Granada, Spain, 2Department of Investigation and Desarrollo de Abbott Laboratories, Camino de Purchil S/ N,
Granada, Spain and 3Institute of Fermentaciones Industriales C.S.I.C., Juan de la Cierva 3, 28006, Madrid, Spain
Hydroxymethylfurfural (HMF), furosine (FUR), lactulose (LU), lysine loss, ascorbic acid and colour
(∆E) were determined in powdered infant formulas stored under nitrogen and oxygen conditions at 20°C
and 55°CC during 15, 30 and 90 days. The indicators of the assay at 20°C showed similar behaviour in
nitrogen or oxygen atmospheres. Changes in furosine and lysine loss after 90 days occurred under
oxygen. Storage at 55°C produced considerable browning. Browning was always greater in nitrogen than
in oxygen. Most of the studied parameters increased with the storage time and are useful in controlling
the extent of browning in powdered infant formulas under adverse storage conditions.
Keywords Colour, Hydroxymethylfurfural, Furosine, Lactulose, Lysine loss, Powdered infant formulas.
*Author for correspondence. E-mail:[email protected]
INTRODUCTION
*Author for
correspondence.
E-mail:[email protected]
© 2002 Society of
Dairy Technology
Infant formulas are designed to be substitutes for
mother’s milk when breast feeding is not possible.
Conventional formulas are based on cow’s milk
products. Children with special requirements
receive specially designed products. The manufacture of infant formulas includes the blending
of components, homogenization, pasteurization,
and spray-drying.
The application of heat during some steps to
obtain powdered infant formulas inevitably gives
rise to reactions between the constituents. One of
the most important reactions that can occur is the
Maillard reaction. Infant formulas have a high level
of lysine, which can react with reducing sugars.
This reaction can also take place during storage,
especially under adverse conditions of temperature
and atmosphere. Because infant formulas can be
the sole source of lysine for babies, the Maillard
reaction is undesirable in these products.1
Other reactions that can take place include the
denaturation of proteins, isomerization and the
crystallization of lactose, which markedly decrease
the solubility of powdered milk.2
The early stages of the Maillard reaction can be
evaluated by the determination of furosine [ε-N(furoylmethyl)-L-Lysine]. This is an amino acid
formed during acid hydrolysis of the Amadori
compounds fructosyl-lisine, lactulosyl-lysine and
maltulosyl-lysine, produced by the reaction of εamino groups of lysine with glucose, lactose and
maltose.1 Furosine is a useful indicator of damage
in milk, prolonged heating or inadequate storage
increasing the level of furosine.3,4
The determination of available lysine is a classic
measure to assess heat damage in foods. In infant
formulas, available lysine has been quantified by
HPLC (high-performance liquid chromatography).5
A rapid and simple measure, based on fluorescence
using o-phtaldehyde (OPA), has been applied to
milk samples by Vigo et al.6 and Morales et al.7
HMF is a classic index of the browning process
in milk products. Depending on the procedure, the
method determines either free HMF or free HMF
plus potential HMF derived from other browning
intermediates. The latter is also called ‘total’ HMF.8
It is well known that the Maillard reaction
causes the milk to become browner with increased
heating. The overall progress of the reaction can be
followed by colour measurements.8
Lactulose is a disaccharide produced from lactose
by isomerization in alkaline solutions and during
the heat treatment of milk.9 According to previous
studies, isomerization of lactose is the predominant reaction during the sterilization or UHT treatment of milk.10 However, the Maillard reaction is
more marked than lactose isomeration during the
storage of milk powders.11
Ascorbic acid is relatively stable to heat treatment, but is rapidly lost during storage when O2 is
present.8
Few data have been found on the progress of
browning reactions during storage of powdered
Vol 55, No 4 November 2002 International Journal of Dairy Technology
171
IDT_049.fm Page 172 Thursday, October 17, 2002 6:17 PM
Vol 55, No 4 November 2002
infant formulas. The present study aims to determine the influence of usual and adverse storage
conditions (temperature, time and atmosphere) on
the browning of powdered infant milk formulas,
using established indicators of heat damage, such
as HMF (free and total), furosine, lactulose, ascorbic
acid, colour and lysine loss.
MATERIALS AND METHODS
Samples
Powdered infant milk formulas were supplied by a
Spanish dietetic food factory. The infant formulas
were produced by spray-drying. The samples were
stored at 20°C or 55°C for 15, 30 or 90 days in
nitrogen (industrial nitrogen, approximately 99%)
or oxygen (air) atmospheres. The composition
of the formulas was given on the label as follows
(g/100 g): proteins (10.6), fat (27.7), carbohydrates
(55.2), minerals (1.8).
Analytical methods
Ascorbic acid (AA)
Half a gram of the sample were dissolved in 5 mL
of deionized water; 10 mL of 10% (w/v) metaphosphoric acid were added, and the whole was
filtered through Whatman paper no. 541. A sample
of 25 µL were injected into the HPLC, fitted with
a reverse-phase column and detection at 254 nm,
according to the method of Romera.12 Duplicate
analysis were carried out using a Waters Chromatograph (Milford, MD), comprising a 717 autosampler, 519 pump, 996 photodiode array detector
and Millennium 2011 chromatography manager.
The chromatographic column was a symmetry C18,
3.5 µm, 150 × 4.6 mm i.d. (Waters, Milford, MD).
The mobile phase was 0.1 m phosphate buffer
pH 3.5 at 1 mL/min.
Free hydroxymethylfurfural (Free-HMF)
One gram of powder infant milk formula was
weighed in a 25-mL centrifuge tube, to which
15 mL of deionized water were then added. The
centrifuge tube was shaken vigorously for 1 min
and the sample was then centrifuged for 10 min
at 5000 r.p.m. The supernatant was clarified
with 1 mL each of Carrez I (potassium ferrocyanide 15% w/v) and Carrez II (zinc acetate 30%
w/v) solutions and the resultant mixture was centrifuged. HMF was determined by reverse-phase
HPLC and UV detection (284 nm), using the
method of Guerra-Hernández et al.13 The HPLC
analysis was carried out as above, except that
the chromatographic column was a Nova-Pak
C18, 5 µm, 150 × 3.9 mm i.d. (Waters Millipore),
and the mobile phase was acetonitrile : water
(5 : 95) at 1 mL/min. Duplicate analyses were
performed.
172
© 2002 Society of Dairy Technology
Total hydroxymethylfurfural (Total-HMF)
One gram of powder infant milk formula was
weighed in a 25-mL centrifuge tube and 10 mL of
deionized water plus 5 mL of 0.3 N oxalic acid
were added. The sample was shaken and then
heated at 90°C for 25 min When the sample had
cooled, 1 mL each of Carrez I and II solutions were
added and the same procedure was followed as for
the HMF determination. Duplicate analyses were
performed.
Furosine (FUR)
One and a half grams of powdered infant milk
formula were reconstituted with deionized water
to 10 mL. Sample preparation and HPLC analysis
were carried out following the method of Resmini
et al.14 Two millilitres were hydrolysed with 6 mL
of 10.6 m HCl at 110°C for 24 h in a Pyrex screwcap vial with PTFE-faced septa. High-purity
helium gas was bubbled through the solution for
2 min. The hydrolysate was filtered through Whatman paper N°541. A 0.5-mL portion of the filtrate
was applied to a Sep-pak C18 cartridge (Millipore)
pre-wetted with 5 mL of methanol and 10 mL of
water. Furosine was eluted with 3 mL of 3 m HCl;
50 µL were then injected into the chromatograph.
Duplicate analyses samples were performed
by ion-pair RP-HPLC on a C8 column (250 mm ×
4.6 mm; Alltech Furosine-dedicated) with a linear
binary gradient. A Dionex chromatograph (DX300) and variable wavelength detector (LDC
Analytical, SM 4000) were used. Acquisition and
processing of data was carried out on a HPChem
Station (Hewlett-Packard).
Calibration was performed by the external
standard method using a commercial standard of
pure furosine (Neosystem Laboratories, Strasbourg,
France).
Lysine loss
The fluorometric technique used was based on the
available lysine methods of Morales et al.,7 and
Medina and García,15 with slight modifications.
The powdered milk formula was reconstituted at
3.5%; 1.5 mL of sodium dodecyl sulphate (SDS;
12% w/v) solution was added to 1.5 mL of the
sample and then refrigerated overnight. Before
analysis, the solution was centrifuged at 4000 r.p.m
and decanted and 1 mL of the supernatant was
mixed with 2 mL of water and 2 mL of freshly prepared OPA reagent (16.4 mg of o-phthaldehyde
in 2.5 mL of 95% methanol, 5 mL of 20% SDS,
25 mL of 0.1 m of borate buffer pH 9.5, 400 µL of
10% β-mercaptoethanol solution, made up to
100 mL with distiller water) with constant stirring
and incubated for 2 min at 25°C. The relative
fluorescence (RF) was measured at 3 min, with
emission and excitation wavelengths of 455 nm and
340 nm, respectively. Quinine sulphate solution
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Vol 55, No 4 November 2002
(5 µg/mL in 0.1 N H2SO4) was prepared daily as
calibration standard set at 100% RF. The mean
blank value was 93.75% RF in nonstored formula.
Duplicated analyses were performed.
Lactulose (LU)
A 1.5-g sample was reconstituted with deionized
water to 10 mL, and 2.5 mL of this reconstituted
sample were gently mixed with approximately
10 mL of ethanol in a 25-mL volumetric flask (so
that denatured protein particles would not remain
attached above the volume mark) and made up to
volume by adding additional ethanol. After a second mixing, the mixture was maintained at room
temperature for 48 h to allow precipitation of
lactose as described by De Rafael et al.16 Four
millilitres of supernatant were mixed with 1 mL
0.5% phenyl β-glucoside in 60% methanol,
evaporated under vacuum at room temperature
and converted to trimethylsilyl derivatives using
N-trimethylsilylimidazole. Lactulose determination
was performed by GC, using a Sigma 3B gas
chromatograph equipped with a 3-m × 1.0 mm i.d.
stainless steel column (Chrompack, Middelburg,
the Netherlands), packed with 2% OV-17 nonsilanized 120/140 Volaspher A2 (Merck, Darmstadt,
Germany), following the method described by
Olano et al.17 Duplicated analyses were performed.
Colour (∆E)
The colour was measured by the CIE L*a*b* colour system, where L* is lightness, a* is redness and
b* is yellowness. The instrument used was a reflec-
Table 1 HMF recovery in the analysis of powdered infant formula (mg/kg)
Added
Total
Detected
Recovered (%)
Free-HMF†
0.9
2.2
4.3
8.6
43.0
86.0
4.1
5.4
7.5
11.8
46.2
89.2
3.9
4.8
7.1
10.3
43.2
76.8
95.1
88.9
94.7
87.3
93.5
88.1
mean = 91.3 ± 3.54
CV(%) = 3.88
Total-HMF‡
0.9
1.9
3.8
9.6
24.0
47.0
107.5
20.2
21.2
23.1
28.9
43.3
66.3
126.8
18.2
18.9
21.2
25.5
39.0
59.3
113.5
90.1
89.2
91.8
88.2
90.1
89.4
89.5
mean = 89.76 ± 1.11
CV(%) = 1.23
†Sample = 3.2 mg /kg; ‡Sample = 19.3 mg/kg
© 2002 Society of Dairy Technology
tance spectrophotometer Elrepho 2000 (Datacolor
S.A., Spain). The colourimetric parameters L*,
a*, b* were referred to illuminant D65 and the
instrument was calibrated using a BaSO4 standard.
Duplicated determinations were performed. The
results were expressed as ∆E, according to the
equation ∆E = (∆L2 + ∆a2 + ∆b2)1/2.18
Statistical analysis
Analysis with the Student’s t-test used a Sigma
Package supplied by Horus Hardware S.A.,
Madrid, Spain.
RESULTS AND DISCUSSION
Preliminary studies
The methodology used in this article has been
applied previously to similar samples, with the
exception of HMF and available lysine.
The study of the recovery of free-HMF and
total-HMF was carried out by the standard addition procedure. Standards were added to reconstituted formula. Six addition levels were used for
free-HMF and seven for total-HMF, corresponding
to the levels found in the samples (Table 1). The
mean recovery for free- and total-HMF was 91.3
and 89.8%, respectively. The accuracy obtained in
powdered infant milk formula was slightly inferior
to that in baby cereals13 but was adequate to show
changes in the HMF of these samples during storage.
The precision of the fluorometric measure of available lysine was assessed on nonstored powdered
formula. The mean value was 96.75 ± 1.73 (n = 10)
and the coefficient of variation 1.85%.
Chemical changes during storage at 20°C
Table 2 shows the results for the different parameters determined for powdered infant formulas
stored at 20°C in oxygen and nitrogen atmospheres
at different times. This is the temperature usually
applied by the industry in their standard shelf-life
studies.
The storage at 20°C in nitrogen and oxygen
atmospheres showed similar behaviour. Differences could only be observed at 90 days. The HMF
indicator did not always follow the expected
sequence, because in the oxygen atmosphere the
free- and total-HMF decreased at 30 and 15 days of
storage, respectively. Our results do not agree with
those reported by Albala-Hurtado et al.19 for solid
infant formula unstored and stored at 20°C for 30
and 90 days, and the HMF values in our study were
approximately four-fold lower than in theirs. However, a similar relationship between total-HMF/
free-HMF (mean 1.8) was obtained in the two
studies.
The initial vitamin C value was 541 mg/kg,
remaining stable in the oxygen atmosphere for
30 days, but showing a 40% decrease at 90 days.
173
IDT_049.fm Page 174 Thursday, October 17, 2002 6:17 PM
Vol 55, No 4 November 2002
Table 2 Chemical changes in powdered infant formula during storage at 20°C
Furosine
(mg/100 g
of protein)
Time
(days)
Free-HMF
(mg /kg)
Total-HMF
(mg /kg)
Ascorbic acid
(mg/kg)
0
2.1 ± 0.09
3.8 ± 0.11
541 ± 14.0
701 ± 15.2
2.3 ± 0.07
1.9 ± 0.10
2.8 ± 0.09
3.0 ± 0.10
3.0 ± 0.09
5.9 ± 0.30
537 ± 17.0
552 ± 16.2
331 ± 10.1
2.3 ± 0.07
2.5 ± 0.10
2.8 ± 0.12
5.4 ± 0.29
3.4 ± 0.12
7.1 ± 0.45
615 ± 17.3
566 ± 13.2
413 ± 10.5
Oxygen
15
30
90
Nitrogen
15
30
90
Furosine
(mg/kg)
Lactulose
(mg/kg)
LU/FUR
∆E
Lysine loss
(%)
805
361 ± 88.3
0.45
—
—
854 ± 9.3
1006 ± 20.6
1054 ± 13.2
980
1155
1210
375 ± 86.3
393 ± 79.5
430 ± 82.5
0.38
0.34
0.36
1.49
1.33
1.22
4.9 ± 0.09
5.6 ± 0.10
6.7 ± 0.11
1027 ± 21.2
1021 ± 19.3
932 ± 10.2
1180
1173
1070
445 ± 86.2
563 ± 75.2
365 ± 90.5
0.38
0.48
0.34
1.94
1.53
2.31
6.0 ± 0.11
8.9 ± 0.16
8.3 ± 0.14
n=2
The loss at 90 days in storage under nitrogen was
only 24%.
In the oxygen atmosphere, furosine increased
during storage. Under nitrogen, however, furosine
had decreased at 90 days of storage, which would
indicate a greater development of the Maillard
reaction under nitrogen conditions. The changes
observed in lactulose content during storage at
20°C under either atmospheric condition were not
really significant.
As shown in Table 2, the level of furosine found
in powdered infant milk formula was higher than
the level of lactulose, due to the more intense Maillard reaction in low-moisture foods.20,21 The levels
of lactulose and furosine found in the powdered
infant milk formula were within the range obtained
by Corzo et al.3 for reconstituted nonfat cow milk
powder, but lower than those found by Henle
et al.22 and Sarriá et al.23 in powdered infant formulas. The lactulose/furosine ratio (LU/FUR) is
another thermal index used to assess the heat
treatment of milk. Our present findings in powdered
infant milk formula showed LU/FUR values
within the range reported by Corzo et al.3 in commercial milks and by Sarriá et al.23 in powdered
infant formula.
Lysine was lost during storage and the loss
increased with storage time. Greater lysine losses
were observed under nitrogen. Calcagno et al.4
found similar lysine losses (determined as furosine) to the present results in solid infant formula
stored at room temperature for 8 months. AlbalaHurtado et al.19 found no changes in available
lysine (determined by HPLC) in solid infant milk
stored at 20, 30 and 37°C during 1–9 months.
Colour difference (∆E) increased after 15 days
of storage under both nitrogen and oxygen atmospheres and differences were not observed at
90 days. The intensity of browning was higher
under nitrogen than under oxygen conditions.
174
© 2002 Society of Dairy Technology
Under oxygen, the Maillard reaction could be
detected and its quantification could be followed
by the lysine loss and furosine indicators. In nitrogen, the lysine loss increased with the time of
storage whereas the furosine began to decrease
after longer storage times. Under nitrogen, only the
lysine loss indicator (measured by OPA reactivity)
is useful.
Chemical changes during storage at 55°C
Table 3 shows the results for the different parameters for powdered infant formula stored at 55°C
under oxygen and nitrogen atmospheres at different times. This is the temperature usually applied
by industry for accelerated studies of shelf-life.
Free and total HMF values increased during
storage, especially under nitrogen. The 90-day
storage in a nitrogen atmosphere produced more
than eight times the free-HMF than that produced
under oxygen.
Ascorbic acid content decreased from 541 mg/
kg to 337 mg/ kg under oxygen. No losses were
observed under nitrogen. Thus, the higher browning under nitrogen is not dependent on ascorbic
degradation.
Furosine increased under oxygen and increased
and then decreased under nitrogen. At the
advanced stage of the Maillard reaction, the furosine level may no longer increase linearly with the
severity of storage conditions.11,24
Losses of lysine were very high under both types
of atmosphere and increased with the storage time.
The losses were higher under nitrogen than under
oxygen.
The lactulose content increased greatly under
both types of atmosphere and was higher under
nitrogen, where it reached 5000 mg/kg. The samples
with LU/FUR values of more than 0.09 showed a
loss of lysine (measured by OPA reactivity) of over
30%.
IDT_049.fm Page 175 Thursday, October 17, 2002 6:17 PM
Vol 55, No 4 November 2002
Table 3 Chemical changes in powdered infant formula during storage at 55°C
Time
(days)
0
Oxygen
15
30
90
Nitrogen
15
30
90
Free-HMF
(mg /kg)
Total-HMF
(mg /kg)
Ascorbic acid
(mg/kg)
Furosine
(mg/100 g
of protein)
Furosine
(mg/kg)
Lactulose
(mg/kg)
LU/FUR
∆E
Lysine loss
(%)
—
—
2.1 ± 0.09
3.8 ± 0.11
541 ± 14.0
701 ± 15.2
805
361 ± 28.3
0.45
2.4 ± 0.08
3.2 ± 0.09
9.2 ± 0.80
26.8 ± 2.70
20.0 ± 2.20
25.1 ± 3.00
592 ± 16.2
449 ± 15.9
337 ± 10.3
2185 ± 54.8
2085 ± 52.3
2936 ± 52.5
2509
2395
3372
784 ± 31.5
1047 ± 51.0
2701 ± 72.0
0.31
0.44
0.50
2.1
6.9
6.0
14.9 ± 0.27
31.5 ± 0.55
35.2 ± 0.63
2.5 ± 0.07
3.6 ± 0.10
68.9 ± 4.10
6.7 ± 0.46
15.9 ± 0.54
116.3 ± 5.90
589 ± 17.1
640 ± 17.5
621 ± 18.2
3401 ± 78.8
5484 ± 92.3
1384 ± 19.8
3906
6298
1590
1273 ± 59.0
5144 ± 163
5443 ± 289
0.33
0.82
3.42
2.3
24.8
42.3
28.0 ± 0.48
51.1 ± 0.97
88.3 ± 1.59
n=2
The intensity of browning, measured as ∆E,
showed very high values under nitrogen after 30
and 90 days of storage, whereas the colour changes
under oxygen remained relatively modest.
The storage at 55°C showed considerable
browning reactions. The browning was always
greater under nitrogen than under oxygen. Thus,
the browning reaction is favoured under nitrogen
conditions.
The free-HMF, total-HMF, lactulose, ∆E and
lysine loss increased with the time of storage and
are useful indicators to follow the extent of the
browning reaction in infant formula under adverse
storage conditions.
Total- and the free-HMF exhibited similar
behaviour. The correlation between them was
r 2 = 0.8378 at 20°C and r 2 = 0.9806 at 55°C. The
determination of one of them (free- or total-HMF)
could be sufficient.
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