Scientia Horticulturae 192 (2015) 180–186

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Internal discoloration of various varieties of Macadamia nuts as

influenced by enzymatic browning and Maillard reaction
Warangkana Srichamnong a,b,∗ , George Srzednicki b
a
Institute of Nutrition, Mahidol University, Phuttamonton, Nakhonpathom 73170, Thailand
b
School of Chemical Engineering, Department of Food Science and Technology, UNSW Australia, NSW 2032, Australia

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

Article history: Brown kernels or internal discolouration (IDC) in various varieties of Macadamia nuts (Macadamia integri-
Received 9 April 2015 folia and Macadamia tetraphylla) occurred through three different pathways: (i) an enzymatic browning
Received in revised form 2 June 2015 reaction, (ii) Maillard reaction and (iii) infection by microorganisms. The phenolic compounds and
Accepted 5 June 2015
polyphenol oxidase (PPO) activities of macadamias were analysed. Brown sections of the same kernel had
higher levels of bound phenolics compared to the white sections, indicating the participation of phenolic
Keywords:
compounds in the formation of brown kernel. The Maillard reaction was studied by determining the
Browning
sugar amount using HPLC. The reducing sugars during drying reacted with kernel proteins causing the
Enzymatic browning reaction
Internal discolouration
formation of brown pigments. Among the various varieties studied, ‘Daddow’ variety showed the least
Macadamia degree of hydrolysis. When kernel was infected with Penicillium aurantiogriseum, the kernel turns brown.
Maillard reaction The internal discoloration in macadamias is the first report that has been explored in our study. The find-
ings of this study have potential to improve the existing postharvest techniques used in the Macadamia
processing industry.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction kernels resulted in significant reduction of oxidative stress and

Macadamia nuts are packed with numerous health benefiting
nutrients, minerals, antioxidants and vitamins that are essential
for optimum health and wellness. Macadamia nuts have sweet
taste and are rich source of monounsaturated oil like oleic acid
(18:1) and palmitoleic acids (16:1) (Murano, 2003). They are widely
grown in Australia, South Africa, Guatemala and United States
of America, especially, in Hawaii (USDA, 2011). Only two species
viz., Macadamia integrifolia and Macadamia tetraphylla and their
hybrid varieties are commercially available as compared to other
species, which have a bitter taste and unsuitable for consumption.
Macadamia nuts contain high content of unsaturated fats, which
are beneficial to health by reducing the low density lipoprotein
cholesterol levels and improving the markers for oxidative stress,
inflammation and clotting tendencies (Griel et al., 2008). From the
review literature, high consumption of unsaturated fat could have
some health benefits, including prevention of diabetes, control of
body weight and prevention of cardiovascular disease (Lovejoy,
2005). Garg et al. (2007) reported that consumption of macadamia
∗ Corresponding author at: Institute of Nutrition, Mahidol University, Phuttamon-
ton, Nakhonpathom 73170, Thailand. Fax: +66 2 441 9344.
E-mail address: warangkana.sri@mahidol.ac.th (W. Srichamnong).
inflammation in human body. In addition, the high concentration of
oleic acid in macadamias is beneficial for decreasing the coronary
heart disease due to the high percentage of unsaturated fatty acids
(Sinanoglou et al., 2014)
The development of browning is associated with Macadamia
nuts, which usually occur during the thermal processing of posthar-
vest treatment. Postharvest treatments are the steps performed
after the nuts are no longer on the tree. Proper postharvest proce-
dures are needed to prevent physical and chemical damage which
can lead to loss of quality. There are generally six postharvest steps
in macadamia nut processing; harvesting, de-husking, thermal
processing, cracking, grading and packaging. The internal dis-
colouration in macadamia nuts causes the formation of off-flavours
and aroma, which in turn leads to economic loss and decrease
the nut quality.The extent of this defect, is largely unquantified
at this stage. As a consequence, it costs the industry an estimated
loss of several million dollars per year due to kernel downgrading
and processing inefficiency. More importantly, the occurrence of
brown centers in nuts sold at the retail level has the potential to
greatly undermine customer confidence and thus reducing repeat
sales. The fundamental factors associated with this defect are still
unknown. Since this problem occurs erratically, therefore the basic
cause of this defect has been difficult to investigate (Lagadec, 2009).
However, the internal discolouration or browning of the kernel cen-

http://dx.doi.org/10.1016/j.scienta.2015.06.012
0304-4238/© 2015 Elsevier B.V. All rights reserved.
 W. Srichamnong, G. Srzednicki / Scientia Horticulturae 192 (2015) 180–186
ter varies from light brown to dark brown depending on the shape
of nuts which ranges from oval to unconformity. The discoloura-
tion occurs mostly at the center but could spread to any locations
in the Macadamia kernel. In general, the colour of Macadamia
nuts varies from white to creamy; therefore the discolouration in
these nuts could be easily detected. Walton and Wallace (2015)
reported that by reducing time period between harvest and de-
husking processing Macadamia at low moisture content (10–12%)
could improve the kernel quality. Whilst, Macadamia nuts stored
at high moisture content and elevated temperature with lim-
ited air circulation showed increased occurrence of brown center
(Walton et al., 2013). Other study conducted by Phatanayindee
et al., (2012) showed that reduction of drying time using heat
pump dryer combined with tunnel drying resulted in improved
kernel quality and reduced IDC in Macadamia nuts. This indicated
that brown kernels could be found during all postharvest treat-
ments from harvesting to packaging stage. In general, the kernel
quality is influenced by internal and external factors, processing
steps and environmental conditions. Furthermore, these factors
could be interrelated, for example the polyphenol oxidase enzyme
could be inactivated during drying, leading to reduced suscepti-
bility to enzymatic browning. Moreover, brown kernels could be
found among fresh, dried and roasted samples. Therefore, it was
hypothesized that browning in Macadamia kernel could be trig-
gered by enzymatic and non-enzymatic browning reactions. These
two biochemical reactions could form brown pigment as their end
product. In addition, microorganisms may also influence brown
kernel development via the production of extracellular enzymes.
Therefore, the aim of this study was to determine the possi-
ble mechanisms of kernel browning and study various factors that
are likely related with quality deterioration based on different cul-
tivars. The information obtained from this study will contribute
to understand the internal discolouration (IDC) and could lead to
preventive measures for maintaining nut quality.
2. Material and methods
2.1. Materials
Macadamia samples were obtained from various plantations in
different seasons located in New South Wales (Deenford planta-
tion, Lismore) and Queensland. The samples included were various
commonly grown varieties including ‘A 38’, ‘246’, ‘816’, ‘842’, and
‘Daddow’. Samples were sorted, graded and cracked. The nuts
obtained were graded into 4 categories (1) nuts with smooth
texture and light colour; (2) nuts with colour defects, uneven
browning or off-colour; (3) nuts with brown centers; (4) dark shriv-
eled nuts. The nuts were classified based on the definitions of
Prichavudhi and Yamamoto (1987). Category 1 nuts were used in all
the experiments, whilst category 2 nuts were used in brown kernel
experiments only. Due to the insufficient quantity of brown nuts,
the brown kernel samples were also obtained from colour sorter at
Macadamia processing company (MPC Ltd, Lismore, NSW). The belt
type colour sorter machine (Alstonville, Australia) is used during
processing step to separate brown colour kernel from white colour
kernel. Therefore, brown nuts used in this study were procured
from both plantations as well as from processor.
2.2. Chemicals and reagents
1, 1-Diphenyl-2-picrylhydrazyl (DPPH), Folin-Ciocalteu
reagent, phenolic compounds (gallic acid, protocatechuic acid,
p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic
acid and syringic acid) and fatty acids standard C12-C20 were
purchased from Sigma–Aldrich (Sydney, Australia). HPLC grade
181
methanol acetonitrile and other solvents were obtained from
Merck and Honeywell (Sydney, Australia). All chemicals and
reagents used in the study were of analytical grade.
2.3. Kernel segmentation
The segmentation of kernels was performed on fresh brown
nuts using sharp knife to mechanically separate the non-brown
and brown sections. The concentration and types of phenolic com-
pounds present in both brown and non-brown sections were
determined separately. Prior to the analysis, the non-brown and
brown section was carefully placed in different tubes (1.5 mL). The
brown kernels samples were divided into 7 groups (A–G) randomly.
Each group included the average data of 10 brown Macadamia nuts.
2.4. Processing
2.4.1. Drying
Drying of nuts were carried out through a series of experiments
using an in-house built cabinet dryer, followed by heat pump dryer
(Greenhalgh, Australia) and finally with vacuum oven (Croydon,
England). Samples were dried in the form of nut in shell (NIS). The
drying conditions in the cabinet varied from 30 to 50 ◦ C and RH
range was 20–40%. Heat pump dryer was operated at 30 ◦ C and
20% RH. Vacuum oven drying was performed at 35 ◦ C at 3.29 kPa.
The drying time of each experiment was 8 days.
2.4.2. Roasting
The roasting experiments were carried out in a convection oven
(Contherm, Australia) at 30, 60, 90, 120 and 150 ◦ C for 30 min.
2.5. Chemical analysis
2.5.1. Moisture content and pH determination
The moisture contents of kernels and shells were determined
by a vacuum oven method 934.01 (18) G (AOAC, 1995). Macadamia
kernels were ground with a coffee grinder and placed in pre-dried
aluminium dishes and dried in vacuum oven at 75 ◦ C for 24 h at
3.29 kPa. The ground Macadamia nut was mixed with MilliQ water
at 1:4 ratio and was used for pH determination using a pH meter at
25 ◦ C.
2.5.2. Enzyme extraction and analysis of polyphenol oxidase
(PPO)
The extraction and analysis method of PPO were performed
according to Srichamnong et al., (2012). Macadamia nuts (10 g)
were defatted twice with 100 mL of hexane and10 mL of petroleum
ether. Defatted sample (5 g) was transferred to a 250 mL conical
flask and mixed with 100 mL phosphate buffer (pH 6.8) containing
5% polyvinylpyrrolidone (PVPP). Extraction time was 8 h at 4 ◦ C.
Samples were then filtered through Whatman No. 41 filter paper
and the filtrate was centrifuged at 4 ◦ C at 20,000 rpm for 30 min.
Supernatant was collected and diluted with acetone (4 × volume)
to precipitate protein. Any remaining liquid was evaporated by
nitrogen flushing. The acetone precipitates were kept at −18 ◦ C for
further analysis. Prior to purification, protein powder was mixed
with 1 mL of 0.1 M phosphate buffer (pH 6.8) and passed through
a syringe filter (0.22 ␮m) and injected into a Bio-Rad (BioLogic LP)
chromatography system with LP Data view v1.03 software. Chro-
matography conditions for purification were programmed with
gradient elution; flow rate was ramped up from 1 to 2 mL/min.
Mobile phase was a binary system: mobile phase A was phosphate
buffer (pH 6.8) and mobile phase B was phosphate buffer (pH 6.8)
with 1 M NaCl. Total run time of 1 cycle was 113 min. Fractions were
collected at 2.5 min interval and subjected to enzyme activity anal-
ysis. Absorbance measurements were recorded with a Spectramax
182 W. Srichamnong, G. Srzednicki / Scientia Horticulturae 192 (2015) 180–186
M2 (Molecular device) spectrophotometer with temperature set to
25 ◦ C.
2.5.3. Fat extraction and isolation of fatty acids in kernels
The fresh oil was methylated according to the method of Bannon
and Craske (1987). Briefly, Macadamia nuts were cracked with a
hand cracker and the kernel was solvent extracted in a soxhlet
apparatus for 6 h with petroleum ether (bp. 40–60 ◦ C) as extrac-
tion solvent. Solvent was evaporated in a rotary evaporator (Buchi,
Switzerland) and then the sample was dried in an oven at 75 ◦ C for
30 min to complete solvent evaporation of the petroleum ether at
the oil surface. Oil was capped with nitrogen to remove traces of
oxygen to minimise lipid oxidation. The fresh oil was methylated
according to the method of Bannon and Craske (1987) with some
modifications. Macadamia oil extract was transferred to a 50 mL
volumetric flask and mixed with 5 mL of 0.25 M sodium methox-
ide in methanol-diethyl ether (1:1 v/v). The mixture was refluxed
for 30 sec and removed from the heat source. A volume of 3 mL of
isooctane and 15 mL of saturated sodium chloride were added. The
aliquot was vortexed for 20 s and saturated NaCl solution was added
to bring the total volume to 50 mL. The mixture was left undis-
turbed for 1.5 h at room temperature. Approximately 2.5 ␮L from
the top layer were collected and injected into a gas chromatograph
connected to a flame ionisation detector.
2.5.3.1. Quantification. The theoretical relative response factor
(TRF), (Perkin, 1993) was used for conversion of raw peak area to
corrected area when analysing fatty acid methyl ester (FAME) by
gas chromatography. Software used was GC Solution.
2.5.4. Sugar extraction
The method was adapted from Wall and Gentry (2007) with
slight modification; 5 g of defatted Macadamia kernel were trans-
ferred into a 50 mL test tube and mixed with 80% aqueous ethanol.
The mixture was vortexed for 3 min, sonicated for 30 min at room
temperature and then heated for 15 min at 70 ◦ C in a water bath.
The resulting mixture was evaporated under a stream of nitrogen
gas to dryness. Extraction was repeated twice. Spectrophotomet-
ric method was performed according to Canizares-Macias, et al.,
(2001) for quantification.
2.5.4.1. Sugar analysis. Sugar analysis (glucose, fructose and
sucrose) was performed by HPLC with a mobile phase of acetonitrile
(ACN) and water (80:20 v/v). Several ratios were initially trialed
including 75:25, 70:30, and 88:12 v/v. The mobile phase was vac-
uum filtered through 0.2 ␮m membrane before using at a flow rate
of 1.5 mL/min with 10 ␮L sample injection volume. The column
used was a Luna 5 ␮ NH2 100A model (Phenomenex, USA) with
PDA detector set at 200–800 nm wavelength followed by Refrac-
tive index detector (Wall and Gentry, 2007). Quantification was
performed by comparing the peak area to known standard curve.
Software used was LC Solution.
2.5.5. Phenolic extraction
The ground samples were defatted twice with hexane and
petroleum ether (bp. 40–60 ◦ C) and defatted samples (5 g) were
transferred into a 50 mL centrifuge tube and extracted with solvent.
The extraction solvent used for extraction of phenolic compounds
was acetone: methanol: water (7:7:6 v/v), with water added to
increase the extraction efficiency of phenolics, as most phenolic
compounds in Macadamia nuts are hydrophilic. In addition, as phe-
nolic compounds exist in various forms in nature, the use of solvent
with different polarity could enhance solubility of phenolics. The
supernatant was sonicated for 15 min which was the optimum time
for high phenolic recovery and then centrifuged at 4000 rpm for
10 min. The acid hydrolysis and base hydrolysis of the resulting
supernatant were conducted to reduce the signal to noise ratio of
the chromatogram and release hydrolysable phenolic compounds.
Acid hydrolysis was conducted by addition of 2 N trifluoroacetic
acid (TFA) while addition of 4 N NaOH was used for base hydrol-
ysis. The mixture was heated in a water bath at 70 ◦ C for 2 h. The
residues were re-extracted again and the supernatants were com-
bined to obtain the free phenolic extract. Bound phenolics were
extracted similarly to free phenolics except that the solvent used
was ethyl acetate: diethyl ether (1:1 v/v) (Naczk and Shahidi, 2006).
Pool supernatant was evaporated to dryness with rotary evapora-
tor and re-dissolved with methanol. These extracts were used for
HPLC and antioxidant analysis. Prior to inject into HPLC, the extract
was filtered through PTFE 0.22 ␮m syringe filter.
2.5.5.1. Phenolic content determination. Total phenolic content (TP)
was measured with the Folin–Ciocalteu reagent and gallic acid as a
standard in a 96 well plate. The amount of solution and reagent used
were optimised and modified from Tsantili et al., (2010). Phenolic
extract (50 ␮L) was pipetted into a well followed by MilliQ water
(90 ␮L). Folin–Ciocalteau (2 N) was diluted to 1 mL in 9 mL aque-
ous solution and added into the same well allowing the reaction
to occur for 8 min. Finally 7.5% sodium carbonate solution (100 ␮L)
was added. The plate was incubated at 25 ◦ C for 2 h before read-
ing the absorbance at 765 (end product) and 280 (protein content)
nm wavelengths. Protein content was monitored due to some pro-
teins which have phenol ring structure will give positive result in
TP assay hence wavelength at 280 was monitored and absorbance
was maintained as low as possible. Total free and bound phenolics
content of brown and white sections within brown kernel samples
were also analysed by this method.
2.5.5.2. Analysis of phenolics by HPLC. Binary mobile phase system
was used in this experiment. The mobile phase A was 0.3% formic
acid aqueous and B was 100% methanol. Gradient mode was as fol-
lows: 0–40 min B concentration 37%, 40–50 min B 100%, 50–70 min
B 100 %, 70–85 min B 6%, and 85–110 min B 37%. Column temper-
ature was 40 ◦ C to reduce mobile phase viscosity and facilitate the
flow of mobile phase which was 0.3 mL/min with 1 ␮L injection
volume of the sample. The column type used was reverse phase
C18 (Gemini, Phenomenex) with an internal diameter of 3 ␮m and
15 cm in length. Quantification and identification were conducted
by comparison to known external standard curve.
2.5.6. DPPH antioxidant activity and melanoidin analysis
The test was performed according to the method described by
Xu et al., (2007) with a slight modification by using methanol as
a solution and phosphate buffer instead of Tris-HCl buffer with
similar pH. The activity was given as absorbance at 517 nm.
2.5.7. Microbiological analysis
One gram of either mould nut in shell (NIS) or mould kernels
sample was homogenised with 9 mL of peptone water. An aliquot of
1 mL was then mixed with 9 mL peptone water in test tube for serial
dilution and vortexed for 1 min. After well mixing, 1 mL of each
serial dilution was pipetted out and dispersed on Dichloran Rose
Bengal Chlortetracycline (DRBC) agar using spread plate method.
After inoculation, petri dishes were incubated at 25 ◦ C for 3 days
for the growth of colonies. The young colonies were used as wet
mount sample. The matured colonies after 6 days of growth were
used for the determination of visible colony morphology. The struc-
ture of the young spores was observed under a microscope (Leica
DM 2500) at 100×. The Leica Application Suite was used for data
analysis.
 W. Srichamnong, G. Srzednicki / Scientia Horticulturae 192 (2015) 180–186 183

0.5

Corretec absorbance at 413 nm

0.4
A38
246
0.3 816
842
Dad
0.2

0.1

0.0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Fig. 1. Polyphenol oxidase activity of different Macadamia kernel of various varieties.

2.6. Statistical analysis

Samples were analysed in triplicate and results were subjected

to statistical analysis, which included determination of the mean
data set of sample and standard deviation as well as analysis of
variation (ANOVA) at the significance level p ≤ 0.05. The differences
were tested using Duncan Multiple Range Test (DMRT) at the sig-
nificance level of p ≤ 0.05.

3. Results and discussion

The three hypotheses which were the basic cause of browning

including: enzymatic browning, non-enzymatic browning and an
extracellular enzyme mediated browning caused by microorgan-
isms. Enzymatic browning occurs principally due to PPO and was
tested in all Macadamia varieties. Non-enzymatic browning reac-
tions are intensified by heating processes (eg. drying and roasting).
Microorganisms also played a role in kernel discoloration as indi-
cated by the experimental results shown below. With exception of
the ‘Daddow’ variety, internal browning was observed in all of the
varieties studied. In the ‘Daddow’ variety, the kernel colour was
slightly yellowish, but not brown.

3.1. Polyphenol oxidase activity in Macadamia kernel

PPO was studied in order to evaluate its potential to catalyse
enzymatic browning reactions in Macadamia kernels. No signifi-
cant difference in PPO activity was observed in different varieties
(Fig. 1). This indicated that all varieties studied have the same level
of browning susceptibility due to enzymatic activity. However, as
shown in Fig. 2A, the ‘Daddow’ variety had lowest phenolic con-
centration compared to other varieties. This indicated that enzyme
activity with the limitation of substrate could result in minimum
or less activity. (Auden and Dawson, 1931) and thus could be cor-
related with other varieties which were more prone to ICD. This
was postulated that ‘Daddow’ variety has the lowest susceptibil-
ity to form brown kernels. These results were in agreement with
the previous study conducted by Walton et al., (2013) which stated
that brown kernels are formed through high metabolic activity and
most of the postharvest browning resulted from enzymatic process.
3.2. Phenolic compounds and antioxidant activity
The ‘A38’ and ‘842’ varieties had the highest phenolic content in
fresh kernels in the following order ‘A38’> ‘842’> ‘246’> ‘816’> ‘Dad-
dow’ (Fig. 2A). The phenolic content of individual brown kernels
was studied. Since, the total phenolic content is represented by both
Fig. 2. Total content of phenolic compounds in fresh and roasted nuts (A) and free
and esterified phenolics in white and brown portions of the same kernels (B). The
letters A–G represent each kernel. Capital letters alone indicate white portion while
small letters followed by (b) indicate brown portion of the same kernel (darker
colour).
free phenolic content and esterified phenolic compounds. Thus,
free and esterified phenolic compounds were analysed separately
because free phenolic alone will not represent total phenolic due
to it can esterified with either protein or polysaccharide as nature
phenol are reactive. The proportion of free and esterified phenolic
compounds was determined in the brown and white sections of
the same kernels. The number of kernels studied was divided into
7 groups (A–G) randomly. Each group included the average data of
10 Macadamia nuts (Fig. 1B). The results showed a random distri-
bution in an average processed batch as the brown kernel samples
contained various varieties including ‘A38’, ‘Daddow’, ‘246’, ‘816’,
‘842’and others.
The total phenolics content of the brown kernels varied between
5290 and10,380 mg gallic acid equivalent (GAE)/kg defatted sam-
ple. Most of the brown kernels had a higher content of total
phenolics in their brown section than in their white section except
in groups E, F and G. The total phenolic content of E, F and G in white
184 W. Srichamnong, G. Srzednicki / Scientia Horticulturae 192 (2015) 180–186

Table 1 Table 2
Sugar concentration in fresh and dried Macadamia kernels. Sugar level in Macadamia kernels roasted of variety ‘246’ at 60, 90, 120 and 150 ◦ C
for 30 min.
‘Variety’drying method Sample (mg/g DW)
Roasting temp. Sucrose Glucose Fructose
Sucrose Glucose Fructose (◦ C) (Sample mg/g DW)
‘A38¢ 41.0 ± 0.06a 3.0 ± 0.01a nd 60 21.4 ± 0.10b 5.53 ± 0.30b 4.8 ± 0.10b
‘A38¢CD 37.0 ± 0.09a 10.0 ± 0.01b 5.0 ± 0.20b 90 19.6 ± 0.20b 5.03 ± 0.20b 4.8 ± 0.10b
‘A38¢HP 37.0 ± 0.03a 10.0 ± 0.30c 5.0 ± 0.30b 120 19.5 ± 0.10b 4.48 ± 0.10b 4.7 ± 0.00b
‘246¢ 23.0 ± 0.30b 2.0 ± 0.01a nd 150 1.55 ± 0.00a 1.55 ± 0.20a 1.0 ± 0.20a
‘246¢CD 28.0 ± 0.10b 13.0 ± 0.01c 7.0 ± 0.30b
‘246¢HP 27.0 ± 0.60b 13.0 ± 0.00c 7.0 ± 0.20b a
Values are means ± SD. Mean separation within columns based on significant
‘816¢ 33.0 ± 0.10b 3.0 ± 0.01a nd difference (p ≤ 0.05).
b
‘816¢CD 30.0 ± 0.30b 15.0 ± 0.10c 9.0 ± 0.10b Each of the three replication consisted of an independent extraction.
‘816¢HP 30.0 ± 0.30b 13.0 ± 0.003c 9.0 ± 0.20b
‘842¢ 44.7 ± 0.40a 2.0 ± 0.01a nd
‘842¢CD 41.3 ± 0.10a 7.0 ± 0.01b 5.0 ± 0.50b was 3.6–5.3 mg/g (dry sample) in comparison with 2.0–4.0 mg/g in
‘842¢HP 40.0 ± 0.60a 7.0 ± 0.01b 5.0 ± 0.50b fresh sample.
‘Dad’ 50.0 ± 0.80c 4.0 ± 0.01a 2.6 ± 0.30a
When NIS was dried either in cabinet dryer or heat pump dryer,
‘Dad’CD 60.0 ± 0.60c 4.0 ± 0.02a 2.6 ± 0.20a
‘Dad’HP 60.0 ± 0.60c 3.0 ± 0.01a 2.6 ± 0.20a
the sucrose was hydrolysed into glucose and fructose (Table 1). In
comparison, after thermal processing, sucrose was not hydrolysed
nd = not detected.
ns = not-significantly different.
in the ‘Daddow’ variety but naturally present in raw nuts, while
CD = cabinet dryer. sucrose was hydrolysed in other varieties (Table 1). In general,
a
Values are means ± SD. Mean separation within columns based on significant sucrose could be readily hydrolysed with acid and the reaction is
difference (p ≤ 0.05). accelerated at high temperature. Goldberg et al., (1989) stated that
b
Each of the three replications consisted of an independent extraction from the the hydrolysis involving non-electrolytes will have a slight depen-
same batch.
c
Statistical analysis was done separately in each drying method since the raw
dence on pH, which depends on ionisation capability of the reactant
materials were not the same. or product. ‘Daddow’ variety had a higher pH compared to the other
varieties, perhaps explaining its slower sucrose hydrolysis.
Reducing sugar content of the roasted sample was decreased
and brown centers were in the range of 6951–10,327, 9919–10,324 to half due to roasting temperature. It then reacted with protein
and 7476–9,385 mg gallic acid equivalent (GAE)/kg, respectively. in the nuts resulted in colour development through Maillard reac-
Approximately 70% of the total brown kernels had higher esterified tion. The change of reducing sugar content at high temperature is
phenolics content in the brown than in the white sections (Fig. 2B shown in Table 2. A detectable increase in sweetness noticed in
(b) column). In addition, 57.1% of the total brown kernels studied the Macadamia nut after roasting may be due to caramelisation
had a lower free phenol content compared to the white section associated with browning development.
(Fig. 2B). The content of esterified phenolic compounds was higher Fructose was not detected in fresh samples except the ‘Daddow’
than that of free phenolic compounds in the same brown kernel variety, but was present in all dried samples. In addition, glucose
(85.8%). Furthermore, brown kernels had a higher total phenolic concentrations in dried samples increased except in dried ‘Dad-
content compared to non-brown kernels (Fig. 2B). This indicates dow’ kernels. Sucrose, glucose and fructose were still present in
that phenolics content plays an important role in the enzymatic the samples roasted at 60 ◦ C (Table 2). When the temperature was
browning mechanism. increased to 90 ◦ C, the sucrose concentration decreased, while glu-
Srichamnong et al., (2013) reported that esterification of phe- cose and fructose concentration increased (Table 2). At 90 ◦ C, the
nolic compounds with associated protein substrates could be Macadamia kernels showed a slightly more intense golden colour
observed by staining Macadamia cells with periodic acid. Further compared to 60 ◦ C. This indicated that the hydrolysis of sucrose into
study of protein content and esterified phenolic structure could be glucose and fructose along with Maillard reactions had occurred.
determined by using NMR which will give more detail of chemical However, after roasting at 120 ◦ C, the concentration of glucose and
structure orientation. fructose decreased compared to those at 90 ◦ C (Table 2). This indi-
The scrape method could be improved to give more accuracy by cated that these two compounds were involved in other chemical
better separation of the brown from white section. There were two reactions. Furthermore, a more intense brown colour appeared at
major phenolic compounds identified in this study. All Macadamia 120 ◦ C. Finally, minute quantities of sucrose, fructose or glucose
varieties studied contained chlorogenic acid (14–25 ␮g/g) and p- were detected after roasting at 150 ◦ C.
hydroxybenzoic acid (3.6–10 ␮g/g) as major phenolic compounds. Absorbance data revealed that total phenolic decreased (Fig. 3B)
The p-hydroxybenzoic concentration was lower than that (24 ␮g/g) and the formation of melanoidin compounds increased as the tem-
measured by Quinn and Tang (1996). In addition, hydroxycinnamic perature and heating time increased (Fig. 3B). In general, the colour
was not detected in this study. However, there were some unknown of the Maillard reaction by-products (MRP) solution was a darker
chromatogram peaks that need to be further identified with mass brown at 120 ◦ C compared to 90 ◦ C. However, heating at high tem-
spectrometry. perature (150 ◦ C) for an increased time period (>30 min) degraded
the antioxidant-MRP formed in the early stage of the reaction. This
3.3. Sugar determination change was reflected by a reduction of absorbance (Fig. 3B).

The purpose of this investigation was to determine the sugar
concentration in the fresh and processed kernel, which can act as a
substrate for the Maillard reaction. Fresh kernels contain two dif-
ferent sugars namely sucrose and glucose and the concentrations
varied according to the variety (Table. 1). The measured sucrose
concentrations were in agreement with literature values (Wall and
Gentry, 2007) in which sucrose concentrations of fresh sample
ranged between 29.5 and 69.1 mg/g while that of reducing sugars
3.4. Fatty acid profiles
The fatty acid concentrations of the brown and non-brown
kernels of variety ‘246’ are shown in Table 3. Majority of fatty
acids were oleic acid (300 and 562 mg/kg kernel) palmitoleic acid
(86 and121 mg/kg kernel) and palmitic acid (43.6 and 63.2 mg/kg
kernel) from non-brown and brown respectively. There was no
significant difference noted among fatty acid profiles. However,
 W. Srichamnong, G. Srzednicki / Scientia Horticulturae 192 (2015) 180–186 185

(A) 1.6 (B) 1.6

Absorbance at 517 nm
Absorbance at 765 nm
1.5 1.5

1.4 1.4

1.3 1.3

1.2 1.2
30 C 60 C 90 C 120 C 150 C 30 C 60 C 90 C 120 C 150 C
Temperature Temperature

Fig. 3. Absorbance indicating total phenolics content (A) and melanoidin production (B) of Macadamia kernels roasted for 30 min at 60, 90, 120 and 150 ◦ C.

Table 3
Comparison of fatty acid composition of brown and non-brown kernel of Macadamia
variety ‘246’.

Fatty acid Concentration (mg/kg kernel)

Brown kernel Non-brown kernel

Palmitic (C16:0) 62.3 ± 12.2

b
40.3 ± 9.0a
Palmitoleic (C16:1)ns 121.7 ± 25.0 86.4 ± 10.4
Stearic (C18:0)ns 25.2 ± 20.8 15.0 ± 4.6
Oleic (C18:1) 561.7 ± 12.4b 300 ± 74.0a
Linoleic (C18:2)ns 9.1 ± 2.7 10.7 ± 2.4
Arachidic (C20:0) 30.5 ± 0.6b 13.1 ± 3.3a
Eicosenic (C20:1)ns 21.5 ± 7.1 14.0 ± 2.5
Behenic (C22:0) 13.0 ± 6.1b 4.5 ± 0.1a

ns = Not-significantly different.
a
Values are means ± SD. Mean separation within lines based on significant dif-
ference (p ≤ 0.05).
b
Each of the three replications consisted of an independent extraction.

the quantity of fatty acids in brown kernels was almost double as

compared to non-brown kernels (Table 3). Thus, fatty acid con-
centrations could be involved in kernel browning. This result was
in agreement with the previous study of Rui et al., (2010), which
stated that fatty acid accumulation resulted in membrane disinte-
gration, hence internal browning could occur. However, the actual
mechanism of the factors which lead to fatty acid accumulation
remains unclear.

3.5. Microorganisms and kernel discoloration

Browning due to microorganisms such as mould was also evi-

denced in this study. Interestingly, not all mould infected samples
turned brown. The ‘816’ and ‘A38’ varieties were stored under simi-
lar conditions as that of ‘842’ and were infected by moulds (greenish
area with filaments). However, they did not produce a brown ker-
nel. Samples that were kept at an elevated temperature (35 ◦ C) and
high moisture content (22% wet basis) showed mould growth and
the kernels turned from white to brown.
The moulds present on NIS and kernels were identified as Peni-
cillium aurantiogriseum, when the colony morphology and spore
structure were compared with a previous study involving pista-
chio, hazelnut, pecan and peanuts (Pitt and Hocking, 1997). Based
on microscopic examination, microorganisms possess biverticillate
type conidiophore, and its shape was slender and in ampulli-
Fig. 4. Mycelium (A), (B) and hypae structure (C) conidia under 100X in colonies on
form (Fig. 4A). Hyphae were septate with branched conidiophores
DRBC agar, 3 days, 25 ◦ C isolated from brown and mouldy Macadamia kernel.
(Fig 4B). In addition, its conidia wall structure was smooth and in
spherical shape (Fig 4C). These microorganisms also produce an
inverted orange pigment. The picture of conidiophores, hyphae and Uncontrolled storage conditions resulted in browning due to
conidia were showed as Fig 4. Thus; we identified similar mould microorganism infection even in unprocessed Macadamia nuts.
species in NIS and kernels. In addition, P. aurantiogriseum has high Therefore, brown colour development in kernels prior to drying
lipolytic activity; and thus infected kernels could experience sig- could be due to mould infection through cracks or the faulty for-
nificant lipid degradation which is undesirable. mation of the nut micropyle. The nuts in the present study were
186 W. Srichamnong, G. Srzednicki / Scientia Horticulturae 192 (2015) 180–186
noticed with the small hole at the micropyle position which could
promote the microbial infection and subsequently browning in ker-
nels.
4. Conclusions
Browning of kernels occurred through three different pathways
(i.e., (i) microorganisms, (ii) Maillard reaction, and (iii) enzymatic
browning) based on the postharvest treatments used. Soon after
the nuts achieve maturity and falls on the ground, it is suscepti-
ble to browning by microorganisms. Microorganism contamination
could occur through the open micropyle or faulty shell formation or
cracks. It is therefore recommended to harvest the nut as soon as it
falls on the ground and use good agricultural practices to minimise
unwanted microbial attack. In addition, the wet husk attached to
the NIS could be a source of microorganisms. Prompt removal of
the husk needs to be strictly followed. Sucrose is hydrolysed into
glucose and fructose. These reducing sugars together with a pro-
tein component can lead to browning induced by the Maillard
reaction during drying. This was evidenced by microscopy work
in which high concentration of proteins were distributed in the
brown kernel sample. Thus kernels with high protein content are
more susceptible to browning. Regardless of sucrose concentration,
the amount of glucose and fructose hydrolysed during drying are
the key determinants in the degree of browning. The third possible
mechanism, enzymatic browning is related to phenol content as
the brown sections of the kernels had higher bound phenolic con-
centrations than the white sections of the same kernel. The brown
sections result from esterification of the phenolic compounds with
associated protein substrates. The ‘Daddow’ variety had the highest
activity of polyphenol oxidase compared to other varieties studied
but it contains lowest phenolic content. Thus, that phenolic con-
tent as well as polyphenol oxidase activity play a role in kernel
discoloration.
Acknowledgements
The authors would like to thank Mr. Cliff James, Mr. Steven
Lee, Brice Kaddatz and Kim Jones for their technical support and
Macadamia Processing Company (MPC) Lismore, Australia for sup-
plying the raw materials. The author would also like to thanks Prof
William Price at University of Western Sydney, Australia for this
guideline and final look for the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.scienta.2015.
06.012
References
AOAC, 1995. Official methods of analysis, Association of Official Analytical
Chemists, 15th ed. AOAC International, Washington D.C.
Auden, A.H., Dawson, R.E., 1931. The hydrolysis of concentrated sugar solution by
invertase. J. Biol. Chem. 25 (6), 1909–1916.
Bannon, C.D., Craske, J.D., 1987. Gas liquid chromatography analysis of the fatty
acid composition of fats and oils: a total system for high accuracy. J. Am. Oil
Chem. Soc. 64, 1413–1417.
Garg, M.L., Blake, R., Wills, J., 2007. Macadamia nut consumption modulated
favourably risk factor for coronary artery disease in hypercholesterlemic
subjects. Food Lipid 24, 583–587.
Goldberg, R.N., Tewarit, Y.B., Ahluwalia, J.C., 1989. Thermodynamics of the
hydrolysis of sucrose. J. Biol. Chem. 246 (17), 9901–9904.
Griel, A.E., Cao, Y., Bagshaw, D.D., Cifelli, A.M., Holub, B., 2008. A Macadamia
nut-rich diet reduced total and LDL-Cholesterol in mildly
hypercholesterolemic men and women. Am. Soc. Nutr. J. 138 (4), 761–767.
Lagadec, D., 2009. Kernel brown centers in macadamia: a review. Crop Pasture Sci.
60, 1117–1123.
Lovejoy, A., 2005. The impact of nuts on diabetes and diabetes risk. Curr. Diab. Rep.
5, 379–384.
Murano, P., 2003. Understanding Food Science and Technology, 1 ed. Thomson
Learning, USA, pp. 10–15 (Chapter 1).
Naczk, N., Shahidi, F., 2006. Phenolics in cereals, fruits and vegetables: occurrence,
extraction and analysis. J. Pharmacol. Biol. Anal. 41, 1523–1542.
Phatanayindee, S., Borompichaichartkul, C., Srzednicki, G., Craske Wootton, J.M.,
2012. Changes of chemical and physical quality attributes of Macadamia nuts
during hybrid drying and processing. Dry. Technol. 30 (16), 1870–1880.
Perkin, G., 1993. Analysis of Fats, Oils and Derivatives. AOCs Press, America
(Chapter 16).
Pitt, J.I., Hocking, A.D., 1997. Fungi and Food Spoilage, 2nd ed. Blackie Academic &
Professional, Melbourne.
Prichavudhi, K., Yamamoto, H. Y., 1987. Effect of drying temperature on chemical
composition and quality of Macadamia nuts. C.M.S. book. 98–104.
Quinn, L.A., Tang, H.H., 1996. Antioxidant properties of phenolic compounds in
Macadamia nuts. JAOCS 73 (11), 1585–1588.
Rui, H., Cao, S., Shang, H., Jin, P., Wang, K., Zheng, Y., 2010. Effects of heat treatment
on internal browning and membrane fatty acid in loquat fruit in response to
chilling stress. J. Sci. Food Agri. 90, 1557–1561.
Sinanoglou, V.J., Kokkotou, K., Fotakis, C., Strati, I., 2014. Monitoring the quality of
gamma-irradiated Macadamia nuts based on lipid profile analysis and
Chemometrics. Traceability models of irradiated samples. Food Res. Int. 60,
38–47, Special Issue.
Srichamnong, W., Wootton, M., Srzednicki, G., 2012. Lipoxygenase and Peroxidase
Activity of Macadamia Kernel after Thermal Processing. ISHS Acta
Horticulturae 943. Asia Pacific Symposium on Postharvest Research, Education
and Extension.
Srichamnong, W., Price, B., Gardner, T., Dean, R., Plougonven, E., Léonard, A.,
Srzednicki, G., 2013. Studies of microstructure of kernels of Macadamia
integrifolia and its hybrids through MRI, X-ray tomography and confocal
microscopy. J. Food Sci. Eng. 3, 503–516.
USDA, 2011. United State Department of Agriculture: Tree nut production in
selected countries. USDA national agricultural statistics services. <http://www.
fas.usda.gov/htp/Hort Circular/2002/02-04/Stats/MAC.pdf> Accessed on 31
March 2010.
Wall, M.M., Gentry, T.S., 2007. Carbohydrate composition and color development
during drying and roasting of Macadamia nuts (Macadamia integrifolia).
Lwt-Food Sci. Technol. 40, 587–593.
Walton, D.A., Wallace, H.M., 2015. The effect of mechanical dehuskers on the
quality of Macadamia kernels when dehusking Macadamia fruit at differing
harvest moisture contents. Sci. Horticult. 182, 119–123.
Walton, D.A., Randall, B.W., Le Lagadec, M.D., 2013. Maintaining high moisture
content of Macadamia nuts-in-shell during storage induces brown centers in
raw kernels. J. Sci. Food Agri. 93 (12), 2953–2958.
Xu, Q., Tao, W., Ao, Z., 2007. Antioxidant of vinegar melanoidins. Food Chem. 102,
841–849.