Food Chemistry 134 (2012) 1461–1467

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effects on starch and amylolytic enzymes during Lepidium meyenii Walpers

root storage
Gerby Giovanna Rondán-Sanabria ⇑, Beatriz Valcarcel-Yamani 1, Flavio Finardi-Filho
Departamento de Alimentos e Nutrição Experimental, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Brazil

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

Article history: The high water content in maca (Lepidium meyenii W.) roots combined with the damage produced during
Received 4 October 2011 or after harvest makes them vulnerable to attack by enzymes and microorganisms. Although starch deg-
Received in revised form 23 January 2012 radation has been extensively studied, in maca roots there is a paucity of research regarding the starch
Accepted 13 March 2012
reserves. In this paper, parameters of starch degradation are shown to be related to the action of amylo-
Available online 21 March 2012
lytic enzymes during storage at room temperature. Over the course of three weeks, the starch and protein
content, soluble sugar, total amylolytic activity, and a- and b-amylase activity were measured. In addi-
Keywords:
tion, the integrity of starch granules was observed by scanning electron microscopy. Despite the evidence
Amylolytic enzymes
Lepidium meyenii Walpers
of dehydration, there were no significant differences (p 6 0.5) in the total starch content or in the activ-
Scanning electron microscopy and starch ities of a- and b-amylase. After the third week the roots remained suitable for consumption. The results
indicate a postharvest latency that can lead to sprout or to senescence, depending on the environmental
conditions.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction to complete the reproductive phase. After the harvest, healthy

Maca (Lepidium meyenii W.) is a native plant of the Andes and
belongs to the family Brassicaceae. It is grown at an altitude of
3500–4500 m (Vilches, 1998). The roots have a similar nutritional
composition to cereal grains, and they contain up to 18% protein
and 76% carbohydrate, as well as possessing high amounts of free
amino acids and a significant amount of minerals, but contain a
small amount of lipids, 2.2%. In addition to being a regular food
and food ingredient, maca is also used as a functional food and folk
medicine to improve the fertility and vitality, among other tradi-
tional therapies (Fahey, Zalcmann, & Talalay, 2001; Ganzera, Zhao,
Muhammad, & Khan, 2002; Gonzales et al., 2006; Ochoa & Ugent,
2001; Quirós & Cárdenas, 1997; Rea, 1994; Zheng et al., 2000).
The propagation of the maca plant is by seeds. The harvest takes
place seven months after planting, when the roots or hypocotyls
are fully extended and ready for consumption. At this point, the
reproductive phase begins with flowering and seed production;
this reproductive phase can be completed within a year, as long
as the environmental conditions are favourable for reproduction.
If environmental conditions during the reproductive phase are
not suitable, a dormancy-like step and then sprouting will occur
⇑ Corresponding author. Address: Av. Prof. Lineu Prestes, 580, Butantã, Sao Paulo,
05508-000 SP, Brazil. Fax: +55 11 30913652.
E-mail address: gerbyrs@usp.br (G.G. Rondán-Sanabria).
1
Present address: Departamento de Tecnologia Bioquímico-Farmacêutica, Fa-cul-
da-de de Ciências Farmacêuticas, Universidade de São Paulo, Brazil
roots remain stable without showing signs of deterioration until
they are dry. When kept at room temperature with low humidity
it is possible to store the roots for a long period (Quirós & Cárdenas,
1997).
While in storage, losses due to mechanical damage, physiologi-
cal changes and diseases by biological vectors can occur. These
losses are affected by intrinsic and extrinsic factors, but they gen-
erally cause a decline in the quality and shelf life of fruits and veg-
etables. Aside from physiological deterioration, chemical and
enzymatic changes that cause the softening of the tissue, loss of
colour, odour, flavour, and nutritional value may also occur (Floros,
1993). The action of amylolytic enzymes in starch granules has
been the subject of several investigations. These studies have
shown differences in the resistance to the action of a-amylase,
not only in the degree of hydrolysis but also in the mode of attack
and the products liberated by hydrolysis. This susceptibility is due
to several factors such as crystalline structure, particle size, amy-
lose and amylopectin contents, as well as the presence of enzyme
inhibitors.
Native grains of potato, corn, and rice subjected to hydrolysis
show a degradation rate that is inversely proportional to the size
of the grain (Floros, 1993; Kim, Kong, Kim, & Lee, 2008). However,
in sweet potato starch, the high amylopectin content is associated
with a high gelatinization temperature and consequently has a low
tolerability to a-amylase attack. Cereal starches are more vulnera-
ble to a- and b-amylase action due to their porous surfaces, in
contrast to tubers with smooth surfaces (Zhang & Oates, 1999).

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.03.056
1462 G.G. Rondán-Sanabria et al. / Food Chemistry 134 (2012) 1461–1467
High enzymatic activities for a- and b- amylase, pectic enzymes
and cellulase were identified in maca roots by setting conditions
for extraction and the reaction temperatures using the response
surface methodology (Sanabria-Rondán, Pires, & Finardi-Filho,
2006).
The physicochemical properties of maca starch show that the
amylose/amylopectin ratio influences the gelatinization and
viscosity parameters of starch granules (Sanabria-Rondán &
Finardi-Filho, 2009). Based on the X-ray diffraction pattern, maca
starch displays a B-type pattern on crystalline structure, is consid-
ered rich in amylose and has similar shapes and sizes; additionally,
the granules are resistant to both acidic and enzymatic hydrolysis,
such as waxi potato and Chinese potato (Jayakody, Hoover, Liu, &
Weber, 2005; McPherson & Jane, 1999). A fundamental character-
istic of native starches from different plant sources is that their
granules and molecular structures influence their physicochemical
and functional properties.
This study investigates the possible changes in the profiles of
carbohydrates and proteins during the storage of maca roots by
amylolytic enzyme actions.
2. Materials and methods
2.1. Materials
Samples of maca roots were collected at randomly in crop areas
seven months after planting in the Department of Junin-Peru, at
4500 m above sea level. The samples were ripe, ready to be con-
sumed, and at the end of the vegetative phase. After harvest, they
were taken to the laboratory and the analyses were performed the
second day after harvest. The samples were monitored every
2 days for 22 days. On the 22 day of storage, a new set of analyses
was performed with a very dehydrated root. Roots were stored in
the same conditions as those that are available for consumption,
at room temperature (20–23 °C) and at a relative humidity above
40%.
The roots were subjected to the quartering system to obtain
representative portions: each portion consisted of five to six maca
roots. The roots were then peeled and cut into small cubes for
analysis. All tests were performed in triplicate. BSA, precision pro-
tein standard, benzamidine and PVP 40,000 were obtained from
Sigma–Aldrich Co., (São Paulo, Brazil). Betamyl and Ceralpha were
purchased from Megazyme (Campinas, Brazil) and Immobiline Dry
Strip (IPG Strip) was obtained from GE Healthcare Bio Sciences (São
Paulo, Brazil). All chemicals used were of analytical grade.
2.2. Preparation of enzymatic extract
Three 10 g samples from the peeled and cut roots were weighed
and 30 ml of phosphate buffer 0.01 M (pH 6.0) was added to each;
the material was homogenised in an UltraTurrax crusher for
approximately 3 min. The homogenate was filtered and centri-
fuged at 10,000 rpm/30 min/4 °C. In parallel, enzymatic extraction
was performed by adding protease inhibitors, i.e. 1% (w/v) soluble
PVP 40,000 and 1 mM benzamidine. The supernatant was dialysed
for 12 h against distilled water at 4 °C and stored in 1.5 ml Eppen-
dorf tubes at 20 °C for further analyses.
2.3. Determination of protein concentration
Proteins were quantified by the method described by Bradford
(1976). Briefly, 1.0 ml of the Bio-Bradford reagent was added to
30 ll of enzymatic extract in a volume of 970 ll water and after
5 min the reaction was read at 595 nm. Bovine serum albumin
(BSA) was used as a protein standard.
2.4. Determination of enzymatic activity
Initially, the total amylolytic activity in the enzymatic extracts
was determined by the formation of the starch–iodine complex
(Street, 1974). Aliquots of 50 ll of enzymatic extract were incubated
in 200 ll of a 0.1% potato starch solution, 500 ll of 0.05 M phos-
phate buffered saline (pH 6.0), and 200 ll of a 0.01 M HCl solution.
After a 15-min incubation at 30 °C, the reaction was stopped by add-
ing 400 ll of iodine solution (10 mM I2 and 14 mM KI). Distilled
water was added to the reaction up to a final volume of 10 ml and
the absorbance was read at 578 nm in a Micronal B582 spectropho-
tometer. One unit of amylase activity (1 U) was defined as the
amount of enzyme needed to hydrolyse 200 mg of starch per ml
of enzymatic extract in the reaction conditions.
Subsequently, specific substrates were employed for detecting
amylase activity. For the b-amylase activity, the detection was per-
formed following the method described by McCleary and Codd
(1989) using the BetamylÒ reagent, which contains p-nitro-
phenyl-maltopentaoside (PNPG5) as a substrate. For determining
the a-amylase activity, the method described by McCleary and
Sheehan (1987) was employed, which uses the CeralphaÒ reagent
containing p-nitrophenyl-maltoheptaoside blocked at the non-
reducing end (BPNPG7) as a substrate.
The reaction of both methods occurs in the presence of a-glyco-
sidase and liberates p-nitrophenol, which develops a yellow colour
under alkaline conditions allowing it to be read at k = 410 nm. For
detection of enzymatic activity, 50 ll of each extract were incu-
bated with 50 ll of substrate (BetamylÒ or CeralphaÒ), and after
incubation at 30 °C for 10 min, the reaction was stopped by adding
750 ll of 1% (p/v) Trizma-base (pH > 10). In order to quantify the
enzymatic activity, a standard p-nitrophenol curve was used. One
unit of enzymatic activity in the reaction conditions (1 UG) was de-
fined as the amount of enzyme that releases 1 mMol of p-nitrophe-
nol per mg of protein per min.
2.5. Electrophoresis
During storage, the protein profile of the enzyme extracts was
accompanied by electrophoresis on polyacrylamide gels, under
non-denaturing (native-PAGE) and denaturing (SDS–PAGE) condi-
tions. Separation under dissociative conditions was performed in
accordance with the method described by Laemmli (1970), which
uses gels with 12% or 15% polyacrylamide in Tris/glycine buffer
(pH 8.8) containing 0.1% SDS.
Separation by native-PAGE was performed in 6.5% polyacryl-
amide gels in the same buffer as above but without SDS. The gels
were stained with Coomassie blue R-250 to detect proteins. Amy-
lase activity was detected with a solution of iodine (10 mM I2 and
14 mM KI) after incubating the gel in phosphate buffer (0.1 M, pH
6.0) containing 1% of potato starch at 30 °C for 3 h.
2.6. Native two-dimensional electrophoresis
Isoelectric focusing (IEF) was performed using 7 cm, 3–10 linear
IPG strips. The strips had been hydrated at room temperature for
17 h, using 30–50 mg aliquots of protein present in the enzymatic
extract, which had been dissolved in 125 ml of buffer (2% CHAPS,
0.5% ampholytes, and 0.005% bromophenol blue). They were then
separated according to their isoelectric point (pI) by IEF using the
Ettan IPGphor III – GE Healthcare system. The strips were covered
with mineral oil to prevent the evaporation of the reagents and the
sample. After focusing, the strips were subjected to a second
dimension to verify the enzymatic activity. Enzymatically active
spots were detected as described above.
 G.G. Rondán-Sanabria et al. / Food Chemistry 134 (2012) 1461–1467
2.7. Isolation of starch granules
Starch was isolated by the method described by Singh and Singh
(2001) with some modifications. Maca roots were manually peeled,
cut into small cubes (about 3 mm edge), and crushed with an
UltraTurrax in distilled water for 3 min. The homogenate was fil-
tered through three layers of a thin layer membrane of cotton
gauze, collected in a beaker, and then centrifuged at 1300g for
15 min. The supernatant was discarded, while the starch precipi-
tate was re-suspended in water and washed several times with
water until the starch became white and until the filtrate was
transparent. The starch was carefully dried under ventilation at
room temperature for 12 h then stored at 20 °C.
2.8. Determination of sugars
The total soluble sugars were extracted from approximately 1 g
of mashed maca sample in liquid nitrogen with 5.0 ml of 80% eth-
anol. The samples were shaken in a water bath at 80 °C for 10 min,
centrifuged at 12,000g for 10 min, and then, the supernatant was
collected in a 25 ml flask. The precipitate was extracted twice. A
1.0 ml aliquot was concentrated with a speed-vac and the residue
was re-suspended in water. The total sugars were determined
using the phenol–sulphuric method (Dubois, Giles, Hamilton, Re-
bers, & Smith, 1956), and the reducing sugars were determined
using a 3,5-dinitrosalicylic acid reagent (DNS), as described by
Bernfeld (1951) who used a glucose standard.
2.9. Determination of total starch
The determination of total starch was performed according to
the method described by Areas and Lajolo (1981) with some mod-
ifications. Approximately a 0.5 g sample was mashed in liquid
nitrogen and homogenised with a solution of 0.5 M NaOH for
5 min. The pH was then neutralised with 5 ml of 0.5 M acetic acid,
and water was added to reach a final volume of 50 ml in a volumet-
ric flask. A volume of 8.0 ml absolute ethanol was then added to
2 ml aliquots to precipitate the starch. These mixtures were then
centrifuged at 12,000g for 15 min.
The starch-containing precipitate was washed with 80% ethanol
(2), dried, and hydrolysed with 2 ml of a solution containing
amyloglucosidase and a-amylase (14.0 and 0.4 U/ml, respectively,
Sigma) in 0.2 M acetate buffer (pH 4.8) at 37 °C for 2 h. The reaction
was stopped by adding 0.6 N perchloric acid. The glucose released
was quantified following the method described by Bernfeld (1951)
using a glucose standard.
2.10. Scanning electron microscopy
The integrity of starch granules isolated during the storage per-
iod was observed using a Quanta 600 FEG scanning electron micro-
scope (FEI brand – Technological Characterisation Laboratory,
Department of Mines and Petroleum Engineering at the Polytech-
nic School of USP). The samples were prepared in ‘‘stubs’’ and set
on a double-sided carbon tape and covered with platinum for met-
allization in the Modular High Vacuum Coating System MED 020
(Bal – Tec). The images obtained were of secondary electrons when
operated at 5 kV.
2.11. Statistical analysis
For statistical analysis of quantitative data obtained from the
study, all variables had their initial variance tested by dispersion
graphs and Hartley’s test. Next, univariate analysis of variance
was performed for all data measured during storage, followed by
1463
Tukey’s test to compare the means. The analyses were performed
using Statistica 7.0 (StatSoft Inc. South America, Tulsa, OK, USA).
3. Results and discussion
3.1. Enzymatic activity and protein concentration
Fig. 1 shows the values measured during storage such as protein
concentration, total amylolytic activity, and a- and b-amylase activ-
ity. The protein concentrations in the enzymatic extracts were 0.90–
1.0 mg/ml (Fig. 1A). The maximum protein concentration was
1.01 mg/ml on day 10, and lowest concentrations were 0.90 mg/
ml on days 6 and 8. At the last observed point, on day 22, an extrac-
tion was carried out on the visibly dehydrated root (30% moisture
compared to 67% on day 2), but these data show that there was no
significant difference (p 6 0.05) in this study period, indicating that,
based on dehydration, there was stability in the protein profile of
maca roots. The SDS–PAGE gel in Fig. 2 shows several bands in all
of the time points, and a similar profile was found throughout the
storage period. The gel bands with higher intensities were marked
with black arrows, as these protein bands indicate the highest con-
centration of protein and could be a type of enzyme, such as an amy-
lase, which have molecular weights greater than 40 kDa. This could
suggest that there was no degradation or increase in the concentra-
tion of some proteins during storage. Protein stability was observed
with the addition of protective agents against proteases in the
extraction solution such as PVP-40 (1%) and 1 mM benzamidine.
The result was as expected, with no changes in protein profiles by
SDS–PAGE or native-PAGE (data not shown).
Variations during the storage period are shown in the total amy-
lolytic activity profile (Fig. 1B). These variations are within the same
order of magnitude: on day 2 of storage, the activity was 0.249 U,
and on day 16, activity was 0.258 U. The highest activity (0.258)
was seen on day 16, while the lowest activity (0.243 U) was seen
on day 12. Despite the small variations between the measurements,
the data showed no significant differences (p 6 0.05). The profile of
activity for a- and b-amylase (Fig. 1C and 1D) was determined using
specific substrates and showed no significant differences between
the measured activities, despite some variations in the data. The
lowest a-amylase activity was detected on day 8 of storage with
0.123 U and the highest activity was on day 2 with 0.137 U. b-Amy-
lase showed the highest activity on storage day 4 with 0.644 U and
the lowest on the second day with 0.634 U. Notably, methodological
differences did not allow numerical comparisons between the total
amylase activity and the separate activity of the enzymes; however,
oscillations are characterised around the initial values, without
pointing to tendencies in the period studied.
Native-PAGE separation allowed us to identify bands with amy-
lase activity. Several tests were performed to standardise the en-
zyme reaction time and concentration of starch. The best view of
the activity was in bands with 1% soluble starch in 0.01 M phos-
phate buffer that were incubated for 3 h at 30 °C (Fig. 3A). This
gel reveals three well-defined bands (A1, A2, and A3) with different
reaction intensities and therefore shows the affinity for the sub-
strate. The A1 band reveals a greater intensity in the hydrolysis
of starch, probably due to the higher affinity of some enzymes
for the substrate. However, it is difficult to visualise the action of
only one or several enzyme isoforms, due to the shared location
of different enzymes in the gel. Because of this, we adapted a meth-
od for the separation of native proteins according to their pI
termed two-dimensional native electrophoresis. After IEF, separa-
tion in the second dimension was performed in a polyacrylamide
gel under native conditions (Fig. 3B). We can see several spots in
the gel with amylolytic activity and a set of spots (ellipse) with
similar intensities and pI that reinforces the hypothesis that band
1464 G.G. Rondán-Sanabria et al. / Food Chemistry 134 (2012) 1461–1467

A 1.1 B 0.27

1.0 0.26

U (amylolytic activity)
0.25

Protein (mg/mL)
0.9
0.24
0.8
0.23
0.7
0.22

0.6 0.21

0.5 0.20
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Days after harvest Days after harvest

C 0.15 D 0.66

0.14 0.65
U (alpha amylase)

0.64

U (beta amylase)
0.13
0.63
0.12
0.62

0.11
0.61

0.10 0.60
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Days after harvest Days after harvets

Fig. 1. Parameters measured for the enzyme extract; (A) protein concentration, (B) profile of amylolytic activity, (C) a-amylase activity, and (D) b-amylase activity. The
symbols represent the mean (n = 3) and the bars represent the standard deviations.

Days after harvest Dayys afteer harvvestt

KDa MW 2 4 6 8 10 14 16 22 2 8 16 22
120
90 A
A1
50
45 A
A2

25
20 A3
A

10
A
Fig. 2. Protein profile of the enzyme extract during storage shown by SDS–PAGE
(15% polyacrylamide) stained with Coomassie blue G-250. Each spot contains 10 lg
of protein. pH 3 10

A1 is formed by a protein complex. We also observed other en-

zymes with amylolytic activity and at the range of lower pI, but
it is necessary to fully explore this methodology to obtain more de-
fined spots. This methodology is still unexplored, but it could help
to identify possible isozymes and to separate them according to pI
and molecular weight or volume to preserve their enzymatic activ-
ity. The same approach was performed on dehydrated cucumber
cotyledons, where the amylolytic activity of isozymes of b-amylase
was preserved and was detected by activity staining using IEF (To- B
daka & Kanekatsu, 2007). These authors suggest that the analytical
method of activity staining employed after two-dimensional native
Fig. 3. Native-PAGE (6.5% acrylamide) for amylase activity incubated with 1%
electrophoresis that was described is quite effective for distinction
potato starch on 0.1 M phosphate buffer stained with iodine. (A) Regular native-
of b-amylase isozymes with similar pI values. Furthermore, this PAGE of the enzyme extracts during storage, and (B) Two-dimensional native-PAGE.
method can be easily linked to proteome analysis.

3.2. Starch and total soluble sugar maturation or storage of certain plants. The levels of starch, total
sugars and reducing sugar in maca roots were analysed. These val-
Some changes in the levels of starch and soluble sugars and in ues were similar throughout the storage period (Fig. 4). The starch
the activity profiles of hydrolytic enzymes occured during the contents on days 1, 16 and 22 of the analysis were 39% and 37% and
 G.G. Rondán-Sanabria et al. / Food Chemistry 134 (2012) 1461–1467 1465

A 44 B 18.0
Total sugar Reducing sugar

Total soluble sugar (%)

42
16.5

Total starch(%)
40
15.0
38

36 13.5

34
12.0
32
10.5
30
0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24
Days after harvest Days after harvest

Fig. 4. Carbohydrate concentration during storage of maca roots on dry weight basis. (A) Levels of total starch, (B) Levels of total soluble and reducing sugars. The symbols
represent the mean (n = 3) and the bars represent the standard deviations.

38%, respectively (Fig. 4A). The total sugars were 17.57%, 16.80%,
and 17.02% (w/w) on days 2, 16, and 22, respectively, and a similar
profile of reducing sugars was expected (Fig. 4B). Statistical
analysis did not show variation (p 6 0.05) during storage, which
indicates an increasing stability of these factors in the root post-
harvest.
Apparently, maca is a plant that does not have starch degrada-
tion by the action of amylolytic enzymes during storage, even
though it has enzymes capable of binding starch as a substrate.
Micrographs acquired by scanning electron microscopy show the
surfaces of isolated granules from the root, where smooth and in-
tact surfaces are observed without showing characteristic attacks
by amylolytic enzymes (Fig. 5). In general, this set of results mea-
sured during the 22 days of storage could explain why maca has an
extended shelf life after harvest; root features are preserved until
they are dry, particularly if they are stored properly at room tem-
perature (19–23 °C).
Similar results were observed when this profile was compared
with other roots and tubers, such as yams (Dioscorea dumetorum)
and sweet potatoes; all three showed no activation of amylolytic
enzymes, resulting in a long shelf life (Ikediobi & Oti, 1983). Yams
can be stored at temperatures ranging from 20 to 28 °C for
29 weeks. During this period, there is low enzyme activity, but
activity rapidly increases after these first nine weeks, resulting in
a gradual reduction in starch content. Similar to the yam, the sweet
potato is also a resistant tuber; it can be stored for 3 months if kept
at 20 °C with controlled humidity (75%). The authors suggest that
the low activity of the tuber in this first period is derived from la-
tency, which may account for the long shelf life of the tuber (Ovon-
o, Kevers, & Dommes, 2010; Panneerselvam, Abdul-Jaleel,
Somasundaram, Sridharan, & Gomathinayagam, 2007).
In some plants, dormancy depends on the species, the weather
conditions during growth, the degree of maturation of the tubers,
the mechanical injuries, and the damage and diseases caused by in-
sects and pathogens in the tubers. Several techniques, including
cutting the tubers, thermal shock, suffocation and use of chemicals
such as gibberellic acid or plant hormones are used to break this
dormancy (Panneerselvam et al., 2007; Silva et al., 2004).
The present results show that the roots might be in latency or a
rest period during the course of the study. This behaviour could be
described as a natural defence of the species, waiting for favour-
able conditions for the plant to reproduce, or falling into senes-
cence. Maca’s growth cycle occurs in two phases: during the
vegetative phase, it grows fast and expansion of reserve organs oc-
curs; during the second phase, the reproductive phase, root sprout-
ing and flower and fruit production occurs. This only happens in
favourable weather conditions, such as in the absence of frost
and high humidity (Quirós & Cárdenas, 1997).
3.3. Scanning electron microscopy
Scanning electron microscopy allowed us to perform a detailed
morphological characterisation of the grain as well as to view the
profile of degradation of the starch granule surface. Fig. 5 shows

A B C D E F

A1 B1 C1 D1 E1 F1

A2 B2 C2 D2 E2 F2

Fig. 5. Scanning electron microscopy of the maca root starch grains monitored during storage: day 2 (A-A2); day 6 (B-B2); day 10 (C-C2), day 14 (D-D2); day 16 (E-E2); and
day 22 (F-F2). The starch contents were 40.29%, 39.15%, 40.06%, 37.69%, 37.97%, and 38.47%, respectively.
1466 G.G. Rondán-Sanabria et al. / Food Chemistry 134 (2012) 1461–1467
F 2
G G
Fig. 6. SEM of native maca starch. (F-F2) Hydrolysed with a-amylase, 19.5 U/mg of starch in acetate buffer pH 4.8 for 2 h at 37 °C. (G-G2) Hydrolysed with the root enzyme
extract (1 ml of enzymatic extract/g starch) in 0.01 M phosphate buffer (pH 6.0) for 2 h at 30 °C.
the micrographs of starch granules isolated from maca roots during
storage. The micrographs present the starch granules in various
sizes and forms, with a predominance of the ovular form. This fig-
ure shows intact granules with smooth surfaces, without any evi-
dence of amylolytic attack by amylase. In addition, there are also
ovular and circular light depressions (white arrows) around the
granules, suggesting that there were layers of starch forming or
that enzymatic hydrolysis was beginning.
The granules show some cracks (white arrows), which might be
damage introduced during sample purification; these cracks could
facilitate the enzymatic attack during the hydrolysis assay. The
micrographs show that there was no apparent degradation during
the 22 days of storage. This absence of degradation was expected
due to the lack of significant differences in the levels of starch
and sugars, which would be indicative of starch degradation. It is
known that a-amylase is an enzyme capable of initiating the deg-
radation of native granules and that low activity releases sub-
strates for the action of other amylolytic enzymes. However,
recent evidence has emerged from studies on Arabidopsis thaliana.
These studies show that transitory degradation on leaves does not
depend on the initial action of a-amylase, suggesting that a still
unidentified endoamylase is present (Smith, Zeeman, & Smith,
2005; Zeeman et al., 2007).
Maca starch granules were hydrolysed in vitro with the enzyme
extract from the root and with pancreatic a-amylase (Fig. 6). These
tests allowed us to identify that the beginning of the enzymatic at-
tack on starch granules is caused by the action of a-amylase. Fig. 6
(F:F2) corresponds to hydrolysis of starch by pancreatic a-amylase,
and showed a greater attack on the depression of the granules
mentioned. These depressions were more visible (black arrows)
and more susceptible to enzymatic attack, and in some cases, they
traverse the granule, resulting in total fractures (arrows white).
By comparing these data with the hydrolysis of starch granules
with the enzymatic extract, similarities in the attack can be ob-
served (Fig. 6G:G2); although attack by the enzymatic extract is
less intense. These data converge to the hypothesis of root latency;
harvest would stop the action of a-amylase and the other enzymes,
resulting in a stable root composition (except for the loss of water)
during storage. In the case of potato starch hydrolysis by the a-
amylase from Bacillus sp., granule degradation occurred from the
surface toward the center, a centripetal hydrolysis, completely
degrading the core (Apinan et al., 2007). However, the authors sug-
gest that it was very difficult to analyse the structural organisation
2
within the degraded part of the granules using SEM, although it
was possible to observe the surface of the granule.
4. Conclusions
During the storage period, the maca roots did not show signifi-
cant variations in the measured parameters (p 6 0.05), except to
loss of water. These results suggest after harvest does the maca
roots enter a period of latency before the senescence, because they
are not in proper ambient for sprouting. The enzyme profiles ob-
tained by native electrophoresis showed several amylolytic en-
zymes with similar pI values, but different molecular weight or
volume, that were not activated in the hypocotyl. The hydrolytic
attack of maca starch granules by the action of the enzymatic ex-
tract and pancreatic a-amylase were alike from the surface to
the core of them, suggesting an important role of the endogenous
a-amylase if it is in contact with the substrate. The physiological
mechanism of the amylolytic enzymes activation in maca hypocot-
yls still need to be investigated.
Acknowledgements
The authors would like to thank CNPq and CAPES for financial
support.
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