Effect of physical and thermal processing upon ... - Wiley Online Library

International Journal of Food Science and Technology 2015, 50, 2443–2450

Original article Effect of physical and thermal processing upon benzylglucosinolate content in tubers of the Brassicaceae maca (Lepidium meyenii) using a novel rapid analytical technique Jing Li,1,2,3 Ying Zou,1,2 Qingrui Sun,2,3 Cheng Yang,2,3 Jinwei Yang4 & Lianfu Zhang1,2,3* 1 2 3 4

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China School of Food Science and Technology, Jiangnan University, Wuxi 214122, China National Engineering Research Center for Functional Food, Jiangnan University, Wuxi 214122, China Tokiwa Phytochemical Co. Ltd., 158, Kinoko, Sakura-Shi, Chiba 285-0801, Japan (Received 10 April 2015; Accepted in revised form 15 June 2015)

Summary

Benzylglucosinolate (BG) was extracted by 70% methanol from maca and purified on acidic alumina column and semi-preparative Cosmosil cholester column. The purified sample was verified as BG by electrospray ionisation mass spectrometry (ESI-MS) and nuclear magnetic resonance spectra. The purity was 98.3% as determined by HPLC-MS. BG content of maca was quantified using external standard method by HPLC. Effect of physical and thermal processing on BG content of maca was investigated. When maca was steamed for 5 min before shredding, no significant difference of BG content was observed during postshredding time, while raw maca lost 57.2% of BG in 24 h. Steamed maca showed no significant loss of BG after drying at the temperature from 20 to 80 °C in 24 h. Thermal degradation was described by the first-order kinetics–Arrhenius equation for BG in the temperature range of 90–100 °C.

Keywords

Benzylglucosinolate, degradation, drying, Maca (Lepidium meyenii), myrosinase, shredding, steam.

Introduction

Maca (Lepidium meyenii) belongs to the family Brassicaceae, originating in 3500–4500 m above sea level in the Andes in South America (Clement et al., 2010). The introduction of maca to China was approved by the China’s Ministry of Health in 2002; now it has been successfully cultivated in Yunnan Province and Xinjiang district (Li & Zhou, 2012). The physiological functions of maca in preventing testosterone-induced prostatic hyperplasia (Gonzales et al., 2007), improving sexual behaviour (Lentz et al., 2007), osteoporosis (Zhang et al., 2006), female fertility (Massoma Lembe et al., 2012; Uchiyama et al., 2014), memory impairment (Rubio et al., 2011) etc., have been widely studied. Glucosinolates (GLs) are sulphur-containing plant secondary metabolites that found in Brassicaceae (Richard, 2001). GL hydrolysis is catalysed by endogenous b-thioglucosidases (EC 3.2.3.1), named myrosinases, which also exist in Brassicaceae (Frederic et al., 2002). The myrosinases are sequestered away from GLs in the growing plant, only coming into contact *Correspondent: Fax: +86-510-85917081; e-mail: lianfu@jiangnan. edu.cn

doi:10.1111/ijfs.12911 © 2015 Institute of Food Science and Technology

when the plant tissue is disrupted by crushing or chewing (Donato & Elizabeth, 2014). Benzylglucosinolate (BG) is the most abundant glucosinolate in maca that accounts for 80–90% of the total GLs (Emilio et al., 2011). The structural formula of BG is shown in Fig. 1. In recent studies, BG is considered as the major functional component in maca and proved to enhance the endurance capacity in male mice (Mayumi et al., 2009). Gonzales et al. (2007) showed that aqueous extract of red maca containing 0.1 mg of benzylglucosinolate can prevent testosterone-induced prostatic hyperplasia. BG also showed in vitro activity against Entamoeba histolytica strain HM1-IMSS (IC50 of 20.4 lg mL1) (Calzada et al., 2003). Because of the functional potential of maca, various kinds of maca products such as maca flour, dried maca plate and aqueous extract of maca appear on the market. However, the determination of functional ingredient of maca products is unclear and confused. Although BG can be quantified using internal standard method, the pretreatment of sample is complicated and time-consuming (Genyi et al., 2001; Emilio et al., 2011). Therefore, a rapid and simple method for determination of BG is in demand. Genyi et al. (2001) showed that BG content was 16.9 lmol g1 dry weight (DW) of fresh maca tuber,

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Determination of benzylglucosinolate in maca J. Li et al.

but in the dried samples, the content decreased to 3.20 lmol g1 DW of dry tuber and 2.70 lmol g1 DW of flour. The decrease in BG might result from heat and myrosinase activity during processing, so clarifying how they affect BG content and finding a suitable method to protect BG from hydrolysis are really important in maca utilisation. Previous studies (Kirsten et al., 2006; Oliviero et al., 2012; Sarvan et al., 2014) observed thermal degradation of other glucosinolates such as glucoraphanin, glucoiberin, glucobrassicin and 4-methoxyglucobrassicin in Brassicaceae; however, the research on thermal degradation of BG in maca has not been reported. The aims of this work were to purify BG as standard from maca, establish a method for rapid determination of BG and investigate the effect of physical and thermal processing upon BG content in tubers of maca. Materials and methods

Plant material

The yellow maca tubers, harvested in January 2014, were purchased from a local supplier (Li Jiang, China). The maca tubers (diameter about 4 cm) were selected and kept at 4 1 °C before experiment. Chemicals

Methanol (HPLC grade), trifluoroacetic acid (HPLC grade), kieselguhr, acidic alumina (pH 4.5), potassium sulphate, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dehydrate, dithiothreitol (DDT), ethylene diamine tetraacetic acid (EDTA) and glycerol were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). D2O and tetraO O O

O

7

3 1

N 0

OH S

O 1'

5' HO

3'

Extraction and purification of BG

Fresh maca tubers were treated with steam (see the section of ‘Enzyme deactivation treatment’ below) for enzyme deactivation, and then cut and crushed. Fifty gram of maca pulp was extracted with 500 mL of 70% methanol in water bath at 80 1 °C for 60 min. The solid particles were filtered and the filtrate was decolorised by kieselguhr. The extraction was concentrated to 10 mL and then added onto an acidic alumina column (100 g). The column was first washed with deionised water (1 L), and then, BG was eluted with 0.1 M K2SO4 (200 mL). Each 10 mL of eluent was collected and detected by LC-MS. The eluents that contained BG were combined and freeze-dried to white solid. Methanol was added to the solid, and the remained K2SO4 was removed by filtration (0.45 lm; Whatman, Hangzhou, China). The solution was concentrated to 10 mL by vacuum-rotary evaporation and filtered through a filter (0.22 lm; Whatman, Maidstone, UK), which was suitable for HPLC analysis. The preparation of BG standard was performed on a semi-preparative cholester column (Cosmosil, 10 mm 9 250 mm, 10 lm) at 235 nm. An HPLC Waters 1525 Separation Module (Waters, Milford, MA, USA) equipped with a binary pump (Waters 1525), a photodiode array detector (Waters 2998) and an autosampler (Waters 2707). The mobile phase consisted of HPLC-grade water and methanol (90:10 v/v, containing 0.05% TFA) was run in isocratic mode; the flow rate was 2 mL min1 and the total run time was 30 min; the injection volume was 500 lL. The elution that contained BG was collected and freeze-dried to white solid. LC-MS and NMR identification of BG

5

S

methylsilane (TMS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

OH

OH Figure 1 Structural formula of benzylglucosinolate (BG).

International Journal of Food Science and Technology 2015

LC separation was using the WATERS ACQUITY UPLC system equipped with a BEH C-18 column (2.1 mm 9 50 mm, 1.7 lm; Waters) with the mobile phase consisted of HPLC-grade water and methanol (90:10 v/v); the flow rate was 0.3 mL min1, and the total run time was 6 min., and the column temperature was maintained at 25 °C. The samples were detected by WATERS MALDI SYNAPT Q-TOF MS equipped with an electrospray ionisation source with negative ion mode: capillary voltage: 3.0 kV; cone voltage: 30 V; source block temperature: 100 °C; desolvation temperature: 400 °C; desolvation gas flow: 500 L h1; cone gas flow: 50 L h1; collision energy: 6 eV; mass range: 100–1500 m/z; detector voltage: 1800 V. The 1H NMR and 13C NMR experiments were performed on AVANCE III 400 MHz solid-state

© 2015 Institute of Food Science and Technology


NMR spectrometer (Bruker, F€ allanden,Switzerland) using D2O as solvent and TMS as internal standard: 13 C NMR (D2O, 100 MHz) d: 162.67 (C-0), 38.20 (C1), 135.08 (C-2), 129.20 (C-3), 128.08 (C-4), 127.57 (C5), 128.08 (C-6), 129.20 (C-7), 81.35 (C-10 ), 71.78 (C20 ), 76.95 (C-30 ), 68.78 (C-40 ), 79.84 (C-50 ); 1H NMR (D2O, 400 MHz) d: 3.227–3.370 (4H, m, H-20 , 30 , 40 , 50 ), 3.600–3.688 (2H, m, H-60 ), 4.119–4.142 (2H, m, H1), 4.705 (1H, d, J10 , 20 10, H-10 ), 7.410 (5H, m, H-30 , H-40 , H-50 , H-60 , H-70 ). Analytical HPLC determination of BG

Analysis of BG was performed on an analytical cholester column (Cosmosil, Tokyo, Japan, 10 mm 9 250 mm, 10 lm) at 235 nm. An HPLC Waters 1525 Separation Module (Waters) equipped with a binary pump (Waters 1525), a photodiode array detector (Waters 2998) and an autosampler (Waters 2707). The mobile phase consisted of HPLC-grade water and methanol (90:10 v/v, containing 0.05% TFA) was run in isocratic mode; the flow rate was 1 mL min1 and the total run time was 30 min; the injection volume was 20 lL. Calibration curves were generated by analysing five different concentrations (0.04, 0.08, 0.12, 0.16 and 0.20 lmol mL1) of standard solution in triplicate. The calibration curve was then constructed as peak area vs. the standard concentrations and the linear relationship determined. Samples were quantified using the standard calibration curve. The method validation was referred to ICH-Q2 (2014), which in terms of linearity, limit of detection and quantification, precision and accuracy. Determination of BG in maca during processing Enzyme deactivation treatment

The selected raw maca tubers were treated by steaming for enzyme deactivation. Water was boiling in a domestic boiler on an electromagnetic oven. Then 100 g of maca tubers in a 500-mL beaker was placed in the covered boiler, which was kept steaming for 1, 3, 5, 10 and 15 min. After treatment, the samples were removed from the boiler, drained and analysed. All samples were first frozen in liquid nitrogen and then immediately pestled in a mortar. Subsequently, these samples were analysed for BG, myrosinase activity and moisture. BG extraction and quantification

In total, 0.5 g samples were extracted with 20 mL of 70% methanol in water bath at 80 1 °C for 60 min. Solid particles were removed from the extract solution. The solution was diluted with distilled water to 50 mL and filtered through a 0.22-lm filter (Whatman, Maidstone, UK) before HPLC analysis. The quantification


of BG content was accomplished by equating peak area from regression equation of the BG standard curve. BG content was expressed as lmol g1 of DW. The moisture determination was performed based on (China, 2010). Myrosinase extraction and activity analysis

The methodology described by Ruud & Matthijs (2004) with some modifications was used. Maca samples were homogenised with 10 mM (pH 7.0) phosphate buffer (containing 1 mM EDTA, 3 mM DDT and 5% glycerol) in a 1:10 ratio (w/v) in ice water bath. Crude myrosinase solution was prepared by filtering solid particles. Myrosinase extract (2 mL) was mixed with BG extract (2 mL) at 40 1 °C for 20 min. A unit of activity (U) was defined as the disappearance of 1 lmol BG per minute. Myrosinase activity of sample was expressed as U g1 DW. Shredding and drying

Processed and unprocessed maca tubers were cut into 3 mm pieces and kept at ambient temperature. BG contents were detected after 0.5, 1, 2, 10 and 24 h. Processed and unprocessed maca tubers were cut into 3 mm pieces, drying, respectively, at 20 1 °C, 40 1 °C, 60 1 °C, 80 1 °C in vacuum for 24 h. After the drying treatment, dehydrated material was powdered by ultrafine grinder (Chuangli Medicine Equipment Factory, Zhejiang, China); BG content, myrosinase activity and moisture were determined. Thermal breakdown

Steamed maca powder (1 g) was transferred to glass tubes and heated in thermostatic oil bath. The time– temperature combinations used for heating are shown in Table 1. The centre temperature of sample was measured with an infrared thermometer. The come-up times were between 10 and 30 s; this time was excluded from the kinetic parameter analysis. The samples were cooled on ice after heating and analysed immediately. Statistics and modelling

Three replicates were used in all the above analysis. One-way analysis of variance and Duncan’s test were Table 1 Heating times (min) and temperatures for maca Temperature (°C)

90 1

100 1

110 1

120 1

Heating time (min)

20 40 60 80 100

10 30 50 70 90

5 10 20 30 40

5 10 15 20 30


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used for analysis of data by the software SPSS 16.0 (SPSS Inc., Chicago, IL, USA) for Windows. Differences at P < 0.05 were considered as significant. Thermal degradation of the BG was described with a first-order reaction mechanism performed according to eqn (1) as follows: C ¼ k t; ð1Þ ln C0 where C is the BG content of the heat-treated maca at time t, C0 is the initial BG content of the maca, t is the heating treatment time, and k is the rate constant. Temperature dependence of the reaction rate constants was described by the rearranged Arrhenius equation: Ea ; ð2Þ k ¼ k0 exp RT where k0 is the reaction rate constant (min1), Ea is the activation energy (kJ mol1 K1), R is the universal gas constant (8.314 J mol1 K1), and T is the absolute temperature (K). A log treatment can be applied to the eqn (2); thus, the model can be transformed to the following equation: Ea : ð3Þ ln k ¼ lnk0 RT Results and discussion

Purification and identification of BG in maca Purification

The acidic alumina (pH 4.5) exhibited the best qualities for separating glucosinolates from other groups (Charpentier et al., 1998). Separation of individual glucosinolates by reverse-phase chromatography is difficult, because these molecules contain strongly acidic sulphate groups that are retained weakly on C18 column (Tory et al., 1996). Common method is accomplished using a sulphohydrolase to remove sulphate group of glucosinolate before analysis by HPLC; however, desulphuration of glucosinolate is time-consuming, which need sulphatase to keep for 16 h (Genyi et al., 2001). Ammonium ions are favourable to be paired with sulphates, and the ion pair behaves like a hydrophobic molecule which can be separated by reverse-phase chromatography. However, the fractions that collected after ion-pair HPLC contain hydrophobic counter ion, which makes both mass spectrometry and nuclear magnetic resonance (NMR) analyses difficult. Therefore, further purification to remove the excess counter ion is required (Tory et al., 1996). These imply that ion-pair HPLC is not suitable for preparing pure BG. In our study,


a method of rapid detection and purification for BG was established by utilising HPLC equipped with Cosmosil cholester column. Cosmosil cholester column is a reverse-phase column that has a better performance on analysis and molecular recognition than C18 column, especially on the separation of phenyl compounds. In addition, Cosmosil cholester column was used for separating polyacetylenic glucoside, galactocerebroside and aromatic compounds, which were similar to BG in structure in the previous study (Kazuhide et al., 2008; Yuriko et al., 2009; Kurimoto et al., 2010). Therefore, we chose Cosmosil cholester column to analyse BG instead of C18 column. Figure 2 shows the HPLC chromatogram (detection wavelength at 235 nm) of the crude extract, and the peak at 10.6 min is identified to be BG. LC-MS and NMR identification of BG

The total ion chromatogram of the purified sample is presented as a single peak (Fig. 3), and the purity of the sample is 98.3%. The relative molecular mass of BG (C14H19O9NS2) is 409, and the main molecular ion 408 (M-1) obtained from MS spectrometric analysis is in accordance with the BG molecular mass. The structural formula of BG is shown in Fig. 1. Compared with the NMR data given from Kiddle et al. (2001), the sample was identified as BG. HPLC quantification was accomplished by equating peak area (detection wavelength at 235 nm) from regression equation, which performed by the standard curve method using the prepared pure BG. The method validation (Table 2) proved that this method is repeatable and selective for the analysis and determination of BG in maca.

BG

0.100 0.090 0.080 0.070 0.060

AU

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0.050 0.040 0.030 0.020 0.010 0.000 –0.010 0.00

5.00

10.00

15.00

20.00

25.00

30.00

min

Figure 2 HPLC chromatogram (wavelength at 235 nm) of the crude extract of maca.



50

BG content (umol g–1 DW)

(b)

1.0

0.8

40

0.6

30

0.4 20 0.2 10

Myrosinase activity (U g–1 DW)

BG content Myrosinase activity

(a)

0.0 0 0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15

Steaming time (min)

Figure 3 LC-MS chromatogram. (a) Total ion chromatography of purified sample; (b) MS in negative mode.

Determination of BG content in maca during processing

In our study, the BG content of raw yellow maca was 47.2 1.9 lmol g1 DW and myrosinase activity was 0.81 0.11 U g1 DW. However, the BG content was 29.1 lmol g1 DW and myrosinase activity was 0.84 U g1 of fresh maca produced in Peru (Emilio et al., 2011). Fresh purple maca, yellow maca and white maca produced in China were investigated, respectively, in BG content of 40.2 lmol g1 DW, 38.6 lmol g1 DW and 73.4 lmol g1 DW (Gan et al., 2012). These imply that significant differences exist in BG content of maca in different regions and colour types. Enzyme deactivation treatment

Steaming is a common way of enzyme inactivation. As shown in Fig. 4, the treatment significantly decreased

Figure 4 Benzylglucosinolate (BG) content (▲) and myrosinase activity (■) after steaming treatment in different time. Data are the means with their standard errors represented by vertical bars for three replicates.

the BG content and myrosinase activity in maca samples (P < 0.01). Myrosinase in steamed maca showed an initial significant decrease of 16% in activity after being treated for 1 min and decreased gradually in 15 min of steaming, as compared with raw maca (P < 0.01). When the myrosinase was completely inactivated at 5 min of steaming, the BG content fell to 41.9 1.9 lmol g1 DW. Glucosinolate (GL) is relatively stable in plant cells, not until plant tissue damage brings myrosinase in contact with the glucosinolates and activates the € enzyme (SchOne et al., 1994). For optimisation of health-promoting properties of maca, it is important that BG is retained during food processing and storage. Therefore, it is necessary to inactivate the myrosinase before performing further processing of maca. Vanessa et al. (2006) showed that the myrosinase activity in cabbage was effectively lost after 7 min of steaming. In our study, 5 min of steaming was the preferred treatment when the myrosinase was completely inactivated.

Table 2 Parameters of method validation

Shredding and drying

Parameters Regression equationa Linear range (lmol mL1) Determination coefficient (r2) LOD (lmol mL1) LOQ (lmol mL1) Precision (R.S.D.) Accuracy (recovery)

y = 6490x – 8373.24 0.04–0.2 0.99906 0.003 (S/N = 3) 0.012 (S/N = 10) 1.28% 98.52%

LOD, limit of detection; LOQ, limit of quantification; BG, benzylglucosinolate. Data are the means for five replicates. a y = peak area, x = concentration of BG in lmol mL1.


Tissue cells of maca were broken when being shredded, and subsequently BG was hydrolysed by myrosinase. As shown in Fig. 5, BG content of steamed maca (10 min of treating) was 31.9 1.5 lmol g1 DW and presented no significant difference after shredding. A significant decrease in BG was observed in raw maca after shredding. Raw maca rapidly lost 25% of BG in the initial 0.5 h of postshredding time and 57% over 24 h. This change was similar to that of total glucosinolate contents of Brassica vegetables after shredding observed by Song & Paul (2007). In 1 h of postshredding time at ambient temperature, BG in raw maca


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f

50

Myrosinase-inactivated maca Raw maca


45 40 35

e

a

a

a

a

a d

30

a

a

cd

bc

b

25

a

20 15 10 5 0 0

1

0.5

2

10

5

24

Post-shredding time (h)

Figure 5 Benzylglucosinolate (BG) content of myrosinase-inactivated maca and raw maca after shredding at ambient temperature (25 °C). Data are the means with their standard errors represented by vertical bars for three replicates. Different letters on the bar mean significantly different (estimated by one-way analysis of variance and Duncan’s test, P < 0.05).

degraded quickly, because hydrolysis occurring immediately as soon as BG contacted myrosinase. The loss of BG in maca slice in 24 h of postshredding time at ambient temperature was more than that of drying at 20 1 °C in vacuum (51%), because higher moisture in the former was more beneficial to the myrosinase hydrolysis of BG. Initial myrosinase activity of raw maca was 0.81 0.11 U g1 DW, while significantly decreased 30% after 24 h of drying at 40 1 °C (Fig. 6).

c

0.9

Myrosinase activity decreased with the rising of drying temperature and was completely destroyed at 80 1 °C. No significant difference (P > 0.05) in myrosinase activity was observed in raw sample and the sample dried at 20 1 °C; nevertheless, myrosinase activity significantly declined to 0.57 0.08 U g1 DW (40 1 °C) and 0.48 0.04 U g1 DW (60 1 °C). No significant difference (P > 0.05) of BG content was observed in steamed maca after drying at various temperature level (Fig. 7). With the increase in drying temperature, the BG content of steamed maca slightly declined. The initial BG content of steamed maca was 41.9 1.9 lmol g1 DW and decreased 1.7%, 4.4%, 7.2% and 10.0%, respectively, after 24 h of drying at 20 1 °C, 40 1 °C, 60 1 °C and 80 1 °C. As shown in Fig. 7, significant differences of BG content were observed in raw maca after drying at different temperature. BG content decreased significantly to the lowest (15.9 1.1 lmol g1 DW) at 40 1 °C, which was the optimum temperature for enzymatic hydrolysis. The remained myrosinase activity in maca also contributed to the degradation of BG when powdering. The BG content of raw maca after 24 h of drying at 80 1 °C (45.1 2.4 lmol g1 DW) presented no significant difference compared to that of the raw maca (47.2 1.9 lmol g1 DW), as myrosinase was totally inactivated at this temperature (Figs 6 and 7). There was no significant difference between the samples at 20 1 °C (20.9 0.9 lmol g1 DW) and 60 1 °C (20.8 1.2 lmol g1 DW). The results indicate that the myrosinase activity has a greater influence on the BG degradation in maca

c

0.8

b

0.7 0.6

b

0.5 0.4 0.3

Myrosinase-inactivated maca Raw maca

c

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1.0

Myrosinase activity (U g–1 DW)

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a

a

a

a

40

c

a

30

b 20

b a

10 0.2 0.1

a

0 Before drying

0.0 Before drying

20

40

60

80

Temperature (°C)

Figure 6 Myrosinase activity of raw maca after 24 h of drying at different temperature. Data are the means with their standard errors represented by vertical bars for three replicates. Different letters on the bar mean significantly different (estimated by one-way analysis of variance and Duncan’s test, P < 0.05).


20

40

60

80

Temperature (°C)

Figure 7 Benzylglucosinolate (BG) content of myrosinase-inactivated maca and raw maca after 24 h of drying at different temperature. Data are the means with their standard errors represented by vertical bars for three replicates. Different letters on the bar mean significantly different (estimated by one-way analysis of variance and Duncan’s test, P < 0.05).



(b)

0.0

–3.5

–0.1

–4.0

–0.2

–4.5

–0.3

–5.0

lnk

ln(C/C0)

(a)

–0.4 –0.5

–5.5 –6.0

ć ć ć ć

–0.6 –0.7

–6.5 –7.0

–0.8

y = –16390x + 38.13 R 2 = 0.9728

–7.5

0

20

40

60

80

100

0.00250

0.00255

0.00260

0.00265

0.00270

0.00275

1/T

Time (minutes)

Figure 8 First-order kinetics for thermal degradation of BG (a) and Arrhenius plot showing the temperature dependence of rate constant of first-order kinetics (b). Table 3 Parameters of model for BG degradation

Temperature (°C)

90 1

100 1

110 1

120 1

k (9103 min1) R2 Ea (kJ mol1)

0.79 0.05 0.9774 136.3 13.1

3.28 0.30 0.9603

12.30 0.97 0.9695

24.74 2.00 0.9681

K, reaction rate; R2, determination coefficient; Ea, activation energy; BG, benzylglucosinolate.

than the temperature below 80 °C during drying. Deactivating the myrosinase is important in processing and utilisation of maca products. Thermal degradation and modelling

We have observed that BG was stable during drying at the temperature below 80 °C. Domestic and industrial processing usually use thermal treatment at the temperature between 90 and 120 °C, which can affect BG content of maca. Based on the proposed degradation kinetics and the estimated parameters, Kirsten et al. (2006) predicted that cooking would cause more thermal degradation to indole glucosinolates (38%) as compared to aliphatic glucosinolates (8%); canning, the most severe heat treatment, would result in significant thermal degradation (73%) of the total amount of glucosinolates in red cabbage. To investigate the effect of heat on the BG, first-order kinetics–Arrhenius equation was employed to describe the BG degradation process upon heating of maca. Figure 8 (a) shows the performance of the first-order kinetics. Parameters of the models are presented in Table 3. The reaction rate constant k of the first-order kinetics-based models was calculated from the slope of ln(C/C0) vs. heating time using linear regression analysis. With the heating temperature increasing, k significantly increases, indicating that higher temperature causes higher degradation rate. Figure 8 (b) presents the Arrhenius equation using k estimated by the first-order kinetics. Activation energy Ea for BG degradation was 136.3 kJ mol1, which was similar to the result calculated by Oliviero et al. (2012). These results imply that domestic


cooking and industrial processing such as boiling, steaming and microwave treatment will result in significant thermal degradation of BG in maca. Conclusions

In this study, BG of maca was separated and quantified by a novel rapid technique. Steaming was introduced to deactivate myrosinase, and the steamed maca presented better preservation of BG than raw maca during shredding and drying. The results clarified the effect of physical and thermal processing on BG content in maca and indicated that myrosinase activity was a key factor that affects BG degradation during processing. Moreover, the change of BG content of steamed maca and raw maca during storage at different condition needs further investigation; hydrolysis products of BG in processing and storage are expected to be clarified. References Calzada, F., Barbosa, E. & Cedillo-Rivera, R. (2003). Antiamoebic activity of benzyl glucosinolate from Lepidium virginicum. Phytotherapy Research, 17, 618–619. Charpentier, N., Bostyn, S. & Coic, J.P. (1998). Isolation of a rich glucosinolate fraction by liquid chromatography from an aqueous extract obtained by leaching dehulled rapeseed meal (Brassica napus L.). Industrial Crops and Products, 8, 151–158. China (2010). Determination of moisture in foods. In: National Food Safety Standard (edited by Department of policy and Regulation of Ministry of Health of China), Pp. 2, 105–112, China: Standardization Administration of the People’s Republic of China.


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