content and distribution of nutritional and non ...analytical grade provided by jt baker...

Jan. 2015. Vol. 2, No.9 ISSN 2311 -2476 International Journal of Research In Agriculture and Food Sciences © 2013 - 2015 IJRAFS & K.A.J. All rights reserved http://www.ijsk.org/ijrafs.html

1

CONTENT AND DISTRIBUTION OF NUTRITIONAL AND NON-

NUTRITIONAL COMPOUNDS DURING GERMINATION OF THREE

MEXICAN FABA BEAN (VICIA FABA) VARIETIES

DULCE M VALDEZ-ANGUIANO1, EDGAR HERRERA-CABRERA

2, GLORIA DÁVILA-ORTIZ

1, B.

DAVE OOMAH3, ANABERTA CARDADOR-MARTÍNEZ

4, CRISTIAN JIMÉNEZ-MARTÍNEZ

1

1Departamento de Graduados e Investigación en Alimentos. Escuela Nacional de Ciencias Biológicas. Instituto

Politécnico Nacional. Prolongación de Carpio y Plan de Ayala. Col. Casco de Sto. Tomás. Del. Miguel Hidalgo.

C.P.11340. México, Distrito Federal. 2Colegio de Postgraduados, Campus Puebla. Km. 125.5, Carr. Fed. Méx.-Pue., Col. La Libertad. 72130. Puebla,

México. 3National Bioproducts and Bioprocesses Program, Pacific Agri-Food Research Centre, Agriculture and Agri-Food

Canada, Summerland, British Columbia, Canada V0H 1Z0. 4Instituto Tecnológico de Estudios Superiores de Monterrey, Campus Querétaro, Av. Epigmenio González No. 500,

Fraccionamiento San Pablo, C.P. 76130, Queretero, México.

Corresponding author: [email protected]

ABSTRACT

Three Mexican faba bean varieties with different flowering growing were evaluated for chemical composition and

trypsin inhibitors (TI), phytic acid (PA) and phenolic contents (PC) to elucidate their distribution in their fractions

(cotyledon, hull and embryo axis) during germination. Germination reduced carbohydrate, lipid, ash and PA

contents, whereas TI levels increased in the cotyledon and hull.

Protein, lipid, ash and PC increased, whereas PA and TI levels decreased in the embryo axis. Changes in most

components depended primarily on the anatomical part analyzed, except PA that was significantly influenced by the

time of germination. Multivariate data analysis performed on 8 components analyzed in this study using principal

component analysis (PCA) and cluster analysis demonstrate that differences in varieties, fractions and germination

time are due to the increase in concentration and distribution of one or more compounds during germination.

Germination is a process with high utility and low cost to increase the nutritional compounds or reduce/eliminate

non-nutritional compounds, nevertheless, this cannot occur in a uniform way in seeds fractions because some of

these compounds increase in concentration. However, this process can increase digestibility and bioavailability of

the protein in cotyledon and embryo axis.

Keywords: faba beans; germination, non-nutritional, nutritional compound, principal component analysis

1. INTRODUCTION

The food industry has developed processing methods

such as germination, an easy and cheap treatment

where seed reserve materials are degraded and used

for the embryo development with the potential to

increase nutritive value, digestibility and

bioavailability of free amino acids, available

carbohydrates, dietary fiber and bioactive compounds

(Dueñas, Hernández, Estrella & Fernández, 2009).

Non-nutritional factors such as phytates are degraded

and used for energy production, vitamin C

concentration increases, while chlorophyll starts to

appear when the plant is exposed to light (Bohn,

Meyer & Rasmussen, 2008; Martine, 2002). During

this process some of these compounds act as storage

components and are transformed in necessary

nutritive elements for plant growth, while other

compounds providing a defense role are unaffected

mailto:[email protected]


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throughout the germination process (Vessal, Palta,

Atkins & Siddique, 2012).

Faba bean (Vicia faba), is of great economic

importance worldwide in the East and Mediterranean

regions; however, Mexico's Puebla state ranks as the

largest producer of dry faba bean(Crépon, Marget,

Peyronnet, Carrouée, Arese & Duc, 2010). Its

nutritional value is high as it provides carbohydrates

and proteins, is a good source of B vitamins,

particularly thiamine, niacin and folate, and minerals

including potassium, phosphorus, magnesium and

zinc, as well as an appreciable amount of iron

(Pastor-Cavada, Juan, Pastor, Alaiz & Vioque, 2011).

Faba bean seed is about 26% protein, which, as other

legumes is cysteine and methionine deficient. A large

proportion of faba bean protein is mainly represented

by the storage proteins globulins (60%), constituted

by legumins and vicilins (Pasqualini, 1991; Vioque,

Alaiz & Girón-Calle, 2012). Faba bean is an

important source of carbohydrates (45 or 68%),

consisting mainly of sugars such as sucrose (1-2%),

oligosaccharides (3.1-4.2%) and starch, which is

located in the cotyledons and represent 80% of total

carbohydrates, while non-starch polysaccharides,

about 17 % are located in the hull as fiber (Campos-

Vega, Guevara-Gonzalez, Guevara-Olvera, Dave

Oomah & Loarca-Piña, 2010; Guillon & Champ,

2002). Moreover, the proportion of lipid in seeds of

V. faba is relatively low ranging from 1.0 to 2.9%,

48.8-50.1% of which are simple lipids (Yoshida,

Tomiyama, Yoshida, Saiki & Mizushina, 2008).

During germination, protein content increases

between 20 and 40% for faba bean and 8 to 17% for

common beans due to enzymatic degradation to

support plant growth, while carbohydrate, fiber and

lipid contents generally decrease by about 6, 15 and

18% respectively, for faba bean seeds (Goyoaga et

al., 2011; Khalil & Mansour, 1995; Ragab, Kijora,

Abdel Ati & Danier, 2010; Sangronis, Rodríguez,

Cava & Torres, 2006).

Legumes seeds such as faba bean contain many non-

nutritional compounds that can limit their

consumption. However, legumes have recently been

considered as functional foods because they contain

many bioactive substances with potential beneficial

effects on health (Frühbeck, Monreal & Santidrian,

1997).

Protease inhibitors are proteins widely distributed in

the plant kingdom and in legume seed they can have

major impact on nutritional value by impairing

protein digestion due to pancreatic serine protease

inhibition. Kunitz and Bowman-Birk are two legume

seed protease inhibitors found in soybean and in

other grain legumes. In faba bean the Bowman-Birk

inhibitors have been characterized (Guillamón,

Pedrosa, Burbano, Cuadrado, Sánchez & Muzquiz,

2008), and consists of 71 amino acids with high

proportion of Cysteine producing 7 disulfide bonds,

in addition the chain possess two independent

inhibition sites, for trypsin chymotrypsin (Birk, 1968;

Fan & Wu, 2005). The activity of these inhibitors

decrease during germination process by 32% for faba

bean and 18 to 33% for other legumes (Frias, Diaz-

Pollan, Hedley & Vidal-Valverde, 1995; Khalil et al.,

1995; Mubarak, 2005).

During germination phytic acid is reduced between

18 and 56% for most legumes and around 35-45% for

faba bean (Domínguez, Gómez & León, 2002;

Ghavidel & Prakash, 2007; Mubarak, 2005; Vidal-

Valverde, Frias, Estrella, Gorospe, Ruiz & Bacon,

1994). Phytic acid has high chelating capacity,

reducing the bioavailability of divalent cations,

proteins or carbohydrates as starch by the formation

of insoluble complexes. These complexes are highly

insoluble over a wide pH range, causing micro-

nutrient or protein deficiencies, but can help in

kidney stone disease or diabetes mellitus treatment

acting as hypoglycaemic for its influence in the blood

response to glucose (Domínguez et al., 2002).

During seed maturation stage condensed tannins

diminish in cotyledon between 10 and 66% for some

legumes and 29 to 50% for faba bean, while

phenolics in hull increase (Al-Numair, Ahmed, Al-

Assaf & Alamri, 2009; Alonso, Aguirre & Marzo,

2000; Chung, Wong, Wei, Huang & Lin, 1998;

Ghavidel et al., 2007).

There are some reports on the effect of germination

on the proteins, carbohydrates, fiber, lipids, ash,

phytates, trypsin inhibitors and phenolics in different

legume seeds during 8 days of germination, but

relatively little is known of the distribution of these

compounds in the different anatomical fractions of

the seed after this time period in faba bean seeds. The

present study focuses on describing the effect of

germination on chemical, nutritional and non-

nutritional composition and the distribution in

separated anatomical fractions (cotyledon, hull and

embryo axis) of three germinated and un-germinated

Mexican faba bean (Vicia faba) varieties.


3

2. MATERIALS AND METHODS

2.1 Materials

Seeds of three Mexican faba bean (Vicia faba)

varieties (Col-160, Col-181 and Col-281), grown in

2007 at Tlachichuca, Puebla with different flowering

cycle were donated by Colegio de Postgraduados

Campus Puebla and stored protected from light at

room temperature.

2.2 Chemicals

Total dietary fiber assay kit, phytic acid sodium salt

hydrate, 5-sulfosalicylic acid dehydrate, Folin &

Ciocalteu’s phenol reagent, gallic acid, BAPNA (Nα-

Benzoil-DL-arginine-4-nitroanilide hydrochlroide),

Trypsin type XI (E.C.3.4.21.4, ≥6000 BAEE

units/mg protein, from bovine pancreas) were

purchased from Sigma-Aldrich (Sigma Chemical Co.,

St. Louis, MO, USA). All other chemical were of

analytical grade provided by JT Baker (Phillipsburg,

NJ, USA).

2.3 Germination assay

The seeds were soaked in 5% hypochlorite solution

(v/v) for 5 min and washed with water three times.

Approximately 50 seeds were placed in plastic

containers with a Phaeozem soil substrate purchased

in Xochimilco, México. The containers were placed

in a free space with temperature between 10-22°C,

and 16 days. Seeds were washed with distilled water

to eliminate soil residues and separated into

cotyledon, hull and embryo axis. Seeds and the

anatomically separated fractions were stored at -

20°C, freeze-dried and ground.

2.4 Chemical proximate analysis

Proximate analysis of the samples including crude

protein (Nx5.83), lipids, ash, and total dietary fiber

were performed according to the official AOAC

methods 954.01, 920.39 923.03, and 985.29

respectively (A.O.A.C., 1995). Carbohydrate content

was determined as the weight difference using crude

protein, lipids and ash content data. Each sample was

analyzed in duplicate and the values were then

averaged.

2.5 Non-nutritional compounds

2.5.1 Phytates

Phyates was determined based on the method

described previously for Vaintraub & Lapteva

(1988). Briefly, samples (0.5 g) were extracted with

3.5% HCl v/v (10 mL) for 1 h, and then centrifuged

(3400 rpm for 10 min at 4°C, Model Z383K, Hermle,

Alemania). After, 1 mL of the supernatant was

diluted with distilled water to 3 mL, mixed with

Wade reagent (1 mL), vortexed (Model M37615Q,

Thermo Scientific Thermolyne) and centrifuged at

3400 rpm for 10 min at 4°C (Model Z383K, Hermle,

Alemania). The absorbance was monitored at 500 nm

with a spectrophotometer (Model 6505, Jenway, UK)

using phytic acid (0-160 µg/mL) prepared in distilled

water as standard. Results are expressed in milligram

equivalent of phytic acid per gram of sample.

2.5.2 Total Phenolic Compounds

Polyphenols were extracted according to Abdel-Aal

& Hucl (1999). Briefly, 1% acidified methanol (10

mL) was added to 0.5 g of flour, mixed for 12-16 h in

the dark at 30°C and centrifuged (5000 rpm, 10 min,

4°C, Model Z383K, Hermle, Alemania). Supernatant

was recovered. Phenolic concentrations were

determined using the Folin-Ciocalteu reagent as

described by Singleton (Singleton, Orthofer &

Lamuela-Raventós, 1999). Briefly, 20 µL of extract

was mixed with 1.58 mL of distilled water and 100

µL solution 1:2 of Folin-Ciocalteu reagent, vortexed

30 s (Model M37615Q, Thermo Scientific

Thermolyne, Alemania), followed by addition of 300

μL 10% Na2CO3. The mixture was incubated for 2h

in the dark at room temperature. The absorbance was

monitored at 765 nm with a spectrophotometer

(Model 6505, Jenway, UK) using gallic acid prepared

in ethanol/water (0-0.5 mg/mL) as standard. . Results

are expressed in milligram gallic acid equivalent per

gram of sample.

2.5.3 Trypsin Inhibitors

Trypsin inhibitors were evaluated based on the

method described by Smith et al. (1980) with

modifications. Enzyme was added in the last step

according to Liu & Markakis (1989) using Nα-

Benzoil-DL-arginine-4-nitroanilide hydrochloride

(BAPNA) as trypsin subtract.

The method consisted of mixing 0.25 g of flour with

12.5 mL 0.01M NaOH for 30s adjusting the pH at

9.4-9.6 with 1M NaOH or 1M HCl, vortexing for 5


4

min (Model M37615Q, Thermo Scientific

Thermolyne), then centrifuged at 3600 rpm for 15

min at 4°C (Model Z383K, Hermle, Alemania).

Supernatant was recovered slowly to prevent

disturbing oil at the surface. Trypsin Inhibitor

Activity (TIA) was calculated based on the

absorbance at 410nm against distilled water and was

expressed in trypsin inhibitor units/ mg protein

(TIU/mg).

2.6 Statistical analysis

Analysis of variance by the general linear models

(GLM) procedure, means comparison by Duncan’s

test Pearson correlation, and variance components

using PROC VARCOMP were performed according

to Statistical Analysis System SAS 9.1 for Windows

All effects were considered random for the variance

component analysis, and calculations were based on

type I sum of squares method. Principal component

analysis (PCA) was performed according to

XLSTAT 2012 for Windows (Addinsoft, NY).

Cluster analysis was performed using SYSTAT 12

version 12.02 for Windows, using hierarchical

clustering with average (linkage) and Euclidean

(distance).

3. RESULTS/DISCUSSION

The varieties differed considerably in their growth

cycle, proportion of anatomical segments (fractions),

and proximate composition during the germination

period. Percent germination, cotyledon, hull and

embryo axis weights (the latter determined as length)

were measured at different experimental conditions.

During the first growth cycle of germination (8 days),

Col-160 and Col-181 varieties had the highest (95%)

and lowest (86%) percent germination, respectively.

Major fractions weights were 0.176 g for embryo axis

of Col-281, 3.26 g for cotyledon of Col-160. After 12

days the embryo axis of Col-281 grew in weight and

length (0.76 g and 5.71 cm), in contrast, this variety

had the lowest cotyledon weight (3.62 g); the highest

cotyledon weight was found in Col-160 (3.94 g). At

the end of germination (16 days) leaves developed in

the embryo axis, with highest percent (98%)

germination and cotyledon weight (3.57 g) observed

for Col-160 variety. Col-281 variety had the highest

weight and embryo length (2.25 g and 14.42 cm).

During the three germination periods hull weights

were similar among the three varieties.

3.1 Proximal composition analysis

The proximate composition was dependent on

varieties, growth cycle, anatomical fraction and

germination time (Table 1). Protein content varied

significantly (p<0.05) among germination days for

different faba bean anatomical fractions, except for

the hulls where significant difference was observed in

only one germination time (Table 1). For example,

protein content in cotyledon of Col-160 increased by

14% after 16 days germination, while the highest

protein increase (21.5%) was observed at 8 days

germination for Col-181 cotyledon.

Protein content of Col-281 cotyledon decreased from

27.65 to 24.81 g/100g at 16 days of germination. Hull

protein content of variety Col-160 decreased

constantly until the final stages of germination (5.36

to 4.49 g/100g). However, the reverse effect was

observed for Col-181 and Col-281 varieties where

hull protein content increased (3.52 to 4.01 and 3.95

to 5.53 g/100g). In the embryo axis protein content

increased gradually with Col-160 and Col-281

expressing the lowest and highest contents,

respectively (Table 1). Khalil & Mansour (1995)

observed similar increase in protein content, while

Bakr (1996) reported 1.5% increase after three days

germination of faba bean seeds. The changes in total

protein content during the germination process may

be due to the enzymatic hydrolysis of storage

proteins present into peptides in cotyledon; these

peptides participate in embryo axis development

through proteins synthesis, thereby elevating protein

concentration in germinated seed.

Carbohydrate content showed significant differences

(p<0.05) among germination times in the different

fractions of each variety. The hull and embryo axis

had the highest and lowest carbohydrate contents

(Table 1). Carbohydrate content decreased

significantly in cotyledons of Col-160 and Col-181

during 16 days of germination. However, reduction in

carbohydrate content occurred during the first 12

days of germination for Col-281 cotyledon. Similar

reduction in carbohydrate content was observed in

the hulls with the highest decrease observed in Col-

281 hulls. Significant decrease (10-12%) was

observed in carbohydrates in the embryo axis of all

varieties during germination (Table 1). Similar to the

behavior of Col-160 and Col-181, Khalil & Mansour

(1995) observed a reduction in carbohydrate

composition in cotyledon of faba bean after three

days of germination; in contrast, Bakr (1996) and

Youssef et al., (1987), reported an increase (54.3-


5

61.6% and 54.3-63.7%) in this component after

germination. These germination effects can be

attributed to the consumption of carbohydrate as

energy through free sugar resulting from

polysaccharide hydrolysis and mobilization in the

cotyledon.

Lipid content differed significantly (p<0.05) among

fractions with the embryo axis and the hull

expressing the highest and lowest lipid content,

respectively (Table 1). During germination, lipid

content decreased at a constant rate in the cotyledon

and hull. For example, lipid content of Col-281

cotyledon decreased from 2.66 to 2.18 g/100g,

whereas an insignificant reduction was observed for

those of Col-160 cotyledons. Hull of Col-281 and

Col-160 varieties had the lowest and greatest

reductions of lipid content at around 40-45%,

respectively. The embryo axis of all varieties showed

significant increase in lipid content (about 39%) at 12

days, followed by a reduction (about 24%) in

relation with the initial concentration, except Col-160

that showed not significant reductions (13.27 to 13.20

g/100g) in relation to 12 days germination (Table 1).

Our results contrast with previous reports (Bakr,

1996; Youssef et al., 1987) where germination had no

effect on faba bean lipid content. Reduction in lipid

content may be due to lipid hydrolysis by

endogenous lipases, these hydrolysis products form

part of the general carbohydrate pool present in the

seed and as such becomes available for various

processes including respiration (Mayer & Poljakoff-

Mayber, 1989).

Total dietary fiber content generally increased

significantly (p<0.05) during germination.. The hull

and the cotyledon were the fraction is the with the

highest and lowest fiber content, respectively (Table

1). At 8 days germination, total dietary fiber

increased in the cotyledons of Col-160 and Col-281.

Thereafter, total dietary fiber decreased significantly

in cotyledons of Col-160 and Col-181 at 12 days

germination followed by an increase at 16 days

germination. Changes in total dietary fiber content in

the cotyledon of Col-281 were not significant during

germination. Similar to carbohydrate content, the hull

had the highest total dietary fiber content compared

to other fractions (≈13-23%). Fiber content increased

in the hulls of Col-160 and Col-181 at 12 days

germination followed by a reduction at day 16.

However, the fiber content in the hull of Col-281

decreased at 12 days and increased at 16 days

germination. Dietary fiber content in the embryo axis

of Col-160 and Col-281 increased then decreased at

12 and 16 days; while significant decrease was

observed in Col-181 embryo axis during all

germination stages (Table 1). Increase in dietary

fiber content has been reported after 96 h of

germination for different legumes (Martín-Cabrejas,

Díaz, Aguilera, Benítez, Mollá & Esteban, 2008).

The ash content in fractions showed significant

differences (p<0.05) in hull and embryo axis (Table

1). Ash content increased significantly in hulls of

Col-181 and Col-281during germination, whereas

minimal changes occurred in Col-160 hull.

In cotyledon of Col-160 and Col-181 ash content

changed minimally from 0 to 16 days of germination.

In Col-281 cotyledon, ash increased significantly

from 2.12 to 3.56 g/100g 16 days. During

development of embryo axis, ash content of Col-160

increased significantly up to the end of germination

(5.78 to 6.65 g/100g); while a reduction occurred in

Col-181 at 12 days followed by an increase at 16

days (5.85 to 5.05 to 6.24 g/100g), both varieties

showed no significant differences between 8 and 12

days, contrary to this, Col-281 showed no significant

increase at 12 days followed by reduction at 16 days

(Table 1). Both increase and decrease in ash have

been reported previously (Bakr, 1996; Khalil et al.,

1995; Youssef et al., 1987) in the same variety. To

further elucidate the variability in faba bean

proximate composition, germination time (treatment)

and fractions were studied in combination of variety

Analysis of variance (Table 2) showed that proximate

composition was dependent on germination time,

fraction, variety and their interactions. Fractions had

the single largest contribution to variation in protein,

carbohydrate, lipid, total dietary fiber and ash

contents. Variety and germination time also

contributed to the variation in carbohydrate and total

dietary fiber contents to a small extent suggesting

that varieties behave differently in these components.

The interaction of variety, germination time and

fraction played a very small part in the variation of

proximate composition evidenced by their low

variance components.


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Table 1. Proximal composition analysis from three germinated and un-germinated of Mexican faba bean varieties

Col-160 Col-181 Col-281

0 8 12 16 0 8 12 16 0 8 12 16

Protein

Cot 23.72

c

25.10b

23.92c

27.12a

24.72c

30.03a 25.98

b

24.65c

d

27.65a

c

26.83c

27.56b

c

24.81d

Hull 5.36ª 3.99bc

5.02ab

4.49ab

3.52bc

3.37bd

3.65ab

4.01ª 3.95d 3.98

cd 5.32

b 5.53

ab

Embry

o ---

35.69c

38.08a

37.45a

b

--- 29.92c 36.95

b 39.16

a --- 30.82

c 37.89

b

41.37a

Carbohydrates

Cot 64.77

a

56.67d

60.64b

56.93c

d

65.92a

54.68d 61.77

b 60.70

c

62.34a

b

61.53ab

c

59.79b

d

62.95a

Hull 78.10

ª

74.52c

70.92d

74.69b

c

80.53

ª

73.01b

d 72.37

d

c 72.78

b

c 80.84ª 74.78ab

74.18c 71.50

d

Embry

o ---

35.48a

29.82c

30.35b

c

--- 42.84ª 35.80c

37.34b

c

--- 45.77a 34.94

c

39.13b

Lipids

Cot 2.28ª 2.00a 1.79ª 1.47ª 1.48ª 1.36

a 1.21ª 1.13

ab 2.66

a 2.38

ab 2.22

b 2.18

b

Hull 0.31a 0.24

ab 0.21

ab 0.17

bc 0.30ª 0.26ª 0.24ª 0.17

ab 0.27

a 0.20

a 0.17

ab 0.16

ab

Embry

o

10.90b

13.27a

13.20a

c

8.36b 10.45ª 6.35c --- 8.18

b 11.39ª 6.38

c

Total Dietary Fiber

Cot 5.90d

13.25c

10.75b

12.15ª 5.52d 11.48

b 8.77

c

11.69a

b

5.24d 6.64

ab 6.99ª 6.51

ac

Hull 13.86d

18.94

b 21.22

a

18.39b

c 13.48

d 21.69b

22.56a

18.77c

13.70d

18.77b

18.12c 19.45

a

Embry

o ---

12.16b

12.91

ª

12.36a

b

--- 13.04a 11.76

b 10.93

c --- 10.27

b 11.20

a 8.75

c

Ash

Cot 3.34ª 2.98ª 2.90ª 2.34ª 2.36ª 2.46ª 2.28a 1.84

a 2.12

cd 2.63

c 3.44

abc 3.56

ac

Hull 2.39a 2.32ª 2.63ª 2.26ª 2.18

b 1.68

cb

1.19bc

d

4.28a 1.25

d 2.78

b 2.22

bc

3.37ab

c

Embry

o --- 5.78

c 5.93

bc 6.65

a --- 5.85

bc 5.05

c 6.24

ab --- 4.96

a 5.59

a 4.38

a

aMeans in a row with different letters between varieties are significantly different (p<0.05) bConcentration are expressed as g/100g sample (dry matter basis)


7

Table 2. Analysis of variance for Nutritional composition analysis of three germinated and un-germinated Mexican

faba bean varieties

df

Mean squares

Protein Carbohydrates Lipids TDFb

Ash

Variety (Cv) 2 4.39a

(0)

58.53a

(0.50)

9.95a

(0.17)

38.68a

(4.85)

1.19

(0)

Treatment (TR) 3 198.74a

(0)

806.97a

(1.35)

27.78a

(0.04)

56.26a

(18.16)

6.35a

(0.54)

Fraction (Frac) 2 5478.59a

(97.8)

6428.90a

(95.73)

486.34a

(90.48)

607.21a

(74.67)

51.16a

(80.04)

Cv*TR 6 2.17a

(0)

9.10a

(0)

1.39a

(0)

2.99a

(0)

0.73

(0)

Cv*Frac 4 5.36a

(0)

28.70a

(0.91)

10.94a

(5.44)

7.27a

(1.33)

2.38a

(3.45)

TR*Frac 5 28.26a

(1.07)

40.90a

(1.55)

4.31a

(1.85)

5.07a

(0.87)

1.02

(0.61)

Cv*TR*Frac 10 8.47a

(1.06)

9.13a

(1.10)

1.25a

(1.81)

3.36a

(4.73)

0.70

(9.07)

Error 33 6.24

(0.07)

0.87

(0.25)

0.07

(0.25)

0.08

(0.24)

0.23

(6.36) aMeans squares are significant at 0.0001 probability levels. Values in parentheses are percent variance components. bTotal Dietary Fiber

3.2 Non-nutritional compounds

Contents of non-nutritional components

(pytochemicals) of germinated faba bean fractions

are shown in table 3. Phytate content differed

significantly (p<0.05) among germination times in

the fractions, with the cotyledon and embryo axis

possessing the highest and lowest phytate contents,

respectively (Table 3).

During germination phytate concentration increased

significantly in cotyledon of the three varieties

between 1 and 21% at 0 and 8 days, with the highest

increase in Col-281. After 16 days germination,

phytate content of cotyledon decreased with Col-181

experiencing the largest reduction (209.23 to 121.79

g/100g). In hull, Col-160 and Col-181 showed

reduction of 40.71% and 22.75% in phytic acid

content. For Col-281 reduction in phytate

concentration occurred at 8 days (2.76%), followed

by an increase at 12 days (5.04%) and a further

reduction at the end of germination (8.72%).

Significant reductions of 17 and 21% occurred in

embryo axis of Col-160 and Col-281 respectively; in

Col-181 the reduction was observed at 12 days

followed by a 7% increase to16 days (Table 3). These

reductions were similar to those reported by Al-

Numair et al., (2009) after 6 days of germination.

Phytic acid reduction during germination may be due

to phytase activity reported in many crops and this

can significantly reduce phytic acid content and

increase bioavailability of phosphorus in the seed that

is transported to the embryo axis for the organic

phosphate synthesis and used as energy source during

germination, or stored for later use by the seed.

Trypsin inhibitory activity differed significantly

(p<0.05) among fractions during germination with

the cotyledon and hull exhibiting the highest and

lowest activity, respectively (Table 3).

The trypsin inhibitors activity in cotyledon increased

significantly by 30-33% at 16 days in Col-160 and

Col-281, 58% in Col-181 at 12 days followed by an

insignificant decrease at 16 days. Trypsin inhibitory

activity was absent in the hull at 0 days due probably

to minimal metabolic process at this stage. However,

during germination trypsin inhibitory activity

increased from 2.85 to 16.96 TIU/mg proteins at the

end to the 16 days for Col-160 hull and over two

folds increase for Col-181 and Col-281. In contrast

trypsin inhibitory activity in the embryo axis

decreased (30-52%) with major reduction in Col-160

and the lowest reduction in Col-181 (Table 3).

Reduction of trypsin inhibitory activity in embryo

axis during germination process may be related to the

presence of diverse endogenous proteases that can

hydrolyzed this inhibitor during development of this

fraction for use as source of sulfur amino acids and

mobilized to cotyledon for protection against

microorganism and insects.


8

Table 3. Changes in non-nutritional factors of three germinated and un-germinated Mexican faba bean varieties

Col-160 Col-181 Col-281

0 8 12 16 0 8 12 16 0 8 12 16

Phytates

Cot 193.75

b

209.89a

168.23c

150.11d

193.75b

209.89a

b

168.23c

150.11d

193.75b

209.89a

168.23c

150.11c

d

Hull 190.13

a

189.80a

b

141.06c

112.73d

199.02b

185.52c

186.67b

c

153.74d

185.02b

179.92c

188.98a

b

172.51d

Embry

o --- 183.38

a

167.40b

152.42c

--- 191.94a 90.67

c 97.25

b ---

192.76a

155.38b

152.42b

c

Phenolics

Cot 24.41d 47.01

c 70.68

b

118.06a

19.79d 40.43

c 39.36

bc 81.71

a 11.60

d 33.31

c 68.72

b 93.28

a

Hull 30.29ª 21.57b 13.20

c 5.73

d 18.01

a 14.45

b 11.07

c 3.24

d 34.91

a 24.95

b 10.36

c 1.99

d

Embry

o --- 80.29

c

155.72b

195.42a

--- 41.68c 94.35

b

164.28a

--- 88.48c 169.80

b 220.51

a

Trypsin Inhibitors

Cot 13.92d 15.85

c 16.94

b 18.21

a 13.92

d 15.85

bc 16.94

ab 18.21

ac 13.92

d

15.85a

c

16.94bc

18.21ab

Hull nd 2.85c 5.13

b 16.96

a nd 7.13

c 8.96

b 17.91

a nd 4.72

c 4.53

bc 10.60

a

Embry

o --- 18.00

a 16.33

b 8.58

c --- 14.31

a 13.12

b 9.88

c --- 12.81

a 8.17

bc 7.35

c

aMeans in a row with different letters among varieties are significantly different (p<0.05) bConcentration of phytates and phenolics are expressed as milligram equivalents of phytic acid and gallic acid , respectively per

100 gram sample (dry matter basis); trypsin inhibitors as TIU/mg of protein. cnd. Not detected

During germination phenolic content in fractions

varied significantly with the embryo axis and the hull

containing the highest and lowest phenolic contents

at 16 days germination (Table 3).

In cotyledon of the three varieties phenolic content

increased significantly with Col-281 variety

expressing the highest increase of eight times the

initial concentration at 16 days compared to other

varieties where 4-5 times increase occurred. In

contrast, phenolic content in hull decreased

significantly, with the highest decrease expressed in

Col-281. In the embryo axis concentration of

phenolics increased constantly during all germination

stages, Col-181 variety had the largest increase of

3.94 times the initial concentration and Col-160 the

lowest (2.43 times the initial concentration) reduction

(Table 3). This increase may probably be due to

pigment synthesis, giving an intense green coloration

in the new plant. However this behavior is unusual in

the embryo axis suggesting that the embryo may be

responding to environmental stress and protecting

against microorganism, insects or other stimulus.

The results of analysis of variance for phytate and

phenolic contents and trypsin inhibitory activity

showed that they were highly dependent on variety,

germination time (treatment), fraction and their

interactions. Germination and its interaction with

variety and fraction had large relative contribution to

variation in phytic acid (42, 36 and 18%,

respectively) (Table 4). For trypsin inhibitors

"Fractions" was the prime contributor (52%) of

variation, while its interaction with germination was

the second contributor (36%) although all main

factors (variety and treatment) and their interactions

had significant effect during germination. "Fractions"

and its interaction with the “Treatment” were the

main contributors (63 and 25%) to variation in

phenolics concentration although all main factors

(Variety and Treatment) and their interactions had

significant effect during germination.


9

Table 4. Analysis of variance for non-nutritional

compounds of three germinated and un-germinated

Mexican faba bean varieties

D

f

mean squares

Phytates

Trypsin

Inhibitor

s

Phenolics

Variety (Cv) 2 343.89

a

(0)

22.72a

(0.48)

3257.35a

(2.20)

Treatment

(TR) 3

9831.06a

(41.88)

140.97a

(0)

16371.31a

(3.92)

Fraction

(Frac) 2

2501.46a

(2.12)

641.15a

(51.90)

63670.45a

(63.06)

Cv*TR 6

1154.53a

(2.47)

5.59a

(0)

216.42a

(0)

Cv*Frac 4

2018.96a

(17.77)

20.84a

(4.56)

1506.47a

(4.98)

TR*Frac 5 675.48

a

(0)

101.02a

(35.93)

7797.89a

(25.06)

Cv*TR*Fra

c 10

887.12a

(35.50)

6.22a

(6.76)

168.42a

(1.55)

Error 33 3.05

(0.51)

0.17

(0.40)

3.57

(0.07)

aMeans squares significant at 0.0001.

Comparison of the protein content of the fractions of

faba bean varieties germinated and un-germinated

(Table 5) showed a significant positive correlation

with phenolics (r= 0.78, p<0.0001), lipids (r= 0.77,

p<0.0001) and ash (r= 0.71, p<0.0001).High

correlations were also observed for lipid and ash

contents (r= 0.85, p<0.0001), phenolics (r= 0.75,

p<0.0001) and ash content with phenolics (r= 0.65,

p<0.0001); this indicates that fractions at the different

germination time with high lipid, ash and phenolic

contents will also have high protein content as in the

embryo axis.

Protein was inversely correlated with carbohydrates

(r= -0.92, p<0.0001) and total dietary fiber (r= -0.71,

p<0.0001), indicating that low protein content will be

expected in fractions that have a low carbohydrate

and total dietary fiber contents as in the cotyledon.

Carbohydrate content was also inversely related to

lipid (r=-0.93, p<0.0001), ash (r= -0.84, p<0.0001)

and phenolic (r= -0.82, p<0.0001) contents,

suggesting that lower lipid content may be a result of

low ash and phenolic contents in some fractions

during germination as in the hulls.

Table 5. Correlation coefficients for nutritional and non-nutritional compounds of three germinated and un-

germinated of Mexican faba bean varieties

Carbohydrates Lipids TDFb

Ash Phytates Trypsin

Inhibitors Phenolics

Protein -0.916***

0.773*** -

0.708***

0.669

*** -0.220 0.500

*** 0.775

***

Carbohydrates -

0.930***

0.399

**

-

0.836***

0.320

* -0.360

* -0.820

***

Lipids -0.294 0.850***

-0.267 0.163 0.745***

TDFb

-0.225 -0.083 -0.433**

-0.365*

Ash -0.313 0.211 0.650***

Phytates -0.111 -0.408***

Trypsin

Inhibitors 0.103

*p<0.01: **p<0.001; ***p<0.0001 (n=66) bTotal Dietary Fiber

The principal component (PCA) was performed on

the 8 constituents analyzed to explore their

interrelationships. The PCA generated two factors

with eigenvalues exceeding 1.0 and two factors

below this value that accounted for 94.5% of the total

variance.

The first component (F1), accounted for 58.87% of

total variance, with positive loadings for

carbohydrates (20%/0.975) and negative loadings for

ash (15%/-0.854), phenolics (15%/-0.854), lipids

(17%/-0.902), and protein (19%/-0.945). The second

component (F2, 17.2%) was influenced by a positive

loading for total dietary fiber (35%/0.696) and a

negative loading for trypsin inhibitory activity (28%/-


10

0.625). The score plot of the first two principal

components accounted for 76% of the total variance

revealing strong differences in seed fractions in faba

bean varieties (Figure 1).

Figure 1. Classification of fractions of faba bean germinated and un-germinated according to principal components 1

and 2. Varieties Col-160, Col-181 and Col-281 are denoted, respectively A, B and D.

Thus, hull of all germinated and un-germinated

varieties had a large content of total dietary fiber and

high positive loadings on both F1 and F2 grouped on

the upper right quadrant (positive) of the plot

diagonally opposite the cotyledon after 12 and 16

days with highest trypsin inhibitory activity and

embryo axis at 8 days with high protein content. The

PCA plot grouped embryo axis of all varieties at 12

and 16 days in the upper left quadrant because it had

the highest concentration of phenolics, lipids and ash,

whereas the cotyledon at 0, 8 of all varieties, and 12

days for the Col-181 with high carbohydrates and

phytates content were located to the lower right

quadrant (Figure 2). In the plot, hull at 12 days of

Col-160 variety (A12T) was positioned away from

the hulls of other varieties because of low

carbohydrate content.

Protein content was correlated with trypsin inhibitory

activity, while these compounds were negatively

associated with total dietary fiber and responsible for

clustering of the hull fraction. Also, phenolics, ash

and lipids attributes were negatively correlated with

carbohydrates and phytates content. This suggests

that faba bean when grouped by fractions and

germination time can be distinguished for their major

nutritional components.


11

Figure 2. Biplot of fractions of faba bean germinated and un-germinated according to principal components 1 and 2.

Varieties Col-160, Col-181 and Col-281 are denoted, respectively A, B and D. The variables PR, ASH, LP, FB, CB,

PY, PH and TI represent protein, ash, lipids, total dietary fiber, carbohydrates, phytates, phenolics and trypsin

inhibitors, respectively.

The dendogram (Figure 3) obtained from cluster

analysis based on the same 8 variables displayed four

discrete clusters. Embryo axis at 16 days of Col-181

variety (181-16E) was separated from the other based

on high phytates and phenolics content and low

trypsin inhibitory activity and lipid contents. Embryo

axis at 8 days and cotyledon at 12 and 16 days of

Col-160 and Col-281(160-12C, 160-16C, 160-8E,

281-12C, 281-16C and 281-8E)

grouped for their minimal distance of their low

carbohydrate, trypsin inhibitory activity and their

high protein, ash and phenolic contents. Embryo axis

at 12 days and cotyledon at 16 days of Col-181 (181-

16C and 181-12E) were also equidistant with similar

low carbohydrate, ash and phytate contents.

Remaining fractions, days of germination of all

varieties were grouped together yielding a distinct

profile.


12

Figure 3. Dendogram of cluster analysis performed

on 8 constituents of fractions of faba bean germinated

and un-germinated

4. CONCLUSIONS

Germination is a process with high utility and low

cost to increase the nutritional compounds or

reduce/eliminate non-nutritional compounds,

nevertheless, this cannot occur in a uniform way in

seeds fractions because some of these compounds

increase in concentration. However, this process can

increase digestibility and bioavailability of the

protein in cotyledon and embryo axis. Experimental

results indicate that germination reduces

carbohydrate, lipid, ash and phytate contents in

cotyledon and hull. In contrast, phenolics increased in

cotyledon and decreased in hulls, while trypsin

inhibitory activity increased in both fractions.

Nevertheless, concentration of different compounds

as proteins, lipids, ash or phenolics compounds

increased in the embryo axis; phytates and trypsin

Inhibitors were reduced; all this can be the result of

the hydrolysis and mobilization of storage material

during germination producing changes in their

concentration, fraction and distribution, resulting in

new molecules which participate in different

metabolic pathways to benefit growth and

development of the new plant.

ACKNOWLEDGEMENTS

We are grateful for the financial support of the

Consejo Nacional de Ciencia y Tencología

(CONACyT) through doctoral scholarship 226867


13

and a scholarship from the Programa Institutional de

Formación de Investigadores (PIFI).

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