Content and Bioaccessibility of Phenolics and Flavonoids in Processed Germinated Brown Chickpea (Cicer Arietinum L.)

Short title: Digestible bioactive components in germinated chickpea

Morteza Oghbaei and Jamuna Prakash*

Department of Food Science and Nutrition, University of Mysore, Mysuru, 570 006, India

Corresponding author: Jamuna Prakash, Department of Food Science and Nutrition, University of Mysore, Mysuru, 570 006, India, E-mail: jampr55@hotmail.com

Citation: Oghbaei M and Prakash J (2021) Content and Bioaccessibility of Phenolics and Flavonoids in Processed Germinated Brown Chickpea (Cicer Arietinum L.). J Nut Sci Heal Diet 2(1): 12-20

Received Date: May 10, 2021; Accepted Date: June 03, 2021; Published Date: June 04, 2021

Abstract

Germination is a traditional pre-process used for improving the cooking and sensory quality of legumes. The study explored the availability of bioactive components in chickpea fortified through germination in mineral fortified soak water. Whole chickpea grains were germinated in water enriched with either iron or zinc at two levels. A portion of grains was dehulled. Both whole and dehulled grains were cooked by two methods, namely, pressure cooking or microwave oven. The sample germinated in plain water served as control. All samples were analyzed for total and in vitro digestible bioactive components. The remaining soak water was also analyzed for presence of bioactive components. The results indicated that the process of dehulling improved the content and digestibility of total phenolics and tannins in fortified chickpea in comparison to control grain. For flavonoids, the results were opposite. In general, cooking reduced the content and digestibility of bioactive components; however, percent digestibility from cooked grains was better. Differences were found in digestibility of bioactive among iron and zinc fortified grains. In conclusion, the dehulling and cooking processes influenced the content and digestibility of bioactive components in mineral fortified germinated chickpea.

Keywords: Cicer Arietinum; Germination; Dehulling; Cooking; Phenolics; Tannins; Flavonoids

Introduction

Legumes are categorized as healthy grains on account of their nutritional composition and presence of several phytochemicals [1]. Evidence regarding their disease prevention role is emerging in many epidemiological, clinical and experimental studies [2, 3]. Many legume seeds have also been reported to exhibit alpha-amylase and alpha-glycosidase inhibition properties [3, 4]. Specifically, chickpea cultivars were associated with 58-62% of alpha amylase inhibitory activity indicative of anti-diabetic properties [5].

Generally dry legume seeds need some processing to convert them to edible form. Thermal treatments such as puffing, roasting, boiling, pressure cooking or frying develop flavors and improve the textural quality and palatability of legumes remarkably. Some legumes are also consumed as germinated grains with or without further processing. Germination improves the digestibility of grains by developing new enzymes, partially breaking down protein and starch and destroying anti-nutrients [6, 7, 8]. During pre- germination process, especially soaking, the bioactive components lixiviate in water and the water could affect the rate and kind of bioactive components which move out of the grain hulls. Germination has also been reported to influence the antioxidant components and activities of legumes [9, 10]. Legumes are also milled to remove the outer portion of grain which improves their cooking characteristics further. Outer layer of grains (husk) is a rich source of bioactive components; hence, dehulling can change its content. Cooking can cause considerable changes in the content of bioactive components and antioxidant activity [11].

Physiological availability of nutrients and bioactive components is a prime determinant of food and nutrition security and human health. Hence, the inherent or externally added constituents of any food should be absorbed for the ultimate utilization by individuals. The bioavailability of any food component is influenced greatly by factors related to food or to the host. The food matrix, the processing conditions, and overall composition of food determines how much of an ingested component is absorbed by the body. For this reason, it is important to study the bioaccessibility of essential constituents from food [12].

Legumes serve as an important source of protein in SouthEast Asian countries where they are part of everyday diet. While as such they are good sources of vitamins and minerals, it is possible to fortify them to improve the micronutrient profile. Iron and zinc deficiencies are commonly seen across all age group, especially in developing countries and are recognized as major public health problems. Fortification of staple foods can be one of the strategies to overcome malnutrition [13]. Apart from industrial fortification of staples, it is also possible to incorporate fortificants at domestic level in traditional processes to improve malnutrition. In the present study, whole chickpea grains were germinated using iron or zinc fortified soak water and thereafter processed further. The objective was to study the effect of dehulling and cooking on total and digestible (bioaccessible) bioactive components in chickpea geminated in mineral fortified soak water.

Materials and methods

Materials

The material used for the study was whole chickpea (Cicer arietinumL.) of Desi variety with a brown color coat purchased from local market in one batch. All experiments were carried out in duplicate/triplicate with analytical grade chemicals and double distilled water. Chemicals were procured from (i) SD Fine Chemicals, India and (ii) HiMedia Company, India. The enzymes used for the digestibility experiments were pepsin (Batch No. 3-0060), pancreatin (Batch No. 0-0864), diastase (Batch No. 0695/195/270511) and papain (Batch No. 0993/493/130811). The dialysis tubing was procured from Sigma Aldrich Co. USA with a molecular mass cut off of 10 kDa.

Methods

Study Design

The study involved fortifying germinated Desichickpea through enriching the soak water with minerals (iron or zinc at two levels), dehulling a part of germinated grains and cooking both whole and dehulled grains with two methods. The control sample was germinated in plain water. All samples were analyzed for total and digestible bioactive components. The soak water, in which grains were soaked prior to germination, was also analyzed for presence of bioactive components.

Germination and cooking of chickpea

For the control sample, a 100g portion of whole chickpea was washed and soaked in 300 ml of distilled water for 10 hours. For the fortified samples, the soak water was enriched with two levels of iron (ferrous sulfate equivalent to 100 or 200 mg of iron) or zinc (zinc sulfate equivalent to 50 or 100 mg of zinc). The soaked grains were transferred to a grain germinator, [wherein grains are kept moist to facilitate germination] and allowed to germinate for 48 hours at normal room temperature (28 ± 5o C) and lighting conditions. The germinated grains were divided into two portions, and one portion was manually dehulled. Manual dehulling was done by gently rubbing the grain between two fingers, since the husk portion was sufficiently loosened on account of soaking and germination, a gentle rub facilitated easy dehusking. A part of this was homogenized well in a blender and kept aside to be used as control and the rest was divided and cooked using two methods, namely pressure cooking (PC) and microwave (MW) cooking. For cooking, 50 ml water was added to 100 g germinated grain as cooking medium. Pressure cooking was done for 10 min on hot plate adjusted for maximum power in a standard pressure cooker with specifications of maximum 15 lbs pressure and 121o C temperature. The microwave (Model BPL-700T, 2,450 MHz, 1,200 watts, from BPL Sanyo Utilities and Appliances Ltd, Bangalore, India) was adjusted to quick set program and 100 g germinated grains were heated in oven continuously for 4 min. The cooking time was standardized by checking the softness of grain by pressing between two fingers. The cooked samples were homogenized with cooking water in a blender and used for further analysis. All analysis was done on freshly prepared samples. Moisture content of all samples was determined using oven drying method [14].

Analysis of bioactive components

To estimate total phenolics, flavonoids and tannin contents in chickpea, extracts were prepared by mixing known amount of sample slurry (raw and cooked) with glass double distilled water. The slurry was shaken for 3 hours and filtered through “Whatman” filter paper No.1 and used for all analysis.

Total polyphenol- A 0.2 ml of sample was mixed with 1.0 ml of Folin-Ciocalteau reagent (10 fold dilutions) and 0.8 ml of 2% Na2 CO3 . The volume was made up to 10 ml using water-methanol (4:6) as diluting fluid. This was allowed to stand for 30 min and absorbance read at 740 nm. Concentration was calculated using tannic acid as standard and the results expressed as mg tannic acid equivalents/100g sample [15].

Total flavonoids- This was estimated following the procedure explained by Arvouet-Grand et al. [16]. A 5.0 ml of 2% AlCl3 in methanol was mixed with 5.0 ml of the extract solution and allowed to stand for 10 min. Absorption was read at 415 nm against a blank sample consisting of extract solution with 5.0 ml methanol without AlCl3 . The total flavonoid content was determined using a standard curve with quercetin and expressed as mg quercetin equivalents/100g of sample.

Total tannins- Colorimetric estimation of tannins is based on the measurement of blue color formed by the reduction of phosphotungstomolybdic acid by tannin like compounds in alkaline solution [17]. A known amount of extract was mixed with 5.0 ml of Folin-Denis reagent and Na2 Co3 solution and made up to 100 ml, mixed well and absorbance read at 760 nm after 30 min. Total tannin content was expressed as mg tannic acid equivalent/100 g of sample.

Digestible bioactive components

In vitro digestibility (also known as bioaccessibility) of total phenols, flavonoids and tannins was determined by simulated gastro-intestinal digestion using pepsin for the gastric stage followed by pancreatin and bile salts for the intestinal stage. The proportion of bioactive compound diffused through a semi-permeable membrane was considered as measure of the digestibility [18]. A known amount of sample was placed in a 250ml Erlenmeyer flask and 70 ml of water was added. The pH was adjusted to 2.0 with 6M hydrochloric acid. After 15 min the pH was checked and readjusted to 2.0, if necessary. A 3.0 ml of freshly prepared pepsin solution (16% pepsin in 0.1M hydrochloric acid) was added, volume made up to 100 ml with water and the samples incubated at 370 C for 2 hours. Then the digests were frozen for 90 min. Titratable acidity was determined in an aliquot containing 5 ml of pancreatin bile extract mixture (0.4% pancreatin and 2.5% bile salt in 0.1M sodium bicarbonate) by titrating against 0.2M sodium hydroxide till the pH of 7.5 was obtained. A 20 ml of frozen digests (after thawing) were subjected to stimulated intestinal digestion by placing the dialysis tubing in Erlenmeyer flasks. The dialysis tubing contained 25 ml of sodium bicarbonate (equivalent to moles of sodium hydroxide determined by titratable acidity). The flasks were incubated in a shaker bath at 370 C for 30 min (till the pH reached 5.0), 5.0 ml of pancreatin bile extract mixture was added and shaken for another 2 hours (till the pH reached 7.0). Then the dialysates were carefully transferred to graduated tubes and volume was measured. The collected dialysates were analyzed for total phenols, flavonoids and tannins as per the aforementioned procedures.

Statistical Analysis

Data were subjected to statistical analysis using statistical software SPSS 15.0 (SPSS Inc., Chicago, IL). Mean and standard deviation for all values were calculated. Data were analyzed using Student’s ́T ́ test to determine significant differences between (i) whole and dehulled samples, (ii) raw and cooked samples and (iii) PC and MW cooked samples. In addition, data were also subjected to analysis of variance to determine the significant differences between all variations of control germinated raw whole and dehulled grains (five variations each) and cooked whole and dehulled grains (ten variations each).

Results and discussion

Effect of dehulling germinated chickpea on total and digestible bioactive components

The total, digestible and percent digestible total phenols (TP), tannins and flavonoids in raw, whole and dehulled chickpea are compiled in Table 1. The TP content of the control whole chickpea reduced significantly after dehulling from 170.2 mg/100g to 155.6 mg/100g, whereas in other variations (fortified grains) dehulling increased TP content in germinated grains. Fortification of grain with minerals influenced the TP content to a large extent and in most of the fortified grains; a higher TP content could be estimated. Digestible TP increased in all variation of chickpea following dehulling and was significant for control and sample with 200 mg Fe. When computed as percent digestibility, the control chickpea had highest value of 81.58 after dehulling, whereas all variations followed an opposite trend of showing higher percent digestibility for whole grains. Among variations, Zn fortified samples had least percent digestible TP. In a similar study with green gram (Vigna radiata) the results were slightly different. Dehulling did not affect the total and digestible phenolics in green gram significantly and among all variations, iron fortification reduced the bioactive components while zinc did not have any effect [19].

Data were further analyzed using ANOVA to examine if differences were significant within all variations of (i) whole and (ii) dehulled grains. The overall observations are as follows, mineral fortified grains had higher phenolic content in comparison to control with one exception of 200 mg iron fortified sample. This could possibly be explained on the basis that minerals bound phenolics, so they could not be leached out in soak water. However, this difference was reduced when bio accessibility of phenolics was examined. Grains with 100 mg iron fortification showed maximum bioaccessibility whereas zinc fortified dehulled grains had least. Similar trend was also seen in percent bioaccessible phenolic constituents. This indicates that zinc binding with phenolics was stronger and not easily broken down by digestive enzymes.

The process of soaking and germination itself can influence the phenolic constituents of legumes. Lopez-Amoros et al. [10] in their study on effect of germination on antioxidant components of beans, lentils and peas reported that germination modified the quantitative and qualitative phenolic composition. These were in turn influenced by the type of legume and the germination conditions. Vadivel et al. [3] also reported an increase in total phenolic constituents of wild legumes on germination. Fouad and Rehab [20] found a progressive increase in the phenolic content of lentils with an increase in sprouting time.

Table 1: Effect of dehulling of germinated chickpea on total and digestible bioactive components (mean±standard deviation per 100g)

Variations

Total 

Digestible

(mg)

(mg)

(%)

Total phenols

A. Distilled water (control)

Whole 

170.2±0.00c

108.6±0.57b

63.79±0.33a

Dehulled 

155.6***±0.00c

126.9*±1.22ab

81.58*±0.78a

B. With 100 mg Fe

Whole 

207.3±2.52a

138.8±3.90a

66.95±1.88a

Dehulled 

234.8*±3.56a

139.7±5.11a

59.48*±2.17b

C. With 200 mg Fe

Whole 

144.0±0.00d

104.8±0.00b

72.80±0.00a

Dehulled 

190.2*±2.82b

121.4**±0.26b

63.86**±0.14b

D. With 50 mg Zn

Whole 

184.4±0.00b

91.3±0.97b

49.51±0.53b

Dehulled 

229.5*±1.86a

103.9±2.09c

45.26±0.91c

E. With 100 mg Zn

Whole 

186.7±1.70b

92.4±8.41b

49.40±4.60b

Dehulled 

225.1*±8.59a

103.0±7.37c

45.74±3.27c

Tannins

A. Distilled water (control)

Whole 

223.8±1.23c

173.7±3.07b

77.60±1.37a

Dehulled 

286.5*±6.31bc

190.8*±0.00a

66.58*±0.00b

B. With 100 mg Fe

Whole 

247.4±1.94a

192.8±3.53a

77.93±1.43a

Dehulled 

268.1*±0.00c

190.9±3.31a

71.20**±1.23a

C. With 200 mg Fe

Whole 

183.0±2.59e

80.3±5.74d

43.87±3.14c

Dehulled 

328.0*±8.65a

91.6*±0.34d

27.94*±0.06e

D. With 50 mg Zn

Whole 

196.9±0.00d

92.3±4.49d

46.88±2.28c

Dehulled 

317.0*±10.15ab

110.9*±1.99c

34.99*±0.62d

E. With 100 mg Zn

Whole 

235.8±2.04b

152.4±3.77c

64.60±1.60b

Dehulled 

315.3*±10.65ab

164.1±3.28b

52.05*±1.04c

Flavonoids

A. Distilled water (control)

Whole 

22.52±1.17a

5.73±0.00a

25.50±0.00a

Dehulled 

18.40±1.97a

3.44*±0.27a

18.77*±1.43ab

B. With 100 mg Fe

Whole 

21.93±2.83a

5.08±0.23a

23.16±1.04a

Dehulled 

15.41±0.00a

2.61*±0.12a

17.04*±0.76b

C. With 200 mg Fe

Whole 

19.72±0.00a

4.83±0.13a

24.52±0.68a

Dehulled 

14.60*±0.67a

2.36*±0.00a

16.20*±0.00b

D. With 50 mg Zn

Whole 

22.56±0.32a

5.61±0.21a

26.21±0.99a

Dehulled 

17.33±1.78a

3.90±0.23a

22.57±1.24a

E. With 100 mg Zn

Whole 

19.93±0.00a

4.81±0.24a

24.20±1.20a

Dehulled 

17.40±0.53a

3.10±0.00a

19.23*±0.00ab

Significant differences between whole and dehulled grains on application of ‘T’ test; * P< 0.05, **: P< 0.01, ***: P< 0.001, values without notation are not significantly different.Differing superscripted small letters in a column indicate significant difference among all variations of (i) whole grains and (ii) dehulled grains.

The tannin content of chickpea increased in all variationsin soak water mediated by presence of external minerals. Digestible tannin increased in chickpea due to dehulling in four samples and increases in variation A, C and D was significant. Variation C (with 200 Fe) showed least tannin digestibility in whole and dehulled grain (80.3 and 91.6 mg/100g respectively). Percent digestible tannin reduced significantly in all variations of chickpea in dehulled significantly on dehulling. The highest tannin in whole (247.4 mg/100g) and dehulled grain, (328.0 mg/100g) was found in variation B and C respectively. Mineral fortification either increased or decreased tannin content of chickpea, and no specific trend was seen. These alterations could be due to loss or retention of tannins in soak water mediated by presence of external minerals. Digestible tannin increased in chickpea due to dehulling in four samples and increases in variation A, C and D was significant. Variation C (with 200 Fe) showed least tannin digestibility in whole and dehulled grain (80.3 and 91.6 mg/100g respectively). Percent digestible tannin reduced significantly in all variations of chickpea in dehulled grain. In a similar study with green gram dehulling did not affect the content and digestibility of tannins, though the fortification with iron had an effect. The iron fortified samples had lesser digestibility compared to control and zinc fortified samples [19]. Application of ANOVA revealed significant differences among tannin content of both whole and dehulled grain variations. The bioaccessibility was in the lower range for mineral fortified samples with exception of samples with 100 mg iron indicating that presence of minerals affected tannin bioaccessibility.

Total, digestible and percent digestibility of flavonoids reduced in chickpea due to dehulling which shows that the husk portion could be good source of flavonoids. The flavonoid content ranged from 14.60-22.56 mg/100g in chickpea which was markedly lesser than TPs and tannins. Reduction of total flavonoids in variation C was significant (by 5.12 mg/100g). Digestible flavonoids reduced significantly in control chickpea and in variations with iron whereas changes in variation with zinc were not significant. Highest percent flavonoids digestibility was seen in variation D (26.21%). The overall percent digestible flavonoids was in lower range in comparison to TP and tannins. Application of ANOVA did not reveal any significant differences among all variations of either whole or dehulled grains for content and bioaccessibility of flavonoids, though differences were observed in percent bioaccessibility.

Sharma et al. [5] analysed the bioactive components of five cultivars of Indian brown chickpea and reported range of values for total phenolics, tannins and flavonoids as 203-255 mg, 170-210 mg, and 31-36 mg/100g respectively. The range of values for all constituents were similar to the present study. A high content of phenolics and flavonoids in the whole grains can be attributed to their high concentration in the hull portion as observed by Kanatt et al. [21], who found that the hull extract of chickpea was a rich source of phenolics and flavonoids.

Effect of cooking on total and digestible bioactive components in germinated chickpea

Table 2 presents the content and digestibility of phenolics, tannins and flavonoids in cooked whole chickpea. The content of TP as well as its digestibility were significantly higher in PC than MW cooked samples in all variations of whole chickpea. Highest TP content (129.7 mg/100g) and digestibility (128.5 mg/100g) was found in PC control sample. Fe fortification at higher level (200 mg) and Zn fortification at both levels resulted in decreased content and digestibility of TP than control. However, both control and Fe fortified variations had better percent digestibility of TP (>90%), whereas Zn fortification reduced it to some extent. This shows that zinc bound phenolic constituents to a larger extent as was seen in dehulled raw sample (Table 1). Application of ANOVA revealed significant differences among all variations considered together, both for content and digestibility of phenolics. However, for percent bioaccessibility, there were no differences among control and iron fortified samples whereas zinc fortified samples were significantly lower.

When effect of cooking was analyzed as comparison between raw and cooked samples, cooking lowered the TP content significantly in all variations, both control and fortified. Digestibility was also lesser, though it was significant only for few variations. Percent digestibility was much higher in comparison to raw for all variations and was marginally significant. To summarize the overall effect, cooking decreased the TP content but improved the availability.

The tannin content was in the range of 127.3-190.7 mg/100g for PC samples and 116.9-198.5 mg/100g for MW cooked samples. MW cooking increased tannin content and digestibility for iron fortified samples but lowered it for Zn fortified samples and differences were marginally significant. The percent digestibility did not follow any specific trend but was higher for control and in lower ranges for mineral fortified samples. When cooked samples were compared with raw, the tannin content was lower, though digestibility was similar indicating that cooking did not influence the availability of tannin in germinated chickpea. Application of ANOVA revealed significant differences in tannin content among all variations. Bioaccessibility was higher in variations A and B followed by E, and then by C and D. Similar trend was seen for percent bioaccessibility of tannins too.

The flavonoid content of whole cooked chickpea was in the lower range in comparison to raw in all variations, though differences were not significant. However, cooking improved the digestibility of flavonoids in control and Zn fortified samples. Percent digestibility was also higher than raw samples. The method of cooking (under pressure or in microwave) did not influence the flavonoids content or availability.A further analysis using ANOVA did not reveal significant differences among all variations put together except for PC sample with 100 mg iron, which was marginally lower for flavonoid content. However, for digestibility, significant differences were seen among all. The differences were lesser in percent digestibility with seven variations showing similar values and the rest in lower ranges.

The data on content and digestibility of bioactive components of dehulled germinated chickpea grains are compiled in Table 3. The phenolic compounds of PC and MW cooked variations were in the range of 118.0-155.5 and 85.3-130.1 mg/100g respectively. MW cooked samples had significantly lesser TP content and digestibility than PC samples. To analyze the effect of cooking, when cooked samples were compared with raw dehulled grains, both TP content and digestibility were significantly lesser, though in terms of percent digestible components, cooked grains were better. Similar observations could be made for tannin content and digestibility, wherein they were much lesser than raw samples but percent digestibility was higher. No specific trend was seen between methods of cooking. The flavonoids followed a pattern similar to whole grains with the content being lesser and percent digestibility being higher than raw grains.

Table 2: Effect of cooking on total and digestible bioactive components in whole chickpea (mean±standard deviation per 100g)

Variations

Total 

Digestible

(mg)

N

(mg)

N

(%)

N

Total phenols

A. Distilled water (control)

PC

129.7±2.62a

*

128.5±2.10a

*

99.12±1.61a

*

MC

107.7*±0.00c

**

99.63*±5.32cd

-

92.49*±4.94a

*

B. With 100 mg Fe

PC

119.9±1.47b

**

115.7±0.00b

-

96.49±0.00a

*

MC

110.4**±0.71c

**

108.9*±1.11c

-

98.681.00a

*

C. With 200 mg Fe

PC

122.0±0.00ab

*

106.1±1.66c

-

86.97±1.37a

*

MC

94.57***±0.00d

**

88.57*±0.68d

*

93.66*±0.71a

*

D. With 50 mg Zn

PC

105.2±0.00 c

**

78.13±1.39e

-

74.11±1.51b

*

MC

90.44***±3.47

***

67.87*±2.06f

*

75.03±2.28b

*

E. With 100 mg Zn

PC

97.17±2.88

*

78.49±2.52e

-

80.78±8.77b

*

MC

82.71*±1.43

*

59.57**±1.04f

*

72.06*±1.25b

*

Tannins 

A. Distilled water (control)

PC

190.7±0.00ab

*

179.50±0.99a

-

93.59±0.00a

-

MC

163.2*±0.63d

**

155.73*±2.61b

-

95.42±1.60a

-

B. With 100 mg Fe

PC

176.1±2.85bc

*

159.20±8.37ab

-

90.40±4.75a

-

MC

198.5*±4.65a

*

149.85*±6.93b

*

75.50±3.49b

-

C. With 200 mg Fe

PC

170.7±4.62cd

-

44.75±0.00d

-

26.21±0.00c

-

MC

171.2*±1.56c

-

56.81*±8.96d

-

33.17*±5.23c

-

D. With 50 mg Zn

PC

127.3±6.62e

*

56.94±0.00d

-

44.73±0.00c

-

MC

116.9*±1.14e

**

52.72*±2.55d

-

45.09±2.17c

-

E. With 100 mg Zn

PC

179.5±3.74bc

*

128.62±1.78c

-

72.04±1.37b

-

MC

168.3*±1.39cd

*

119.29*±3.63c

-

70.89±2.17b

-

Flavonoids

A. Distilled water (control)

PC

16.80±2.24a

-

6.32±0.08ab

-

37.74±0.45a

-

MC

18.40±0.00a 

-

6.64±0.00a

*

36.20±0.00a

-

B. With 100 mg Fe

PC

13.20±1.13b

-

4.12±0.22def

-

31.28±1.74a

-

MC

15.89±0.51a

-

4.58±0.00de

-

28.83±0.00bc

-

C. With 200 mg Fe

PC

14.68±1.63a

-

4.04±0.08f

-

26.92±1.28bc

-

MC

14.77±0.90a

-

3.79±0.24g

*

25.71±1.59c

-

D. With 50 mg Zn

PC

17.42±1.48a

-

6.23±0.21ab

-

35.86±1.14a

-

MC

16.90±0.00a

-

5.86±0.06bc

-

34.79±0.31a

-

E. With 100 mg Zn

PC

15.62±0.78a

-

5.16±0.17cd

-

33.07±1.15a

*

MC

17.35±1.23a

-

5.35±0.00c

-

30.83*±0.00ab

-

PC: pressure cooked; MW: microwave cooked. Significant differences between PC and MW cooked grains on application of ‘T’ test; * P< 0.05, **: P< 0.01, ***: P< 0.001, values without notation are not significantly different. Column N shows significant differences between raw (Table 1) and cooked grains. Differing superscripted small letters in a column indicate significant difference among all variations together.

The data were also analyzed using ANOVA for all variations put together and the results showed significant differences among variations for phenolic content. Again, there was no specific trend but zinc fortified samples had lower content and digestibility. Similar trend was seen for tannins also. The differences were lesser in flavonoid content and digestibility. These differences can possibly be explained by the presence of varying amount of mineral uptake by the grains during germination (the process being further influenced by dehulling), which in turn influences the extent of

Table 3: Effect of cooking on total and digestible bioactive components in dehulled chickpea (mean±standard deviation per 100g)

Variations

Total 

Digestible

(mg)

N

(mg)

N

(%)

N

Total phenols

A. Distilled water (control)

PC

118.0±0.00c

*

122.1±1.59a

-

103.1±0.92a

**

MC

109.1***±3.79c

*

94.35*±1.89c

*

86.49**±1.73b

-

B. With 100 mg Fe

PC

155.5±2.26a

*

118.1±2.84ab

*

75.96±1.83c

*

MC

130.1*±2.16b

**

109.6*±0.88b

*

84.24**±0.68bc

*

C. With 200 mg Fe

PC

125.4±2.07bc

*

89.53±2.33d

*

71.39±1.87d

-

MC

106.1*±1.20c

**

83.71*±1.74d

*

78.93*±1.64bc

*

D. With 50 mg Zn

PC

119.6±1.58c

**

102.3±2.12c

-

85.54±1.77bc

*

MC

105.5*±6.72c

**

77.64**±2.15e

-

73.57*±2.03d

*

E. With 100 mg Zn

PC

117.9±5.44c

**

86.71±0.00d

-

73.52±0.00d

*

MC

85.28**±1.37d

**

69.87*±1.21f

-

81.96*±1.40bc

*

Tannins

A. Distilled water (control)

PC

198.4±2.28b 

*

178.9±1.00a

-

90.16±0.50a

*

MC

171.8*±0.56d

*

144.1*±2.64c

*

83.86**±1.54a

*

B. With 100 mg Fe

PC

202.9±4.39ab

*

158.6±0.92b

*

78.14±0.45bc

-

MC

214.5*±0.00a

*

153.8*±6.26bc

*

71.73*±2.92c

-

C. With 200 mg Fe

PC

210.9±3.91ab

*

118.5±3.82de

*

56.20±1.23d

*

MC

202.1*±0.00ab

*

88.92*±2.47g

-

44.00*±1.22e

*

D. With 50 mg Zn

PC

202.0±3.46ab

*

100.9±1.54f

-

70.24±1.51c

*

MC

185.7*±1.03c

*

107.2±2.19ef

-

57.74*±1.25d

*

E. With 100 mg Zn

PC

177.4±2.18d

*

126.5±3.45d

*

71.33±1.95c

*

MC

169.8*±1.32d

*

147.5*±5.74bc

-

86.87*±3.38ab

*

Flavonoids

A. Distilled water (control)

PC

15.30±1.32ab

-

5.39±0.15a

-

35.22±0.99ab

**

MC

16.41±0.00a

-

6.01±0.25a

-

36.66±1.54a

**

B. With 100 mg Fe

PC

10.41±0.72bc

*

4.03±0.15bc

*

38.80±1.40a

**

MC

12.40*±0.00ab

-

4.31±0.20b

*

34.82*±1.58ab

**

C. With 200 mg Fe

PC

8.74±1.72d

-

3.20±0.06c

*

36.60±0.69a

**

MC

11.23*±1.15b

-

3.72±0.05c

*

32.19*±1.52b

**

D. With 50 mg Zn

PC

12.82±0.00ab

-

4.59±0.15b

*

35.92±1.30ab

**

MC

12.88±0.72ab

-

4.29±0.10b

*

33.37±0.76b

**

E. With 100 mg Zn

PC

10.40±0.00cd

*

3.23±0.09c

-

31.29±0.8b

**

MC

12.83±0.74ab

-

4.18±0.11b

*

32.74±0.91b

**

PC: pressure cooked; MW: microwave cooked. Significant differences between PC and MW cooked grains on application of ‘T’ test; * P< 0.05, **: P< 0.01, ***: P< 0.001, values without notation are not significantly different. Column N shows significant differences between raw (Table 1) and cooked grains. Differing superscripted small letters in a column indicate significant difference among all variations together.

binding of bioactive compounds. Since the presence of minerals was more by adsorption, a larger variability in the content and digestibility of bioactive constituents was seen in dehulled grains in comparison to whole grains. The data on mineral content and bioaccessibility of these samples is available in a recently published paper from same study [22].

Xu and Chang [11] analyzed the total phenolic contents of soaked and cooked chickpea and reported that soaking and pressure boiling reduced the total phenols by 3.0 and 30.5% respectively, while losses were nil on steaming. Vadivel et al. [3] also analyzed the total free phenolics in ten types of soaked and cooked wild legumes and reported a mean reduction of 38% of total phenolics on cooking. A reduction in phenolic constituents on soaking and cooking of Cassia hirsute L., an Indian underutilized legume was also reported by Vadivel et al. [4]. The results confirm our findings, wherein a considerable loss of phenolics on cooking was observed. The observations were similar to green gram wherein cooking reduced content of total bioactive constituents, however the availability was better from cooked grains [23].

The overall observations indicate that both the content and digestibility of bioactive components reduced significantly among all variations of chickpea on cooking. Losses were greater in dehulled grains and in Zn fortified samples, however, percent digestibility of bioactive components improved on cooking. According to Parada and Aguilera [24], thermal processing increases the digestibility through softening cell wall and release of compounds from food matrix. In our study, we found that though cooking reduced the content of bioactives, of the available constituents a higher percentage could be absorbed.

Bioactive components in soak water

Since, some of the bioactive components are water soluble and there is a possibility that these could also be dissolved in the soak water, we also analyzed the remaining soak water for total phenolics, tannins and flavonoids, and interestingly a considerable amount of these could also be measured in left over water (Table 4). Total phenols were in the range of 20.51-23.54 mg/100ml of water for control and Zn fortified samples. For iron fortified samples, there were variations with one sample having lesser and the other having more total phenols than the control. These variations can be explained on the binding ability of iron, though it did not follow any specific trend. For tannins, most of them were similar with one iron fortified sample showing lower value. Flavonoids did not show any difference and the amount was low. These results confirm the findings that process of soaking the grains for germination or added minerals can influence the bioactive components through leaching out or physical binding process.

Table 4: Bioactive components (mg per 100 ml) in soak water (plain and fortified) originated from leaching during soaking period

Variations

Total phenols

Tannin

Flavonoids

A. Distilled water (control)

23.54±0.94

31.95±0.35

3.82±0.69

B. With 100 mg Fe

13.34*±1.12

18.71*±0.31

5.43 ns±0.78

C. With 200 mg Fe

28.26*±1.43

30.82 ns±0.86

4.41 ns±0.92

D. With 50 mg Zn

20.75ns±0.65

25.15 ns±2.15

1.98 ns±0.06

E. With 100 mg Zn

20.51ns±0.58

29.69 ns±0.81

2.68 ns±0.00

Significant differences between control and fortified soak water on application of ‘T’ test; * P< 0.05, ns: not significant.

Conclusion

The overall results show that chickpea grains were rich sources of phenolic compounds with a high digestibility in whole and dehulled grains. Mineral fortification increased the total phenolics and tannin content as well as digestibility on dehulling, though flavonoids were much lower. The content and digestibility of bioactive components was lesser in cooked chickpea; however, percent digestibility was better in cooked grains. Iron fortification during germination did not affect the digestibility of bioactives significantly, though Zn fortified samples showed lesser digestibility. The soak water remaining after the removal of grains for germination showed the presence of bioactive components indicating that a part of these were leached out in the soaking medium.

Funding information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest statement

Authors declare that they have no conflict of interest with anyone with respect to research reported in this paper.

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