Physicochemical Attributes and the Impact of UV-B Irradiation on the Black Gram (Vigna mungo (L.) Hepper.

M. Priyadharshana¹, M. Girija¹, V. Smitha¹, M. A. Badhsheeba² and V. Vadivel^*1

¹Department of Botany, V.O. Chidambaram College (Affiliated to Manonmaniam Sundaranar University, Tirunelveli), Tuticorin – 628 008, Tamil Nadu, India

²Department of Biotechnology, Kumararani Meena Muthiah College of Arts and Science, Adyar, Chennai-600 020, Tamil Nadu, India

*Corresponding Author V. Vadivel Article History Received: 10.07.2022 Accepted: 20.07.2022 Published: 30.07.2022 Abstract: Grain legumes, which are interchangeably called pulses in the Indian sub-continent, have occupied an important place in the daily diets of the population of the region due to their nutritional potential, particularly as rich sources of proteins. In the present study, physicochemical attributes and the impact of UV-B irradiation on the black gram (Vigna mungo (L.) Hepper) were documented. The hundred seed weight of the black gram is higher than certain common legumes cultivated in India. The seeds posse good physicochemical properties which can be incorporated into human diets not only as protein supplements but also in processed food such as weaning, backed and soup products. In this paper, we investigated the changes in morphological, photosynthetic and physiological responses of the black gram seedlings to UV-B radiation. In the present study, the results showed that the growth of black gram was adversely affected under UV-B exposure with the response depending on the duration of exposure. Maximum reduction of growth i.e., plant height, internodal length, leaf area, plant fresh weight, plant dry weight, plant water content, chlorophyll a, b and total chlorophyll, was noted under the higher duration of UV-B irradiation. Keywords: Black gram, Impact of UV-B irradiation, Physicochemical properties.

INTRODUCTION

Man has always selected the types of food crops with some consideration for their nutritional and functional attributes, although these have not been properly understood. In this context, grain legumes, which are interchangeably called pulses in the Indian sub-continent, have occupied an important place in the daily diets of the population of the region due to their nutritional potential, particularly as rich sources of proteins. The supplementation of cereals with high protein legumes is considered to be one of the best solutions to protein-calorie malnutrition, particularly in developing countries (Singh & Singh, 1992).

Because of the increasing utilization of grain legumes in composite flours for various food formulations, their functional properties are assuming greater significance. Their functional characteristics provide a set of data, which gives information on the fields of application in food formulations (Hermansson, 1979). These can be used as a guideline in product development, especially in wheat-legume composite flours, where proteins are used as major functional ingredients (Luz Fernandez & Berry, 1989). The functional properties are provided not only by the proteins of flours but also by complex carbohydrates and other grain components such as pectins and hemicelluloses (Martinez, 1979). In recent years, functional foods are assuming greater importance and have attracted the attention of food processors, marketers and consumers (Reilly, 1994).

Functional properties are physicochemical properties, which give information on how a particular ingredient (for example protein, or carbohydrate) will behave in a food system (Kinsella & Melachouris, 1976). Voluminous literature exists on the nutritional composition of grain legumes. This includes protein and carbohydrates, vitamins, minerals and antinutritional factors of grain legumes (Singh & Singh, 1992). But only limited information is available on the functional properties of grain legumes (Singh, 2001). Hence, in the present study, some of the functional or physicochemical properties of the grain legume, black gram (Vigna mungo L.) are evaluated.

Some disadvantages of grain legumes are their time-consuming preparations and the presence of antinutritional factors such as protease inhibitors, lectins, amylase inhibitors, polyphenols and oligosaccharides (Gupta, 1987). Fortunately, these compounds are efficiently destroyed or removed by soaking, germination and cooking at the household level or during industrial processing. Perhaps the above-mentioned factors are responsible for the steady decline in the consumption of grain legumes in many counties. However, at present, a renewed interest exists in developing countries and in developed societies in vegetable protein sources inclined to ‘natural’ foods which may stimulate a higher consumption of grain legumes.

Phenolics are known to bind proteins and reduce their availability. On the contrary, it has been suggested that consumption of low levels of certain antinutrients may produce health benefits while avoiding some of the adverse effects associated with their large intake (Jansman, 1993). Because of this, in the present investigation, an attempt has been made to deduct the quantity of total free phenolics in the raw seeds of black gram (Vigna mungo L.).

Phenolic metabolites function to protect the plants against biological and environmental stresses and therefore are synthesized in response to pathogenic attacks such as fungal or bacterial infection or high-energy radiation exposure such as prolonged UV exposure (Briskin, 2000). According to (Matern & Kneusel, 1988), the first step of the defence mechanism in plants involves a rapid accumulation of phenols at the infection site, which restricts or slows the growth of the pathogen. The total phenol status of plants has been correlated with host resistance to a variety of diseases and insects (Eleftherianos et al., 2006). In this contest, in the present study, five lots of black gram seeds are exposed to UV-B radiation for different time intervals (i.e., control, 15min, 30min, 45min and 60min). Then total free phenolic content of the seed is determined.

Lower yields of certain cash crops and undesirable effects in agriculture may result due to increased UV-B stress (Caldwell et al., 1998). Many studies have been conducted on the possible biological effects of enhanced UV-B exposure on higher plants at the species level (Greenberg et al., 1997). The results of these studies are varied, but the most commonly documented impacts of UV-B exposure are damage to DNA, proteins, and lipids, alteration in growth and morphology, and decreased rate of photosynthesis. In this context, five lots of black gram seeds are exposed to UV-B radiation for different time intervals (i.e., control, 15min, 30min, 45min and 60min) and after that, they are grown in plastic trays.

Another five lots of black gram seeds are grown in plastic trays and after germination, 35 days old seedlings are exposed to UV-B irradiation for different time intervals (i.e., control, 15min, 30min, 45min and 60min). From both the seedlings (nearly 40days after germination), chlorophyll contents (mg per g of fresh weight), leaf area (cm²), plant height (cm), number of leaves per plant, internodal length (cm), fresh weight of the plant (g), dry weight of the plant (g) and water content of the plant (g) are recorded.

MATERIALS AND METHODS:

Procurement of Materials:

The seed material, Vigna mungo (L.) Hepper was procured from the local market, Tuticorin and was stored in airtight plastic containers at room temperature (25°C±2°C) for further studies.

Experiment 1:

From the plastic containers, 500g seed materials were taken out and used for the following physicochemical parameter analysis.

100 - Seeds Weight (g):

One hundred seeds were taken at random three times and weighed separately. The average weight of 100 seeds was recorded in grams.

Density (Sood et al., 2002):

One-hundred-gram seeds were weighed accurately and transferred to a measuring cylinder. Then 100ml distilled water was added to it. Seed volume was recorded as total volume (ml) – 100ml. Density was recorded as ml per seed.

Hydration Capacity (Sood et al., 2002):

Seeds weighing 100g were counted and transferred to a measuring cylinder of 500ml capacity and 100ml water was added. The measuring cylinder was covered with aluminium foil and left overnight at room temperature. The next day, seeds were drained, superfluous water was removed with filter paper and swollen seeds were reweighed. Hydration capacity per seed was determined using the following formula.

Hydration capacity =

Hydration Index (Sood et al., 2002):

The hydration index was calculated as below

Hydration index =

Swelling capacity (Sood et al., 2002):

Seeds weighing 100g were counted; their volume was noted and soaked in 350ml water overnight. The volume of the seed before and after soaking was noted with the help of a graduated cylinder. Swelling capacity per seed was determined using the following formula.

Swelling capacity =

Experiment 2:

Twenty-gram seed material was taken out from the plastic container and was powdered in a Wiley Mill to pass a 60-mesh screen and stored in screw-capped bottles at room temperature and was designated as raw seed powder.

Four lots of seed materials (each lot 20g) were taken out from the plastic containers and put in Petridishes and were exposed to UV-B radiation in the following time intervals.

• First lot seed was exposed to UV-B radiation for 15min

• Second lot seed was exposed to UV-B radiation for 30min

• Third lot seed was exposed to UV-B radiation for 45min

• Fourth lot seed was exposed to UV-B radiation for 60min

After the above treatments, the seeds were powered in a Wiley Mill to pass a 60-mesh screen and stored in screw-capped bottles at room temperature and the seed powder obtained by this method was designated as UV-B radiation-exposed seed powder.

Raw as well as treated seed material powders were used for estimating total free phenolics.

Extraction and Estimation of Phenols:

• Extraction (Maxson & Rooney, 1972):

One gram of seed flour was taken in a 100ml flask, to which 50ml of 1% (v/v) HCl in methanol was added. The samples were shaken on a reciprocating shaker for 24h at room temperature. The contents were centrifuged at 10,000 x g for 5min. The supernatant was collected separately and used for further analysis.

• Estimation of phenols (Sadasivam & Manickam, 1992):

One millilitre aliquots of the above extract were transferred into a test tube, to which, 1ml folin-ciocalteu's reagent followed by 2ml of 20% (w/v) Na₂Co₃ solution was added and the tubes were shaken and placed in a boiling water bath for exactly 1min. The test tubes were cooled under running tap water. The resulting blue solution was diluted to 25ml with distilled water and the absorbance was measured at 650nm with a help of a UV-visible spectrophotometer. The amount of phenols present in the sample was determined from a standard curve prepared with catechol. A blank containing all the reagents minus plant extract was used to adjust the absorbance to zero. The average value of triplicate estimations was expressed as g 100g^-1 of the seed flour on dry weight basis.

Experiment 3:

Five lots of seed materials (each lot 20g) were taken out from the containers and put in Pertidishes and were exposed to UV-B radiation in the following time intervals.

• The First lot was treated as a control

• The second lot was exposed 15min to UV-B radiation

• The third lot was exposed 30min to UV-B radiation

• The fourth lot was exposed 45min to UV-B radiation

• The fifth lot was exposed 60min to UV-B radiation

After that, the seeds were soaked in tap water for 12h and sowed in plastic trays. Thirty-five days after sowing, morphological traits and leaf chlorophyll contents were evaluated.

Experiment 4:

Five lots of seeds were taken out from the containers (each lot 50g) and were soaked in tap water for 12h and then sowed in plastic trays. Twenty-five days after germination (DAG) the plants were exposed to UV-B radiation in the following time intervals:

• The plants raised in the first tray were designated as control

• The plants raised in the second tray were exposed 15min to UV-B radiation

• The plants raised in the third tray were exposed 30min to UV-B radiation

• The plants raised in the fourth tray were exposed 45min to UV-B radiation

• The plants raised in the fifth tray were exposed 60min to UV-B radiation

• Morphological traits and leaf chlorophyll content were recorded after seven days.

Morphological Traits:

• Leaf Area:

Three leaves were randomly harvested from each treatment of UV-B light exposed seeds and UV-B light-exposed plants and their leaf area (cm²) was recorded by using a graph sheet.

• Plant Height, Number of Leaves and Inter-nodal Length:

Three plants were selected randomly from each treatment and their height (cm), the number of leaves per plant and inter-nodal length (cm) were recorded.

• Measurement of Dry Matter (DM) and Plant Water Content (WC) :

Three plants were randomly taken from the plastic trays as the samples for DM and WC analysis. The weight of fresh matter (FM) of the collected plants was immediately measured using a portable electronic balance (minimum scale: 0.001g). The plants were oven-dried at 130°C for 2h to measure the dry matter (DM) present. The water content of each plant was calculated by subtracting DM from FM (Catchpole & Wheeler, 1992)

• Leaf Chlorophyll Content Estimation (Arnon, 1949):

One gram of leaf material was taken from the leaf of each treatment and was ground separately with a chilled pestle and mortar in diffuse light with the addition of 20ml of 80% (v/v) cold acetone and the homogenate was centrifuged at 5,000 rpm for 5 minutes. Transferred the supernatant to a 100ml volumetric flask. Again, grounded the residue with 20ml of 80% (v/v) cold acetone, centrifuged and transferred the supernatant to the same volumetric flask. Repeated this procedure until the residue was colourless. The mortar and pestle were thoroughly washed with 80% (v/v) cold acetone and collected the clear washings in the volumetric flask. Made up the volume to 100ml with 80% (v/v) cold acetone. The absorbance of the solution was taken at 645, 663 and 652nm against the solvent (80% cold acetone) blank. Calculated the amount of chlorophyll present in the extract by using the following formula and expressed in mg chlorophyll per g tissue

mg chlorophyll a/g tissue = 12.7 (A 663) – 2.69 (A 645) x

mg chlorophyll b/g tissue = 22.9 (A 645) – 4.68 (A 663) x

mg total chlorophyll/g tissue = 20.2 (A 645) + 8.02 (A 663) x

Where,

A = Absorbance at a specific wavelength.

V = Final volume of chlorophyll extract in 80% acetone.

W = Fresh weight of tissue extract.

Statistical Analysis:

100-seed weight, density, swelling capacity, hydration capacity, hydration index, total free phenolics, plants height (cm), leaf area (cm²), inter-nodal length, DM, WC and leaf chlorophyll were estimated on triplicate determinations. Estimates of mean and standard error for the above-stated parameters were calculated with help of a calculator.

RESULTS AND DISCUSSION:

Data on physicochemical attributes of black gram (Vigna mungo) seeds are given in Table 1. Hundred seeds’ weight recorded in V. mungo seem to be higher than that of earlier investigations in Lens culinaries (Ereifej & Shibli, 1995); Vigna radiata, V. mungo, V. sinensis and V. umbellata (Hira et al., 1988) and V. radiata (Kochhar & Hira, 1997). Haq (1983) suggests that geographic distribution can account for much of the observed variation within a species. The present results are also in agreement with the suggestions of Haq (1983), Vadivel et al. (1998) and Vadivel & Janardhanan (2000). Seed density, hydration capacity and swelling capacity of the presently investigated V. mungo seem to be low compared to earlier investigations in Vicia faba (Sharma & Sehgal, 1992) and Arachis hypogea (Abulude et al., 2006). El Tabey (1982) showed consumers’ preference for beans which absorb more water.

Table 1: Data on physicochemical traits ^a

Physiochemical traits	Vigna mungo
100-seed weight (g)	36.775 ± 0.28
Density (ml/seed)	0.330 ± 0.34
Swelling capacity (ml/seed)	0.441 ± 0.68
Hydration capacity (g/seed)	0.404 ± 0.48
Hydration index (g/seed)	1.081 ± 0.54

a – values are mean ± standard error of the mean of triplicate determinations

Data on total free phenolics of raw seeds of black gram is presented in Table 2. The phenolic content reported in black gram seems to be low compared to earlier reports in Vigna mungo (Rani & Hira, 1998); V. mungo and V. radiata (Babu et al., 1988) and V. unguiculata (Laurena et al., 1987). In general, in the present study, the variability in the content of phenols for the same species may be related to genetic origin, geographical source, level of soil fertility and ecological factors. This is in agreement with an earlier report (Vadivel & Janardhanan, 2000). The phenolic compounds are water-soluble (Uzogara et al., 1990) and are moreover concentrated in seed coats (Bressani et al., 1988). They can be eliminated by dehulling, soaking and heat treatment or cooking process (Singh & Singh, 1992).

Table 2: Effect of different time exposure of black gram seeds to UV-B radiation on total free phenolic contents ^a

Time (min)	Phenols (mg g-1 seed flour)
Control	1.475 ± 0.052
15	1.538 ± 0.024
30	1.698 ± 0.027
45	1.704 ± 0.017
60	1.784 ± 0.025

a – values are mean ± standard error of the mean of triplicate determinations

In the present study, among the four treatments, 60min exposure of seeds to UV-B exhibited the highest percentage increase in the contents of phenols (Table 2). Recently, the phenolic compounds found in the legumes are considered to be natural antioxidants (Dueñas et al., 2002; López-Amorós, 2006), representing an important group of bioactive compounds in foods, and may prevent the development of many diseases such as atherosclerosis and cancer (Formica & Regelson, 1995). As a consequence of this activity, the presence of phenolic compounds in food has in recent years come to be viewed in a positive light by both scientists and consumers and has resulted in a push to procure food with specific beneficial effects such as functional foods. Phenolic, compounds not only effectively prevent oxidation in foods; they also act as protective factors against oxidative damage in the human body (Haslam, 1998). Hence, in the presently reported phenolics are not antinutrients but they acted as antioxidants. The presence of these antioxidants provides added value to the pulses. This will lead to commercial utilization of pulses as sources of these compounds and a better acceptance of them not only as sources of nutrients but also as a storehouse of antioxidants, which help in the maintenance of the body against oxidative damage caused by pollutants, sunlight etc.

The effects of UV-B treatment on plant height, internodal length, leaf area, plant fresh weight, plant dry weight, plant water content, chlorophyll a, b and total chlorophyll contents are shown in Tables 3 and 4. Plant height and leaf area decreased with increased UV-B exposure time. At 60min exposure of seeds to UV-B, plant height decreased by 69.73 %, internodal length decreased by 65.39%, leaf area decreased by 83.69%, plant fresh weight decreased by 67.84%, plant dry weight decreased by 54.71%, plant water content decreased by 71.61%, chlorophyll a decreased by 67.63%, chlorophyll b decreased by 32.20% and total chlorophyll decrease by 60.87% versus the control (Table. 3).

Table 3: Effect of different time exposure of black gram seeds to UV-B radiation on morphological traits and chlorophyll contents ^a

Traits	Time (min)
	Control	15	30	45	60
Plant height (cm)	19.03 ± 0.95	17.90 ± 0.30	15.10 ± 0.42	13.83 ± 0.83	13.27 ± 0.52
Internodal length (cm)	2.63 ± 0.72	1.90 ± 0.15	1.83 ± 0.42	1.73 ± 0.24	1.72 ± 0.30
Leaf area (cm2)	15.58 ± 89.14	15.04 ± 23.46	13.14 ± 48.68	14.37 ± 20.67	13.04 ± 18.76
Plant fresh weight (g)	1.987 ± 0.89	1.778 ± 1.67	1.627 ± 1.27	1.487 ± 0.98	1.348 ± 1.45
Plant dry weight (g)	0.435 ± 1.45	0.378 ± 0.34	0.342 ± 0.56	0.267 ± 0.78	0.238 ± 0.78
Plant water content (g)	1.55	1.40	1.28	1.22	1.11
Chlorophyll a (mg/g fr.wt)	1.73 ± 0.11	1.46 ± 0.11	1.38 ± 0.10	1.21 ± 0.22	1.17 ± 0.29
Chlorophyll b (mg/g fr.wt)	0.59 ± 0.10	0.43 ± 0.27	0.38 ± 0.40	0.25 ± 0.43	0.19 ± 0.44
Total chlorophyll (mg/g fr.wt)	2.30 ± 0.12	1.81 ± 0.22	1.74 ± 0.36	1.58 ± 0.13	1.40 ± 0.04

a – values are mean ± standard error of the mean of triplicate determinations

At 60min exposure of seedlings to UV-B, plant height decreased by 85.06%, internodal length decreased by 60%, leaf area decreased by 69.08%, plant fresh weight decreased by 62.80%, plant dry weight decreased by 61.54%, plant water content decreased by 63.05%, chlorophyll a decreased by 63.77%, chlorophyll b decreased by 48.84% and total chlorophyll decrease by 59.72% versus the control (Table. 4).