Dissecting Proteomic Estimates for Enhanced Bioavailable Nutrition during Varied Stages of Germination and Identification of Potential Genotypes in Chickpea

Food legumes are mostly cultivated worldwide and unique resource of nutritional proteins in the food of people livelihood in tropical and subtropical areas with a total harvested area of 95.720198 million hectares, 964 kg/hectare yield and 92.277859 million tonnes total production (FAO, 2018). The chickpeas (Cicer arietinum) also known as garbanzo beans (Spanish-speaking countries and the US) Gram, Chana, Bengal gram (India), Chickpea (English), Hommes, Hamaz (Arab world), Nohud, Lablabi (Turkey), Shimbra (Ethiopia), are the 2^nd most essential kind of food legume after dry beans with a total harvested area of 17.814502 million hectares, 965.1 kg per hectare yield and 17.192188 million tonnes total production and first in the Mediterranean basin, among pulse crops (FAO, 2018). It is cultivated in several countries with largest harvested area of 11.899185 million hectares by India followed by Australia, Pakistan, Russian Federation, Turkey, Iran, Myanmar, United States of America, Ethiopia, Mexico, Canada, Argentina, United Republic of Tanzania etc. (FAO, 2018). Currently, India represents as the largest producer of chickpeas accounting for around 70% of the global production followed by Australia, Turkey, Russian Federation, United States of America, Ethiopia, Myanmar, Mexico, Pakistan and Canada are measured as the top ten major world producers (FAO, 2018). Among the top ten exporting countries, Australia  represents the biggest exporter of chickpeas accounting for more than one-third of the total global export volumes (PRNewswire March 15, 2017) with an amount value of 1329.2 Million US$ followed by Mexico, Argentina, United States of America, Canada, India (143.596 Million US$), Russian Federation, Ethiopia, United Arab of Emirates and Turkey (FAO, 2017). However, India, irrespective of having the locus standing of 6^th top most exporter, holds the 1^st rank accounting for around one-fifth of the total global import volumes with an amount value of 1311.335 Million US$ amongst top ten importing countries followed by Pakistan, Bangladesh, United Arab Emirates, Algeria, Turkey, Iran, Saudi Arabia, Spain and United States of America (FAO, 2017).
       
Chickpea is considered a healthy vegetarian food due to its dietary values, therapeutic features and health benefits recognised since Roman times. Chickpea is used in Uyghur traditional medicine to treat hypertension and diabetes on the post production glucose rise in healthy or diabetic persons and in ayurvedic medicine to treat disorders of the blood, skin and other organs (Jenkins et al., 2012). The Romans noted that preparations of chickpea seeds or leaves were beneficial in the treatment of menstrual and urinary disorders, warts, jaundice and to soothe gout pains. In traditional Persian medicine, chickpea is prescribed for cancer and other diseases and is thought to increase libido and breast milk production (Javan et al., 2017). In India, the acidic secretions of chickpea leaves were used in traditional preparations to treat indigestion and constipation. As cosmetic preparation, a paste containing chickpea flour is applied to the skin of babies to improve the complexion (Patil et al., 2001). 
       
The amount and composition of protein in chickpeas varies depending on variety, plant growing period, process of examination, environmental circumstances, in addition to geographic places (Sahu et al., 2020). Accessibility of data on the effect of germination on the proximate composition, variety and germination circumstances such as temperature, light, humidity and time of germination is very important (Kuo et al., 2004). Thus, in the light of above background, the current research was executed with a hypothesis to complement our previous studies (Sharma et al., 2013a, 2013b, 2016, 2018 and Misra et al., 2016), analyse and assess to discover the highest amount of bioavailable digestible protein and other nutrients, most suitable germination stage, enzymatic activities and potentiality of genotypes under investigation for future utilization.

The present series experiment as 6^th one was undertaken to find out the impacts of varying seed seedlings germination stages at 0, 3 and 6 days on observed estimates of protein contents in 12 potential genotypes of chickpea including Pusa-256 as standard (control) check in continuation to our already reported five interconnected experimental studies. The experimental material consisted of twelve potential chickpea genotypes viz; BG-1058, BG-1053, BG-2024, BG-1092, BGD-112, Pusa-212, Pusa-256 and SBD-377 developed by Indian Agricultural Research Institute, India; H-82 developed by Chaudhary Charan Singh Haryana Agricultural University, Hisar, India; JG-74 developed by JNKVV, Jabalpur, India; ICC-11378 developed by ICRISAT, Patencheru, India having their origin source in India and ILC-3279 developed by ICARDA, Syria with origin source in Russian Federation. These genotypes were chosen from chickpea germplasm consisting of more than 2000 genotypes grown in the research field during 2018-19 observing standard agronomical practices by us at IARI, New Delhi, India on the basis of our inferences withdrawn from various unorganised observations noticed over a decade. The genotype specific seeds were used for preparation of experimental samples for analysis of protein content during 2019-20.
 
Soaking of chickpea seeds
 
Twenty-five healthy seeds of each gram variety were washed with tap water followed by immersion in distilled water in thoroughly washed and autoclave sterilized petri dishes of 12 cm in diameter and 2 cm depth for 24 h at room temperature arranged in completely randomized design (CRD) with three replications. After pouring off the soaking water, the seeds were immersed for one minute in a solution containing 0.001% Benlate (methyl-1-butyl carbamoyl-2- benzimidazole carbamate) followed by thorough washing with distilled water, spreading uniformly on petri dishes lined with spongy / blotting paper and then placed in a laboratory with sufficient humidity at a temperature of 20-25°C.
 
Seed germination
 
The soaked chickpea seeds were wrapped in already 0.001% Benlate solution treated appropriate paper (germi-test paper). Germination was carried out in a germination chamber with enough moisture at a temperature of 20-25°C in the dark. Germination was accomplished by daily distilled water spraying with adequate humidity at a room temperature on each CRD arranged petri dishes in three replications. On “0”, “3” and “6” days of germination times, sprouts were separated from the seeds. The seeds were manually dehulled and cotyledons were obtained. The air-dried cotyledons were ground to pass through a 60-mesh sieve, defatted in n-hexane (1:8), filtered and dried at room temperature. The defatted flours obtained were used for total protein analyses.
 
Estimation of protein content (%)
 
The protein assessment was made by the method (Lowry et al., 1951) using bovine serum albumin as a standard. One gram of dry seed powder was taken for analysis of protein content and absorbance was recorded at 660 nm and protein content (%) was estimated.
 
Statistical analysis
 
Complete randomized design with three replications derived quantitative data on 12 chickpea potential accessions for two years were pooled and subjected to standard statistical analysis as per statistical package PAST, Paleontological Statistics (Version 3.0).
               
Similarity indices were analysed as per Jaccard’s coefficient followed by cluster analysis as per UPGMA using DARwin 6.0.0 software.

The “0” day seedling germination stage
 
The findings at “0” day seedlings germination stage for the protein substances amongst the twelve genotypes were found to be in the range of 18.89 to 28.62% (Table 2). The genotypes namely BG-1058, BG-1053, BG-2024, BG-1092, BGD-112, H-82, ILC-3279, JG-74, ICC-11378, Pusa-212 and SBD-377 were found to be statistically significant at “P” 0.1% level of test of significance. Maximum protein content was noticed in the genotype ICC-11378 (28.62 %) followed by genotypes BG-1092 (28.46%) and JG-74 (27.80%) with increase of 50.95%, 50.12% and 46.62% respectively, over the average protein (18.96%) content of “0”, “3” and “6” days stages of standard check Pusa-256.
 
The “3” days seedling germination stage
 
During the investigation at “3” days seedlings germination stage, the protein contents in chickpeas were found in the range of 18.93 to 28.78 % (Table 2). The genotypes BG-1058, BG-1053, BG-2024, BG-1092, BGD-112, H-82, ILC-3279, JG-74, ICC-11378, Pusa-212 and SBD-377 were found to be statistically significant at “P” 0.1% level of test of significance at par with “0” day seedlings germination stage as presented in subsection 3.1. Maximum protein content was observed in the genotype BG-1092 genotype (28.78 %) followed by genotypes ICC-11378 (28.66%) and JG-74 (27.95%) with increase of 51.79%, 51.16% and 47.42% respectively, over the average protein (18.96%) content of  “0”, “3” and “6” days stages  of standard check Pusa-256.
 
The “6” days seedling germination stage
 
The investigation findings at “6” days seedlings germination stage for protein contents in chickpea demonstrated a range of 18.93 to 28.78% (Table 2). The genotypes viz; BG-1058, BG-1053, BG-2024, BG-1092, BGD-112, H-82, ILC-3279, JG-74, ICC-11378, Pusa-212 and SBD-377 were also found to be statistically significant at “P” 0.1% level of test of significance at par with “0” and “3” days seedlings germination stages as presented in subsections 3.1 and 3.2. Maximum protein content was recorded in the genotype BG-1092 genotype (28.84%) followed by genotypes ICC-11378 (28.71%) and JG-74 (27.96%) with increase of 52.11%, 51.42% and 47.47% respectively, over the average protein (18.96%) content of  “0”, “3” and “6” days stages  of standard check Pusa-256.
 
The average protein estimates of all stages
 
Overall average protein estimates of all stages for “0”, “3” and “6” days germinated seeds of chickpea in present investigation were found in the range of 18.96 to 28.69 percent (Table 2). Maximum protein content was observed in BG-1092 (28.69%) followed by ICC-11378 (28.66%) and JG-74 (27.90%) with increase of 51.32%, 51.16 % and 47.15% respectively, over the average protein (18.96%) content of all the stages of standard check Pusa-256.
 
Similarity / dissimilarity indices and cluster analysis
 
According to the Jaccard’s matrix coefficient genotypes BG 112 and ILC 3279 having least matrix coefficient i.e., 0.01 were found to have maximum protein percentage 26.31 and 26.30 respectively. Cluster analysis revealed three clusters (Fig 1), Cluster I was having only one genotype (P 256) having least protein percentage (18.96). Cluster II consists of seven genotypes of which BG 1092, JG-74 and ILC 3279 having maximum protein percentage and lowest Jaccard’s matrix value 0.01 were placed most closely in the dendrogram. In the Group III there are only four genotypes of which BG 2024 and Pusa 212 are more closely placed and it has 0.01 matrix coefficient and 24.17 and 24.19 Protein percentage respectively.


       
Chickpea is a known source of good quality protein and multipurpose legume widely used around the world. The twelve potential genotypes included in the present investigation differed significantly at morphological (Kumar et al., 2017), physiological, biochemical and molecular (Bhardwaj et al., 2014) levels indicating their diverse nature and characteristics. The highly significant varietal and genotypic differences found at all three stages of seedlings germination along with average of all stages for the trait protein under investigation (ANOVA Table 1) authenticate the potential of material and experimentation. The protein contents at “0”, “3”, “6” days seedlings germination stages along with their averages as presented in the Table 2 divulged that the chickpea genotypes BG-1092, ICC-11378 and JG-74 demonstrated significant increase in the protein contents and the maximum protein content (28.84%) was recorded in BG-1092 accounting for 51.11 % increase over the control, when grown up to “6” days seedlings germination stage. Seed germination is a natural activity that facilitates transportation of reserve materials and increases the bioavailability of minerals. Germination persuades important nutritional, biochemical and sensory alterations in nutrient by increasing their metabolism. It upgrades the digestibility of grains, increases deterioration of certain anti nutrients and enhances bioavailability of certain nutrients by minimizing the effect of obstructive factors viz; trypsin inhibitors, oxalates, phytates and certain fibres. It also minimizes certain oligosaccharides which affects flatulence thereby improving overall nutritive value of the products, amylase, protease and other enzymatic activities. Mostly Chickpea proteins are supposed to be storage proteins which serve as reserves of metal ions and amino acids that are mobilized and utilized for the maintenance and growth throughout seed germination, influencing the solubility properties. The alterations after 3 days of germination demonstrated extensive protein deterioration at the end of the process. The proteolytic processes might be associated with albumin content leading to escalation till fourth day followed by a gradual decline after sixth day of germination. This proteolytic activity may be followed by a gradual decline in trypsin inhibitor activity during the sprouting period. Chickpea germination may not modify significantly the in vitro digestibility of the protein fractions, except for the major globulin structures that may be increased to values near to those of casein, rendering it to be the more susceptible to the enzymatic activities (Uppal and Bains, 2012).




       
Proteolytic processes of the chickpea albumin content, using chickpea total globulin and casein as substrates (1:5 enzyme: substrate ratio), showing an escalation till fourth day, followed by a gradual decline on the sixth day germination. The chickpea seeds albumin to globulin ratio (3:5) was the reason for the higher action of the acid proteases than obtained in vitro. This upgradation in proteolytic activity appeared to have been followed by a gradual decline in trypsin inhibitor activity during chickpea seed germination (Neves and Lourenco, 2001).
       
The storage protein degradation and the escalations in proteolytic processes observed in germinating chickpea seeds are in agreement with the previous studies. The decline in enzymatic systems with caseinolytic processes have also been observed during germination of different leguminous species. The degradation of storage proteins and synthesis of enzymes at different times of germination as a characteristic of the seed after 7 days of germination of V. faba, C. arietinum and L. termes have also been observed (Ahmed et al., 1995).
       
The findings of this investigation at “0” day seedlings germination stage for the protein substance amongst the twelve genotypes indicate that genotypes ICC-11378, BG-1092 and JG-74 have potentials to be utilized for varietal nutritional enhancement programmes for the protein. The findings at “3” days seedlings germination stage leads to an inference that germinated seeds contained enhanced digestible protein and also confirmed the findings of “0” day seedlings germination stage. However, the order of potentiality was re-aligned for BG-1092, ICC-11378 and JG-74 as potential resources. The findings at “6” days seedlings germination stage for protein contents is re-validating the findings of “3” days seedlings germination stage that germinated seeds contained more protein to be consumed as digestible human nutrition and also reassured the findings of “0” and “3” days seedlings germination stages that the genotypes ICC-11378, BG-1092 and JG-74 have potentials to be utilized in varietal nutritional enhancement breeding programmes for the nutrition protein. However, the order of potentiality as potential resources coincided at par with “3” days seedlings germination stage as BG-1092, ICC-11378 and JG-74.
       
The several findings in conjugation with our already reported five experimental studies lead to an inference that in view of the food requirements across the diversified sections of the society, overall nutrient and proximate compositional analysis, the potential chickpea genotypes can be an economic and alternative source of protein and other essential micro and micro nutrients that could alleviate protein, iron and zinc malnutrition in developing countries. We conclude that the “6” days seedlings germination stage gave highest nutritional protein content for human consumption probably due to enhanced duration of germination that increased the enzymatic activity resulting in breakdown of complex nutrients into simpler one as noticeable increase in protein content in contrary to the anti-nutrient components showing decrease in their contents and overall improvement in the availability of easily digestible protein nutrient across the seed germination stages and the genotypes BG-1092, ICC-11378 and JG-74 having higher proteins and other micro nutrients on the basis of our serial studies confirm their potentials to be utilized in breeding for varietal nutritional enhancement programmes.
       
Hence, it is recommended that the “6” days seedlings stage of chickpea should be utilized as an ideal source of easily digestible protein for human nutrition and the newly identified closely related potential genotypes BG-1092, ICC-11378 and JG-74 should be exploited for human nutrition as well as in hybridization programmes for harnessing their genetic potential in varietal nutritional enhancement of breeding projects for protein and other nutritional components.

Germination duration positively correlated with enzymatic activities indicating that germination improves the nutritional value of the chickpea grains due to the enzymatic degradation of carbohydrate, protein and fats. Our hypothesis was confirmed and we found the “6” days chickpea sprouts as best stage for highest digestible protein and also identified the closely related genotypes BG-1092, ICC-11378 and JG-74 as resource for highest nutritional protein content along with other micro nutrients suitable for human consumption particularly for infants, children, expecting mothers during high protein requirement stages. These genotypes have potentials to be utilized in varietal nutritional enhancement breeding programmes for the protein.  Hence, we recommend that the “6” days seedlings germination stage chickpea seeds should be utilized as an ideal source of easily digestible protein for human nutrition in general and Govt. sponsored food schemes in particular and the newly identified potential genotypes BG-1092, ICC-11378 and JG-74 should be exploited for human nutrition as well as in hybridization programmes for harnessing their genetic potential during varietal nutritional enhancement breeding projects for the protein and other nutritional components.