Agronomic Biofortification of Amaranthus dubius with Macro Nutrients and Vitamin A

Agronomic biofortification of Amaranthus dubius with carbohydrates, proteins and Vitamin A using Spirulina platensis is reported in the present preliminary study. S. platensis was applied basally to field and its influence on germination, concentration of proteins, carbohydrates, chlorophyll, carotenoids and antioxidant activity of leaves were assessed in CO1 variety of A. dubius. Biofortification of the nutrients were evaluated on 5th and 20th day. Germination was optimum at 0.005 % of S. platensis as a fertilizer and the agronomic fortifying agent. Germination in control was 82 % and 95 % in 0.005 % of S. platensis fortification. The concentration of total chlorophyll in control and biofortified leaf on 20th day were 85.6 mg/g and 325 mg/g of dried leaf. The concentration of proteins was 71.93 mg/g and 450 mg/g on 20th day in control and fortified leaves. S. platensis applied field produced leaves fortified with 1.27 % of vitamin A aldehyde. Antioxidant activity of control leaf was 34.85 % and it was significantly increased in fortified leaves to 87 %. Thus the study confirms the fortification of leaf with proteins, carbohydrates and vitamin A.


INTRODUCTION
Malnutrition affects millions of people every year globally. Macro and micronutrient malnutrition affect the quality of living and leads to death ultimately. Due to urbanization and industrialization, major proportion of agricultural lands is lost. The fertility of the cultivable land is progressively decreasing due to the improper management of agrochemicals. Hence the yield obtained is deficient in many macro and micro nutrients. In developing countries, more than 40% of preschool children are anemic and 38% of pregnant women are affected by micronutrient malnutrition or "hidden hunger" [1]- [2]. Protein is the macronutrient that supplies essential amino acids. WHO/FAO/UNU consultation has revised the daily average protein requirement for adult as 0·83 g/kg/d. This is 10% more than the recommendations by 1985 [3]. Protein intake in rural population is insufficient in terms of digestible quality [4]. In addition to protein deficiency many preschool children suffer from Vitamin A deficiency. Globally about one third of pre-school children are suffering from vitamin A deficiency [5]. Biofortification for macro and micronutrients is an attractive tool to improve the nutritional status of food. It can be accomplished by traditional plant breeding, recombinant technology and agronomic practices. Success of biofortification depends on the type of crop being fortified, its yield attributes and consumption by vast majority of population, simplicity of the measures to be adopted for fortification and economic benefit assured to the farmers. S. platensis is rich in proteins, carbohydrates, lipids, minerals, vitamins, essential fatty acids, phenolic acids and tocopherol. From Mayan Civilization onwards S. platensis is consumed as a food. In addition to its nutritional significance, S. platensis is well known for its antioxidant, anticancer and immuno modulatory activities [6]. In spite of its nutritional and medicinal significance, the consumption is very low because of its unpleasant unique odor. Biofortification of a leafy vegetable with the nutrients from S .platensis is an attractive solution, as the quantity of leaf consumed is more and bioaccumulation in leaf is greater than other parts of the plant. Amaranthus dubius belongs to the economically important family Amaranthaceae. The leaves of the plant are recommended as a good food with medicinal properties for young children, and lactating mothers. It grows under any type of soil. It has the high photosynthetic rate [7]. Hence, the objective of the present study is to fortify A. dubius with protein, carbohydrates, carotenoids, vitamin A and antioxidants by exploiting S. platensis as a fertilizer and a fortifying agent.

Chemicals
All chemicals were purchased from High media, Mumbai, India. Solvents used were of analytical grade. The seeds of A. dubius (CO 1) was purchased from the local market.

Preparation of field for plant growth
The black soil in the College campus was used for the field trial. Two weeks before the sowing of seeds, the field was ploughed well and used. Two plots of 4x4 feet were prepared in completely randomized block design. All treatments were executed in triplicates. One plot served as a control where no fortification was done. The treatment plot included fortification with 5g of S. platensis flakes as a basal application. Leaf samples were collected on 5th and 20th day for the analysis of total carbohydrates, proteins, chlorophyll, carotenoids and antioxidant activity. The leaf samples on 20th day were analyzed by GC-MS.

Biochemical analysis of leaves
Seed germination was determined by the method of ISTA [8]. Total carbohydrates was estimated by the method of Hodge and Hofreiter [9]. total chlorophyll and carotenoids were estimated by the method of Arnon [10] and proteins by Lowrys method [11]. Total antioxidant activity of the leaves were assessed by using DPPH assay [12].

GC-MS of A. dubius leaves
The ethanol extract of leaves were used for GC-MS analysis in Clarus 500 Perkin Elmer. Elite-5MS column was used. The flow rate of carrier gas was maintained at 1ml /min. All the experimental were replicated thrice. The results are expressed is the mean ± standard error. At the lower concentration of 0.001 %, the germination was 87 %. As the concentration was increased above 0.01 %, inhibitory effect was observed. This is due to the high concentration of nutrients that osmotically blocked imbibition and absorption of water. Enzymes used to mobilize reserved nutrients in the endosperm are utilized only when the hydrolytic enzymes are activated by water. So reduced availability of water reduced the germination rate. Minerals present in S. platensis served as cofactors for majority of hydrolytic enzymes. This accelerated the metabolic rate culminating in increased germination. Synthesis and secretion of IAA in S.platensis was reported [13]. Fig. 2a shows the influence of S.platensis on the concentration of total chlorophyll. The concentration of total chlorophyll in leaves on 5th day in control was 49.5 mg/g of dried leaves. In S. platensis treated plants the concentration was remarkably increased to 155 mg/g. On 20th day also significant difference between control (85.6 mg/g) and treatment (325 mg/g) was observed. Increased availability of Mg and nitrogen in the soil obtained from S. platensis has facilitated the synthesis of chlorophyll in leaves. Porphyrin is the major constituent of chlorophyll. Availability of nitrogen in the soil increases the synthesis of proptoporphyrin and then into chlorophyll. Association between the soil nutrients and the concentration of photosynthetic pigments, photosynthetic rate, yield characteristics and nutritional profile of the yield were reported [14].  In biofortified leaves, it was significantly increased to 650 mg/g and 1500 mg/g on 5 th and 20 th day respectively. th and 20 th day was 31.5 mg/g and 71.93 mg/g respectively. In biofortified leaves, it was significantly increased to 115 mg/g and 450 mg/g on 5 th and 20 th day respectively. Increased availability of nitrogen has increased the synthesis of proteins [14]. Proteins are indispensable for the structure and function of cells. Increase in the concentration of proteins indicates the positive impact of biofortification. Influence of organic farming in increasing the concentration of carotenoids and phenolic compounds were well studied [15]. Fig. 3b shows the influence of S. platensis on the concentration of carbohydrates in leaves. The concentration of carbohydrate on 5 th and 20 th leaves, it was significantly increased to 405.3 mg/g and 723.98 mg/g on 5 th and 20 th day respectively. Increase in the expression of starch biosynthesis enzymes like adenosine diphosphate-glucose pyrophosphorylase, starch synthase and starch branching enzyme after the application of organic fertilizer is observed by Song et.al [16].  and 20 th day was 28.45 % and 55 % in control respectively. In biofortified leaves, it was significantly increased to 55 % and 87% on 5 th and 20 th day respectively. Carotenoids are well known for their antioxidant activity.Fortification of leaves with S.platensis has increased the concentration of carotenoids on 5 th and 20 th day. Polyphenolic constituents in A. dubius and their antioxidant characteristics was studied by Moyo et.al [17]. All are collectively responsible for the significant increase in the antioxidant activity of fortified leaves.  In addition, it showed the presence of thunmbergol, 2H-Pyran 2-(7heptadecynyloxy) tetrahydro, octadecanoic acid, hexadecanoic acid etc. Presence of thunbergol a diterpene in leaves was reported by Adams [18]. Presence of stearic acid in S.platensis was reported earlier. Stearic acid and its analogue are potent antidepressants and possess antimicrobial activity [19].The role of hexadecanoic acid in the structure and function of cell is described well [20]. Vitamin A is important in preventing night blindedness. It plays a key role in phototransduction.It plays a significant role in improving the immune system. Fortification in economically inexpensive foods that are consumed in good quantities are a viable solution to avoid vitamin A deficiency [21].

CONCLUSION
The preliminary study proved that S.platensis can be used as a fertilizer to augment plant growth and metabolism. Simultaneously it can be used as an agronomic fortifying agent. Economically inexpensive leafy vegetables can be fortified with the essential macronutrients, micronutrients and Vitamin A. An elaborate study is required to understand the mechanism of fortification in a large scale field study.