DESC:FIELD OF THE INVENTION
The present invention relates to an instant fortified beetroot-millet soup formulation. More particularly, the present invention discloses a instant, healthy, ready-to-cook iron fortified soup formulation of beetroot and millets through integration of fortification strategy with various processing technologies, for the management of anemia.

BACKGROUND OF THE INVENTION
Anemia is a long-term micronutrient deficiency where the number of red blood cells or the hemoglobin concentration is below normal, due to which the capacity of the body to carry oxygen to the tissues is reduced. Iron Deficiency Anemia (IDA) is the most common form affecting millions of people for decades and is the leading cause of various metabolic diseases, contributing to disability and death of vulnerable populations (International Institute for Population Sciences (IIPS) and ICF, 2021; World Health Organization, n.d.-b). Iron plays a significant role in the body by acting as an oxygen carrier and for myoglobin synthesis, red blood cell formation, and brain development. Iron deficiency causes lack of oxygen in the tissues which is evident as tiredness, fatigue and shortness of breath, being a primary indication of anaemia. Other visible symptoms of anaemia include pale or yellowish skin, lightheadedness or dizziness, weight loss, cold hands and feet etc. Anaemia may be caused by several factors including nutrient deficiencies caused by inadequate diets, inadequate absorption of nutrients, infections (e.g. malaria, parasitic infections, tuberculosis, HIV), inflammation, chronic diseases, gynaecological and obstetric conditions, and inherited red blood cell disorders. The most common nutritional cause of anaemia is iron deficiency, although deficiencies in folate, vitamin B12 and vitamin A are also important causes.

According to the World Health Organization (WHO), anaemia is a serious global public health problem that particularly affects young children, menstruating adolescent girls and women, and pregnant and postpartum women. Most recent estimates suggest a global prevalence of 30.7% in women, 35.5% in pregnant women and 43% in children. The gravity of the problem can be gauged from the fact that one in four individuals aged 10–24 years (~430 million), around the world suffer from anaemia, with the highest prevalence found in low- and middle-income countries.

According to the National Family Health Survey 5 (2019-21), the prevalence of anaemia in India is estimated to be 25% in men (15-49 years) and 57% in women (15-49 years). The comparative data of past years shows an increased prevalence of anemia in all women by 4%. As the years between childhood and adulthood represent a sensitive period for developmental, physiological and behavioural changes, anaemia in this formative phase of life can reduce work capacity, impair neurocognitive and pubertal development and increase susceptibility to infections. Anaemia affects overall health, survival, productivity, income and development. The prevalence of anaemia in India since last 50 years is primarily attributed to iron and folic acid deficiency, however, deficiencies of other micronutrients such as vitamin B6, and vitamin B12, widely prevalent in India, especially in children, (as indicated by the comprehensive national nutrition survey (CNNS) 2016–2018 report) is also a contributory factor.

Dietary preferences are considered to play a major role in aggravating nutrient deficiencies especially iron deficiency. Indian population predominantly consumes a vegetarian diet which may have antinutrients that can interfere with iron absorption. Moreover, the intake of animal sources food, having higher bioavailability of iron, is low in India, due to which intake and/or absorption of iron and other nutrients is inadequate for generation of blood cells (hemopoiesis). Thus, dietary preferences restrict the nutrient availability of the population and underscores the need for planning diets involving food synergy to enable micronutrient absorption for maximizing nutrient bioavailability.

Food-based approach is considered as the most desirable and sustainable method of preventing anaemia and other nutritional deficiencies which is based on enhancing dietary diversity, as the key intervention, through increased availability and accessibility of iron/micronutrient rich foods. Of the various strategies recommended by the World Health Organisation/Food Agriculture Organisation (2006) guidelines to combat micronutrient deficiencies, food fortification has been considered as one of the most efficacious and economical approach, so much so that fortification remains the only cost-effective strategy to address malnutrition to a mass level and among nutritionally challenged communities around the world.

WHO has defined Fortification as “the practice of deliberately increasing the content of one or more micronutrients (i.e., vitamins and minerals) in a food or condiment to improve the nutritional quality of the food supply and provide a public health benefit with minimal risk to health”. Accordingly, research around the world is primarily focused on the methodologies for fortification of food with nutrients which are not readily available for absorption in the body. Fortification strategies for combating iron deficiency anaemia entail the supplementation of food with iron rich ingredients wherein iron and folic acid are readily absorbed in the body. Iron is available from animal and plant sources in the form of heme and non-heme iron. The body absorbs and utilizes heme iron more than non-heme iron due to the presence of certain dietary components in the plant sources. Compounds like citric, ascorbic, tartaric, and malic acids in plants enhance iron absorption, while oxalates, calcium, phytates, and certain polyphenols reduce it. Iron inhibitors bind the ferric form of iron and make it unavailable for the conversion to ferrous form and thereby prevent its absorption, whereas iron enhancers counteract this binding process and accelerate the absorption in the body.

Among the plant-based sources, beetroot (Beta vulgaris), has gained attention for its rich nutritional profile, including iron, ascorbic acid, folic acid, nitrates, saponins, triterpenes, carotenoids, alkaloids, polyphenols, and flavonoids. Studies have shown that beetroot supplementation in food can improve iron deficiency anemia and enhance the oxygen-carrying ability of erythrocytes, increasing folate levels and controlling birth defects, and relaxing smooth muscles. Additional benefits of beetroot include its antioxidant activity, anti-inflammatory properties, action toward carcinogens, and protective effects on the liver and kidney. Due to these nutraceutical benefits, it has been incorporated into versatile products including cereal-based products, milk and milk products, meat and meat products, and films and coating. It is used in various forms such as juice, powder, pomace or pomace powder, extract or extract powder, residue, and peel, and processed using different technologies while maintaining the sensory quality of products. Processing of beetroots such as steaming, pressure-cooking, pressure-steaming, steeping, steaming, boiling, and drying etc. not only enhances sensory characteristics but also boosts certain nutritional compounds like phenols and antioxidant value while reducing antinutritional factors like the oxalate content.

To make most of the iron content of beetroot accessible to the consumer, it is necessary to counter the antinutrients like oxalates, tannin, phytates, and certain polyphenols which limit the iron availability. To achieve this objective, the processing methods and forms of beetroot are subjected to the optimization process to maximize nutrient bioavailability in food.

Globally millets constitute an important staple crop, ensuring food and nutritional security since its grains are a good source of protein, micronutrients and phytochemicals. While sorghum and most of the millets contains about 10% protein, 3.5% lipids, finger millet contains 12-16% protein and 2-5% lipids. Little millet, a category of millets, is an important climate resilient crop and rich in nutrients similar to major cereal crops, providing 314 Kcal of energy, 10.13 g of protein, 65.55 g of carbohydrates, and 7.72 mg of crude fibre per 100 g. In addition, it is a rich source of micronutrients, particularly iron and zinc, thus making them an ideal choice in fortification strategies to combat iron and zinc deficiency. However, the bioavailability of iron in millets is influenced by the presence of non-synergistic nutrients, which is less readily absorbed compared to heme iron found in animal productsFortification strategies involving combination of millets with of food with other food items to create a food synergy facilitating the availability of nutrients in readily accessible form has been the focus of diverse approaches to combat major nutrient deficiencies around the globe.

In one such effort, a Chinese patent application no. CN-113598377-A titled “Composition for improving anemia, functional food thereof, preparation method and application” discloses a composition, based on Chinese traditional medicine, for improving anemia, and the method of preparing the composition. The composition includes bovine bone marrow polypeptide powder, red date powder, bovine blood protein polypeptide powder, medlar powder and fructo-oligosaccharide. The components of the formulation act synergistically to improve anaemia. However, the method does not assess the effect of iron inhibitors present in the composition and does not validate the ingredients and method for iron absorption. Moreover, this formulation is restricted to a vegan diet.

Another patent application no. EP-2420243-A1 titled “Compositions obtainable from red beetroot juice to promote iron absorption and blood forming” discloses a composition obtained by extraction of a complex of biologically active substances having molecular weight up to 10000 Da from the red beetroot (Beta vulgaris) juice, supplemented with ascorbic acid and citric acid in a synergistic approach. The method focused on enhancing iron absorption by supplying beetroot juice itself along with addition of ascorbic and citric acid without testing for antinutritional factors which may inhibit iron absorption.

Patent application no. WO-2020136690-A1 titled “A Composition for Treating Anemia” discloses a pharmaceutical composition designed to treat anaemia by enhancing intestinal iron absorption in mammals, comprising dry fruit of a plant from Malvaceae family, and an acid. Since it is a pharmaceutical composition, it primarily acts as an iron supplement which may have mild side-effects including nausea, vomiting, constipation, etc. upon consumption along with reduced acceptability among society.

Existing fortification strategies are majorly focused on staple foods and the mass population. There is a lack of fortification strategy on a market basis. Current research on iron fortification to treat anaemia utilizes a combination of ingredients to enhance absorption of iron and micro- nutrients in the body. However, none of these techniques validate the level of absorption of individual ingredients and fail to assess the effect of iron inhibitors and iron enhancers present in the ingredients which may interfere with iron absorption.

OBJECT OF THE INVENTION
In order to obviate the drawbacks of the existing state of the art, the present invention discloses an instant iron fortified beetroot-millet soup formulation for the management of iron deficiency anaemia.

The main object of the present invention is to provide an instant, healthy, ready-to-cook soup formulation with iron fortification, prepared by combining beetroot and millets through integration of fortification strategy with various processing technologies, for the management of iron deficiency anemia (IDA).

Another object of the invention is to provide an instant, iron fortified beetroot millet soup powder, formulated to preserve the original nutrients.

Another object of the invention is to provide a method for preparing an instant iron fortified beetroot-millet soup formulation with optimized processing methods and form of beetroot based on nutrient retention, antinutritional factors and nutraceutical properties.

Yet another object of the invention is to optimize the processing of millet malt to be used in the iron fortified beetroot millet soup, based on its functional properties.

Yet another object of the invention is to undertake a comparative assessment of iron bioavailability and iron bio accessibility of the developed iron fortified beetroot-millet soup powder with the formulation prescribed in Ayurveda (ancient medicinal practices) for the management of anemia.

Another object of the invention is to provide an instant iron fortified beetroot-millet soup formulation devoid of synthetic additives and possessing enhanced iron absorption value.

Yet another object of the invention is to provide an instant iron fortified beetroot-millet soup powder which is made available in an attractive package to enhance its shelf life.

SUMMARY OF THE INVENTION
The present invention discloses an iron fortified beetroot-millet soup formulation comprising beetroot and little millets, both having good nutraceutical property and acting synergistically to enhance the iron absorption in the formulation and making the iron readily accessible to the consumer. The formulation is intended for use as fortified food in treatment of Iron Deficiency Anaemia (IDA). The invention also discloses the method of preparing the said soup formulation. The unique feature of the invention is that the process for preparing the soup formulation is validated at each step based on the synergistic approach towards iron absorption, to overcome a major lacuna in the current fortification strategies.

The primary ingredients in the soup formulation comprise beetroot and little millet, which is rich in nutrients and antioxidants. Beetroot variety locally available in the market and found to be potent in nutraceutical properties, is selected as the fortification food source. Different forms of beetroot such as juice, residue, and juice with residue, intended for use in the formulation were subject to nutrient analyses and the best form selected based on the interaction of nutrients on iron absorption. Different processing methods, namely Pressure processing, Steaming and Open Pan processing were tested based on their effect on bioactive compounds, nutrients and antinutrients present in beetroot. The best form and processing method of beetroot was selected based on the criteria of nutrient retention, nutraceutical properties, and reduced antinutrients. Thereafter, little millet in the form of malt was added to the beetroot as a thickener for soup formulation. The processing of little millet malt was optimized based on its functional properties. The enhanced functional properties of malt processing ensure uniform, stable, smoother, bioavailable, and digestible products with improved shelf-life and stability. This optimization is useful for product formulation by substituting little millet malt as starch additives which makes the little millet malt highly soluble and digestible and suitable for instant foods, infant nutrition, or hydrolysis-based applications.

Finally, the prepared beetroot-millet soup was mixed with spices and optimized for iron fortification. The formulation was then processed into an instant, ready-to-cook form through different dehydration technologies. The efficacy of each of these technologies was tested for their effect on nutrient retention in the final formulation. Of all the methods, freeze drying was considered to be the most suitable drying method for preparing the soup formulation into a ready-to-cook powder form by preserving the nutrients in their original state as well as by improving the organoleptic properties of the final product. The instant fortified beetroot-millet soup powder formulation was then subject to iron bio accessibility and bioavailability assay. The prepared formulation was packaged into attractive packages to enhance its shelf life.

Additionally, the iron fortified beetroot-millet soup powder formulation was compared with an Ayurveda formulation. As per the guidelines of ‘Ayush Aahar’- food prepared with the recipes, ingredients and processes according to the method described in the authoritative books of Ayurveda listed under ‘Schedule A’. The developed iron-rich formulation is instant, easy-to-prepare, ready-to-cook, and timesaving, with nutraceutical properties that caters to the healthy food choices of the consumers.

BRIEF DESCRIPTION OF DRAWINGS:
Fig. 1: depicts the flow chart of the iron fortified beetroot millet soup preparation and standardization
Fig. 2a: depicts the Mean value of the effect of thermal processing methods on
DPPH, ABTS, H2O2, TPC, and TFC in beetroot juice with residue variant
Fig. 2b: depicts the Standard deviation of the effect of thermal processing methods
on DPPH, ABTS, H2O2, TPC, and TFC in beetroot juice with residue variant
Fig. 3a: depicts the Mean value of the effect of thermal processing methods on
DPPH, ABTS, H2O2, TPC, and TFC in beetroot juice variant
Fig. 3b: depicts the Standard deviation of the effect of thermal processing methods
on DPPH, ABTS, H2O2, TPC, and TFC in beetroot juice variant
Fig. 4a: depicts the Mean value of the effect of thermal processing methods on
DPPH, ABTS, H2O2, TPC, and TFC in beetroot residue variant
Fig. 4b: depicts the Standard deviation of the effect of thermal processing methods
on DPPH, ABTS, H2O2, TPC, and TFC in beetroot residue variant
Fig. 5: depicts the Effect of processing on sensory parameters of different beetroot variants
Fig. 6: depicts the Optimization of processing methods and beetroot forms based
on iron absorption
Fig. 7: depicts the flow chart of processing of little millets to malt and assessment
of the functional properties
Fig. 8: depicts the effect of dehydration methods on antioxidant activity
Fig. 9: depicts the effect of dehydration methods on nutraceutical properties

DETAILED DESCRIPTION OF THE INVENTION:
The present invention discloses an iron fortified beetroot-millet soup formulation and its method of preparation. The iron fortified soup carries the nutritional contents of beetroot and little millets both having good nutraceutical property which act synergistically to enhance the iron absorption in the formulation and makes the iron contents of beetroot and millets readily accessible to the consumer. The steps from preparation to packaging of iron fortified beetroot-millet soup includes the phases of: processing of beetroot and millets; mixing the contents in optimum amounts and preparing the soup; optimizing the nutritional quality of the fortified soup formulation; processing of the soup formulation in powder form and packaging of the finished product. A flow chart of the soup preparation and standardization has been depicted in Fig. 1.

PREPARATION OF BEETROOT:
Selection and processing of beetroot:
The dark purplish-red colored beetroot (Ooty-1 variety) was procured from the local market in Coimbatore, India. After primary cleaning, the skin was peeled and sliced into 4 mm-thick uniform slices and separated into four equal beetroot sample portions, as described below, to optimize the best form of beetroot for use in the soup formulation:
(a) Raw form: The beetroot samples were taken as raw and ground with water (beetroot: water ratio of 1:0.5). It is then divided into three variants, such as Raw Juice (RJ), Raw Residue (RR), and Raw Juice with Residue (RJ+R).
(b) Pressure-cooking: Each variant was pressure-cooked for 6±2 minutes at 120±5 °C (beetroot: water ratio of 1:0.5). The pressure-cooked samples were processed using a domestic juice extractor, and separated into Pressure-cooked Juice (PJ), Pressure-cooked Residue (PR), and Pressure-cooked Juice with Residue (PJ+R) after grinding with water used for pressure cooking.
(c) Steaming: Beetroot samples were steamed for 20±5 minutes at 100±5 °C (beetroot: water ratio of 1:10) using a stainless-steel steamer. Steamed beetroot was then ground with 50 ml of water used for steaming and then separated into Steamed Juice (SJ), Steamed Residue (SR), and Steamed Juice with Residue (SJ+R).
(d) Open pan cooking: Beetroot samples were subjected to open pan-cooking for 45±5 minutes at 100±5 °C (beetroot: water ratio of 1:4). After grinding with the cooked water, the samples were separated into Open pan-cooked Juice (OJ), Open pan-cooked Residue (OR), and Open pan-cooked Juice with Residue (OJ+R).
The juice and residue separation from each processing method were filtered using a muslin cloth of grade 90. Double-distilled water (DDW) was used for every processing, including cleaning and cooking.

Determination of antioxidant activity:
The antioxidant activity was determined by 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and hydrogen peroxide (H2O2) methods and expressed as IC50 value. For this, 1 mg/ mL stock solution of the sample extract was prepared in methanolic solution, and different concentrations of 10, 20, 40, 60, 80, and 100 µg/ mL were prepared for antioxidant activity determination.

For the DPPH assay, 3 mL of 0.1 mM DPPH (Sisco research laboratories Pvt. Ltd.) prepared in methanol was added to 2 mL of each sample extract from different concentrations. The absorbance was recorded at 517 nm in the Double beam UV-Visible spectrophotometer (LABMAN-LMSP-UV1900) after 30 minutes of incubation in the dark.

For the determination of H2O2 radical scavenging activity, 2 mM H2O2 (Spectrum reagents and chemicals Pvt. Ltd.) solution was prepared by adding an equal volume of 7.4 pH 0.2 M phosphate buffer. The buffer was made with 50 mL of 0.2 M potassium dihydrogen phosphate and 39.1 mL of sodium hydroxide solution (0.2 M). 600 µL of H2O2 was added to 100 µL of each sample concentration and absorbance measured at 230 nm after 30 min of incubation in the dark at room temperature.

ABTS assay was performed by adding 192 mg of it to 50 mL of DDW to prepare 7 mM of ABTS and mixing it with an equal volume of 2.45 µM potassium persulphate and keeping in dark for 12-16 h to produce radical cation. The absorbance value of the ABTS solution was modified to 0.700±0.02 at 734 nm by diluting with DDW. 3 mL of ABTS solution was added to 100 µL of different sample concentrations and absorbance recorded at 734 nm after 15 min using a UV-Visible spectrophotometer.

Determination of total phenolic content:
Total phenolic content (TPC) was estimated by the Folin-Ciocalteu (F-C) method. 1 mL of the sample was taken from the 1 mg/ mL stock solution, diluted with 10 mL of DDW, and 1.5 mL of F-C reagent (Sisco research laboratories Pvt. Ltd.) added. 4 mL of 20% Na2CO3 was added to the mixture after 5 minutes of incubation at room temperature. The solution was made up to 25 mL, and the absorbance was read at 765 nm after 30 min incubation in the dark. A calibration curve was prepared using a standard solution of gallic acid (R2= 0.99). The result was expressed as mg of Gallic Acid Equivalent/100 g of sample (mg GAE/100 g).

Determination of total flavonoid content (TFC):
In TFC, 0.5 mL of methanolic extract from the stock solution (1 mg/mL) was treated with 1.5 mL of 95% methanol and 0.1 mL of 10% aluminum trichloride, 1 M potassium acetate, and 2.8 mL of DDW were added. The solution was mixed well and incubated for 30 minutes in the dark. The absorbance was read at 415 nm by a double-beam UV-Visible spectrophotometer (LABMAN-LMSP-UV1900). A calibration curve was prepared using a standard solution of quercetin (R2= 0.99). The result was expressed as mg of Quercetin Equivalent/100 g of sample (mg QE/100 g).

Determination of Saponin, nitrate, betanin, and vulgaxanthin:
The determination of saponin was performed using the method using diosgenin as a standard solution. 0.2 mL of the sample was mixed with 0.80 mL of methanol and 0.35 mL of 8% vanillin in ethanol. 1.2 mL of sulfuric acid was added to the mixture and kept at 60 °C for 10 minutes in a water bath (Labtech, LTWBD-1D). The mixture was cooled and absorbance measured at 544 nm in a double-beam UV-Visible spectrophotometer by keeping methanol as blank. For nitrate determination, 10 mL DDW was added to 100 mg sample and incubated at 45 °C for an hour followed by centrifugation at 5,000 rpm for 15 minutes. 0.2 mL supernatant was mixed with 0.8 mL of 5% salicylic acid, which was prepared in concentrated sulfuric acid and the solution was kept for 20 minutes. 19 mL of 2N NaOH was added to the solution slowly to raise the pH above 12, cooled and absorbance was measured at 410 nm.

The determination of betanins and vulgaxanthin was done by adding 0.7 g of sample to 50 mL of phosphate buffer (1.79 g of sodium hydrogen phosphates was mixed with 1.36 g of potassium dihydrogen phosphates and 7.02 g of sodium chloride in DDW, and made up to 1L), mixed for 20 min at 2000 rpm in a vortex shaker and the absorbance of supernatant was read at 476, 538, and 600 nm by keeping buffer as blank. The absorbance value for red dye (B) at 538 nm and mg of betaxanthin per 100 gm of sample was determined from Equation 1 and Equation 2:
EB=1.095×(E538-E600) Equation 1
B=(c×EB)/(1120×a×b) Equation 2

where E538 is the absorbance at 538 nm, E600 is the absorbance at 600 nm, and 1.095 is an absorption coefficient at 538 nm resulting from impurities present, c is the mass of the sample with buffer in mg, 1120 (-) value resulting from the absorbance of a 1% betanin solution measured at 538 nm, a is the sample weight (g) and b is the dry matter content (g/g dm).
The absorbance value for yellow dyes (W) at 476 nm and mg vulgaxanthin /100 g sample was determined by Equation 3 and Equation 4:
EW=(E476-E538 )+(0.667×EB) Equation 3

W=(c×EW)/(750×a×b) Equation 4

where E476 absorbance at 476 nm, E538 absorbance at 538 nm and EB is the absorbance value for red pigments. 750 (-) value resulting from the absorbance of a 1% betanin solution was measured at 476 nm.

Fig. 2a illustrates the impact of various processing methods on antioxidant activity using DPPH, ABTS, and H2O2 assay (IC50 value) among juices with residue variants, while the standard deviation is represented in Fig. 2b. The values are represented by a color gradient from dark to light, indicating the range from lowest to highest. The lowest values showed the highest antioxidant potential. From the figure, it was identified that the PJ+R and SJ+R exhibited better free radical scavenging activity towards DPPH and ABTS. Meanwhile, RJ+R and SJ+R showed better activity towards H2O2. Concerning the juice variant (Fig. 3a and Fig. 3b), pressure and steam-cooking showed significant (p<0.05) activity towards DPPH with an IC50 value of 39.34±0.55 and 38.48±0.35 µg/mL. OJ showed better activity towards ABTS with an IC50 value of 23.29±0.26 µg/mL. Moreover, RJ and PJ exhibited significant (p<0.05) activity towards H2O2 radicals. Compared to juice with residue and juice, the OR variant revealed greater activity towards DPPH, ABTS, and H2O2 free radicals with an IC50 value of 58.30±0.12, 72.52±0.19, and 26.74±0.23 µg/mL as depicted in Fig. 4a and Fig. 4b.

The antioxidant activity towards a free radical varies depending on the potential compounds present in the plant foods, mainly phenols and flavonoids. TPC, TFC, nitrates, and ascorbic acid contribute substantially to the antioxidant activity of beetroots. The results revealed in Fig. 3a and Fig. 3b depict that steam-processed beetroot juice has a high TPC content of 702.81±6.95 mg GAE/100 g of sample compared to other processing methods. Among juice with residue samples, the pressure-cooked sample showed greater phenolic content of about 666.66±6.95 mg GAE/100 g of sample, as depicted in Fig. 2a and Fig. 2b, whereas there was a reduction in phenolics in beetroot residue after processing as depicted in Fig. 4a and Fig. 4b. This indicates that thermal processing enhanced the TPC level compared to the raw variant, except in the residue. Similar trends were observed in TFC except for the OJ+R.

The food matrix, temperature, processing time, and types have been shown to be the major factors influencing the phenolics and flavonoids which exist either in a free state or bound to the cell membrane in plant-based food. Thermal processing breaks down the cellular compounds in fruits and vegetables and releases the bound phenolics and flavonoids. It degrades the enzymes peroxidases and polyphenol oxidases responsible for the oxidation of phenolics. Thermal processing has both positive and negative impacts on the phenolic and flavonoid compounds. It either causes a transformation of certain phenolic compounds or their degradation due to hydrolysis, dimerization, epimerization, oxidation, and polymerization. In addition, thermal treatment weakens the ester linkage of certain phenolics like p-coumaric acid with lignin and hemicellulose and made them available in esterified forms. In the present invention, it was observed that phenolics were available more in juice variant due to the extraction of the bound phenolics and there was a reduction in residue variant due to degradation. The various types of thermal processing enhanced the TFC among different beetroot variants by hydrolyzing the bound form in the cell matrix and transforming glycosides to aglycones.

Similar to TPC and TFC, nitrates too can scavenge reactive oxygen species. Dietary nitrates are the exogenous source for endogenous nitric oxide synthesis, which has potential towards lowering blood pressure, cardiovascular diseases, and oxidative stress. Research studies revealed that beetroots are a remarkable source of dietary nitrates, and the presence of nitrates is one contributing factor to the health benefits of beetroot. Higher amounts of nitrates were observed in the RJ+R sample (200.10±0.10 mg/100 g), as shown in Table 1. However, thermal processing reduced the nitrate content in beetroot juice with residue and the residue variant, whereas it was enhanced in the juice variant after processing due to its solubility. Saponins are another bioactive compound present in plants as a part of their primary protection from pathogens. These compounds possess hypolipidemic activity, antifungal activity, virucidal activity, antimicrobial activity, and hypoglycemic activity. They are considered phytochemical compounds due to their pharmacological activities. Beetroot is a notable source of saponin, and the thermal processing of beetroot was found to have significant (p<0.05) changes in saponin content. In the present invention, an increment of saponin was observed from 12.66-17.33 mg/100 g in juice with residue, 2-15 mg/100 g in juice, and 4-15.33 mg/100 g in residue variant, as shown in Table 1. This might be due to the inclusion of cooked water for the grinding process.

Table 1 Effect of food processing on nutraceutical properties in various forms of beetroot
Different processing methods & forms of
Nutraceutical Properties


beetroot Saponin (mg/100 g) Nitrates (mg/100 g) Betanins (mg/100 g) Vulgaxanthin (mg/100 g)


RJ+R 12.66±0.57de 200.10±0.10a 21.43±0.03e 29.90±0.10d
PJ+R 15.66±0.57ab 157.37±0.17c 17.28±0.02f 23.19±0.03g
SJ+R 17.33± 0.57a 124.62±0.17e 12.41±0.03j 20.76±0.07i
OJ+R 15.66±1.15ab 111.87±0.17g 39.84±0.04b 47.60±0.24b
RJ 2.00±0.01g 50.00±0.10i 15.49±0.02h 23.50±0.04fg
PJ 13.33±0.57cd 171.12±0.17b 13.17±0.09i 18.97±0.11j
SJ 10.66±0.57ef 174.12±0.17b 3.60±0.10l 11.80±0.08k
OJ 15.00±1.00bc 118.50±0.25f 11.59±0.06k 21.80±0.14h
RR 4.00±0.01g 125.60±0.49e 34.79±0.03c 43.03±0.38c
PR 9.00±1.00f 137.12±0.17d 22.47±0.04d 28.64±0.05e
SR 11.66±0.57de 107.87±0.17g 16.11±0.10g 23.83±0.14f
OR 15.33±0.57abc 64.50±0.35h 43.11±0.04a 54.37±0.15a
a-l means values with the same letters are not significantly different according to the Tukey test at p < 0.05. Data represents the mean value±standard deviation of three replicates. RJ+R: Raw juice with residue; PJ+R: pressure-cooked juice with residue; SJ+R: Steamed juice with residue; OJ+R: Open pan-cooked juice with residue; RJ: Raw juice; PJ: Pressure-cooked juice; SJ: Steamed juice; OJ: Open pan-cooked juice; RR: Raw residue; PR: Pressure-cooked residue; SR: Steamed residue; OR: Open pan- cooked residue.

Determination of antinutrients:
Phytate was determined by first weighing a sample having 5-30 mg of phytate in the Erlenmeyer flask. The sample was then extracted in 50 mL of 3% TCA for 30 minutes by mechanical shaking, and then for 45 minutes by hand. It was then centrifuged for 10 minutes at 10,000 rpm in a centrifuge (Remi, R-24), and 10 mL of an aliquot was treated with 4 mL ferric chloride. The samples were heated for 45 minutes in a water bath and centrifuged for 10-15 minutes followed by decanting the clear supernatant and washing the precipitate twice by dispersing it with 20-25 mL of 3% TCA. It was then heated for 5-10 minutes and centrifuged again. The solution was filtered and washing repeated with DDW. The precipitate was dispersed in 10 mL of DDW and treated with 1.5 N NaOH, made up to 30 mL volume with DDW and heated in boiling water for 30 minutes followed by filtering and washing the precipitate with hot DDW and discarding the filtrate. The precipitate was dissolved in 40 mL 3.2 N hot nitric acid into a 100 mL volumetric flask and washed with several portions of DDW. The solution was made up to 100 mL with DDW and a 5 mL of aliquot was transferred to another 100 mL volumetric flask, which was then diluted to 70 mL followed by addition of 20 mL of potassium thiocyanate (Sisco Research Laboratories Pvt. Ltd.). The absorbance value was read at 480 nm within 1 minute.

Tannin was estimated by the vanillin-hydrochloride method in which 1 gram of the sample was mixed with 50 mL of methanol and kept for 20-28 h. I mL of the supernatant after centrifugation was mixed with 5 mL of vanillin-hydrochloride reagent, which was made by mixing an equal volume of 8% HCl and 4% vanillin in methanol and absorbance read at 500 nm after 20 minutes. A calibration curve was prepared using a standard solution of tannic acid (R2= 0.98). The result was expressed as mg of Tannic Acid Equivalent/100 g of sample (mg TAE/100 g).

Oxalates were also determined for the samples by the method of Stoleru et al., in which two grams of the powdered sample was mixed with 190 mL of DDW and 10 mL of 6N HCl, and the mixture was digested for an hour at 100 °C. The sample was cooled and diluted to 250 mL with DDW and filtered and to a 50 mL of filtrate, 10 mL of 6N HCl was added which was then placed in a water bath and evaporated till it reached half its volume. The precipitate was filtered and washed again using DDW and made the volume made up to 125 mL. 3-4 drops of methyl indicator was added to the mixture and treated with ammonium hydroxide concentrate till it turned to pale yellow. It was then heated to 90 °C, cooled and filtered. 10 mL of 5% calcium chloride was added to the solution while boiling with constant stirring and kept in the dark for twelve hours and filtered. The precipitate was then washed with hot DDW and dissolved in 30 ml of water and concentrated sulfuric acid mixture (3:1). It was then warmed at 80 °C for 10 minutes, cooled and titrated with 0.5% KMnO4 until the pink color persisted for 1 min. 1 mL KMnO4 is equivalent to 2.24 mg oxalate.

Various processing methods play a vital role in determining the nutritional and antinutritional properties by altering their compositions. Processing, like soaking, cooking, boiling, roasting, steaming, and pressure cooking, has a significant influence on nutritional characteristics. The optimization studies in the present invention focused on the nutritional compounds iron, calcium, ascorbic acid, citric acid, folate, and ß-carotene, which are illustrated in Table 2a and 2b.
Table 2a: Effect of food processing on antinutritional compounds in various forms of beetroot
Different processing methods & forms of beetroot Oxalates (mg/100 g) Tannin (mg tannic acid equivalent/100 g) Calcium (mg/ 100 g)
RJ+R 171.46±13.39a 172.83±2.88h 4.72±0.14a
PJ+R 126.93±12.93bc 116.16±2.88j 2.67±0.08d
SJ+R 126.93±12.93bc 400.5±0.50e 2.02±0.12e
OJ+R 104.53±12.93cde 455±0.50c 2.66±0.17d
RJ 149.33±12.93ab 322.83±2.88f 1.52±0.05f
PJ 126.93±12.93bc 167.83±2.88h 2.71±0.10d
SJ 119.46±12.93bcd 528±0.50a 4.52±0.21ab
OJ 74.66±12.93e 515±0.50b 2.23±0.12e
RR 186.66±12.93a 92.83±2.88k 2.34±0.19de
PR 104.53±12.93cde 137.83±2.88i 3.78±0.01c
SR 104.53±12.93cde 446.5±0.50d 4.34±0.12b
OR 82.13±12.93de 315±0.50g 4.72±0.06a
a-l means values with the same letters are not significantly different according to the Tukey test at p < 0.05. Data represents the mean value±standard deviation of three replicates. RJ+R: Raw juice with residue; PJ+R: pressure-cooked juice with residue; SJ+R: Steamed juice with residue; OJ+R: Open pan-cooked juice with residue; RJ: Raw juice; PJ: Pressure-cooked juice; SJ: Steamed juice; OJ: Open pan-cooked juice; RR: Raw residue; PR: Pressure-cooked residue; SR: Steamed residue; OR: Open pan- cooked residue

Determination of ascorbic acid and citric acid:
Ascorbic acid was determined by the spectrophotometric method. About 0.5-5 g of sample was treated with 25-50 mL of 4% oxalic acid solution, filtered, and a 10 mL aliquot was transferred to a volumetric flask. It was then dehydrogenated using bromine water, and a few drops of 10% thiourea were added to it to remove the excess bromine. The brominated sample was made up to a known volume with 25-50 mL of 4% oxalic acid solution. 1 mL sample was made up to 3 mL using DDW and treated with 1 mL of 2,4-Dinitrophenylhydrazine (Sisco Research Laboratories Pvt. Ltd.). The mixture was mixed well and incubated at 37 °C for 3 hrs. and the orange-red osazone crystal formed was dissolved in 7 mL of 80% sulfuric acid. Absorbance was read at 540 nm using a double-beam UV-visible spectrophotometer (LABMAN-LMSP-UV1900). The absorbance was calculated from the calibration curve (R2=0.99) using ascorbic acid as a standard.
Citric acid estimation was carried out according to the method in which 0.2 g of powdered samples were diluted to 500 mL, and then 10 mL of the diluted sample was titrated against 0.05 N NaOH using phenolphthalein as an indicator till a pink color persisted.

Determination of calcium, iron, folate, and ß-carotene:
Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES), Shimadzu ICPE-9800 series, was used for iron estimation. 0.5-1.0 g of the samples were weighed and dried at 110 °C. It was then ignited in a muffle furnace at 550 °C for 3 hrs. till all the carbon is oxidized. The ash was then made up to 100 ml with dilute nitric acid (2%), followed by preparing a standard calibration using 0.1-10 mg/L of standard in nitric acid (R2=0.99). Atomic Absorption Spectrophotometry (AAS), pinnacle 500 series, was used to determine calcium after dry digestion in a muffle furnace. The sample was ignited in a muffle furnace at 450 °C for 5 h and then heated for 30 minutes with a watch glass by adding 40 mL of diluted HCl (1:1), and then 30 minutes without a watch glass. The sample was cooled and added to 10 mL diluted HCl again, filtered into a 100 mL standard flask, and the volume made up. A blank was prepared without the sample and a working standard of calcium of 5 to 25 µg/mL was prepared for the AAS method.

For the determination of folate, the powdered sample was transferred into a 100 mL volumetric flask, exactly equivalent to a laboratory mixture containing 25 mg of meclizine hydrochloride and 2.5 mg of folic acid. About 60 mL of methanol was added and sonicated for 15 minutes. The solution was filtered through Whatman No.1 filter paper and the residue washed with methanol and absorbance was noted at 540.0 nm against a blank. A calibration curve was prepared with standard folic acid solution (100 µg/mL). The sample was taken in a 150 mL glass stoppered Erlenmeyer flask for the beta-carotene estimation and was treated with water-saturated n-butanol and kept overnight for complete carotene extraction followed by filtering the contents through the Whatman No. 1 filter paper. The optical density of the clear filtrate was measured at 440 nm using a spectrophotometer against water-saturated n-butanol as a blank. A calibration curve was made using the carotenoid standard (5 µg/mL).

All the data was analyzed using Minitab version 17 (trial version) with the nutraceutical, anti-nutritional, and nutritional properties as responses. Each experiment was performed in triplicate, and the results were expressed as mean values ± standard deviation. A one-way analysis of variance was carried out to identify the statistically significant factors at p<0.05, followed by a Tukey pairwise comparison test to compare the treatments. Optimization to identify the formulation with the best nutraceutical and nutritional properties, including iron, was performed using response optimizer functionality in Minitab (trial version). Regression models for various responses with cooking method, presence or absence of juice, and residue as factors, obtained from factorial analysis, were utilized for this. The optimized results were validated by evaluating predicted fit with experimental results, and by statistical evaluation by analysing the standard error fit, 95% of confidence interval, and 95% of predicted interval.

Table 2b: Effect of food processing on synergistic nutrients in various forms of beetroot
Different processing methods & forms of beetroot Synergistic nutrients
Ascorbic acid citric acid (%) Iron Folate ß-carotene (µg/100 g)
(mg/100 g) (mg/100 g) (µg/100 g)
RJ+R 3.43±0.05i 0.43±0.05cd 2.29±0.04a 31.33±0.57d 12.33±0.57f
PJ+R 4.97±0.02g 0.83±0.11a 1.36±0.02f 37.00±1.00ab 19.00±1.00ab
SJ+R 7.76±0.02b 0.42±0.02cd 1.50±0.03e 38.33±0.57a 21.00±1.00a
OJ+R 8.18±0.02a 0.05±0.02f 1.60±0.02d 38.33±0.57a 20.33±0.57ab
RJ 3.33±0.05i 0.51±0.09bc 0.71±0.04h 30.00±1.00d 12.00±1.00f
PJ 5.38±0.02f 0.27±0.04de 0.35±0.04j 34.00±1.00c 15.33±0.57de
SJ 7.01±0.02c 0.42±0.02cd 0.26±0.01k 29.66±0.57d 12.66±0.57f
OJ 6.01±0.02e 0.04±0.02f 0.56±0.01i 34.66±0.57c 16.66±0.57cd
RR 3.93±0.05h 0.25±0.04e 1.93±0.03b 25.66±0.57e 5.00±0.01g
PR 2.01±0.02j 0.60±0.08b 1.06±0.03g 30.33±0.57d 13.33±0.57ef
SR 6.9±0.05c 0.17±0.02e 1.75±0.04c 34.33±0.57c 15.33±0.57de
OR 6.55±0.05d 0.04±0.02f 1.81±0.02c 36.00±1.00bc 18.66±0.57bc
a-l means values with the same letters are not significantly different according to the Tukey test at p < 0.05. Data represents the mean value±standard deviation of three replicates. RJ+R: Raw juice with residue; PJ+R: pressure-cooked juice with residue; SJ+R: Steamed juice with residue; OJ+R: Open pan-cooked juice with residue; RJ: Raw juice; PJ: Pressure-cooked juice; SJ: Steamed juice; OJ: Open pan-cooked juice; RR: Raw residue; PR: Pressure-cooked residue; SR: Steamed residue; OR: Open pan- cooked residue

Iron is an essential micronutrient, which acts as an oxygen carrier for myoglobin synthesis, red blood cell formation, and brain development, and its deficiency is a leading factor for anemia. It was observed that Iron was retained more in open pan processing as compared to other thermal methods, especially in the residue variant, which may be due to the increased release of iron from its complex forms due to the elongated time for cooking. A higher level of iron was observed in RJ+R, around 2.29±0.04 mg/100 g. The processes of pressure cooking, steaming, and open pan processing reduced the iron level by approximately 40%, 34%, and 30%, respectively, compared to RJ+R among juice with residue, whereas there was a reduction in the iron content in RJ by around 68.99% and 15.72% in RR. In the juice and the residue variants, processing reduced 21-63% and 6.2-45% of iron.

Beetroot is a good source of calcium, and there was a substantial difference in calcium content across various beetroot variants after processing, as indicated in Table 2a. The juice with residue variants showed a detrimental effect on calcium after processing, whereas, it was increased in the juice variant and the residue variant, and a higher amount was observed in the residue variant after processing. Food processing significantly (p<0.05) increased the level of folate and ß-carotene in various beetroot variants. The concentration of both nutrients was higher in juice with the residue variant, around 37-38.33 µg folate/100 g and 19-21 µg ß-carotene/100 g after processing due to release from the bound form and making it more extractable.

Ascorbic and citric acids are the organic acids that have synergistic effects on iron absorption in the body. However, ascorbic acid is sensitive to temperature and oxygen, leading to its degradation during heating. The optimization studies of the present invention found an increase in ascorbic acid due to thermal processing, as indicated in Table 2b. Ascorbic acid levels in steamed and open pan-cooked beetroot were observed to be around 6.9-7.76 and 6.01-8.18 mg/100g respectively, while pressure-cooked beetroot contained 2.01-5.38 mg/100g. Furthermore, there was a significant variation (p<0.05) in different variants of beetroot. It was higher in the juice with residue and the residue variants compared to the juice variant. Heat, exposure to air, and leaching into water are the major causes of ascorbic acid loss. The inclusion of cooked water for grinding and less exposure to air might be the reasons for better ascorbic acid retention. Greater citric acid degradation was observed in the studies, mainly in the open pan processing. PJ+R found to preserve citric acid around 0.83±0.11% as compared to other processing methods. From these observations, it was evident that the food processing and various variants have a significant influence (p<0.05) on organic acids.

Similar to the nutritional compounds, antinutrients are naturally present in plant foods and can interfere with the absorption of essential nutrients in the body. The presence of antinutrients, oxalates, tannins, and phytates have been observed to exhibit an inhibitory effect on minerals, including iron. The phytate concentration in beetroot is observed below the non-detectable limits, whereas the presence of tannin and oxalates was observed in beetroot, as represented in Table 2. Tannins are polyphenolic compounds found in plant foods and act as antinutrients. Tannin level was enhanced after steaming and the open pan method, whereas there was a reduction in pressure-cooked one. A significant difference (p<0.05) was observed in tannin levels among different processing methods and beetroot variants. A higher tannin content was observed in the juice variant, particularly in SJ, around 528 mg TAE/100 g. Oxalates are available in plant foods and are abundant in green leafy vegetables. They form insoluble complexes with calcium and magnesium and soluble complexes with sodium and potassium thus making them unavailable for absorption in the body. Beetroot is considered to be a good source of oxalates; hence, it is required to reduce them through processing. Accordingly, it was observed that processing reduced the oxalate content in different beetroot variants, it was higher in the raw residue variant, around 186.66±12.93 mg/100 g, however, there was an almost 39% reduction in juice with residue, a 50% reduction in juice variant, and a 56% reduction in residue variant.

Determination of color:
The color of the samples was analyzed by a portable color meter NH310 (Shenzen Three NH Technology Co Ltd) using CIE lab coordinates, and was expressed as L*, a*, and b* values.

A higher L* represents more lighter color (L=100 indicates light and L=0 indicates dark). In a*, positive values move toward the red coordinates and negative values toward the green coordinates. In case of b*, positive values move toward the yellow coordinates and negative values toward the blue coordinates. The L*, a*, and b* values of RJ+R were 0.01±0.0, and a similar result was obtained for SJ+R, RJ, and SJ. Whereas RR showed 12.54± 0.02, 10.03± 0.02, and 0.43± 0.01as L*, a*, and b* values respectively. The L* represents the lightness, and it was reduced in the order of OJ+R>SR>RR>PJ+R> PJ>OR>OJ with values 15.90± 0.01, 15.58± 0.02, 12.54± 0.02, 12.26± 0.01, 11.83± 0.04, 9.15± 0.02, and 6.88± 0.01 respectively. The a* represents the red/ green coordinates, and it was higher in the order of OJ>OR>PJ>PJ+R>RR>SR>OJ+R with values 17.24± 0.01, 14.73± 0.01, 10.55± 0.02, 10.35±0.03, 10.03± 0.02, 9.46± 0.01, and 8.46± 0.01 respectively. The b* indicates the yellow/ blue coordinates, and it was decreased in the order of OJ (4.30± 0.01)>OR (2.85± 0.02)>SR (2.25± 0.01)>OJ+R (1.21± 0.01)>PJ (0.46± 0.01)>RR (0.43± 0.01)=PR (0.42± 0.02) =PJ+R (0.40±0.01).

The steam-cooked juice with residue and juice showed similar results to raw variants, and lower lightness indicated that it was extremely dark in color, with other coordinates being nearer to zero. The pressure-cooked and open-pan methods showed better results of dark color with red coordinates. Among them, OJ, OR, PJ, PJ+R, and PR resulted in dark, strong red color along with a slight yellow color. Considering this data, thermal processing showed a positive effect on the physical characteristics and color of different beetroot variants.

The characteristic dark red color of beetroot is due to the presence of compound betalains, a nitrogen-containing water-soluble pigment, the red-violet color is attributed to betacyanin, while the yellow orange is exhibited by betaxanthin. Betalains possess remarkable therapeutic potential, anti-tumor properties, anti-carcinogenic properties, hepato-protective, and regulate vascular homeostasis, however, they are less stable when exposed to thermal treatment and processing time, and the presence of oxygen and enzymes degrades the compound. This might be due to the decarboxylation, isomerization, or epimerization reactions. The highest concentration of these pigments was found in the OR, with values of approximately 43.11±0.04 and 54.37±0.15 mg/100 g, respectively. Thus, these pigments were found to be better retained in the residue compared to the juice with the residue and juice variants.

Evaluation of sensory parameters:
All samples (RJ+R, PJ+R, SJ+R, OJ+R, RJ, PJ, SJ, OJ, RR, PR, SR, and OR) were sensory evaluated by a panel of 30 semi-trained members from the Food Science background from whom volunteer consent was collected with the concerned department's ethical committee. The test used for evaluation was a 9-point Hedonic Scale (1-Dislike extremely to 9- like extremely) for appearance, flavor, texture, and overall acceptability. Each panelist was presented with a maximum of four samples a day, and samples were randomly assigned along with code numbers.

The sensory scores attained from different beetroot processing and variants ranged between 3±2.45- 7.6±0.55, as indicated in Fig. 5, since most of the variants and processing were disliked by the panel members. Sensory parameters have a significant role in determining consumer acceptance. The ANOVA results revealed that there are no significant (p<0.05) differences among the different processing and variants. Even though Fig. 5 represents that panel members disliked the residue and the juice with residue variants, they were acceptable of the raw juice variant. Among the juice variants, they accepted RJ and PJ, and mostly beetroot is supplemented in the form of raw juice. Although RJ and PJ were sensory-wise more acceptable, they showed a low profile of iron by around 0.71±0.04 and 0.35±0.04 mg/100 g. This suggests that the conversion of beetroot into value-added products can be done to improve its sensory acceptability.

Iron bioavailability determination:
The iron bioavailability of a sample has a significant role in the body as it defines how much iron is absorbed and reaches the targeted tissue to accomplish its bioactivity. Bio accessibility measures the amount of iron released from the food matrix for absorption. Iron bioavailability of the optimized sample was determined by the INFOGEST method proposed. The sample was exposed to oral, gastric, and intestinal phases for digestion. In the oral phase, 5 g of sample was mixed with simulated salivary fluids at a ratio of 1:1 (w/w) with salivary amylase at 37 °C, pH 7 for 2 minutes. The oral bolus was then treated with simulated gastric fluids (1:1) and enzymes, pepsin, and lipase at pH 3 for 2 h. The gastric chyme was then incubated for 2 h at pH 7 by mixing with simulated intestinal fluids, bile salts, and pancreatic enzymes. The sample after intestinal digestion was subjected to the Human colon carcinoma (Caco-2) cells, which were obtained from the National Centre for Cell Sciences. It was then suspended in 1 mL of culture media containing the filtered sample and incubated in an atmosphere of 5% CO2 and 95% air at 37 °C for 24 hrs. The cells were removed from the surface of the plate using trypsin and centrifuged at 2000 rpm for 2 minutes at 4 °C. It was then rinsed with 1 mL of ice-cold phosphate-buffered saline (pH 7.2) after eliminating the supernatant and centrifuged again at 1000 rpm for 1 minute at 4 °C and the supernatant removed. The cells were treated with 1 mL of cytosol buffer (pH 7.5), and the solution was sonicated in a probe sonicator and centrifuged at 14000 rpm for 25 minutes at 4 °C. The supernatant from the centrifugation was used to determine iron content by Inductively Coupled Plasma Mass Spectrometry (ICPMS). The sample (2 mL) was treated with conc. HNO3, HCl, and a few drops of H2O2. It was then digested for 1 h at 180 °C in a microwave digestion system and then diluted and subjected to ICPMS. Iron bioavailability in the optimized sample, PJ+R showed an approximately 4.37±0.05% bioavailability in Caco-2 cells.

The optimization studies revealed favorable results with respect to the nutritional parameters of beetroot for incorporation in the soup formulation, as indicated in Fig. 6. The process of steaming was found to be more advantageous in retaining nutraceutical compounds, antioxidant activity, total phenols, flavonoids, and nitrates. Pressure-cooking was found to reduce anti-nutrients and retain other nutritional compounds like folic acid, ß-carotene, ascorbic and citric acid. The juice with residue variant of beetroot yields maximum benefits under processing, whereas iron was retained more in the residue variant. The above results highlight the significance of strong evidence-based scientific data for the best processing methods and variants. The final optimized results were focused on iron absorption factors in the body. It revealed that pressure-cooked juice with residue variant is effective in maximizing nutritional and bioactive profiles and reducing anti-nutritional properties. It reduced key inhibitory compounds, oxalates, calcium, and tannin by approximately 42-26%, while enriching nutritional compounds like saponin, ascorbic acid, citric acid, folic acid, and ß-carotene, with an average increase of 67%. Despite reductions in nutritional compounds such as nitrates, betanin, and vulgaxanthin, including iron, the overall nutritional improvements make PJ+R a highly favorable option for maximizing beneficial compounds and promoting health benefits, followed by PJ+R, SJ+R, and OJ+R which were found to be better alternative processes and variants based on nutritional significance. Whereas the sensory-based analysis showed that people preferred the raw and pressure-cooked juice variants, which are lower in iron content.
Since the optimized result (PJ+R) showed a lower sensory acceptability and was found to have lower bioavailability, it was decided to develop a iron fortified beetroot millet soup formulation to enhance the bioavailability and consumer acceptability.

OPTIMIZATION OF MILLETS:
Selection and preparation of raw material:
Whole little millet grains of ATL-1 variety was procured from Tamil Nadu Agricultural University, India. The germination process was conducted followed by drying at 50±1°C for 4-5 h. For the malting, millet was ground with water at a ratio of 1:2 after removing the rootlets and shoots. The malt was then processed at different times and with different water content.

Estimation of nutraceutical properties:
The nutraceutical properties of little millets were assessed by their DPPH, ABTS and H2O2, TPC. TFC, and saponin values.
Determination of nutritional and antinutritional properties:
The little millets were tested for Ascorbic and citric acid, Folate, ß-carotene, iron, calcium, and vitamin B12 and Phytates, oxalates and tannin contents.

The germinated malt exhibited greater antioxidant activity around 49.44- 74.36 µg/mL in IC50 assays, compared to whole grain. The whole little millet grain showed an IC50 of 123.54-232.77 µg/mL. However, TPC and TFC showed a significant decrease after malting. The TPC of whole little millet and malt was 818.59±1.18 and 680.080±6.12 mg GAE/100 g. The TFC of whole little millet grain and malt was 618.08±4.30 and 341.529±4.73 mg QE/100 g. The malting reduced the TPC by approximately 20% and the TFC by 80%. It was observed (that though the ascorbic content was negligible in whole little millet grain whereas, it was present in the malt around 1.866 ±0.15 mg/ 100g. The citric acid showed an increment of about 81% post germination and malting. A similar trend was observed in minerals iron and calcium, wherein malting enhanced iron and calcium from 1.00-1.02 ±0.01 mg/ 100 g and 0.05 - 1.42 ±0.01 mg/100 g respectively. However, post germination, there was a detrimental effect on folate, ß-carotene, and vitamin B12. The folate, ß-carotene, and vitamin B12 in germinated malt were 11.33±1.52, 38.33±2.08, and 1.33±0.57 µg/ 100 g, respectively, in whole grain it was 33.33±1.52, 76.00±2.00, and 7.33±0.57 µg/ 100 g, respectively. This might be due to the release of water-soluble compounds in the water during soaking. There was a reduction in phytates from 76.66±2.88 to 41.66±2.88 mg/ 100g of sample after malting. The same trend was observed in tannin, which was reduced from 26.16±2.88 to 21.16±2.88 mg TAE/100 g. Oxalic acid has been shown to have impact on minerals by forming complexes with calcium, iron, magnesium, ammonium, and sodium ions, making them unavailable. Oxalates showed an impact similar to phytate after malting, it reduced from 4.213±0.06 to 1.486±0.12 mg/ 100 g. Reducing these compounds is believed to enhance miner bioavailability, protein digestibility, palatability, and functional properties which positively impact final product formulation.

Determination of physical and functional properties:
(a) Swelling Capacity (SC)
Known sample of 0.1 g was mixed with 10 ml of DW and kept at 60±2 °C for 30 min with continuous stirring followed by centrifugation at 1600 rpm for 15 min and calculated by Eq. (1).
SC= (Weight of precipitate)/(Weight of sample) (1)
(b) Solubility Index (SI)
0.5 g of the sample was treated with 10 ml of DW and kept at 60±2 °C for 30 min in a water bath without stirring followed by centrifugation at 1600 rpm for 10 min and collecting 5 ml of the supernatant. Contents were dried and weighed and SI was calculated using Eq. (2)(3).
SI=(Weight of soluble starch)/(Weight of dried sample) (2)

% SI=SI×100 (3)
(c)Water Absorption Capacity (WAC)
1gm sample was mixed with 10 ml of DW and kept at 30±2 °C for 30 min followed by centrifugation at 3000 rpm for 15 min and calculated by Eq. (4).
WAC= (Weight of flour bound to the water (g))/(Weight of sample (g)) (4)
(c) Dispersibility
10 gm sample was mixed with 100 ml of DW and collected in a 100 ml measuring cylinder. The contents were then kept for 3h.
Dispersibility (%) = 100-settled particle (ml) (5)
(e)Viscosity
10% of the sample was kept at 30±2 °C for 30 min to determine cold paste viscosity. The same amount of the sample was treated at 95±2 °C for 30 min to determine cooked paste viscosity at 100 rpm with a spindle number LV 62 using a Brookfield viscometer.
(f) Freeze-thaw stability
A sample of 3% (w/v) concentration was heated in a boiling water bath for 30 minutes and cooled to RT. The paste was then weighed and kept at -20 to -15 °C for 24 h. The sample was weighed and centrifuged, and the upper layer was discarded. The freeze-thaw stability is expressed in the percentage of separated water.
(g) Sediment volume (SV)
2%, w/v sample was heated in a boiling water bath for 30 minutes and left to cool in a 100 ml stoppered measuring cylinder for 24 h. The SV was expressed in ml/ 100 ml.
(h) Paste clarity
0.5% of the sample was heated in a boiling water bath for 30 minutes with continuous stirring followed by cooling and noting the transmittance at 650 nm using DW as blank in a UV-visible spectrophotometer (LABMAN-LMSP-UV1900).

All the functional properties were assessed by a central composite design with alpha value 1 (face-centered) and various combinations were obtained by 2 factors and 2 levels of factorial designs. The cooking time (5 and 15 minutes) and water content (1 and 2 times) were two independent variables, and functional properties were the response variables. A total of 13 runs were carried out with five replications of center points. All the data was analyzed using Minitab 17 and Design-expert 13 (trial versions). Each experiment was performed in triplicates, and the results were reported as mean values ± standard deviation. The Mann-Whitney U test, a non-parametric method was performed to determine significance among whole grain and malt due to the violation of normality assumption. P value at a 90 % level of significance is considered as significant. The Kruskal-Walli’s test was performed, followed by post-hoc analysis using Dunn’s test to determine the significance of time and water content in each response at p< 0.05.

It was observed that the SI increased from 3.98 to 18.41% after malting and there was an increment in the same after processing and was higher in 15 minutes of cooking similar amount of water. These results indicate the changes in starch molecules while soaking, germinating, malting, and processing. The dispersibility also showed a similar trend as water solubility, it increased by around 23% after malting, whereas there were variations in dispersibility after processing. The 15 minutes of cooking with similar amounts of water showed better dispersibility. The cooked and cold paste viscosity determines the ability of food material when it behaves during cooking conditions and after cooling. The cooked and cold paste viscosity in the present study showed a similar trend as dispersibility. It was reduced after processing, which might be due to the degradation of amylopectin and the presence of more amylose while heating. Cooking for 5 and 15 minutes with similar amount of water were found to yield better cooked and cold paste viscosity. Freeze-thaw stability was expressed in terms of separated water percentage; the lower separated water directly means higher freeze-thaw stability and the retrogradation tendency should be lower. No significant change was observed in freeze-thaw stability after malting. Whereas there was a significant difference while processing malt, the percentage of separated water was increased while increasing the water content as expressed in Table 3 The higher freeze-thaw stability was observed in the malt after 10 minutes of cooking with 1 time of water content.
Table 3: Optimization of little millet malt processing based on the functional properties
Independent variables Response variables
Time A (minutes) Water content B (times) Swelling capacity (g/ g) Solubility index (%) Water absorption capacity (g/g) Dispersibility (%) Cooked paste viscosity (cP) Cold paste viscosity (cP) Freeze thaw stability Paste clarity (% of transmittance) Sediment volume (ml/ 100 ml)
10 1 5.51±0.41bc 55.44±14.27ab 6.80±0.40ab 62.00±2.00ac 20.58±0.32ab 10.42±0.18ab 0.30±0.01bd 1.75±0.01ac 9.66±0.58bd
5 3 7.54±0.24ac 22.48±4.68ac 5.37±0.33ac 23.33±2.31bd 12.20±0.17ac 6.70±0.28ac 0.70±0.02ac 2.46±0.01ab 47.00±1.00ac
10 2 2.06±0.27bd 31.87±4.03ac 3.86±0.02ac 60.33±0.58ac 12.80±0.10ac 3.03±0.25ac 0.45±0.03bd 2.53±0.01ab 21.33±0.57bd
10 2 5.80±0.52bd 22.57±2.33ac 4.52±0.14ac 38.66±1.15ac 12.60±0.10ac 4.50±0.40ac 0.46±0.01bd 2.57±0.01ab 16.33±0.57bd
5 1 9.02±0.05ac 48.74±2.19ab 7.86±0.03ab 40.00±2.00bc 25.60±0.32ab 17.10±0.30ab 0.47±0.01ad 1.87±0.02ac 22.33±0.57ad
10 2 4.24±0.27bd 43.27±3.89ac 4.06±0.08ac 33.33±2.31ac 11.56±0.35ac 5.52±0.41ac 0.32±0.10bd 2.46±0.01ab 13.66±1.53bd
15 2 6.00±0.30ad 47.39±11.98ac 5.19±0.55ac 66.66±2.51ac 17.00±1.00ac 3.80±0.17ac 0.55±0.01ad 2.34±0.02ab 38.00±1.00ad
15 1 9.01±0.05ac 59.05±3.88ab 7.46±0.61ab 71.33±1.15ac 27.00±0.92ab 15.85±0.08ab 0.48±0.01ad 1.99±0.01ac 26.66±0.58ad
10 2 2.92±0.18bd 35.85±3.91ac 3.07±0.02ac 31.33±1.15ac 10.20±0.30ac 3.40±0.17ac 0.47±0.02bd 2.15±0.03ab 11.00±1.00bd
10 2 2.27±0.14bd 40.65±6.04ac 3.06±0.08ac 57.33±1.15ac 10.60±0.33ac 2.30±0.17ac 0.36±0.02bd 2.41±0.02ab 11.33±0.57bd
10 3 5.17±0.63bc 22.00±2.27ac 3.25±0.04ac 37.00±1.00ad 12.00±0.46ac 2.14±0.05ac 0.70±0.01bc 2.76±0.01ab 41.66±0.58bc
15 3 6.97±0.08ac 32.89±8.34ac 5.67±0.03ac 46.33±1.53ad 16.60±0.55ac 3.30±0.30ac 0.79±0.01ac 2.62±0.01ab 43±1.73ac
5 2 7.39±0.27ad 23.85±3.92ac 5.26±0.21ac 33.33±1.15bc 14.40±0.30ac 8.40±0.30ac 0.51±0.01ad 2.00±0.01ab 37.33±0.57ad
Results are the mean of triplicate values ± Standard deviation. a-b Denote significant values at p<0.0.05. First letter represents significance in time and second letter represents significance in water content.

The visual appearance of the final formula depends on transparency and opacity, and here it is expressed as paste clarity in the percentage of transmittance. The malting and processing of the malt reduced paste clarity compared to whole grain due to the concentration of more particles and the presence of starch. Malting enhanced the Sediment Value (SV), which has a key role in product development. The SV is increased by settled particles, hence it affects the visual appearance of the final product. Higher SV enhances the starch to form paste and gel. Hence reduce the percentage of transmittance. A higher SV was observed in 5 minutes of cooking with 3 times of water content and lower in 10 minutes of cooking with same amount of water content. The malting process reduced SC and WAC by 36% and 23%, respectively, compared to whole grain (Table3), whereas there was an increment in SC and WAC while increasing the water content and time of cooking during processing.

The little millet malt exhibited better functional properties after processing. These properties are significantly influenced by the water content and time of cooking while processing. Polysaccharides like guar gum and maltodextrin play an essential role in food formulation due to their functional properties. These characteristics have a crucial role in formulation like bread, cookies, jellies, instant formulas, and beverages. 15 minutes of cooking at a 1:1 millet: water was found to yield better swelling capacity, solubility, water absorption capacity, dispersibility, cooked and cold paste viscosity along with less freeze-thaw stability, paste clarity, and sedimentation volume with a desirability 84%. In addition, 5 minutes of cooking at a 1:1 millet: water yielded better results, and the reduced time of processing will be useful for large-scale production with a desirability 76%. High solubility and digestibility make the little millet malt suitable for instant foods, infant nutrition, or hydrolysis-based applications. The above scientific findings will be useful for product formulation such as weaning foods, gluten-free products, and functional beverages by substituting little millet malt with starch additives. These findings indicate the value of germination as a natural, cost-effective method to enhance the quality and functional attributes of little millet malt, contributing to its potential utilization in the development of uniform, stable, smoother, bioavailable, and digestible products with improved shelf stability.

FORMULATION AND OPTIMIZATION OF THE IRON FORTIFIED BEETROOT MILLET SOUP BASED ON SENSORY EVALUATION AND FUNCTIONAL PROPERTIES:
A mixture design approach was employed by Design-Expert version 13 (trial version) to optimize the iron fortified beetroot millet soup formulation, considering the pressure cooked beetroot juice with residue and millet malt cooked for 15 minutes at a 1:1 ratio of millet to water as the primary variables and ferrous fumarate (iron fortificant) as a process variable. A mixture design is a type of experimental design used when the factors under study are components of a mixture; here, primary ingredients of the soup, beetroot and millet, are the components of the mixture. In the mixture design, the proportions of components must always sum to 100% (or 1.0).

Beetroot and millet were tested in varying proportions (0–100 mL), while ferrous fumarate was added based on FSSAI recommendations for fortified cereal products (e.g., breakfast cereals, pasta, noodles), which specify an added iron content of 1.4–2.7 mg/100 g. The ferrous fumarate contains 28% elemental iron, the quantity added at levels ranging from 4.88 mg to 9.41 mg per 100 g of sample to meet this regulatory guideline.
For each 100 mL of soup, the following spices were incorporated to enhance flavor:
• Ginger: 0.5 g
• Garlic: 3 g
• Pepper: 1.5 g
• Clove: 0.1 g
• Fennel: 1.5 g
• Cinnamon: 0.2 g
• Salt: 1 g

The iron fortified beetroot millet soup formulation was optimized based on sensory analysis and functional properties. Table 4 summarizes the results of the optimization studies. The sensory evaluation was done using a 9-point hedonic scale (1 = Dislike extremely, 9 = Like extremely), assessing appearance, aroma intensity, spice concentration, flavor, texture, and overall acceptability, and also based on viscosity and TSS. Sensory evaluation was conducted by a panel of 30 semi-trained members with a background in Food Science. Each panelist was presented with up to four samples per day, randomly coded to minimize bias. Results have been depicted below:

A total of 10 runs were there in the optimization process. The results from 9-point hedonic scale revealed that, the beetroot: millet ratio of 75:25 and 9.41 mg/100 g ferrous fumarate showed better appearance, intensity of aroma, spice concentration, and flavor. The beetroot: millet ratio of 50:50 and 9.41 mg/100 g ferrous fumarate got texture wise acceptability. The sensory panel suggested overall acceptability to 75:25 beetroot: millet ratio of 9.41 mg/100 g ferrous fumarate. Moreover, to sensory properties, the viscosity and total soluble solids (TSS) were tested, which represent the flow of liquid and soluble components. The viscosity and TSS was enhanced while increasing the concentration of beetroot in the soup formula. For the optimization process, all these parameters were kept at maximum and the optimized results revealed that a 74:26 beetroot: malt ratio with addition of 9.41 g/100 g ferrous fumarate showed better sensory acceptability with a desirability of 84%.

Table 4: Optimization of the iron fortified beetroot-millet soup formula based on sensory evaluation and functional properties
Run Component 1 Component 2 Factor 3 Response Variables
Beetroot (mL) Little millet malt (mL) Ferrous fumarate (mg/100 g) Appearance Intensity of aroma Spice concentration Flavor Texture Overall acceptability Viscosity (cP) TSS (% Brix)
1 25 75 9.41 6.8±2.11aa 6.75±1.07bb 6.75±1.16aa 6.75±1.37ba 6.65±1.49bb 6.5±1.43bb 60±7.50bb 7.45±0.07ab
2 50 50 4.88 7.2±1.58aa 6.8±1.10bb 6.5±1.43aa 6.7±.52bb 7.2±0.95aa 6.75±1.45aa 201±3.00aa 5.65±0.07ab
3 50 50 9.41 7.6±1.35aa 7.1±1.37bb 7.05±1.28aa 7.15±1.35bb 7.6±1.23aa 7.15±1.39aa 199.50±8.50aa 5.65±0.07ab
4 100 0 9.41 7.45±1.43aa 6.6±1.67bb 5.7±2.00bb 5.95±1.64ba 6.45±1.76bb 5.95±1.57bb 846.00±6.00aa 10.15±0.07ab
5 75 25 4.88 7.35±1.18aa 7.35±1.35aa 6.95±1.73aa 7.3±1.56aa 7.35±1.39aa 7.15±1.49aa 526.50±5.82aa 8.35±0.07ab
6 100 0 4.88 7±1.78aa 6.65±1.34bb 5.6±1.73bb 5.75±1.94ba 6.1±2.12bb 5.55±1.95bb 904±4.00aa 10.15±0.07ab
7 75 25 9.41 7.8±1.32aa 7.35±1.27aa 7.3±1.30aa 7.25±1.21aa 7.45±1.32aa 7.4±1.23aa 448.50±1.64aa 8.35±0.07ab
8 0 100 9.41 5.65±2.08bb 6.65±0.99bb 6.5±1.36aa 6.2±1.36ba 6.5±1.36bb 5.85±1.49bb 0bb 4.85±0.07ba
9 0 100 4.88 5.90±2.10bb 6.85±1.14bb 6.75±1.44aa 6.65±1.53ba 6.65±1.39bb 6.25±1.52bb 0bb 4.85±0.07ba
10 25 75 4.88 6.70±2.00aa 6.50±1.91bb 6.40±2.26aa 6.50±2.04ba 6.50±1.91bb 6.15±2.30bb 48±2.46bb 7.45±0.07ab
Results are the mean of triplicate values ± Standard deviation. a-b Denote significant values at p<0.0.05. First letter represents significance with respect to beetroot and second letter represents significance with respect to malt.

DEHYDRATION OF IRON FORTIFIED BEETROOT-MILLET SOUP FORMULATION:
The selected iron fortified beetroot millet soup formula was dried using freeze drying, tray drying, solar drying at normal condition, and solar drying at controlled conditions, wherein:
- Freeze drying was done at -50±2 °C; 20.06-35.75 Pa (vacuum pressure) for 18±2 h and 7.93±1.46 g of powder was obtained from 100 mL of soup.
- Tray drying was done at 50±5 °C for 4±1 h, and 5.51±1.16 g of powder was obtained from 100 mL of soup.
- Solar drying was done at normal temperature was done at 30.3-79.9°C; 8-30.3% relative humidity for 6±1 h, and 8.26±0.76 g of powder was obtained from 100 mL of soup.
- Solar drying at controlled temperature was done at 50±5 °C; 11.6-22.8% relative humidity for 15±2 h, and 8.65±0.85 g of powder was obtained from 100 mL of soup.
The nutraceutical properties, synergistic and non-synergistic compounds and functional properties of the dehydrated soup powder were evaluated by keeping unfortified beetroot millet soup powder as a control and have been presented in table 5.
Estimation of nutraceutical properties:
The nutraceutical properties of dehydrated iron fortified beetroot-millet soup powder were assessed by their DPPH, ABTS and H2O2, TPC. TFC, saponin, betanin, and vulgaxanthin values.
Determination of nutritional and antinutritional properties:
The dehydrated iron fortified beetroot-millet soup powder were tested for Ascorbic and citric acid, Folate, ß-carotene, iron, calcium, and vitamin B12 and Phytates, oxalates and tannin contents.

The antioxidant activity was found to be retained more in freeze-dried samples, which is similar to the control sample, followed by solar dried samples at controlled conditions (Fig 8). Saponin and nitrates were observed to be retained more in the freeze-dried samples. Similar trends were observed in ascorbic acid, iron, folic acid, and ß-carotene (Fig 9). The total phenolic and flavonoids were observed more in the solar dried samples at controlled conditions. The anti-synergistic compounds, such as oxalates, phytates, and tannins, were observed more in freeze dried samples. Whereas calcium is found to be more in tray dried one. The freeze-dried samples showed more similarity with the control, and the nutritional and nutraceutical potential was higher in the freeze-dried samples.

The functional properties of instant powder play a significant role in the final product formulation. Here, the functional properties were estimated by bulk density, tapped density, Carr’s index, Hausner ratio, solubility, wettability, hygroscopicity, and dispersibility (Table 4).

Estimation of bulk density, tapped density, carr’s index, and Hausner ratio
A known quantity of dried powder was taken in a 10 mL graduated cylinder and volume occupied was observed to calculate the bulk density by weight of the sample per unit volume of sample. The same sample was tapped for 5 min (32 taps per min approximately) and final volume was recorded and tapped density was calculated. Carr’s index was calculated from the equation [(tapped density-bulk density)/tapped density] *100. The Hausner ratio was calculated from the equation tapped density/bulk density.

Estimation of solubility, wettability, hygroscopicity, dispersibility, and rehydration ratio
Solubility was determined by blending 1 g of powder with 100 mL of DW and centrifuged for 5 min at 3000 rpm. Then oven dried at 105 °C for 5 h and calculated the weight difference. Wettability is the time required for 1 g of powder deposited on the liquid surface to completely submerge in 400 mL of distilled water at 25°C.
For the determination of hygroscopicity, samples of each powder (approximately 1 g) were placed at 25 °C in a glass desiccator prepared with NaCl saturated solution (75.29% RH). After one week, samples were weighed and hygroscopicity was expressed in %. Dispersibility was estimated by mixing 1 g sample with 10 mL DW at 25°C. The sample was stirred vigorously for 15 s, making 25 complete movements back and forth across the whole diameter of the beaker. The reconstituted powder was poured through a 212 µm sieve into a preweighed aluminum pan. The pan with the sieved sample was dried at 105°C for 4 h.
For the rehydration ratio, 100 mL of distilled water at 30°C and 100°C was added to 5 g of powder and weighed at regular time intervals until the difference between consecutive weights was insignificant.

Determination of color:
The color of the samples was analyzed by a portable color meter NH310 (Shenzen Three NH Technology Co Ltd) using CIE lab coordinates, and was expressed as L*, a*, and b* values.

Bulk and tapped density relate to handling and packaging; higher values indicate less packaging space and better handling. The solar-dried samples at controlled conditions were observed to be higher in bulk and tapped density. Carr’s index and Hausner ratio are the factors that determine the flowing nature of the powder. Carr’s index, <15% indicates excellent flow, 15-25% indicates good flow, and >25% indicates poor flow. All the powdered samples exhibited good flow behavior, except for the tray-dried one, which showed excellent flow behavior. The Hausner ratio, 1.00–1.11, indicates excellent flow, 1.12–1.18 good flow, 1.19–1.25 indicates fair flow, 1.26-1.34 indicates passable flow, and >1.34 indicates poor flow. All the powdered samples lie in fair flow except the tray-dried one, which showed excellent flow behavior. Solubility represents the dissolving nature of the powder in water. The solar dried sample at controlled conditions showed higher solubility (Table 5), around 22.66±1.53%. Wettability is defined as the time taken for powder particles to get wet when sprinkled over water. For an instant powder, the wettability time should be lower and which means faster wetting. All the powdered samples showed a wettability of 33-66 seconds to disperse in water. Moreover, all the powdered samples showed better hygroscopicity, dispersibility rehydration ratio. All the samples showed better functional characteristics.

The color values represented in table 6 showed that better retention of color occurs in all dehydration methods except in the tray-dried one. The lightness value was higher in all dried samples except in the tray-dried one. The tray dried one showed a very dark, reddish, slightly yellow color. The control sample showed a bright reddish, intense, moderately yellowish colour with a high chroma value. The fortification process changed the colour intensity of the powdered samples. The freeze drying and solar drying at controlled conditions showed better colour, darker, strongly red, and almost no yellow with a chroma value nearer to the control. The freeze-dried samples showed a better retention of nutritional, nutraceutical, and functional characteristics (Tables 5a & 5b). Hence, the freeze-dried samples were packed in a multilayered silver package for scaling up the final product.

Table 5a: Nutraceutical, nutritional, and antinutritional compounds of dehydrated iron fortified beetroot millet soup powder
Parameters UFS FDFS TDFS SDFS CSDFS
TPC (mg GAE/100 g) 662.65±12.05b 678.71±6.95b 678.71±6.95b 357.42±6.95c 819.27±6.95a
TFC (mg QE/100 g) 51.91±12.52b 54.64±4.7b 32.78±8.19c 49.18±8.19bc 191.25±4.73a
Ascorbic acid (mg/100 g) 10.50±0.43b 13.33±0.38a 9.53±0.32b 7.43±0.72c 10±0.41b
Citric acid (%) 32.20±0.00a 32.20±0.00a 12.88±0.00b 6.44±0.00c 32.20±0.00a
Iron (mg/100 g) 0.46±0.01d 2.15±0.02a 1.81±0.01c 1.97±0.01b 1.94±0.01b
Folate (µg/ 100 g) 75.33±0.57c 86.66±1.52a 71.00±1.00d 81.33±1.52b 65.00±1.00e
ß-carotene (µg/100 g) 50.00±1.00ab 52.00±1.00a 45.00±1.00c 49.00±1.00b 38.66±0.57d
Oxalates (mg/100 g) 78.4±15.84a 78.4±15.84a 56±15.89ab 56±15.89ab 33.6±15.89b
Tannin (mg TAE/100 g) 3134.50±5.00a 3132.83±10.00a 1574.5±7.64d 2592.83±2.88b 1684.5±5.00c
Phytates (mg/100 g) 92.33±2.51a 94.33±2.08a 81.00±1.00b 76.66±1.53c 61.00±1.00d
Calcium (mg/100 g) 0.82±0.03d 0.86±0.01d 1.54±0.01a 1.25±0.04b 1.08±0.02c

UFS: Unfortified soup; FDFS: Freeze-dried fortified soup; TDFS: Tray-dried fortified soup; SDFS: Solar-dried fortified soup; CSDFS: Controlled solar-dried fortified soup. Results are the mean of triplicate values ± Standard deviation. a-d Denote significant values at p<0.0.05.

Table 5b: Functional properties of dehydrated iron fortified beetroot millet soup powder
Treatments Moisture content (%) Bulk density (kg/m³) Tapped density (kg/m³) Flowability & Cohesiveness Solubility (%) Wettability (s) Hygroscopicity (%) Dispersibility (%) Rehydration ratio at 30 °C Rehydration ratio at 100 °C
Carr’s Index (CI) Hausner ratio (HR)
UFS 7.02±0.32b 588±16.09c 673.40±22.48c 12.66±1.15b 1.14±0.02b 17.66±1.15b 66.66±5.77a 1.31±0.02a 76.87±1.85b 6.86±0.00b 11.92±0.01b
FDFS 7.38±0.63b 588.00±16.09c 705.81±27.17c 16.66±1.15a 1.20±0.02a 18.33±1.53b 33.33±5.7b 1.35±0.01a 88.86±2.44a 8.85±0.01a 14.19±0.02a
TDFS 13.31±0.46a 654.33±26.63b 696.09±28.33c 6.00±0.00b 1.06±0.00b 17.33±2.08b 61.66±7.63a 1.31±0.02a 76.21±2.45b 4.99±0.00c 6.34±0.01c
SDFS 5.59±2.47b 662.33±9.07b 794.82±6.96b 16.66±1.15a 1.20±0.02a 19.66±0.57ab 43.33±7.63b 1.37±0.05a 71.68±2.38b 4.19±0.01e 5.89±0.01d
CSDFS 9.32±0.55b 728.00±28.58a 873.55±29.70a 16.66±1.15a 1.20±0.02a 22.66±1.53a 33.33±5.7b 1.29±0.03a 83.75±1.21a 4.55±0.01d 5.90±0.01d

UFS: Unfortified soup; FDFS: Freeze-dried fortified soup; TDFS: Tray-dried fortified soup; SDFS: Solar-dried fortified soup; CSDFS: Controlled solar-dried fortified soup. Results are the mean of triplicate values ± Standard deviation. a-d Denote significant values at p<0.0.05.

Table 6: Color analysis of dehydrated powder
L* a* b* c* h0
UFS 24.11±0.41b 47.02±0.69a 4.72±0.08b 47.32±0.57a 5.73±0.03b
FDFS 20.64±0.32c 45.86±0.60ab 0.25±0.22e 45.86±0.60ab 0.44±0.07d
TDFS 6.79±0.72e 43.40±0.82c 2.77±0.09c 43.49±0.82bc 3.65±0.08c
SDFS 28.88±2.91a 39.44±0.88d 10.30±0.55a 40.77±0.80c 14.64±0.93a
CSDFS 19.91±0.35d 44.86±0.60bc 1.25±0.22d 40.51±2.13c 4.03±0.17c

UFS: Unfortified soup; FDFS: Freeze-dried fortified soup; TDFS: Tray-dried fortified soup; SDFS: Solar-dried fortified soup; CSDFS: Controlled solar-dried fortified soup. Results are the mean of triplicate values ± Standard deviation. a-d Denote significant values at p<0.0.05.

Packaging has a fundamental role in ensuring safe delivery of goods throughout supply chains to the end consumer in good condition. The flexible plastic packaging made of multilayer film (PET/Al foil/LDPE) where the aluminium foil is the barrier layer to avoid the permeation of gases, moisture and loss of aroma has potential application and gained more attention. Moreover, it has lower cost of production than the glass jars and higher mechanical resistance, which reduces product losses in filling lines, storage, point of sale and final disposal. Here, the multilayered silver package consists of three layers of 12-micron polyester, 12-micron metalized polyester, and 75-micron low density polyethylene, which possess multi-barrier protection with enhanced shelf stability of the product.

The bio accessibility and bioavailability of the freeze-dried powder were tested and compared with a product from the Ayurvedic formula mentioned in the “Ayush Aahar”. In Ayurvedic medicine, the condition known as Pandu roga is often correlated with IDA due to the shared clinical manifestation of generalized pallor (Panduta). Ayurveda describes the human body as composed of seven dhatus (tissues), sustained by Agni (metabolic fire), which governs the transformation and balance of bodily substances. Imbalances in one dhatu can affect others, contributing to disease pathogenesis. Pandu Roga, as detailed in the Charaka Samhita, is classified as a Rasapradoshaja disorder involving vitiation of the Rasavaha Srotas (nutritive channels). It predominantly involves a Pitta-dominant Tridoshaja imbalance, with features resembling Kapha vitiation. The disease arises from impaired transformation of Rasa Dhatu (nutritive fluid) into Raktadhatu (blood tissue), leading to both quantitative and qualitative blood deficiencies, analogous to the decreased hemoglobin or red blood cell count observed in IDA. Ayurvedic management focuses on restoring doshic balance, targeting affected channels, and correcting metabolic dysfunction to enhance iron assimilation and alleviate anemia.

The classical Ayurvedic formulation Punarnava Patrasaka, described in Pakadarpanam, comprises hogweed (Boerhavia diffusa) leaves, bitter guard (Momordica charantia var. muricata) leaves, dry ginger (Zingiber officinale), rock salt, and ghee. It is traditionally noted for its pallor-alleviating property, aligning with the FSSAI Ayurveda Aahara Regulation 2021, which endorses classical formulations listed in Schedule A. This formulation showed a 63% iron bio accessibility and 3% bioavailability, whereas the iron fortified beetroot-millet soup powder formula showed 23% iron bio accessibility and 11% bioavailability. Bio accessibility refers to the fraction of a nutrient or bioactive compound that is released from the food matrix in the gastrointestinal tract (mainly during digestion) and becomes available for absorption in the intestine. Bioavailability is the fraction of an ingested nutrient or bioactive compound that is absorbed, transported, and utilized by the body at its site of physiological function or storage. The Ayurvedic formulation showed a better release of iron from the formula, whereas the absorption was relatively reduced. This might be due to the presence of antinutrients in the formula. Whereas the instant, ready-to-cook iron fortified beetroot millet soup formula showed a better absorption of iron in the body as compared to the Ayurvedic formula.
,CLAIMS:1. An instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation for managing iron deficiency anaemia (IDA), said soup formulation comprising:
- Beetroot as a source of iron and bioactive compounds
- Little millets as a source of starch, proteins, lipids and antioxidants,
- Ferrous fumarate as fortificant,
- Spices and condiments, for enhancing the flavour,
wherein, the optimized components of the formulation act synergistically through integration of fortification strategy to enhance iron absorption in the said formulation thus making the iron readily accessible for improving iron deficiency anaemia (IDA).
2. The instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 1, wherein the primary ingredients in the soup formulation comprise of beetroot and little millet.
3. The instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 1, wherein form and processing method of beetroot is optimized based on the criteria of nutrient retention, nutraceutical properties, and reduced antinutrients.
4. The instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 1, wherein little millets are used in malt form.
5. The instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 1, wherein pressure-cooked beetroot juice with residue and little millet malt in an optimized combination is processed with ferrous fumarate to prepare the soup formulation.
6. A process for preparing the instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 1, the process comprising the steps of:
- processing the beetroot juice with residue by pressure-cooking,
- processing the little millets to prepare malt,
- processing the malt by heating with an equal amount of water for 15 minutes
- combining the processed beetroot juice with residue and little millet malt in an optimum ratio along with a pre-determined concentration of ferrous fumarate, spices, and condiments to obtain the instant, healthy, ready-to-cook iron fortified beetroot-millet soup
- dehydrating the soup formulation to obtain the instant, healthy, ready-to-cook iron fortified beetroot millet soup powder (IBMSP)
7. The process for preparing the instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 6, wherein a 74:26 beetroot: malt ratio and 9.41mg. ferrous fumarate is used for the best sensory acceptability.
8. The process for preparing the instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 6, wherein the pressure-cooked beetroot juice with residue and the little millet malt is cooked for 15 minutes at a 1:1 ratio of millet to water.
9. The process for preparing the instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 6, wherein freeze-drying, tray drying, and solar drying techniques are used for dehydrating the soup formulation.
10. The process for preparing the instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 6, wherein ferrous fumarate was added at levels ranging from 4.88 mg to 9.41 mg per 100 g of the formulation.
11. The process for preparing the instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 6, wherein the beetroot juice with residue and little millet malt are cooked at temperatures ranging from 70-95±5 °C.
12. The instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation as claimed in claim 1, has 23% iron bio accessibility and 11% iron bioavailability
13. The instant, healthy, ready-to-cook iron fortified beetroot-millet soup formulation has iron bio accessibility and iron bioavailability greater than that of the Ayurvedic formulation Punarnava Patrasaka, described in Pakadarpanam, which is mentioned for anemia.