1.       Introduction

The postpartum period extends from childbirth to six weeks after delivery. This stage is regarded as a critical phase in a woman’s reproductive life [1]. To safeguard maternal health, adequate nutrition is essential for

restoring the physiological and nutritional demands of the mother, while supporting the infant development [2].

During lactation, inadequate dietary intake can deplete nutrient stores and compromise maternal health. Breast milk composition remains relatively stable in the case of malnourished mothers, but it leads to the higher risk of

comorbidities and mortality among them [3]. Maternal nutrition is crucial for promoting the health and well-being of mothers and their children. Adequate nutrition not only reduces the risk of anemia, gestational diabetes, and hypertension in them but can also prevent stunting, wasting, and undernutrition in their children [4] Thus, it highlights the need for nutritional awareness and functional food formulations required for the lactating mothers.

Despite its importance, nutritional attention during the postpartum period is often directed primarily towards infants, with insufficient emphasis on maternal health and dietary needs [5] This gap in care and attention emphasizes the need to explore culturally relevant and accessible dietary practices that can meet the nutritional needs of mothers. Since ancient times, medicinal plants were primarily utilized for dietary recipes to enhance the nutrition of pregnant and lactating mothers. Although these recipes may provide nutritional benefits, limited scientific evidences are available regarding their impact on women health. In recent times, traditional recipes have been losing popularity due to modern food practices and the loss of intergenerational knowledge associated with it.

To bridge the gap associated with the imbalance in mother’s nutritional requirements during postpartum period, traditional dietary practices have been known to support the maternal health recovery. Madhuca longifolia, commonly known as Mahua is one of the galactagogue food source that can enhance the milk production and secretion [6] Mahua is a multipurpose tree whose edible flowers are rich in sugars, vitamin C, calcium, phosphorus, amino acids, fats and bioactive compounds. Mahua flowers are utilized in various traditional foods such as halwa, kheer, meethi puri, and barfi [7][8]. Eleusine coracana commonly known as Ragi or Finger millets is another nutrient rich source containing calcium, iron, amino acids and dietary fibre [9]

Therefore, the present study aimed to formulate a functional food, Mahua-Ragi Nutri-Balls (MRNB) shown in Figure 1, using traditional ingredients, mahua flowers and ragi millet, to help meet the nutritional needs of the postpartum women. The study also supports Sustainable Development Goal (SDG) 3 by promoting health and well-being while scientifically validating traditional dietary practices.

The present study aims at the development and standardization of MRNB through sensory analysis. The evaluation includes proximate and mineral analysis and its contribution for the Recommended Dietary Allowance (RDA) per serving. Further assessments include the antioxidant activity analysis of the control and treatments, and shelf-life study of the standardized product.

2.  Materials and Methods

The present work was carried out at the Food Laboratory of the Department of Family and Community Sciences, University of Allahabad, Prayagraj, Uttar Pradesh.

2.1  Materials

Dried mahua flowers, ragi millet, wheat flour, sesame seeds, jaggery, dry fruits, and ghee were procured from the local markets of Prayagraj. Mahua flowers were washed, sun-dried, and ground into flour. Ragi millet grains were washed, soaked overnight, dried at 35°C using a hot air oven, and then ground into flour. All ingredients were visually inspected and selected free from visible contamination and mechanical damage.

2.2  Experimental Trials

The experiment included a control and three treatments of Mahua-Ragi Nutri-Balls (MRNB) namely T1, T2, and T3, as shown in Figure 2.

The experimental design of MRNB is presented in Table 1. In the formulation of control, ragi flour and wheat flour were used in equal proportion, while mahua flower powder was totally absent. In treatment T1, 7g of ragi flour and 6g of mahua flower powder was used. In treatment T2, ragi flour was lowered to 5 g and mahua flower increased to 8 g. Lastly, in treatment T3, ragi flour was further reduced to 3 g and mahua flower maximum quantity to 10 g.

Thus, the formulation involved progressive substitution of ragi and wheat with increasing levels of mahua flower powder, while the quantities of sesame seed flour, almonds, walnuts and jaggery were kept constant.

2.3  Preparation of Mahua-Ragi Nutri-Balls (MRNB)

Preparation of Mahua-Ragi Nutri-Balls are shown in Figure 3.

2.4  Sensory evaluation

Sensory evaluation of the control and treatment formulations was conducted using a 9-point hedonic scale. 15 Semi-trained panelists assessed all the sensory parameters and overall acceptability under controlled laboratory conditions. Sensory scores were statistically analysed to determine the standardized treatment.

2.5  Proximate Analysis and Mineral Analysis

The proximate of the standardized MRNB treatment was determined using standard analytical methods mentioned in AOAC (2005) [10]. For mineral estimation iron and calcium were tested as per the standard procedure described by the AOAC (2005).

2.6     % RDA contribution per serving by the standardized MRNB treatment

The percentage contribution of RDA per serving by the standardized MRNB treatment towards daily requirements of lactating women was calculated using the RDA report [11]

2.7  Estimation of the antioxidant activity

2.7.1  Sample preparation for the antioxidant activity Control and treatments of MRNB were ground to powder using a mixer grinder, dried at 40°C for 4h, and the dried samples were solubilized in 70% methanol for the further analysis.

2.7.2  Total Phenolic Content (TPC)

The total phenolic content of the extracts was measured using modified Folin-Ciocalteu micro-method [12]. In brief, 20 µL of each extract was mixed with 1 mL of distilled water and 100 µL of Folin-Ciocalteu reagent. The mixture was incubated at 38°C for 3 minutes, after which 280 µL of 25% (w/v) sodium carbonate solution and 600 µL of distilled water were added. The samples were kept in the dark at room temperature for 1 hour, and absorbance was recorded at 765 nm using a UV-Visible spectrophotometer. A blank consisting of distilled water and Folin-Ciocalteu reagent without extract was used as a reference. TPC was expressed in terms of gallic acid equivalents (GAE).

2.7.3  DPPH Free Radical Scavenging Assay

The free radical scavenging activity of the extracts were evaluated using the DPPH assay with minor modifications [13]. For the assay, 100 µL of extract was mixed with 150 µL of DPPH solution (2.5 mg DPPH in 100 mL methanol) and 3 mL of methanol. The sample was incubated for 20 minutes, and absorbance was measured at 515 nm. The percentage of radical scavenging was calculated as:

2.7.4  ABTS Free Radical Scavenging Assay

ABTS radical scavenging activity was determined following standard procedures [14]. The radical cation was produced by mixing a 7 mM ABTS stock solution with 2.4 mM potassium persulfate in a 1:1 ratio. This mixture was kept in dark at room temperature overnight. Subsequently, 1 ml of the extract was added to each 2 ml of the ABTS solution. After a 30 min incubation period, the absorbance of the samples was measured at 745 nm where Abs control is the absorbance of ABTS radical in

methanol and Abs sample is the absorbance of ABTS radical solution mixed with sample extract/standard. The control was prepared by using methanol in place of the sample and scavenging activity was calculated using:

where 𝐴control is the absorbance of the reagent without extract, and 𝐴sample is the absorbance with the extract.

2.8  Shelf-life analysis

Standardized treatment of MRNB was stored at room temperature in an airtight container. To evaluate the shelf-life, sensory evaluation was conducted at 1st week, 2nd week, 3rd week and 4th week using 9-point hedonic scale to score various sensorial parameters such as colour, texture, taste and odour. The sensory evaluation was performed by 15 semi-trained panellists.

2.9  Microbial analysis

Microbiological evaluation of the standardized treatment of MRNB included Standard Plate Count (SPC) and Yeast and Mould Count (YMC). Analyses were performed according to the standard protocols [15]. Results were assessed against acceptable microbiological limits for ready-to-eat foods.

2.10   Statistical Analysis

The data were expressed as mean ± S.D for triplicate readings. Statistical analyses were performed using SPSS Software (Version 20). Intergroup comparison was performed using one-way ANOVA. When significant differences were detected, Tukey's Honestly Significant Difference (HSD) post hoc test was used for pairwise comparisons of treatment means. The observations were considered statistically significant at p ≤ 0.05.

3.        Results and discussion

3.1  Sensory Evaluation

The mean scores of organoleptic acceptability of a control and different treatments of MRNB shown in Table 2 and Figure 4. The control had a mean score of overall acceptability of 7.80 ± 0.33, whereas among all the formulations developed, T3 received the highest

overall acceptability with mean score of 8.50 ± 0.42, which was significantly higher than that of the control.

Since, T3 was selected as the standard treatment based on its sensory acceptability, it was further analyzed for proximate and mineral composition for the study.

Table 2: Sensory evaluation score of control and treatments of MRNB

Figure 4. Organoleptic acceptability of treatments of MRNB and control

3.2   Proximate and mineral composition

Table 3: Nutritional composition of standardized treatment of MRNB (T3)

Values are expressed as mean ± standard deviation (SD) of three replicates (n = 3).

The nutritional composition of the standardized treatment (T3) of MRNB is shown in Table 3. Among all nutrients, carbohydrates constituted the highest proportion (46.736 g/100g) and dietary fibre (13.12 g/100g) followed by fat,

moisture, protein and ash content. The calcium and iron contents were recorded as 116.53 mg/100g and 5.70 mg/100g, respectively. The use of nutrient-dense ingredients such as mahua flowers, ragi, dry fruits etc., in the formulation of MRNB suggests its potential nutritional benefits for lactating women and may also be suitable for children during growing phase.

3.3. % RDA contribution per serving by the standardized MRNB treatment (T3)

The analysis of % RDA contribution shows that the per serving (25g) standardized treatment (T3) of MRNB provides a good amount of dietary fibre (13.12% of the RDA) and iron (6.19% of the RDA) for lactating women (Table 4), along with moderate amounts of energy, protein, and fat. Therefore, the study suggests that product may contribute to daily nutrient requirements when consumed as part of a balanced diet.

Table 4: % RDA contribution per serving standardized treatment of MRNB (T3) for Lactating women

Note: All the above calculations have been done taking sedentary women as reference. According to updated RDA 2024 report, for lactating women minimum level of total fat intake per day is 20% of total energy per day. As per recommendations, minimum intake of 100-130g of Carbohydrates per day should be ensured for ages 1year and above. No additional total dietary fibre allowances are given for pregnant and lactating women as intakes in these population were like that of Non-Pregnant, Non-Lactating women (NPNL). Therefore, the dietary fibre calculation is same as normal adult women.

3.4   Antioxidant activity and Total Phenolic Content Analysis

Table 5: Antioxidant activity and Total Phenolic Content across different treatments and control

Values are expressed as mean ± standard deviation (n = 3). Means within the same column with different superscript letters differ significantly according to Tukey's HSD test (p < 0.05).

Figure 5. Graph showing DPPH Activity among control and treatments of MRNB

Figure 6. Graph ABTS Activity among control and treatments of MRNB

Figure 7. Graph Total Phenolic Content among control and treatments of MRNB

DPPH and ABTS radical scavenging activity, and the total phenolic content of control and treatments are shown in Table 5. A significant increase in the antioxidant activity was observed in all three parameters with increasing treatment level. Treatment T3 with the highest incorporation of mahua flowers exhibited highest DPPH activity (18.82 ± 0.06% inhibition), ABTS activity (24.59 ± 0.14% inhibition), and Total Phenolic Content (13.43 ± 0.07mg GAE/g), whereas the control recorded the lowest values for DPPH Activity (3.04 ± 0.02% inhibition), ABTS Activity (5.90 ± 0.04% inhibition) and Total Phenolic Content (6.46 ± 0.05mg GAE/g).

One-way ANOVA confirmed that treatments significantly affected DPPH, ABTS radical scavenging activity and the total phenolic content. Tukey's HSD test further showed that all treatment means differed significantly (p < 0.05) from one another, as indicated by the different superscript letters. The increasing antioxidant values observed across the treatments coincided with increasing mahua flower incorporation (Figure 5, 6 & 7). According to a reported study by Singh et al. (2021), mahua flower extracts have shown total phenolic content (25.3 ± 1.0 mg GAE/g FW), which might support its role in the radical scavenging activity of the formulated product [16].

3.5  Shelf-life study

Table 6: Sensory evaluation of the standardized treatment of MRNB (T3)

The observations of sensory evaluation of the standardized treatment of MRNB (T3) from 1st week to 4th week (Table 6) are as follows:

•  1st week: It can be observed that taste and aroma ranked the highest when compared to colour and texture which were acceptable.

•  2nd week: Compared to the 1st week, colour was ranked high maybe because of absorption of ghee by MRNB while taste, aroma and texture was still the same.

•     3rd week: Taste and aroma scores were declined slightly. Colour and texture scores remained the same.

•     4th week: Score of aroma, taste and texture declined noticeably day by day. It may be because of the oxidation of fats and dry fruits. Score of colour was same.

3.6  Microbiological study

   Table 7: Standard Plate Count and Yeast &Mould Count of standardized treatment of MRNB (T3)

         ND means not detected.