Comparative study of innovative blends prepared by fortification of date powder to alleviate child malnutrition

Abstract Child malnutrition is one of the biggest problems in developing countries with higher level of food insecurity. Pakistan is 5th largest producer of date fruit; therefore, its processing and products should be explored in various dimensions. Being rich source of minerals and sugars, it can contribute in weaning foods in a good manner. In current study, three blends were prepared with specific proportions of spray‐dried date powder and rich in specific proportions. They were compared with each other and control (free of date powder). The nutritional profile of the formulations revealed that 100 g of each formulation included all macronutrients in compliance with the requirements of Food and Agriculture Organization for weaning formulations; moreover, they are enriched with minerals due to presence of date powder. The iron contents reached up to 12.74 ± 0.16 mg/100 g. The phosphorus, zinc, and potassium contents also increased with the increase in date powder subsequently. Physicochemical properties exhibited in compliance with the requirement of the weaning foods. The protein quality was assessed both in vivo and in vitro. Amino acid profiling indicated that the limiting amino acid in F1 and control were lysine but in F2 and F3 were threonine. This is suggested that date powder might contain good quality protein that was further explained in biological studies, the formulations that contained higher amount of date powder reveled better PDCAAS score 86.76 ± 4.5, true digestibility 84 ± 4.36, biological value 69.45 ± 0.69, net protein utilization 73.82 ± 1.46, and protein efficiency score 1.18 ± 0.07. The sensory evaluation revealed that F2 showed better result in overall acceptability. Thus, date powder is suggested to be used as good constituent that can fortify mineral contents and sugar contents of the weaning foods.


| INTRODUC TI ON
The food use to complement breast milk either semisolid or solid from the age of six months is called as weaning food (Agostoni et al., 2008). Weaning foods are characterized as easily chewable and digestible foods with great energy density and possessing low bulk (Onweluzo & Nwabugwu, 2009). Traditionally, cereals are considered as most useful tools for fulfilling the energy and nutritional requirements of the children being caloric dense. But cereals are mainly deficient of lysine; an essential amino acid, but as they are full of other sulfur containing amino acids like methionine and cysteine, they can be used as complementary ingredient instead of a complete baby food itself (Ahmad et al., 2014). Moreover, The commercially available weaning foods that are either imported or produced locally have quite higher cost due to sophisticated packaging, excessive processing, promotions, and marketing (Carvelho, Kenney, Carrington, & Hall, 2001;Fewtrell et al., 2017). The commercial weaning foods are not affordable for low-income mothers, so homemade recipes are mostly preferable.
Plant-based complementary formulations are mostly protein deficient, if they have adequate protein, the antinutrient contents in plants may not suffice available protein and they also suppress the absorption of many macro-and microminerals (Gibson, Perlas, & Hotz, 2006). The iron and calcium requirements should be fulfilled from complementary diets up to 50%-75%, likewise more than 25%-50% protein requirements of infants should be fulfilled from weaning food (Caulfield, Huffman, & Piwoz, 1999;Qasem, Fenton, & Friel, 2017). Therefore, it is the need of the hour in developing countries to produce a complementary food from locally available underutilized resources that is complete in all respects, for instance, it must be energy dense, having all nutrients according to RDA of growing children in order to fight against the problem of malnutrition among children of low socio-economic groups (Sattar et al., 2013;White, Bégin, Kumapley, Murray, & Krasevec., J., 2017). Fruits are the source of immediate energy supply coupled with all sufficient amounts of minerals, vitamin, and handful of dietary fiber that is indispensable for a healthy gastrointestinal tract (Igah, 2008).
A blend of date powder and rice supplemented with whey proteins and whole milk powder could meet the requirements of current situation to cope the malnutrition issues in infants. The rice is documented to be easily digestible with low fiber, highly caloric density, and second most widely produced staple food in Pakistan therefore considered for the current study. Different blends were tried by researchers using brown rice, mung bean, and soybeans with yoghurt as dairy source and calculated the nutrient contents. Higher the contents of cereals and legumes, proteins and fat contents became higher (Munasinghe, Silva, Jayarathne, & Sarananda, 2013).
Therefore, as date powder contained three to four percent proteins and the extent to which utilized in the recipe was insufficient for infant requirements. Therefore, whey proteins being reliable and cheap protein source were used. Whole milk powder contained all those ingredients that were necessary for the growth and development of the growing infants. It was enriched with high-quality proteins that could complement with the low protein and other nutrients enriched date flesh to produces a complete weaning food. Therefore, the aim of the study was the development of such nutritional blend for weanling kids that can fulfill their all requirements regarding proteins, essential amino acids, and micronutrients. Date powder blended with whey protein, whole milk powder, and rice flour could be the best source of food in their weanling age.

| Procurement of raw material
Whole milk powder was taken from Nestle, high-quality brown rice from super market. Analytical grade chemicals were purchased from Merck, Oxide, and AppliChem.

| Preparation of formulations
Rice flour slurry was prepared and cooked for 10 min, cooled, spread on a tray in the form of thin layer (1-2 mm) and oven-dried (2 h at 90ºC). The flakes obtained were grounded in a blender. Spray-dried date powder of aseel (Raza et al., 2019), whole milk powder, whey protein concentrate, and all the ingredients were added and mixed well and finally sieved through 60mesh screen. The finished product was stored at 5ºC in polyethylene bags for further analysis.

| Compositional analysis
Nutritional profile for ash, moisture, crude fat, protein, and NFE was done through respective methods (AOAC, 2000).

| Mineral profiling of weaning foods
The procedure described in AOAC (1990) method No. 3.014-016 was adopted to determine the mineral content. Na, K, was determined on flame photometer according to AOAC (2000). Whereas, Phosphorus contents was determined by spectrophotometry according to method given by Gul and Safdar (2009

| Caloric value
Gross energy value was measured by guidelines described in AOAC (2006) using Oxygen Bomb Calorimeter (C-2000, IKA WERKE).
Energy value of foods as a whole and per portion was determined. Passmore and Eastwood (1986) proposed guidelines that were followed for assessment of macromolecules energy by physiological fuel values implementing Atwater's conversion factors (4x% protein, 9x% fat). Additionally, nutrient density for respective proteins and fats was calculated per 100kcal (Food & Agriculture Organization/ World Health Organization, 2006

| Physicochemical characteristics
The density was measured by adding specific amount of the weaning food in 100 ml volumetric flask and expressed as mass per unit volume. The sedimentation method was applied for determination of reconstitution index of complementary diets (Osundhunsi & Aworha, 2002). For reconstitution index, measured sample (1 g) was mixed with 10 ml distilled water and allowed to stand at ambient temperature (30 ± 2°C) for 30 min. The mixture was centrifuged for 30 min at 3,000 g. Water absorption was examined as percent water bound per gram weaning food (Sosulski, Garatt, & Slinkard, 1976). Rapid Visco Analyzer (Newport scientific, RVA surper 4, Australia) was used to assess the viscosity of 40% slurry of the weaning food. It was expressed as centipoise (Bukusuba, Muranga, & Nampala, 2008). In vitro starch digestibility and in vitro protein digestibility were determined according to respective methods (Ekwere, Igile, Ukoha, Mbakwe, & Anegu, 2017;Souilah et al., 2014).

| Amino acid contribution
The one to two years infant's requirement for amino acids was calculated on an average basis (Reeds & Garlick, 2003;Food and Agriculture Organization/World Health Organization/United Nations University, 2007). The daily amino acid contribution (%) from weaning food was assessed according to the guidelines of Egounlety (2002).

| Protein score
Essential amino acid score was recorded for complementary foods following the instructions of Food and Agriculture Organization/ World Health Organization/United Nations University (2007) for preschoolers, the lowest score was expressed as protein score (Plahar & Hoyle, 1991).

| PDCAAS value
Protein digestibility corrected amino acid score (PDCAAS) was calculated according to method of Kannan, Nielsen, and Mason (2001) by multiplying true digestibility of respective formulation and lowest value of amino acid score as per given in equation:

| Housing of rats
For rats modeling, 40 weanling male rats were purchased from National Institute of Health (NIH), Islamabad, and kept in animal room, Department of Pharmacy, Government College University of Faisalabad. The rats were divided into four groups, 10 in each group.
The rats were fed on formulation specified for a period of four weeks. The room temperature was maintained at 25ºC, and humidity was 50 ± 5% kept constant throughout the housing period with light and dark cycle of 12 hr. Urinary excreta, feces, and spilled diets were gathered throughout the study period, the urinary nitrogen and fecal nitrogen were calculated through kjeldahl apparatus. At the end of trial, rats were decapitated after keeping them overnight fast. The bodies of rats were weighed after removing of all organs and dried in oven for the calculation of Nitrogen assimilated in their bodies.

| Formulation intake
Feed intake of each group was recorded by subtracting spilled plus offered diet from the total feed consumed on daily basis during the study period (Wolf & Weisbrode, 2003).

| Body weight gain
Total gain in body weight of individuals was calculated. Then mean of each group was taken to measure the growth index for specified formulations, the gain in body was calculated with interval of 8, 16, 21, and at the termination of the study period (Seena, Sridhar, Arunb, & Young, 2006).

| Protein quality evaluation
The quality of protein was assessed according to method established by Ingbian and adegoke (2007)  Nitrogen balance study was done after getting the nitrogen contents from spilled diets, urinary excreta, feces, and dried rat bodies.
The calculation of true digestibility (TD), biological value (BV), and net protein utilization (NPU) was done using nitrogen contents.

| Blood sampling
After overnight fasting, the rats were anesthetized and blood collected by jugular vein cannulation (Parasuraman, Raveendran, & Kesavan, 2010) into EDTA-coated sterile containers. For ALP test, the container must not be coated with EDTA.

| Liver and kidney functioning test
Serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate amino transferase (AST) enzymes are the marker of liver function tests and were performed to evaluate current condition of the liver. Similarly, urea and creatinine quantitation are the markers of kindey functions and were performed in semi-automatic analyzer (Mini techno, USA) using individual kits for each assay.

| Sensory evaluation
All the formulations were evaluated for various sensory characteristics by trained taste panel following 15 cm unstructured line for apparent and taste characteristics (Meilgaard, Civille, & Carr, 2007). The format for sensory evaluation was comprised of three parts; dry mix evaluation for visible characteristics; apparent characteristics of reconstituted formulations for color, texture, aroma, ease of preparations, thickness, and smoothness while third portions constitute evaluation of taste attributes like sweetness, flavor, taste, and overall acceptability. Periodic evaluation was done in Food analysis lab, Institute of Home and Food Sciences, GCUF. The formulations were offered to the panelist in transparent cups, water for reconstitution in cups was also provided while water to clear the taste between the sample evaluations was also provided with plain water. The samples were provided randomly to rate their acceptance by marking a cross on the line for all sensory traits. The obtained data by using metric scale were converted to numerical scores.

| Statistical analysis
The obtained data of each parameter were subjected to factorial design using Statistical Package (Statistix 8.1.) Levels of significance were determined through ANOVA using CRD and means were compared through LSD.

| Proximate composition
All three formulations showed highly significant difference among the protein levels ( Table 1). The quantity of proteins in control weaning food was 10.6 ± 0.3 g/100 g while F1 exhibited a better quantity of 14.9 ± 0.7 with subsequent decrease in the rice quantity and increase in date powder in F2 and F3 the quantity of proteins became lower and showed the values of 12.6 ± 0.4 and 10.6 ± 0.4 g/100 g.
The crude fat contents of formulations varied significantly, the lowest amount was assessed in control 8.95 ± 0.25 while F1, F2, and F3 with values 14.4 ± 0.5%, 13.3 ± 0.5%, and 12.8 ± 0.3% respectively. The crude fiber contents in control were 2.52 ± 0.06%, while it showed an increasing trend in the formulations as F1 contained 3.4 ± 0.1% and the F2 and F3 3.9 ± 0.3% and 4.2 ± 0.0, respectively. This increase was due to higher concentrations of date powder in F2 and F3 as compared to F1. The ash content increases significantly among the formulations. As the ash percentage in control was 2.86 ± 0.05 and F1, F2, and F3 were 3.7 ± 0.0%, 4.2 ± 0.0%, and 5.3 ± 0.0%, respectively. The value of NFE represents all digestible carbohydrates. They also revealed an increasing trend as the date powder increases even 10%. In control NFE was assessed as 62.4 ± 0.46% and in F1, F2, and F3 were 65.1 ± 0.2%, 66.2 ± 0.4% and 67.1 ± 0.2%, respectively. All these formulations despite of variation among each other are still in agreement with the Food and Agriculture Organization/World Health Organization (2006)

| Minerals
The values (Table 2) revealed the range of calcium varied between 324.75 ± 0.13 for F2 to 3.27.85 ± 3.37 and the control formulation contained 189.19 ± 0.84 mg/100 g of calcium. But iron content in the controlled formulation was 4.37 ± 0.08 mg/100 g while in investigative formulation it varied between 12.40 ± 0.40

| Water holding capacity (WHC)
The means ± SD showed that control and F2 formulations are having nearly equal water holding capacities with values 0.88 ± 0.05 ml/g and 0.85 ± 0.03 ml/g, respectively. While with the higher contents of date powder, the WHC in F3 were increased to 0.94 ± 0.04 ml/g and the lowest were reported in F1 formulation with value 0.71 ± 0.03 ml/g.
According to Usman, Bolade, and James (2016) found much higher levels of WHC of weaning foods, and the reason for higher WHC was only cereal and soybean-based formulations, higher the protein contents in some gruels, higher will be the WHC. In a similar study, WHC of weaning food from sorghum was assessed as 0.44 ± 0.14 ml/g (Lalude & Fashkin., 2006).

| Viscosity
Higher viscosities are associated with larger gruels, difficulty swal- were compared with the commercial diets but their viscosity was much lower than that of the commercial food, that is, it was ranged from 200 cP to 220cP while in control it was 3030cP (Onweluzo & Nwabugwu, 2009).

| Bulk density
The mean values of all formulations significantly differ in F2 and

| Nutrient density
The quantification of the major nutrients and their expression on calorie basis with respect to the total energy uptake from the weaning food can be helpful to determine whether the particular weaning food can provide the individual nutrients according to standards/as per physiological requirements (Mosha & Vicent, 2004). The results found significant difference among the protein contents provided by the three formulations and control, whereas the fat contents were higher is subsequent formulations compared to control that has value of 2.74 ± 0.16 g/100 kCal; protein contents of control were 3.54 ± 0.07. Among all formulations, the highest fat and protein content was provided by F1 with quantity of 3.58 ± 0.12 g/100 Kcal and 3.71 ± 0.16 g/Kcal of fat and proteins respectively.
According to Food and Agriculture Organization/World Health Organization (2006), the energy provision in a weaning food from protein and fat must not exceed to 5.5 g/100 Kcal and 4.5 g/100 kcal, respectively. The findings in the current study are in parallel with these guidelines.

| In vitro protein digestibility
The significant variations were observed among the protein digestibility values obtained from control and that of formulations. Among

| In vitro starch digestibility
In current study, brown rice was precooked before making slurry for drying therefore the findings are according to the formulations showed good IVSD values. All three formulations did not show significant difference. Comparatively lower value was observed in control.

| Amino acid profile
The quality of protein is mostly assessed by the number and quantity of essential amino acid present in the protein. The quantity of essential amino acids was very much crucial in proteins for better quality evaluation. The essential amino acid being lack of their precursors in the body is indispensable to be provided by external source for better growth and development particularly in infancy where development of better immune system is equally important (Adeyeye & Afolabi, 2004;Wu, 2010). Lysine is mostly the limiting amino acid in most of the cereal-based weaning foods, similarly maize is deficient source of tryptophan and other sulfur containing amino acids, so the combinations of legumes and cereals were used that proved good source of such limiting amino acids (Ijarotimi & Olopade, 2009;Mune, Minka, Mbome, & Etoa, 2011.

| Amino acid score
The findings showed better amino acid scores by F1 and F2 formulations compared to F3, the amino acid score of the control was also almost equivalent to that of F1 and F2. These finding revealed that a good combination of date powder with rice flour could generate a quality weaning food that provide good quantity of essential amino acids. The F1 and control were showing lysine as limiting amino acid which could predict the higher concentration of rice flour which is deficient of lysine. Threonine was found as limiting amino acid in F2 and F3 formulations.
Similar results were found by many scientists who used cereal as staple food for the preparation of weaning foods (Juliano & Hicks, 1996). It is evident from literature review that processing techniques like cooking and soaking could also affect the availability of lysine contents (Seena et al., 2006). Moreover, the second limiting amino acid was threonine in F1 and control and in F2 and F3 s limiting amino acid was lysine. So, all three formulations contain lysine and threonine in lesser quantities compared to the other amino acids (Table 4, Figure 1).

| PDCAAS value
The concept of protein deficiency corrected amino acid score was first time adopted in 1993 in order to evaluate the protein quality by comparing it with another reference protein on the basis of delivery of indispensable amino acids in the human body, and simultaneous determination of true digestibility of the test protein and its ability to fulfill the requirement of essential amino acids.
It is obvious from the  (Muoki, de Kock, & Emmambux, 2012). In another study, the commercial and locally prepared complementary foods were administrated to Sprague Dawley weanling rats for nutritional and biological assessment revealed almost equal PDCAAS score that proposed about the effectiveness of local produce for the production of weaning foods because they are not less than the industrially prepared commercial diets (Amagloh et al., 2015;Mosha & Vicent, 2004).

| Biological evaluation of weaning foods
The nutritional assessment was observed through proximate composition and amino acid profiling of the test formulations and comparison in the above section was quite evident. The biological study gave us more deep insight of those in vitro indices. The biological study was subdivided into three sections where Nitrogen balance study was carried out by calculating true digestibility (TD %), biological value (BV %), and net protein utilization (NPU %). Nutrient

TA B L E 4 Amino Acid Concentrations in all formulations
F I G U R E 1 Amino Acid score for all formulations. *Aromatic Amino acid (Phenylalanine + Tyrosine). **Sulfur containing amino acids (Methionine + Cysteine). ***LAA (Limiting Amino acids) response growth study was carried out by calculating PER (protein efficiency ratio), NPR (net protein ratio), and RNPR (relative net protein ratio).
Mean squares for nitrogen balance included true digestibility (TD%), biological value (BV), and net protein utilization (NPU) study showed highly significant variations among all three formulations and control as well as minimum value of TD in F2 formulation was observed with value 79 ± 1, whereas the F1 and F3 generated 84 ± 4.36%, and 80 ± 1.1% respectively. The TD of control was equal to the F1. But according to Food and Agriculture Organization guidelines (2012)  African yam beans revealed their biological value was equal to 59.5% (Onwuka, Catherine, Jude, & Ayalogu, 2010). The biological value or the rates at which protein deposited in the body have direct relationship between dietary intake of proteins and nitrogen retention for the subjected sources of proteins that further strengthen the importance of biological value. This is then used to calculate the average dietary protein requirement (Reeds & Garlick, 2003

| Kidney functioning tests
The urea and creatinine determination are the key indicators for monitoring the renal functioning. All four groups that were fed on formulations were tested for these indicators. There was very little fluctuation in the results. The weanling animals usually required higher nitrogen contents and maximum assimilation of nitrogen from the proteins for body buildups. And urea contents in blood are due to deamination of proteins. Consequently, less quantity of urea was noticed in the samples. The values for urea were ranged between 11.95 ± 0.63 to 13.30 ± 0.8 mg/dl, a similar trend was observed in creatinine where values were ranged between 0.32 ± 0.2 to 0.33 ± 0.02 mg/dl. Mean squares in Table 5 also showed nonsignificant difference among the groups fed on formulations and control as well.
These results are in the normal range of urea and creatinine, so it is evident from the results that formulations had no adverse effect on the rats therefore, safe to consume (Table 5).

| Liver functioning tests
The serum levels of alanine phosphatase (ALP), aspartate transami- spectively. When these results were analyzed they were found in the safe limit. According to Sime, Quick, Saleh, and Martin (2007) the safe limit for monitoring of hepatocytic integrity was established ranging from 123.1 ± 7.67 to 183.8 ± 9.18 U/L in male SD rats and 72 ± 16 to 94 ± 32 U/L for female SD rats for ALP, whereas 88 ± 18.2 to 111 ± 50.6 U/L in males and 80 ± 9 to 89 ± 14 in females for AST, whereas for ALT normal range for male SD rats were ranged from 38 ± 3.9 to 48 ± 7.8 IU/L and for females ranged between 44.50 ± 7.37 to 53.70 ± 15.38. Current findings are similitude to the normal range of hepatic enzymes which is evident that no hepatocytic activity took place by the administration of the test formulations and results were also in concordance with the control fed experimental SD rats.

| Sensory evaluation
The sensory evaluation of weaning mixes is very crucial because the consumer's acceptance is of prior importance in value addition and processing of food items. In the current study, weaning formulations were prepared from the date powder with the addition of different fortificants. Due to dark color of the date powder the color of dry mix was light brown in the first formulation due to higher contents of rice flour and whole milk powder. For color, texture, and ease of preparation, the control scored minimum score compared to the formulations. The highest position for color was fetched by F1 A similar trend was followed by the texture where value ranged from 11.72 ± 0.29 to 12.53 ± 0.33 for F1. For ease of preparation, the highest score was obtained by F2 with value 13.08 ± 0.38, the significant difference was exhibited by all the formulations. The value for smoothness and thickness was varied from 10.38 ± 0.23 to 11.56 ± 0.26, 10.49 ± 0.26 to 11.63 ± 0.28. The highest value of sweetness among the experimental formulations was in F3 with value 11.66 ± 0.23, it is evident from the sweetness contents that higher was the date powder contents higher will be the sweetness level. The taste and flavor or F2 formulations were observed as the best with values 12.61 ± 0.26 and 11.57 ± 0.28 among the formulations, the control has scored the best for flavor and on no second for taste with very little difference. The overall acceptability of the control was noted with value 12.72 ± 0.58 and among formulations; the highest acceptability was earned by the F2 formulation, that is, 12.55 ± 0.40.
The lowest value for overall acceptability was noted in F3 formulation. It can be deduced that the higher thickness, darker color, and little bit bitter flavor can contribute to the overall acceptability of the F3 formulation due to higher contents of the date powder ( Figure 2).

| Conclusion
The amelioration of weaning food to uplift child health required novel approaches. The effort made in current study showed good results regarding weaning blends. Date powder contributed to protein contents, improve the micromineral and amino acid profile of the blends clearly, and the control with no date powder was lacking good protein quality, good amino acid score, and mineral contents.
Therefore, date powder blending for weaning mixes preparation was proved in current study.