INTRODUCTION

Malnutrition, especially due to protein-poor diet is very common in many parts of the world and is emerging as one of the major health problem that is being faced by developing countries of the Asian subcontinent. The problem has caught the attention of policy makers and researchers, as it was well reported that about 870 million people in the world are undernourished in terms of dietary energy supply in 2012 in which about 17.6 % from the South Asian population is undernourished (FAO, 2012). Considering the growth and development of children; weaning is the most important and critical stage. Weaning is the process of expanding the diet to include food and drinks other than breast milk or infant formula as it is the period of infant vulnerability (Sajilataet al., 2008). To be suitable for the feeding of young children, the cereal-based weaning foods are prepared in liquid form by dilution with a large quantity of water, thereby resulting in more volume but with a low energy and low nutrient dense food (Sanniet al., 1999; Egounlety, 2002; Munasinghe et al., 2013). Hence, such products are often poor in nutritional value as they lack essential amino acids such as threonine, lysine and tryptophan (Egounlety, 2002; Munasinghe et al., 2013). Several strategies have been used to improve the nutritive value of weaning foods, namely malting, dry roasting, milling, steaming, boiling, and sprouting which are some simple processes being used in the production of weaning foods (Wikramanayaka, 1996;Munasinghe et al., 2013). Milk and cereal-millet based complementary foods appear to be unique in the sense that they can deliver a multitude of nutrients to children and complement each other as well. During the last decade, the utilization of microbial sources, particularly microalgae like cyanobacteria as a nonconventional source of food and protein seems promising (Pulz and Gross, 2004; Rodrigues et al., 2015) and has a long history. Knowing their physicochemical characteristics is fundamental for the selection of the most suitable microalgae to specific food technology applications and consequently successful novel foods development (Batista et al., 2013). Of all the blue-green algae, Spirulina (Arthospira) platensis/maxima have received the greatest interest (Kamenidou et al., 2011, Mishra et al., 2015). Spirulina is virtually immune from problems of contamination and indirectly attest to its safety and has been proven safe for human consumption and have numerous health benefits (Ciferri, 1983, Karkos et al., 2010). A combination of probiotics and prebiotics has also been employed to enhance the health benefits of infant formulae referred as synbiotics. The dietary supplement market for probiotics/synbiotic is gaining momentum at a very fast pace over the globe, hence, regulation of these products is a must for consumers safety (Sarkar, 2011). It has been reported that Spirulina could improve the quality of fermented dairy products via increasing the rate of in vitro development of several strains of microorganisms present in them (Bhowmik et al., 2009) and due to its complex composition it might be used for supplementation in synbiotics formulations (Finamore et al., 2017). Charalampopoulos et al., (2002) developed a functional weaning food using novel cereal components as substrates and developed dietary fibre, prebiotics and probiotics. For commercial purposes, it is especially fruitful and important to isolate or search for strains endowed with more favourable characteristics like faster growth, better biomass yield, higher specific cell constituents, adaptability in stress, etc. Thus, a systematic screening of two different strains of Spirulina grown under two different mild stress condition i.e. Zn²⁺/ UV-B radiation was performed to identify the most suitable strains in terms of biomass and phytochemical production. In India, we have traditional products touted as functional but have little scientific validation. It was envisaged that preparation and standardization of Spirulina-supplemented milk and cereal-based weaning food in combination with fermentation with curd culture for flavour enhancement would offer the potential for the development of a simple and nutritious product with some probiotic efficacy made from locally available under-utilized raw materials. Spirulina can be considered safe in healthy subjects, even though sensory characteristics of a functional food are important as per consumer acceptance of the product (Finamore et al., 2017). It was therefore, planned to carry out feasibility studies on the development of such a product, with sensory attributes forming the basis of the study.

MATERIALS AND METHODS

Raw materials

Pearl millet

Wholesome, insect free, pearl millet (Pennisetumglaucam) grains with an average mass of each kernel 8.9 mg of hybrid variety were procured from local market of Allahabad district, Uttar Pradesh (Fig. 1A). The pearl millet grains were germinated for 24 h (Fig. 1B) for removal of antinutritional factor and to increase the digestibility.

Figure 1. Whole Pearl millet grains (A); Sprouted pearl millet grains (24 h) (B).

Curd culture procurement and preparation of curd

Mesophilic mixed strain curd culture NCDC-167 (Lactococcuslactis ssp. lactis, Lactococcuslactis ssp. cremorisand Lactococcuslactis ssp. lactisbiovar. diactetylactis in 1:1:1 ratio) was supplied by the National collection of Dairy cultures (NCDC), Dairy microbiology division, National Dairy Research Institute, Karnal, Haryana, India. Curd was prepared by inoculating 1 % of curd culture NCDC-167 in skimmed milk and incubated at 37 °C for 24 h.

Other ingredients

Salt, cumin seed powder andskimmed milk was procured from the local market.

Harvesting of Spirulina powder grown in optimized laboratory conditionsand maintenance of axenic culture of Spirulina maxima

Pure culture was harvested in liquid broth media under broad spectrum antibiotic (ex. gentamycin) to prevent the growth of other class of bacteria as per our previously set protocol (Mishra and Prasad, 2015). The culture was maintained in Zarrouk’s medium (Zarrouk, 1966) at 27±2 ^°C under the photosynthetically active radiation (PAR) of 75 μmol photon m^-2 s^-1 with a 16/8 h of light/dark period. The next step in the study was to optimize the growth behaviour of S. maxima in two different stresses i. e. Zn²⁺/UV-B. The cells were exposed to varying concentration of Zn²⁺ (i.e. 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 8.0 and 10.0 mg L^-1) and UV-B (i. e. 0.05, 0.1, 0.2, 0.4, 0.6 and 0.8 µmol photon m^–2 s^–1 for 4 h in a day). On the basis of above preliminary growth experiments, a mild range of Zn²⁺ stress i.e. 2.5 as stimulatory dose, 3.5 as moderate dose and 4.5 mg L^-1 as high dose were selected for exposure of Zn²⁺ by adding them to the growth medium. Similarly for UV-B stress; 0.05 was selected as stimulatory dose, 0.2 as moderate dose and 0.6 µmol photon m^–2 s^–1 as high dose and denoted as UV-B_1, UV-B₂ and UV-B_{3, ­}respectively for further analysis of physiological and biochemical characteristics of the Spirulina species (unpublished data). S. maxima grown under mild stress of Zn²⁺ and UV-B₁ in the Ranjan Plant Physiology and Biochemistry Laboratory was selected for the product development. As the cultures were grown in the liquid medium it was harvested using filter paper and washed with distilled water 3-4 times, then dried in hot air oven at 50 °C for complete removal of moisture. Then it was preserved in poly bags and kept in air tight container at -20 °C in a deep freezer.

Preliminary phytochemical screening of Spirulina powder

The mass cultured cyanobacteria was separated from the medium by centrifugation at 8000 rpm for 10 min and pellets were dried at 50 °C for 24 hours. The dried Spirulina sample was ground into a fine powder by using mortar and pestle. Powdered sample was immersed in methanol solvent (10 ml) and placed in a shaking incubator for 24 hours at 25 °C. The solvent extracts were concentrated under reduced pressure at 40 °C by vacuum evaporator. The concentrated sample used for the presence of phytochemicals such as carbohydrates, steroids, terpenoids, saponins, alkaloids (Sofowora, 1993), amino acids, tannins and flavonoids (Trease and Evans, 2002).

Processing of pearl millet grains

Soaking: The pearl millet grains were cleaned and washed with water and soaked in water: 0.1 % formaldehyde solution in the ratio of 1:3 for 24 h at room temperature.

Germination: After draining the soaking water, the grains were placed between the folds of muslin cloth drying tray and then the grains were allowed to germinate at room temperature (24-35 °C) for the period of 24 h (Akubor and Obiegbuna, 1999). Water was sprinkled intermittently to moist the cloth and paper (Khetarpaul and Chauhan, 1989; Narasimha and Malleshi, 2003; Hassen et al., 2006).

Drying: The germinated grains were dried at 55–60 °C for 8h (upto moisture content 5-6%). The rootlets dried grains were removed by scrubbing with hands (Abbey and Berezi, 1988).

Dehusking: The germinated and dried grains were coarsely ground in a low speed mixer for 5 min to remove husk (Hassen et al., 2006).

Cooking: The pearl millet grains were cooked in addition with water, salt and cumin seed powder in pressure cooker for 15-30 min (Ejeta et al., 1987).

Standardization of the method for the development of Spirulina-supplemented milk and cereal-based weaning food

Optimization of particle size of the pearl millet

The dried pearl millet grains were subjected to grinding at low speed mixer for different time periods: without grinding, 10 and 5 min to prepare 3 samples A, B and C, respectively. All the three samples were soaked in water for half an hour and then cooked in a pressure cooker for 30 min with 5 % salt, 0.8 % cumin powder and sufficient amount of water. After cooking the samples were cooled to room temperature and then given to panel of semi trained judges for sensory evaluation.

Optimization of quantity of water to pearl millet ratio

The dried and dehusked pearl millet grains were cooked as detailed in the previous section using different pearl millet grain to water ratios of 1:6 (B), 1:8 (C) and 1:9 (A). After cooking, the samples were cooled to room temperature and then given to panel of semi trained judges for the sensory evaluation.

Optimization of incubation time for fermentation

The product was prepared using the pearl millet to water ratio optimized previously. The cooked pearl millet weaning food was cooled to room temperature. It was then mixed with skimmed milk curd prepared previously and kept ready (skimmed milk cultured with curd culture at the rate of 1 % at temperature of 40 °C) (El Hag et al., 2002; Hassen et al., 2006). The weaning food was then kept in an incubator at 29 °C for a different time: without fermentation i.e. 0 h (C), 8 (B), 12 (D), and 14 h (A) for the preparation of four samples. After fermentation the samples were heated in a microwave oven for 1 min and then given to panel of semi trained judges for the sensory evaluation.

Preparation of Spirulina-supplemented milk and cereal-based weaning food

The product was prepared using step by step standardized protocol after a number of trials. The cooked pearl millet weaning food was cooled to room temperature. It was then mixed with skimmed milk curd prepared previously and kept ready (skimmed milk cultured with curd culture at the rate of 1% at temperature of 30 °C). This food was then added with Spirulina powder at the rate of 0.5% (C), 1.5% (D), 3% (A) and without addition of Spirulina (B) for the preparation of four samples and kept in incubator at 29 °C for 12 h for fermentation. After that the samples were heated in a microwave oven for 1 min and given to panel of semi trained judges for the sensory evaluation.

Physicochemical analysis (proximate composition)

The raw material and final product were analyzed for moisture, protein, fat, carbohydrates, ash and crude fibre content by following the standard methods of AOAC (1984). Along with proximate composition the colour value was also determined by X-rite colorimeter that was calibrated using a white reference standard tile, as described by Rangannaet al., (2005).

Evaluation ofthe probiotic efficacy of developed pearl millet weaning food

Prior to sensory evaluation Spirulina supplemented pearl millet weaning food was evaluated for its probiotic efficacy after 12 h incubation at 29 °C. In brief, a loop of weaning food suspension in normal saline was streaked on nutrient blood agar plate, Mueller-Hinton agar plate and MacConkey agar plate at subsequent time interval of incubation.

Sensory evaluation and statistical analysis

The Spirulina-supplemented and non-supplemented milk and cereal-based weaning food was evaluated for its sensory characteristics by a panel of 15 semi trained judges using a 9-point hedonic scale (Stone et al., 1974). Three replicates of each sample from the three trials of each experiment were used for statistical analysis and the values are reported as mean±SD. Statistical analysis of the present investigation was subjected to the analysis of variance using Duncan’s multiple range test (DMRT) at p<0.05 significance level using SPSS-16.0 software.

RESULTS AND DISCUSSION

This study was carried out in two phases to conduct the feasibility trials on Spirulina- supplemented weaning food based on milk-cereal foods. The first phase was concerned with the optimization of the formulation and processing parameters for developing fermented pearl millet weaning food. In the second phase, optimization of Spirulina supplementation in the developed fermented pearl millet weaning food for sensory and nutritional characteristics was carried out. Recent studies on the positive role of low fluence rate of UV-B in Spirulina spp. has been reported and it was found that there was enhancement in biomass production, photosynthetic pigments, non-enzymatic antioxidants, total phenolic contents, and antioxidant potential (Kumar et al., 2016). Similar to the above studies an attempt was done in the present study to adapt the Spirulina species under mild stress condition (2.5 mg L^-1 of Zn²⁺ or UV-B₁) to enhance their chemical constituents. Thereafter such mild stress-adapted S. maxima was harvested by filtration to remove growth medium and dried to make powder. This powder was further utilized for supplementation in weaning food for its value addition. Results obtained are presented hereunder.

Quality of raw material

The chemical composition of pearl millet flour prepared from raw grains of 24 h-sprouted pearl millet flour is shown in Table 1.

The moisture content of raw pearl millet flour ranged from 9.00 to 9.45 % with an average of 9.26 %. The fat, protein and ash content varied from 5.85 to 6.70, 9.32 to 9.85 and 1.33 to 1.69 % with average values of 6.16, 9.55 % and 1.54 %, respectively. The carbohydrate content calculated by difference ranged from 72.86 to 77.00 % with average value of 74.39 %. The moisture and fat content of 24 h-germinated pearl millet grain ranged from 9.15 to 9.75 % and 10.29 to 10.68 % with average values of 9.50 and 10.39 %, respectively. The protein, ash and carbohydrates had average values of 10.43, 1.63 and 72.71 % with a slight varying range of 10.22 to 10.55, 1.52 to 1.75 and 70.31 to 75.00 %, respectively. The results of proximate composition of pearl millet were in agreement with the report of Hulse et al., (1980) who regarded pearl millet as ‘nutracereal’ in view of its unique nutritional profile and myriad of phytochemicals such as fat, vitamins and minerals (Ojokoh et al. 2015).

Preliminary phytochemical screening of crude extract of Spirulina maxima

The results of preliminary phytochemical screening of methanolic extract of S. maxima grown in optimized mild stress condition i.e. S. maxima grown in 2.5 mg L^-1 of Zn²⁺ and exposure of UV B₁ showed the presence of phytochemicals with positive test for carbohydrates, amino acids, tannins, steroids, terpenoids, flavanoids, phenols, alkaloids, and a negative test for the saponin presence (Table 2).

Clean safety profile of the methanolic extract of S. maxima grown under mild stress was suggested as it was found positive for the presence of phytochemicals and negative for the presence of saponins. The lethal dose (LD 50) of Spirulina has not been determined and unlike other cyanobacteria it exempts any kind of cyanotoxins, thus has been regarded as a GRAS product (Generally Recognized as Safe) FDA (Marles, 2011). So, it is clear that the long-term dietary supplementation of Spirulina does not pose any health risk if consumed in moderation (Yang et al., 2011).

Sensory attributes of pearl millet weaning food

Effect of grinding on pearl millet weaning food

Results showed that there was no significant difference in colour, appearance and flavour score for all type of grain size studied (Fig. 2). The study revealed that the weaning food prepared from the pearl millet grain which was ground for 5 min (C) was maximum as for overall acceptability is concerned.

Figure 2. Effect of grinding time on sensory characteristics of pearl millet weaning food. Values are means (± SD). Means not sharing a common superscript letter in a column are significantly different at P<0.05 as assessed by Duncan’s multiple range test.

A= without crushing, B= crushing for 10 min and C= crushing for 5 min

Figure 3. Effect of pearl millet to water ratio on sensory characteristics of pearl millet weaning food. Values are means (± SD). Means not sharing a common superscript letter in a column are significantly different at P<0.05 as assessed by Duncan’s multiple range test.

Ratio with water during cooking: A=1:9, B=1:6 and C=1:8

Figure 4. Effect of incubation time on sensory characteristics of milk cereal based weaning food. Values are means (± SD). Means not sharing a common superscript letter in a column are significantly different at P<0.05 as assessed by Duncan’s multiple range test. A= 14 h, B= 8 h, C=without fermentation and D= 12 h.

Effect of water quantity on pearl millet weaning food

There was no significant difference in consistency and flavour score for all levels of water studied. The study revealed that the weaning food prepared from the pearl millet grains cooked with water at a ratio of 1:8 (C) was found to be most desirable in terms of overall acceptability (Fig. 3).

Effect of addition of curd culture on weaning food as a producer of probiotic activity

Results showed that there was no significant difference in colour and appearance score for all incubation time intervals studied. The study revealed that the pearl millet weaning food in which curd culture was added and then incubated for 12 h (D) was found to be optimal for overall acceptability concerned and better than other three samples (Fig. 4).

It was reported that endogenous enzyme phytase of pearl millet contributed significantly to the reduction of the phytate content of fermented pearl millet flour which was dependent on pH and temperature of fermentation. It was inferred that the fermentation process released unidentified polyphenols and converted phycocyanin to phycocyanobilin (Patel and Goyal, 2013). Based on the results, it was suggested that fermented Spirulina can be a potent supplement for weaning food due to presence of lactic acid bacteria i.e. Lactococcuslactis, Streptococcusthermophilus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus bulgaricus(Gupta et al.,2017)Despite numerous beneficial properties of probiotic bacteria, it is also useful to mention their safety and efficacy in formula supplemented with probiotics. Suitability of probiotics for premature infants or in infants with immune deficiency must be evaluated because of limited data regarding the mechanisms of probiotic action, appropriate administrative regimens, and probiotic interactions (Sarkar, 2011) (Fig 5).

Along with this the total plate count was also found to be more (1.17x10⁹ -1.04x10¹⁰ CFU g^-1 DM) in Spirulina-supplemented weaning food than in the non-supplemented food (1.03x10⁹ CFU g^-1 DM). As it was previously reported by Bhowmik et al., (2009) that dry algal biomass of Spirulina (10 mg ml^-1) stimulates proliferation of probiotic lactic acid bacteria to 86%.

Effect of supplementation of Spirulina powder in pearl millet weaning food

The sprouted and dried pearl millet grains ground for 5 min and then cooked with water ratio of 1:8. Prepared pearl millet weaning food was fermented by adding curd culture at the rate of 1 %. This food was then added with Spirulina powder at the rate of 0.5 % (C), 1.5 % (D), and 3% (A), and without addition of Spirulina (B) for the preparation of four samples and kept in incubator at 29 °C for 12 h for fermentation. The statistical analysis of different sensory score revealed that there was no significant difference in flavour, consistency, mouthfeel and acidity except colour and appearance score which also affected the overall acceptability of the product. The study revealed that the pearl millet weaning food supplemented with 1.5 % (D) Spirulina powder and then incubated for 12 h was found to be the best-ranked as far as overall acceptability was concerned and was better than other three samples (Fig. 6).

Figure 6. Effect of supplementation of Spirulina powder on sensory characteristics of milk cereal based weaning food. Values are means (± SD). Means not sharing a common superscript letter in a column are significantly different at P<0.05 as assessed by Duncan’s multiple range test. A= 3 %, B= without Spirulina, C=0.5 % and D= 1.5 %.

To develop milk and cereal-based weaning food supplemented with Spirulina, ingredients such as pearl millet, water, skimmed milk, curd culture, salt, cumin seed and S. maxima powder (grown under mild stress condition) were selected and their quantity in the formulation were standardized in a preliminary trial. According to PFA (2008) milk and cereal-based complementary foods commonly referred to as weaning food or supplementary foods based on milk (Sabikhi, 2007), cereals and/or legumes (pulses), soybean, millets, nuts and oilseeds could be processed to low moisture content and then fragmented to permit dilution with water or milk. As it was reported that the pearl millet (family: Poaceae) ranks sixth most important grain and used in preparation of weaning food by people living in arid land zones(Baltensperger, 2002). The supplementation of S. maxima powder in weaning food was done in combination with fermentation (12 h). Fermentation has been found to increase pepsin digestibility of millet protein, decrease the concentration of phytic acid and polyphenols with improvement in the availability of minerals (Khetarpaul and Chauhan, 1989, Inyang and Zakari, 2008).

Final product formulation

From the results obtained above, it is evident that pearl millet grains ground for 5 min, cooked with a grain to water ratio of 1:8, added with 1% curd culture and incubated for 12 h for fermentation and added Spirulina powder at the rate of 1.5 % was found to be most acceptable on the basis of sensory evaluation. The overall schematic representation in the form of flow chart for the development of Spirulina supplemented weaning food is given in Fig. 7.

Figure 7. Schematic diagram of the standardized process for the development of Spirulina-supplemented milk and cereal-based weaning food.

Proximate composition of developed product

The results of proximate composition of developed Spirulina supplemented pearl millet weaning food are given in Table 3.

There were three samples analyzed for proximate composition i.e. weaning food without Spirulina supplementation denoted as sample A, weaning food supplemented with Spirulina powder (UV-B₁) denoted as sample B and with Spirulina powder (2.5 mg L^-1 of Zn²⁺) denoted as sample C (Fig. 8).

Figure 8. Spirulina-supplemented milk and cereal-based weaning food (final product).

Results showed that the dry matter appeared to be same in the samples A and B whereas sample C exhibited slightly less value. The protein, ash, carbohydrate and crude fibre contents were higher in samples B and C as compared with sample A except fat. Total plate count of the samples B and C was also found to be higher as compared to sample A. The Spirulina supplemented pearl millet weaning food formulated by the protocol as above was analysed for its proximate composition as detailed in Table 3. According to FAO/WHO (1982) a minimum protein content of 15 % is required for maximum complementation of amino acids in foods and for growth. In this study, all the three formulations met the protein demandsfor 1-3 year old children (15-21 %, w/w) (Table 3). The ash content indicated the mineral content of the products was higher in the Spirulina-supplemented weaning foods (1.257-1.316%, w/w) than in the weaning food without Spirulina supplementation (1.185%, w/w).Acceptable ash content of weaning foods, given by the recommendations of the Protein Advisory Group (PAG) (1972) should not exceed 5%. Formulated foods studied in the current study were within this limit as well (Table 3). The fat content of a food sample can affect its shelf life stability, because fat can undergo oxidative deterioration, which leads to rancidification and spoilage. Hence, a food sample with high fat content is more liable to spoilage than one with a lower fat content (Amankwahet al., 2009). The PAG (1972) recommends that the fat content of a weaning food should not exceed 10 % due to oxidative deterioration. Thus, the total fat content of formulated product ranging from 3-4 % will not affect its shelf life (Table 3). For weaning foods acceptable fibre content should not exceed 5 % according to the PAG (1972) recommendations becausehigh fibre content decreases the digestibility of the particular food for weaning children. The crude fibre content (0.788- 1.258 %, w/w) of the evaluated formulated food was within this acceptable range (Table 3). In the present study, 24 h-germinated pearl millet grains was used as the main carbohydrate supplier which helped to maintain the carbohydrate content (66-70 %) over 65 % as the recommendations made by PAG (1972).

CONCLUSION

Pearl millet weaning food is a traditional food of India mainly in rural areas. Great prospects exists in India for value addition and improving health benefits of cereals by combining with milk and milk by-products and applying advanced technologies for their processing and preservation. Therefore, in order to enhance the flavour and digestibility of traditional pearl millet weaning food, an attempt was made to conduct feasibility studies on the acceptance of fermented pearl millet weaning food (made on the milk and cereal-based weaning foods employing similar technology). It may be concluded that the standardized product consisted of one part of pearl millet ground for 5 min; cooked (1:8), 5 % salt and 0.8 % of cumin powder. Cooked weaning food was mixed with 1 % curd culture, 1.5 % Spirulina powder and incubated for 12 h (fermentation). The standardized product consisting of this formulation scored 8.14 scores on 9 point hedonic scale as per overall acceptability is concerned. The proximate chemical composition of Spirulina supplemented milk cereal based weaning food was found to be richer as compared to non-supplemented weaning food. This study was exploratory in nature and sheds light on segments based on panellists’ knowledge regarding the sensory attributes of milk and cereal-based probiotic food of Spirulina, but further in-depth research is required on its shelf life determination, development of such product at industrial scale, consumers’ preference and purchasing behaviour and attitudes towards the product. This will enable the future use of microbes and microbial fermentations for a widening area in food technology and in functional and clinical food areas.

ACKNOWLEDGEMENTS

We are thankful to Dr.LathaSabhiki, senior scientist, Dairy Division, National Dairy Research Institute, Karnal, Haryana for providing the culture of NCDC167.

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