3.1. Formulation of organic millet laddus
The various ingredients that were used for the formulation of foxtail millet laddu include foxtail millet, ground nut, horse gram, jaggery, ghee, honey, cardamom and cinnamon. The final product composition was analysed using Design expert software, which generated 30 different experiments using four different variables namely foxtail millet, ground nut, horse gram and jaggery. The response values were shown in the Table 1. Each experiment had generated different formulations by altering the composition of ingredients involved in the product. Therefore all the formulations were prepared and subjected to further analysis based on the organoleptic parameters such as appearance, taste, softness and solubility. Statistical values and Analysis of variance were represented in the Supplementary Table 1–4. The results obtained from the experiments conducted on organoleptic properties were represented in the Table 2 and the results were fed into the software.
Table 1
Experimental Design for mixture design and responses for laddu production using Central Composite Rotatable Design (CCRD)
S.No | Std | Run | Factor 1 | Factor 2 | Factor 3 | Factor 4 | Softness | Appearance | Taste | Solubility |
1 | 12 | 1 | 80 | 40 | 8 | 80 | 9 | 5 | 6.4 | 9 |
2 | 14 | 2 | 80 | 20 | 12 | 80 | 4.6 | 7 | 8 | 9.5 |
3 | 29 | 3 | 60 | 30 | 10 | 60 | 9 | 7.5 | 8.3 | 9.1 |
4 | 3 | 4 | 40 | 40 | 8 | 40 | 7 | 6.5 | 8 | 2.5 |
5 | 30 | 5 | 60 | 30 | 10 | 60 | 9 | 8.5 | 7.8 | 9.5 |
6 | 23 | 6 | 60 | 30 | 10 | 20 | 6 | 5.7 | 6.6 | 4.6 |
7 | 26 | 7 | 60 | 30 | 10 | 60 | 7 | 5.1 | 7 | 8.4 |
8 | 4 | 8 | 80 | 40 | 8 | 40 | 3 | 8.4 | 8.2 | 5.3 |
9 | 8 | 9 | 80 | 40 | 12 | 40 | 2 | 8.3 | 6.4 | 9 |
10 | 9 | 10 | 40 | 20 | 8 | 80 | 4.92 | 6.3 | 9 | 4.9 |
11 | 17 | 11 | 20 | 30 | 10 | 60 | 6 | 3.5 | 4.3 | 7 |
12 | 20 | 12 | 60 | 50 | 10 | 60 | 7 | 9.5 | 10 | 2 |
13 | 19 | 13 | 60 | 10 | 10 | 60 | 2.77 | 9 | 6.7 | 6 |
14 | 15 | 14 | 40 | 40 | 12 | 80 | 6.3 | 2.7 | 5.4 | 6.2 |
15 | 2 | 15 | 80 | 20 | 8 | 40 | 4.02 | 4.9 | 1.8 | 2.34 |
16 | 22 | 16 | 60 | 30 | 14 | 60 | 6.9 | 9.5 | 6.7 | 8.1 |
17 | 11 | 17 | 40 | 40 | 8 | 80 | 7.52 | 4.5 | 7.8 | 8.6 |
18 | 24 | 18 | 60 | 30 | 10 | 100 | 6.6 | 2 | 8 | 6.6 |
19 | 7 | 19 | 40 | 40 | 12 | 40 | 5 | 5 | 2 | 4.3 |
20 | 27 | 20 | 60 | 30 | 10 | 60 | 8 | 9.2 | 5 | 5.9 |
21 | 21 | 21 | 60 | 30 | 6 | 60 | 9.9 | 8.3 | 8 | 3.7 |
22 | 6 | 22 | 80 | 20 | 12 | 40 | 5 | 8.3 | 9 | 9 |
23 | 25 | 23 | 60 | 30 | 10 | 60 | 8 | 5.6 | 4 | 7 |
24 | 13 | 24 | 40 | 20 | 12 | 80 | 4.6 | 4.5 | 4.98 | 6.3 |
25 | 18 | 25 | 100 | 30 | 10 | 60 | 0 | 4 | 8 | 7 |
26 | 28 | 26 | 60 | 30 | 10 | 60 | 7.8 | 6 | 4 | 4 |
27 | 16 | 27 | 80 | 40 | 12 | 80 | 7 | 7.4 | 9.2 | 4.8 |
28 | 1 | 28 | 40 | 20 | 8 | 40 | 6.7 | 5.6 | 9 | 1 |
29 | 5 | 29 | 40 | 20 | 12 | 40 | 9 | 6 | 2 | 7 |
30 | 10 | 30 | 80 | 20 | 8 | 80 | 3 | 7.7 | 3.2 | 7 |
*Factor 1: Foxtail millet; Factor 2: Groundnut; Factor3: Horse gram; Factor4: Jaggery. All are mentioned in gram (g) |
Table 2
Physical evaluation of the prepared laddu
Response | Prediction | SE Mean | 95% CI low | 95% CI high | SE Predicted |
Softness | 7.76983 | 0.40 | 6.92 | 8.62 | 1.04 |
Appearance | 5.74265 | 0.56 | 4.56 | 6.93 | 1.45 |
Taste | 5.72945 | 0.57 | 4.54 | 6.91 | 1.76 |
Solubility | 5.58391 | 0.63 | 4.27 | 6.89 | 1.95 |
Table 3
Optimized concentration of food ingredients against different response using Design expert software
S.No | Name | Level | Low Level | High Level |
1. | Foxtail Millet (g) | 42.99 | 40.00 | 80.00 |
2. | Ground nut (g) | 37.49 | 20.00 | 40.00 |
3. | Horse Gram (g) | 10.30 | 8.00 | 12.00 |
4. | Jaggery (g) | 60.03 | 40.00 | 80.00 |
3.2. Interaction of Various ingredients against the Response factor by Two factorial Interactions
The present study revolves around optimizing the medium composition by using the design expert software. The optimization is mainly based on analysing the interaction between the ingredients used and the response of the product based on the softness, appearance, solubility and taste. Use of RSM has resulted in the better understanding of the possible interaction between various ingredients used in the product. The significant interaction between variable has improved the total acceptability of the product as in the case of Pranaw et al. 2014. The study done by Lungmann et al. 2006, the mixture design predicted as optimized media by design expert was most superior when compared to other media in terms of final result. Similarly the optimum result from the design expert software for laddu production is preferable over other composition.
In the Fig. 1(a) represents the analysis of taste performed against foxtail millet and horse gram. From the analysis it was found that an equal proportion of foxtail millet and horse gram were required to enhance the taste of the laddu. Whereas from Fig. 1(b) it found that, there is not any notable interaction between ghee and foxtail millet in the event of enhancing the taste. With respect to the Fig. 1(c) and 1(d) it was found that a specific interaction between jaggery and horse gram as well ground nut and jaggery is required in order to enhance the taste. But it need not to be an equal proportion, a slight range nearer to the midpoint is found to be the best.
The final predicted composition of the laddus based on analysing all the results were given in the Table 4. The final predicted composition from the software was considered as the optimum concentration and the same concentration was used for doing further experiments. The products gave a good impression which was inferred from the experimental response values. This is in parallel relationship with the study done by Aboulfazli, (2015) where the sensory analysis test was based on the attributes such as color, texture, flavour and taste and none of the product was reported as poor and all the products had a good impact on the review panel.
Table 4
Final composition of the probiotic millet laddu
S.No | Name | Level |
1. | Foxtail Millet (g) | 42.99 |
2. | Ground nut (g) | 37.49 |
3. | Horse Gram (g) | 10.30 |
4. | Jaggery (g) | 60.03 |
5. | Ghee(ml) | 2.75 |
6. | Honey(ml) | 2.55 |
7. | Cardamom(g) | 0.24 |
8. | Cinnamon(g) | 0.235 |
9. | Lyoplilized culture(g) | 1 |
10. | Inulin (g) | 0.1 |
Softness = -1.76792 + 0.158354*Factor 1 + 0.436875*Factor 2 + 0.736458*Factor 3 -0.131313*Factor 4 + 0.001181*Factor 1 * Factor 2 + 0.001281*Factor 1 * Factor 3 + 0.002178*Factor 1 * Factor 4 -0.033687*Factor 2 * Factor 3 + 0.006381*Factor 2 * Factor 4 -0.003469*Factor 3 * Factor 4 -0.003252*Factor 1² -0.008296*Factor 2² +0.012292*Factor 3² -0.001190*Factor 4²
Appearance = + 8.42083 + 0.073958* Factor 1 -0.161250* Factor 2 -2.39375*Factor 3 + 0.348125* Factor 4 + 0.001531* Factor 1 * Factor 2 + 0.015156* Factor 1 * Factor 3 + 0.000359* Factor 1 * Factor 4 -0.007187* Factor 2 * Factor 3 -0.002906* Factor 2 * Factor 4 -0.006406* Factor 3 * Factor 4 -0.002148* Factor 1² +0.005156* Factor 2² +0.107031* Factor 3² -0.002086* Factor 4²
Taste = + 38.17433–0.531521* Factor 1 + 0.113625* Factor 2 -3.26479* Factor 3 -0.078437* Factor 4 + 0.003119* Factor 1 * Factor 2 + 0.050656* Factor 1 * Factor 3 -0.000747* Factor 1 * Factor 4 -0.026188*Factor 2 * Factor 3 + 0.000256*Factor 2 * Factor 4 + 0.013719* Factor 3 * Factor 4
Solubility = -42.30867 + 0.083854* Factor 1 + 0.585875* Factor 2 + 3.94229* Factor 3 + 0.436687* Factor 4 -0.000669* Factor 1 * Factor 2 + 0.002906* Factor 1 * Factor 3 -0.001022* Factor 1 * Factor 4 -0.055187* Factor 2 * Factor 3 -0.000269* Factor 2 * Factor 4 -0.032594* Factor 3 * Factor 4
Response surface plots on different evaluation parameters like softness, taste, appearance and solubility were shown in the Fig. 2–5. ANOVA statistical analysis for four different factors were calculated by Design expert software in Supplementary Table 1, 2,3,4 and it was found to be significant and fit model. Quadratic equation for the prediction the optimum point was obtained according to the CCRD design. Desirability value of 0.7 was obtained using CCRD model of RSM. R2 value was found to be 0.998 and P-value of (< 0.0001) had obtained, which indicated a better agreement between the actual and predicted values of probiotic laddu physical evaluation and confirmed a significant interpretation of the mathematical model.
3.3. Probiotication of millet laddus
The final composition of the prepared laddu was given in the Table 4. The inference from the table depicts that the major nutritional content of the product was mainly offered by the four main ingredients that were taken as variables in the design expert software and the other ingredients serves as supplements that either enhances or improves the quality of the final product.
3.4. Addition of lyophilized Lactobacillus acidophilus
Millet laddus were prepared in the composition as mentioned in the Table 4. 1 g of lyophilised culture was added and different formulations of laddus were prepared for evaluation of nutritional and stability analysis as Fig. 7. 1g of the lyophilized sample of Lactobacillus acidophilus was added to 150g of the probiotic millet laddu and the final concentration of the culture in 1g of the probiotic millet laddu was found to be 10.52 ± 0.04 log cfu/g.
3.5. Addition of Microencapsulated Lactobacillus acidophilus
To the millet laddu composition mentioned in the Table 4, 1g of Lactobacillus acidophilus encapsulated with gum arabic was added, the total colony count of microencapsulated Lactobacillus acidophilus in 1 g of the laddu was found to be 9.97 ± 0.03 log cfu/g.
3.6. Addition of probiotics in the form of curd
The third method probiotics in the form of curd was directly incorporated into the final product, the total colony count of curd incorporated laddu was found to be 10.34 ± 0.03 log cfu/g.
3.7. Evaluation of the prepared laddu against nutritional and stability
Analysis and evaluation of the final product was done on the basis of organoleptic, physical/chemical and microbiological methods.
3.8. Nutritional analysis
Nutritional content of the 4 different kinds of laddu including conventional laddus were given in the Table 5. The formulated millet laddus were found to contain major nutritional components that could satisfy all the requirements for a healthy diet. Appreciable quantity of protein and mineral found in the product could increase the nutritional value of the product. The conventional laddu showed very high content of carbohydrate and cholesterol as 101.44g and 30.23g respectively whereas other important nutrition’s were presented in very less content than the foxtail laddu. Overall mean among the nutrient composition of the three different formulations of laddus were found to be 18.196. Comparison of the mean difference among the different groups was mentioned in Table 6. Lizia and John (2014) developed a millet based high fibre biscuits and nutritional properties for the same was analysed. The total protein, carbohydrate, fat and fibre of the high fibre biscuit was tested and it was found to be 8.62 g, 68.05 g, 13.10g and 19.2 g per 100 g of the product, whereas the probiotic millet laddu have nearly the same nutritional composition as that of high fibre biscuits except for the fibre content. The total protein, carbohydrate, fat and fibre of the millet laddus was 11.133 g, 64.466 g, 15.133 g and 5.06 g per 100 g of the product. The amino acid profile is balanced and the dietary fiber content is very high compared to other cereals. Nutritional composition of Foxtail millet per 100 gm is fat (4.3 gm), minerals (3 gm), protein (12.3 gm), calcium (31 mg %), carbohydrate (60.9 gm), phosphorous (290 mg%) and dietary fibre (14 gm) was found in Zainab et al., 2019. The nutritional analysis of the pearl millet laddu showed that the moisture 12.6 ± 0.2%, protein 9.9 ± 2.8 g, fibre 2 ± 2.6 g, fat 4.2 ± 0.5 g, carbohydrate 69 g was slightly similar to that of Uttara et al., 2017. The total protein, carbohydrate and fat content of the millet laddu were similar to the fibre biscuits with little variation in the composition which is acceptable. Verma et al., 2015 explained that the foxtail and barnyard millet laddu had comparatively high protein content, 5.00 and 3.41%, respectively and low carbohydrate content in both Foxtail and barnyard millet based laddu.
Table 5
Analysis of nutrient composition of the three different formulations of laddus
Nutrition | Conventional Laddu (g) | Lyophilized probiotic Laddu (g) | Probiotic Encapsulated Laddu (g) | Curd incorporated laddu (g) | Mean | SD |
Carbohydrates | 101.44 ± 2.8 | 96.7 ± 1.4 | 97.5 ± 0.8 | 97.01 ± 1 | 98.16 | 2.21 |
Proteins | 14.39 ± 0.2 | 16.7 ± 2 | 16.8 ± 1.6 | 16.75 ± 0.4 | 16.16 | 1.18 |
Fats | 23.18 ± 1.2 | 22.7 ± 0.4 | 22.9 ± 2.2 | 22.74 ± 1.2 g | 22.88 | .013 |
Cholesterol | 0.030 ± 0.0008 | 0.0039 ± 0.0005 | 0.0040 ± 0.0007 | 0.0040 ± 0.0014 | .01048 | .614 |
Fibre | 6.95 ± 1.6 | 7.6 ± 0.4 | 8.45 ± 0.4 | 7.61 ± 0.7 | 7.6525 | .6148 |
Sodium | 0.041 ± 0.0004 | 0.0256 ± 0.0003 | 0.0295 ± 0.0004 | 0.0260 ± 0.0008 | .03053 | .0072 |
Potassium | 0.545 ± 0.0012 | 0.6251 ± 0.0006 | 0.627 ± 0.0008 | 0.6266 ± 0.002 | .60592 | .0407 |
Calcium | 0.029 ± 0.0002 | 0.0841 ± 0.0013 | 0.0951 ± 0.0014 | 0.0853 ± 0.0004 | .07338 | .0299 |
Mean | 18.325 ± 0.73 | 18.054 ± 0.53 | 18.301 ± 0.63 | 18.106 ± 0.413 | 18.196 | |
Std. Dev. | 34.634 ± 1.05 | 32.940 ± 0.76 | 33.177 ± 0.85 | 33.046 ± 0.49 | | 31.79 |
* Composition /150g of the laddu |
Table 6
Comparison between the groups of different laddu samples with respect to nutrition
S.No | Samples | Mean difference |
1 | Conventional Laddu | Lyophilized probiotic Laddu | 0.271 |
2 | Conventional Laddu | Probiotic Encapsulated Laddu | 0.024 |
3 | Conventional Laddu | Curd incorporated Laddu | 0.2191 |
4 | Lyophilized probiotic Laddu | Probiotic Encapsulated Laddu | 0.2458 |
5 | Lyophilized probiotic Laddu | Curd incorporated Laddu | 0.0516 |
6 | Probiotic Encapsulated Laddu | Curd incorporated Laddu | 0.1942 |
- |
3.9. Stability analysis
The laddu incorporated with lyophilized culture of Lactobacillus acidophilus was analysed for the total colony forming units during the storage at 4°C and at room temperature. Figure 6. depicts the total colony forming units over the period of two months during the two different storage conditions. During the initial storage period the total cfu in both the conditions were same and it was around 8.20 ± 0.1 log cfu/g, but once the storage time increases there is a significant decrease in the colony count. On storing the product up to three weeks there is a slight difference in the viability between the two storage conditions. After three weeks there is a great difference in the colony count between two storage conditions. At the last week the total cfu at 4°C was 6.10 ± 0.09 log cfu/g and cfu at room temperature was around 5.41 ± 0.08 log cfu/g. The loss in viability of the lyophilised Lactobacillus acidophilus culture during the storage period of two months was explained in the Fig. 6. From the result it is obvious that storing the product at 4°C was found to be the best condition for maintaining the viability of the freeze dried product when compared to storing the product at room temperature. On storing the product at 4° and room temperature in closed container, comparatively a high rate of survival of probiotic microorganism Lactobacillus acidophilus was observed at 4°C. The obtained result was in accordance with that of the Chen et al. (2008) where storing the product at 4° and 25°C in laminated pouch and glass bottle, the best result was obtained when the product is stored at 4°C in glass bottle. In accordance to the Selvi et al., 2013, The total plate count showed slight elevation on storage and ranged between 2.4 to 3.4 * 104, 0.7 to 1.2 * 104 at the end of the storage period of 180 days.
The laddu incorporated with microencapsulated Lactobacillus acidophilus were also analysed at both the storage conditions. The analysis of total colony count was done by studying the release kinetics of encapsulated bacteria at these storage conditions and it is represented in the Fig. 6. The initial count of bacteria in carrier incorporated laddu was found to be 9.97 ± 0.03 log cfu/g. The study on release kinetics of encapsulated bacteria revealed that the initial viable colony count of 9.97 ± 0.03 log cfu/g in laddu gradually declined and reached a value of 7.43 ± 0.02 log cfu/g at 4°C and 6.97 ± 0.02 log cfu/g at room temperature. The result obtained is in concordance with Talebzadeh and Sharifan 2016 where alginate-chitosan encapsulated Lactobacillus acidophilus showed better viable number of bacteria at 7°C when compared to 25°C in probiotic jellies. The total colony count obtained for the microencapsulated laddu was far higher than that of the laddu incorporated only with lyophilised Lactobacillus acidophilus. The result revealed that encapsulation of Lactobacillus acidophilus using gum arabic before lyophilisation provides protection of the organism during the storage period. It is inferred that the total amount of bacteria retained in the laddu can be protected by microencapsulation before lyophilisation. Thus, instead of simple lyophilisation microencapsulation of Lactobacillus acidophilus before lyophilisation could aid in the protection of bacteria from various stresses during the storage period and could possibly increase the life span of incorporated probiotics. The number of free cells were 6.4 * 107 at first and 4.6 * 104 after last day. Encapsulated were 6.8 * 107 at first and 5.7 * 105 after last day Microencapsulation plays protective role and the bacterial strains is survive longer than the free coating cells. (Farzad, 2020)
3.10. Antioxidant activity
The result of formulated probioticated laddu was analysed for various antioxidant capacity. DPPH radical activity revealed that probiotication of laddu showed moderate capacity of capturing hydrogen free radicals were listed in Table 7. The different formulation of probiotication was performed in which encapsulated laddu predicted the highest percentage of antioxidant potential of 25.39 ± 0.47% followed by curd incorporated and Lyophilized probiotics. Hydrogen free radicals might be captured in highest content by slow release of encapsulation process as well as phenolic substances present in the bacteria and foxtail millet. Ferric reducing anion power assay has carried out to identify the potential of reducing capacity of radical ions in to neutral. The similar trend was also obtained for FRAP assay also compared to other formulation. Highest absorption of reducing capacity was exhibited in encapsulated probiotic laddu as 23.78 ± 0.43%. H2O2 assay was performed to analyse the capturing ability of hydroxyl ions in which curd incorporated laddu showed much potential followed by encapsulated and lyophilized probiotics. Total phenolic content of all the formulations were studied to predict the correlation among them. There was a strong significant correlation was observed in antioxidant capacity as well as phenolic substances in the laddu which indicates the phenolic content is much responsible for producing antioxidant activity. Since the laddu is an eatable, the main focus is towards the taste. Therefore all the ingredients were analysed to test their effect on the taste using two factorial interaction method of design expert software. The products gave a good impression which was inferred from the experimental response values. Comparison between the groups of different laddu formulations with respect to antioxidant activity was represented in Table 8. This is in parallel relationship with the study done by Aboulfazli et al., 2015 where the sensory analysis test was based on the attributes such as color, texture, flavour and taste and none of the product was reported as poor and all the products had a good impact on the review panel. Similarly Savita et al., 2020 has shown that major free phenolic acid among vanillic, ferulic, gallic and caffeic acid is protocatechuic acid (45 mg/100 g) Ofosu et al., 2020 reported that among the millet grains evaluated, barnyard and finger Italian millet exhibited the highest DPPH radical scavenging activity of 359.6 and 436.25 µg/mL respectively.
Table 7
Antioxidant activity of various formulation of probioticated laddu
Antioxidant assays | Control | Foxtail Millet | Probiotic | Lyophilized probiotic Laddu | Probiotic Encapsulated Laddu | Curd incorporated Laddu | Mean | SD |
% Inhibition | | |
DPPH | 8.23 ± 0.43 | 12.34 ± 0.18 | 9.34 ± 0.24 | 15.45 ± 0.23 | 30.12 ± 0.87 | 17.12 ± 0.63 | 15.43 | 7.96 |
FRAP | 6.23 ± 0.12 | 6.59 ± 0.22 | 12.73 ± 1.3 | 22.12 ± 0.12 | 23.78 ± 0.43 | 18.38 ± 0.54 | 14.97 | 7.64 |
H2O2 | 11.13 ± 0.3 | 13.17 ± 0.67 | 12.34 ± 0.366 | 18.24 ± 0.32 | 25.39 ± 0.47 | 27.12 ± 0.21 | 17.89 | 6.93 |
Mean | 8.53 | 10.7 | 11.47 | 18.6 | 26.43 | 20.87 | 16.1 | |
SD | 2.46 | 3.58 | 1.85 | 3.34 | 3.29 | 5.44 | | 7.19 |
N | 3 | 3 | 3 | 3 | 3 | 3 | | |
Total phenolic (µg) | 120 | 140 | 135 | 134 | 178 | 123 | 138.33 | 20.86 |
Table 8
Comparison between the groups of different laddu formulations with respect to antioxidant activity
Samples | Mean difference |
Control | Foxtail Millet | 2.17 |
Control | Probiotic | 2.94 |
Control | Lyophilized probiotic | 10.07 |
Control | Probiotic Encapsulated | 17.9 |
Control | Curd incorporated laddu | 12.3 |
Foxtail Millet | Probiotic | 0.77 |
Foxtail Millet | Lyophilized probiotic | 7.9 |
Foxtail Millet | Probiotic Encapsulated | 15.73 |
Foxtail Millet | Curd incorporated laddu | 10.17 |
Probiotic | Lyophilized probiotic | 7.13 |
Probiotic | Probiotic Encapsulated | 7.82 |
Probiotic | Curd incorporated laddu | 2.27 |
Lyophilized probiotic | Probiotic Encapsulated | 7.82 |
Lyophilized probiotic | Curd incorporated laddu | 2.27 |
Probiotic Encapsulated | Curd incorporated laddu | 5.55 |
During the storage period, the laddus incorporated with curd showed least stability and are highly susceptible to contamination as they developed white patches on the surface during both the storage conditions. The white patches might have been developed due to the increased proliferation of microorganisms during the storage period. Since the curd incorporated laddus contains more colony forming units of microorganisms than the recommended dose of probiotics, they are further not used as the probiotic food as they might affect the consumers. This was similar to the study by Yuvarani and Anitha, 2016. The cooked multigrain ladoo contains 618 µg/100g of antioxidant content. The raw ingredients which were used to prepare multigrain ladoo and their antioxidant level are as follows; finger millet 268 µg/100g, foxtail millet 340 µg/100g, wheat 197 µg/100g, cardamom powder 182 µg/100g, ghee 80.1 µg/100g, horse gram sprouted and green gram sprouted together 662 µg/100g, jaggery 115 µg/100g.