Formulation and validation of probioticated foxtail millet laddu as a source of antioxidant for biological system using response surface methodology

Abstract

Probiotics play a critical role in supporting a healthy gut microbiome, which significantly impacts overall health and well-being. While there has been an increase in the availability of probiotic foods in recent years, there may still be limited options and accessibility in certain regions. This study focused on formulating a traditional Indian sweet called laddu enriched with millet and Lactobacillus acidophilus. The formulation of laddu ingredients was optimized using Design Expert software to create an optimal product for testing. The probiotic Lactobacillus acidophilus culture was incorporated into the laddu in three forms: lyophilized, microencapsulated powder, and natural curd. The probiotic foxtail laddu was selected based on specific criteria such as color, odor, and texture. The nutritional analysis revealed that the laddu contained approximately 64.46 g of carbohydrates, 15.13 g of protein, and 5.06 g of fat per 100 g of laddu. A microbial count analysis was performed over a two-month storage period to assess the viability of the incorporated Lactobacillus acidophilus. The results showed that the lyophilized and microencapsulated culture demonstrated good viability, with counts of 6.10 ± 0.09 log CFU/g and 7.43 ± 0.02 log CFU/g, respectively, when stored at 4 °C. In comparison, storage at room temperature resulted in counts of 5.41 ± 0.08 log CFU/g and 6.97 ± 0.02 log CFU/g at the end of the storage period. Based on the findings, the probiotic millet laddu developed in this study has the potential to be a value-added food product that can enhance the overall health of consumers. Incorporating probiotics into traditional food items like laddu offers a convenient and enjoyable way to promote gut health and improve the product’s nutritional value.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01188-8.

Introduction

Food provides the body with the energy it needs to maintain a healthy body [1]. The food habits of the present scenario are becoming very dangerous, leading to the deterioration of health and causing health-related problems. According to Myles [2], improper food habits and their supplements created the concept of unhealthy life and sources of diseases like protein-energy malnutrition, vitamin A deficiency, iodine deficiency disorders, nutritional anemia, and diet-related non-communicable diseases like obesity, cardiovascular disease, stroke, diabetes, and other diseases. People must consider their eating habits to maintain physical and mental health. In this context, probiotic foods have gained significant popularity as people become more aware of the importance of a healthy gut microbiome [3].

Probiotics are often called good and beneficial bacteria that are naturally found in our body and also occur in some food supplements in adequate amounts to confer health benefits [4]. Thus, probiotics represent a standard group of functional foods and are defined as live supplements with microorganisms, with a proven advantage to the host by improving the intestinal microbial balance of the host organism. Probiotics include various types of bacteria which pose different benefits [5]. However, most probiotic bacteria come under two groups, namely Lactobacillus and Bifidobacterium. Probioticated foods can come in various forms, including dairy products like yogurt, kefir, and fermented milk drinks, as well as non-dairy alternatives such as fermented vegetables, tempeh, and certain types of sourdough bread. These foods undergo a fermentation process where live probiotic bacteria are added or naturally occur, resulting in the proliferation of these beneficial bacteria [6].

According to Divya et al. [7], probiotic benefits include strengthening the immune system, potential antagonistic activity against pathogenic gastrointestinal microorganisms, decreasing cholesterol accumulation, improving bowel regularity, and maintaining individual intestinal microbiota. Probiotics improve the patient’s health disorders such as diarrhea, inflammatory diseases, gastroenteritis, bowel syndrome, and various types of cancer. Probiotics enhance the nutritional value and the digestibility of raw products. It improves the sensory characteristics and enhances the functional qualities of foods and beverages [8]. Combining prebiotics with probiotics, often called synbiotics, is associated with increased gut health. When prebiotics and probiotics are combined, the prebiotics act as a fuel source for the probiotic bacteria, helping them survive and thrive in the gut. The prebiotics essentially serve as a “nourishing” substrate for the probiotics, enhancing their effectiveness and colonization in the gut. This symbiotic relationship between prebiotics and probiotics can result in improved colonization of the beneficial bacteria, increased production of short-chain fatty acids (SCFAs) through fermentation, and a positive impact on gut health [9]. Prebiotics include inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), resistant starch, and beta-glucan. These prebiotics are abundant in various fruits, vegetables, legumes, and whole grains. Notably, whole grain-based products like wheat, barley, and millet have garnered increasing consumer attention as valuable sources of prebiotics [10].

Millets are a diverse group of grasses widely grown worldwide as cereal crops and grains, and it is used as feed for both livestock and human. Millets are highly nutritious compared to major crops such as wheat and rice [11]. It is rich in dietary fiber, vitamins, iron, calcium, and other minerals [12]. Foxtail millet is the second most widely cultivated species of millet. According to Jali [13], foxtail millet releases glucose slowly and steadily in the blood without affecting the body’s metabolism. The prevalence of diabetes is limited among the population consuming foxtail millet. Earlier studies on household processing methods like malting of pearl millet and finger millet may reduce protein content but improve protein efficiency ratio (PER) and enhance the bioavailability of micronutrients by lowering antinutrients [14]. The application of finger millet in making rice balls and pearl millet in baking roti together with wheat was demonstrated by Seetha et al. [15]. Zainab et al. [16] stated that foxtail millet could be used to manufacture various food items such as bread, cakes, traditional foods, weaning foods, popped, extruded, roller-dried, flaked products, and noodles. The foxtail millet rice can be used instead of rice in the preparation of all the traditional products like bisibele bath, chakkali, pongal, dosa, idli, and laddus. The prebiotic-like effects of dietary fibers found in millets are among the significant advantages of millet-based food products [17]. The development of a consumable food product incorporating synbiotics based on foxtail millet involves multiple food processing stages. Ensuring the viability of probiotics throughout the various stages of food processing, transportation, and storage presents a significant challenge in the development of millet-based synbiotics.

Encapsulation is a mechanical or physicochemical process mainly used to increase the viability of bacteria by adding a protective agent. It is mainly used in food processing industries dealing with probiotics to increase the probiotic bacteria’s survival in various environmental conditions during storage [18]. The shell material is generally made up of polysaccharides, polymers, fats, and waxes. The selection of shell material is a critical factor that determines the stability of microorganisms present inside the core [19]. Polysaccharides such as alginate, pectin, gum arabic, and starch are used as the shell material to encapsulate probiotic organisms. Among these materials, gum arabic is widely used in the food industry. Gum arabic (acacia) is a complex mixture of polysaccharides and glycoproteins obtained from Acacia senegal. It is used in the food industry for various applications such as encapsulation, emulsification, gum candies, thickening agent, and confectionary treats. It is highly soluble in water and does not cause any intestinal problems when consumed [20].

In this study, a healthy probioticated foxtail millet laddu was formulated using response surface methodology (RSM). The optimized factors included the quantities of foxtail millet, ground nut, horse gram, and jaggery required to prepare the laddu. The quantities of other ingredients, such as ghee, honey, cardamom, cinnamon, probiotic culture (Lactobacillus acidophilus), and inulin, were kept constant. The responses for constructing the RSM model for laddu preparation were softness, appearance, taste, and solubility. After optimizing the selected factors, additional trials were carried out by adding probiotic bacteria in three forms: pure lyophilized culture, gum arabic-encapsulated lyophilized culture, and lyophilized curd. These trials aimed to determine the form that retained the highest viability of probiotics. Furthermore, the release kinetics of the microbe from laddu from the encapsulated matrix and the antioxidant properties of the laddu were studied. Antioxidants help control cell damage caused by various free radicals in the biological system. Free radicals, unfilled electrons, can induce cell damage and lead to human degenerative diseases. Foxtail millets contain approximately 71% of polyphenols, such as flavonoids, tannins, and phenolic acids, in free form, making them a good source of antioxidants. The present study utilized the edible millet-based South Indian laddu to capture and eradicate free radicals in the human body. The incorporation of suitable probiotic culture in foxtail millet laddu aims to enhance the immune system and provide antiradical activity.

Materials and methods

Formulation of foxtail laddus

The product was formulated using the following ingredients: foxtail millet, groundnut, horse gram, jaggery, ghee, honey, cardamom, and cinnamon. The ingredients were optimized and analyzed for the significant level with the help of Design Expert software version 7.0 (Stat-Ease Inc., USA). Among the ingredients used, foxtail millet, ground nut, horse gram, and jaggery were taken as input variables, while all other factors were kept constant. Four variables were analyzed using the Design Expert software, which generated 30 experiments. All 30 formulations of millet laddus were prepared in the laboratory for further analysis [21]. The various responses of products predicted the analysis. All the foxtail laddu was added with prebiotics of inulin (0.1%) to improve the growth of probiotics [22] and as encapsulating agents in the different formulations.

Collection and maintenance of strains

Pure lyophilized culture of probiotic microorganism Lactobacillus acidophilus NCIM 5306 was purchased from the National Collection of Industrial Microorganisms, Pune, India. Pure cultures were stored at 4 °C, and the strains were frequently sub-cultured in MRS media (procured from HiMedia Laboratories Pvt. Ltd., Mumbai, India) to maintain the viability of the culture for subsequent usage. The strains were inoculated in 100 ml of MRS media for mass production of Lactobacillus acidophilus strains, and it was incubated at 35 °C for 24 h to obtain the log phase cultures.

Probiotication and formulation of millet laddus

Millet laddus were prepared based on the optimized composition obtained from the Design Expert software. The millet and horse gram were washed and soaked in water overnight. The water was drained, and they were spread on a cloth-lined plate and allowed to cool. Then, the millet and horse gram were dry roasted in a heavy pan on a low flame for 6–7 min until their color changed to brown. Groundnuts were dry roasted and ground with whole cardamom and cinnamon to a fine powder. Melted jaggery, ghee, and honey were added gradually until the millet mixture came together. Finally, the lyophilized culture and inulin were added and mixed well. Small laddus were made from the mixture and stored in an airtight container for further analysis [23]. The final volume of laddu was found to be 150 g. Then, probiotics were added based on three different formulations to study the release kinetics of Lactobacillus acidophilus.

Addition of free form of lyophilized Lactobacillus acidophilus (formulation I).

The broth was freeze-dried using a lyophilizer (Penguin Classic Plus, LARK, India) to obtain the dried powder of the culture. The colony-forming units were analyzed in the dried powder by serial dilution followed by pour plate techniques [24]. The lyophilized culture of Lactobacillus acidophilus with a colony count of 8.52 ± 0.04 log CFU/g was added into the millet laddu in an appropriate proportion of 1 g/150 g of the laddu (Majeed et al., 2016). Thus, the ratio of the addition of lyophilized culture to the final product was 1:150. The final product was packed in closed containers and stored at 4 °C and room temperature.

Addition of microencapsulated Lactobacillus acidophilus (formulation II).

Gum arabic was used as the protectant material for the encapsulation of Lactobacillus acidophilus. Gum arabic and sucrose in the ratio of 8:2 are taken to form a total volume of 60 g and were solubilized in 200-ml distilled water at a temperature of 45 °C. The percentage of the carrier in water is 30%. The solution is stirred until the solution is cooled down to room temperature to obtain carrier media [25].

L. acidophilus was cultured in 100 ml of MRS broth and incubated for a period of 24 h at room temperature. After incubation, the culture broth was centrifuged at 6000 rpm for 5 min. The supernatant was discarded, and the pellet was washed with 50-ml phosphate-buffered saline and recentrifuged. The supernatant was discarded, the pellet was resuspended in carrier media, and the solution was homogenized for 30 min at room temperature [26]. The carrier media containing Lactobacillus acidophilus was lyophilized for 12 h to get a powder. The powdered samples were stored at 4 °C. Probiotic millet laddu was prepared by adding 1 g of the obtained powder into the optimized concentration of millet laddu obtained from the Design Expert software.

Addition of probiotics in the form of curd (formulation III)

Curd is one of the rich sources of natural probiotics, and it was freeze-dried for 10 h to get a fine powder [27]. Then, 1 g of dried natural curd powder containing probiotics was added to the foxtail laddu with the same proportion of 1 g/150 g.

Evaluation of the final products

Analysis and evaluation of the final products were done based on organoleptic, physical/chemical, and microbiological methods [28]. Organoleptic parameters used for analysis were appearance, taste, softness, and solubility [29]. The obtained statistical values were analyzed using Design Expert 7.0 to calculate the final product’s acceptability.

Nutritional analysis

Chemical parameters such as carbohydrates, proteins, fats, and other nutritional contents were analyzed using various experiments in the laboratory. The total carbohydrate, protein, cholesterol, and fat content were analyzed using anthrone, Lowry, Zak, and acid hydrolysis methods [30]. The total dietary fiber content was analyzed by the McCleary method. The total sodium, potassium, and calcium were estimated using a flame photometer (Model-381, Electronics India, Ltd., India).

Stability analysis

The products’ overall stability was also studied to determine the best product [21]. Microbiological parameters include analyzing the number of microbes during storage [31]. The prepared laddus were stored in closed containers at 4 °C and room temperature for 2 months. The total colony-forming units from both storage conditions were analyzed weekly during the storage period.

Analysis of bacterial release kinetics in encapsulation

The release kinetics of probiotic bacteria from the encapsulation was studied under two different storage conditions (4 °C and room temperature); 1 g of laddu from each storage period was taken and transferred into 10 ml of sterile saline solution (0.9% NaCl), and it was shaken for 1 h at 30 °C. The total amount of bacteria released into the solution was determined by the plate count method on MRS agar [32]. The experiments were conducted in triplets to monitor the release kinetics.

Evaluation of the antioxidant activity of formulated laddu

The following methods carried out the determination of the antioxidant activity of the formulated laddu; 1 mg/ml of each probioticated laddu formulation was taken to analyze antioxidant assays. DPPH radical scavenging activity was determined for three probioticated laddus formulations using the protocol outlined by Khalaf et al. [33]. Millet and probiotics were kept as a control. Ascorbic acid was used as a standard. FRAP assay was done using a method described by Oyaizu et al. [34]. The scavenging activity of hydrogen peroxide was determined using a method stated by Ruch et al. [35]. The total phenolic content of the formulated laddus was determined using Folin–Ciocalteau reagent with gallic acid serving as a standard [36, 37].

Results and discussion

Formulation of organic millet laddus

The ingredients used to formulate foxtail millet laddu include foxtail millet, ground nut, horse gram, jaggery, ghee, honey, cardamom, and cinnamon. The final product composition was analyzed using Design Expert software, which generated 30 experiments using four variables: foxtail millet, ground nut, horse gram, and jaggery. The response values are shown in Table 1. Each experiment 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 organoleptic parameters such as appearance, taste, softness, and solubility. Statistical values and analysis of variance were represented in Supplementary Tables 1, 2, 3, and 4. The results obtained from the experiments conducted on organoleptic properties are represented in 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 grams (g)

Table 2.

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	Lyophilized culture (g)	1
10	Inulin (g)	0.1

Interaction of various ingredients against the response factor by two factorial interactions

The present study revolves around optimizing the medium composition using Design Expert software. The optimization mainly analyzes the interaction between the ingredients used and the product’s response based on the softness, appearance, solubility, and taste. The use of RSM has resulted in a better understanding of the possible interaction between various ingredients used in the product. The significant interaction between variables has improved the product’s total acceptability, as in the case of Pranav et al. [38]. In the study by Lungmann et al. [39], the mixture design predicted as optimized media by Design Expert was superior to other media in terms of the final result. Similarly, the optimum result from the design expert software for laddu production is preferable over other compositions.

Figure 1a represents the analysis of taste performed against foxtail millet and horse gram. The analysis found that an equal proportion of foxtail millet and horse gram was required to enhance the taste of the laddu. Figure 1b found no notable interaction between ghee and foxtail millet in the event of enhancing the taste. Figure 1c,d found that a specific interaction between jaggery and horse gram and ground nut and jaggery is required to enhance the taste. However, it need not be an equal proportion, but a slight range nearer to the midpoint is the best.

Fig. 1.

Interaction of various ingredients against the response factor by two factorial interactions: (a) foxtail millet and horse gram, (b) ghee and foxtail millet, (c) jaggery and horse gram, and (d) jaggery and ground nut  

The final predicted composition of the laddus based on analyzing all the results is given in Table 2. The final predicted composition from the software was considered the optimum concentration, and the same concentration was used for further experiments. The products gave a good impression which was inferred from the experimental response values, and the results observed are in agreement with the study done by Aboulfazli [40], where the sensory analysis test was based on attributes such as color, texture, flavor, and taste and none of the product was reported as poor and all the products had a good impact on the review panel. The equations connecting the individual response with the independent variable are given below.

$$\mathbf{S}\mathbf{o}\mathbf{f}\mathbf{t}\mathbf{n}\mathbf{e}\mathbf{s}\mathbf{s}=-1.76792+0.158354\ast \mathrm{Factor}1+0.436875\ast \mathrm{Factor}2+0.736458\ast \mathrm{Factor}3-0.131313\ast \mathrm{Factor}4+0.001181\ast \mathrm{Factor}1\ast \mathrm{Factor}2+0.001281\ast \mathrm{Factor}1\ast \mathrm{Factor}3+0.002178\ast \mathrm{Factor}1\ast \mathrm{Factor}4-0.033687\ast \mathrm{Factor}2\ast \mathrm{Factor}3+0.006381\ast \mathrm{Factor}2\ast \mathrm{Factor}4-0.003469\ast \mathrm{Factor}3\ast \mathrm{Factor}4-0.003252\ast \mathrm{Factor}{1}^2-0.008296\ast \mathrm{Factor}{2}^2+0.012292\ast \mathrm{Factor}{3}^2-0.001190\ast \mathrm{Factor}{4}^2$$ 1

$$\mathbf{A}\mathbf{p}\mathbf{p}\mathbf{e}\mathbf{a}\mathbf{r}\mathbf{a}\mathbf{n}\mathbf{c}\mathbf{e}=+8.42083+0.073958\ast \mathrm{Factor}1-0.161250\ast \mathrm{Factor}2-2.39375\ast \mathrm{Factor}3+0.348125\ast \mathrm{Factor}4+0.001531\ast \mathrm{Factor}1\ast \mathrm{Factor}2+0.015156\ast \mathrm{Factor}1\ast \mathrm{Factor}3+0.000359\ast \mathrm{Factor}1\ast \mathrm{Factor}4-0.007187\ast \mathrm{Factor}2\ast \mathrm{Factor}3-0.002906\ast \mathrm{Factor}2\ast \mathrm{Factor}4-0.006406\ast \mathrm{Factor}3\ast \mathrm{Factor}4-0.002148\ast \mathrm{Factor}{1}^2+0.005156\ast \mathrm{Factor}{2}^2+0.107031\ast \mathrm{Factor}{3}^2-0.002086\ast \mathrm{Factor}{4}^2$$ 2

$$\mathbf{T}\mathbf{a}\mathbf{s}\mathbf{t}\mathbf{e}=+38.17433-0.531521\ast \mathrm{Factor}1+0.113625\ast \mathrm{Factor}2-3.26479\ast \mathrm{Factor}3-0.078437\ast \mathrm{Factor}4+0.003119\ast \mathrm{Factor}1\ast \mathrm{Factor}2+0.050656\ast \mathrm{Factor}1\ast \mathrm{Factor}3-0.000747\ast \mathrm{Factor}1\ast \mathrm{Factor}4-0.026188\ast \mathrm{Factor}2\ast \mathrm{Factor}3+0.000256\ast \mathrm{Factor}2\ast \mathrm{Factor}4+0.013719\ast \mathrm{Factor}3\ast \mathrm{Factor}4$$ 3

$$\mathbf{S}\mathbf{o}\mathbf{l}\mathbf{u}\mathbf{b}\mathbf{i}\mathbf{l}\mathbf{i}\mathbf{t}\mathbf{y}=-42.30867+0.083854\ast \mathrm{Factor}1+0.585875\ast \mathrm{Factor}2+3.94229\ast \mathrm{Factor}3+0.436687\ast \mathrm{Factor}4-0.000669\ast \mathrm{Factor}1\ast \mathrm{Factor}2+0.002906\ast \mathrm{Factor}1\ast \mathrm{Factor}3-0.001022\ast \mathrm{Factor}1\ast \mathrm{Factor}4-0.055187\ast \mathrm{Factor}2\ast \mathrm{Factor}3-0.000269\ast \mathrm{Factor}2\ast \mathrm{Factor}4-0.032594\ast \mathrm{Factor}3\ast \mathrm{Factor}4$$ 4

Response surface plots on different evaluation parameters like softness, taste, appearance, and solubility are shown in Figs. 2, 3, 4, and 5. ANOVA statistical analysis for four factors was calculated by Design Expert software in Supplementary Tables 1, 2, 3, and 4, and it was found to be a significant model. A quadratic equation for predicting the optimum point was obtained according to the CCRD design. A desirability value of 0.7 was obtained using the CCRD model of RSM. The R² value was found to be 0.998, and a P-value of < 0.0001 was obtained, which indicated a better agreement between the actual and predicted values of the response and confirmed the significance of the mathematical model constructed.

Fig. 2.

Response surface plot showing the effects on the softness of probiotic millet laddus

Fig. 3.

Response surface plot showing the effects on the appearance of probiotic millet laddus

Fig. 4.

Response surface plot showing the effects on the taste of probiotic millet laddus

Fig. 5.

Response surface plot showing the effects on solubility of probiotic millet laddus

Probiotication of millet laddus

The final composition of the prepared laddu is given in Table 2. The inference from the table depicts that the main nutritional content of the product comes from the four main ingredients that were taken as variables in the Design Expert software, and the other ingredients serve as supplements that either enhance or improve the final product's quality.

Addition of lyophilized Lactobacillus acidophilus

Millet laddus were prepared in the composition as mentioned in Table 2; 1 g of lyophilized culture was added, and different formulations of laddus were prepared for evaluation of nutritional and stability analysis as Fig. 7; 1 g of the lyophilized sample of Lactobacillus acidophilus was added to 150 g of the probiotic millet laddu, and the final concentration of the culture in 1 g of the probiotic millet laddu was found to be 10.52 ± 0.04 log CFU/g.

Fig. 7.

Different formulations of millet laddus for evaluation of nutritional and stability analysis

Addition of microencapsulated Lactobacillus acidophilus

To the millet laddu composition mentioned in Table 2, 1 g of Lactobacillus acidophilus encapsulated with gum arabic was added, and 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.

Addition of probiotics in the form of curd

In the third method, probiotics in the form of curd were directly incorporated into the final product. The total colony count of curd-incorporated laddu was 10.34 ± 0.03 log CFU/g.

Evaluation of the prepared laddu against nutritional and stability

The final product was analyzed and evaluated based on organoleptic, physical/chemical, and microbiological methods.

Nutritional analysis

The nutritional content of the four different kinds of laddu, including conventional laddu, is given in Table 3. The formulated millet laddus contained major nutritional components that could satisfy all the requirements for a healthy diet. The appreciable quantity of protein and minerals found in the product could increase the nutritional value of the product. The conventional laddu showed a very high content of carbohydrates and cholesterol of 101.44 g and 30.23 g, respectively, whereas other important nutritions were presented in very few quantities when compared to foxtail millet laddu. The overall mean among the nutrient composition of the three different formulations of laddus was found to be 18.196. Lizia and John [30] developed millet-based high-fiber biscuits, and their nutritional properties were analyzed. The total protein, carbohydrate, fat, and fiber content of the high-fiber biscuit was tested, and it was found to be 8.62 g, 68.05 g, 13.10 g, and 19.2 g/100 g of the product, whereas the probiotic millet laddu has nearly the same nutritional composition as that of high-fiber biscuits except for the fiber content. The millet laddu’s total protein, carbohydrate, fat, and fiber content was 11.133 g, 64.466 g, 15.133 g, and 5.06 g/100 g of the product. The amino acid profile is balanced, and the dietary fiber content is very high compared to other cereals. The nutritional composition of Foxtail millet per 100 g is fat (4.3 g), minerals (3 g), protein (12.3 g), calcium (31 mg), carbohydrate (60.9 g), phosphorous (290 mg), and dietary fiber (14 g) [16]. The nutritional analysis of the pearl millet laddu showed that the moisture content of 12.6 ± 0.2%, the protein content of 9.9 ± 2.8 g, the fiber content of 2 ± 2.6 g, the fat content of 4.2 ± 0.5 g, and the carbohydrate content of 69 g which was slightly similar to that of Uttara et al., 2017. The millet laddu’s total protein, carbohydrate, and fat content were similar to the fiber biscuits, with little variation in the acceptable composition. Verma et al. [41] explained that the foxtail and barnyard millet laddu had a comparatively high protein content of 5.00% and 3.41%, respectively, and low carbohydrate content in both foxtail and barnyard millet-based laddu.

Table 3.

Analysis of the 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
Fiber	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: 150 g of the laddu

Stability analysis

The laddu incorporated with the lyophilized culture of Lactobacillus acidophilus was analyzed 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 two months during the two different storage conditions. During the initial storage period, the total CFU in both conditions was the same, and it was around 8.20 ± 0.1 log CFU/g, but once the storage time increased, there was a significant decrease in the colony count. On storing the product for 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 the two storage conditions. In the final 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 lyophilized Lactobacillus acidophilus culture during the storage period of two months was explained in 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 freeze-dried product’s viability compared to storing the product at room temperature. On storing the product at 4 °C and room temperature in a closed container, a high survival rate of the probiotic microorganism was observed at 4 °C. The result observed shows many similarities to Chen et al. [21] when storing product at 4 °C and 25 °C in a laminated pouch and glass bottle; the best result was obtained when the product was stored at 4 °C in a glass bottle.

Fig. 6.

Effect of storage time on (a) lyophilized Lactobacillus acidophilus-incorporated laddu and (b) microencapsulated Lactobacillus acidophilus-incorporated laddu

The laddu incorporated with microencapsulated Lactobacillus acidophilus was also analyzed at both storage conditions. The total colony count was analyzed by studying the release kinetics of encapsulated bacteria at these storage conditions and is represented in Fig. 6. The initial bacterial count in microcarrier-incorporated laddu was 9.97 ± 0.03 log CFU/g. The study on the 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 [42], where alginate-chitosan-encapsulated Lactobacillus acidophilus showed a high number of viable 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 those incorporated only with lyophilized Lactobacillus acidophilus. The result revealed that encapsulation of Lactobacillus acidophilus using gum arabic before lyophilization protects the organism during storage. It is inferred that the total amount of bacteria retained in the laddu can be protected by microencapsulation before lyophilization. Thus, instead of simple lyophilization, microencapsulation of Lactobacillus acidophilus before lyophilization could aid in the protection of bacteria from various stresses during the storage period and possibly increase the life span of incorporated probiotics. The free cells were counted as 6.4 × 10⁷ at first and 4.6 × 10⁴ after the last day of storage. In encapsulated form, the count was 6.8 × 10⁷ at first and 5.7 × 10⁵ after the last day of storage. Microencapsulation is protective, and the bacterial strains survive longer than the cells without encapsulation (Fig. 7) [43].

Antioxidant activity

The result of formulated probioticated laddu was analyzed for various antioxidant capacities. DPPH radical activity revealed that probioticated laddu showed a moderate capacity to capture hydrogen free radicals, as listed in Table 4. A 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 largely due to the slow release of microbes from the encapsulation matrix and phenolic substances present in the bacteria and foxtail millet. Ferric reducing anion power assay has been carried out to identify the potential of reducing radical ions’ capacity to neutralize. A similar trend was obtained for the FRAP assay. The highest reduction capacity absorption was exhibited in encapsulated probiotic laddu as 23.78 ± 0.43%. H₂O₂ assay was performed to analyze the capturing ability of hydroxyl ions in which curd-incorporated laddu showed much potential, followed by encapsulated and lyophilized probiotics. The total phenolic content of all the formulations was studied to predict their correlation. A strong correlation was observed between antioxidant capacity and phenolic substances in the laddu, indicating that phenolic content is much more responsible for producing antioxidant activity. Since the laddu is edible, the main focus is on the taste. Therefore, all ingredients were analyzed to test their effect on the taste using two factorial interaction methods of Design Expert software. The products gave a good impression which was inferred from the experimental response values. The results observed are in good agreement with Aboulfazli et al. [40], where the sensory analysis test was based on attributes such as color, texture, flavor, and taste, and none of the products was reported as poor and all the products had a good impact on the review panel. Similarly, Savita et al. [8] have shown that the major free phenolic acid among various millet varieties is protocatechuic acid (45 mg/100 g). Bello et al. [44] reported that among the millet grains evaluated, barnyard and finger Italian millet exhibited the highest DPPH radical scavenging activity of 359.6 µg/mL and 436.25 µg/mL, respectively.

Table 4.

Antioxidant activity of various formulations 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

During the storage period, the laddus incorporated with curd showed the least stability and were highly susceptible to contamination as they developed white patches on the surface during both 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 contain more colony-forming units of microorganisms than the recommended dose of probiotics, they are not recommended for the probiotication of food as they might affect consumer health. Yuvarani and Anitha [45] proposed the same and observed that the cooked multigrain laddu contains 618 µg/100 g of antioxidant content. The raw ingredients which were used to prepare multigrain laddu and their antioxidant level are as follows: finger millet = 268 µg/100 g, foxtail millet = 340 µg/100 g, wheat = 197 µg/100 g, cardamom powder = 182 µg/100 g, ghee = 80.1 µg/100 g, horse gram sprouted and green gram sprouted together = 662 µg/100 g, and jaggery = 115 µg/100 g.

Conclusions

Foxtail millet and Lactobacillus acidophilus have a very good potential to have a synergistic beneficial effect to serve as a wholesome food. As the millet is rich in fiber, catechin, and quercetin, it controls cholesterol and blood sugar levels and boosts liver and kidney function. The probiotic bacteria incorporated in foxtail millet have a good potential to serve as a functional food. Thus, the probiotic millet laddu might serve as a value-added food product for the consumption of the public and might improve the public’s general health. The physical evaluation of the formulated food product showed superior softness, appearance, taste, and solubility at 7.769, 5.742, 5.729, and 5.583, respectively. It has exhibited that better physical evaluation was attained at a concentration of 42.99 g of foxtail millet, 37.49 g of groundnut, 10.30 g of horse gram, and 60.03 g of jaggery. It was identified that the four variables demonstrated a notable effect on the formation of antioxidant-rich probiotic laddu using foxtail millet and have noticed positive relationships with all the variables. The mathematical model with an R² value of (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. The microencapsulated L. acidophilus-incorporated laddu indicated the outstanding stability of 7.43 ± 0.02 log CFU/g at 4 °C compared to other methods of probiotic addition. The DPPH radical activity affirmed the potential antioxidant activity of 25.39 ± 0.47% at the microencapsulation method. Thus, the present study concluded that probioticated laddu has more potential to be used in traditional food products. It can be produced on a large scale for commercial sales as a probiotic snack, which also abides by the current trend of the health-conscious society of switching to a balanced nutritional food diet. Enhancements can be implemented to refine the developed laddu’s flavor profile, texture, and appearance to cater to individual preferences and tastes.

Supplementary information

Below is the link to the electronic supplementary material.

Author contribution

Rubavathi Subbaiyan: formal analysis and writing – original draft; Ayyappadasan Ganesan: supervision, writing – review and editing; Venkatramanan Varadharaj: validation, writing – review and editing; Philip Robinson Jeyachandran: methodology, writing – review and editing; Harini Thangavel – construction of figures.

Data availability

The raw reads generated in this study were discussed in the “Results and discussion” section and supplementary tables.

Declarations

Ethics approval

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Conflict of interest

The authors declare no competing interests.