With the emergence of fortified foods, there is a worldwide increase in health awareness and interest in adding herbs as prized food additives in dairy and food products. Due to the increased consumer awareness and interest to follow healthy nutrition and dietary strategy in achieving health benefits from foods beyond their basic nutrition, the market for value added functional foods has expanded [1]. Herbs have multifarious role such as food flavorings, preservative and medicinal ingredients and various herbs are documented for their therapeutic properties such as, anti-oxidative, anti-hypertensive, anti-inflammatory, anti-diabetic and anti-microbial properties [2].

Some herbs that have been used in the past are Moringa plant (Moringa oleifera) is used to improve weight in malnourished children by adding Moringa powder to food. Drinking of Moringa tea relieves headache, heart burns and gastritis. Yoghurt is one of the most popular fermented milk products worldwide and has gained widespread consumer acceptance as a healthy food. It provides an array of nutrients in significant amounts, in relation to its energy and fat content, making it a nutrient-dense food. In particular, yoghurt could provide the body with significant amounts of calcium in a bioavailable form. Furthermore, yoghurt has many health benefits beyond the basic nutrition it provides, such as improved lactose tolerance, a possible role in body weight and fat loss, and a variety of health attributes associated with probiotic bacteria [3]. Yoghurt is an excellent source of protein, calcium, iodine, phosphorus, riboflavin (vitamin), thiamin (vitamin) and vitamin, and a valuable source of folate, niacin, magnesium and zinc. Yoghurt provides many of the nutrients needed for optimal bone health such as calcium, protein, magnesium, zinc and phosphorus.

Hibiscus sabdariffa commonly named as “red sorrel” or “Roselle” is a member of malvaceae family. Different extracts from Roselle play a crucial role in treating different medical problems including many cardiovascular disorders, helmenthic disease and cancer. The plant also acts as an anti-oxidant and used in obesity management [4]. Moringa leaves contain seven times the vitamin C of oranges, four times the vitamin A of carrots, four times the calcium of milk, three times the potassium of bananas and two times the protein in yoghurt.

Cymbopogon citratus commonly known as “Lemon grass” or “fever grass” is an aromatic perennial tall grass with rhizomes and densely tufted fibrous roots. Nutritionally, lemon grass is a good source of vitamins A and C, folic acid, magnesium, zinc, copper, iron, potassium, calcium and manganese. Lemon grass has been used as a food ingredient, in cosmetics and as folk medicines in several regions of the world. Yoghurt from cow’s milk is lacking in certain phytochemicals such as carotenoids, flavonoids, anthocyanins, isothiocyanates, saponins, tannins, lutein and zeaxanthin, that play functional roles in the body. The lack of these phytochemicals has contributed to certain disease conditions such as cancer, heart disease, inflammation, ulcers and diabetes. The main aim of this study was to produce and evaluate yoghurt formulated with aqueous extracts of Roselle calyx (Hibiscus sabdariffa), Moringa leaves (Moringa oleifera) and Lemon grass leaves (Cymbopogon citratus). Therefore, the addition of herbs would add phytochemicals to the yoghurt to make a functional yoghurt. These phytochemicals would improve the nutritional status of the yoghurt and when taken into the body improves one’s immune system. The use of indigenous herbs would reduce the cost of production as they are readily available.

2.  Materials and methods

2.1 Raw materials

The raw materials were whole milk powder, granulated sugar, stabilizer (CMC), yoghurt culture (Lactobacillus bulgaricus and Streptococcus thermophilus), dried Roselle calyx, fresh Moringa leaves and fresh Lemon grass leaves.

2.1.1  Sample procurement

The whole milk powder, granulated sugar, stabilizer (CMC) and dried Roselle calyx were procured from Ogige market, Nsukka, Enugu State. The fresh Moringa leaves and fresh lemon grass leaves were obtained from farms. These leaves were authenticated by the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka. The yoghurt culture was purchased from the International Bakery and Confectionary market, Ogidi/Ogwunike Onitsha Metropolis, Anambra State.

2.2 Sample Preparation

2.2.1  Processing of aqueous Roselle leaf drink (Hibiscus sabdariffa)

Sixty grams of dried Roselle calyces were cleaned by picking off dirt, foreign matters and washed with cold water. The calyces were boiled in 300mL of water for 30 minutes to ensure proper extraction of the juice from the calyces. The juice was filtered using a sieve to remove the Roselle calyces from the aqueous extract [5]. The production of the Roselle juice is summarized in Scheme 1.

Scheme 1. Processing of Roselle calyx extract [5]      

2.2.2  Processing of aqueous Moringa leaf (Moringa oleifera)

Sixty grams of fresh Moringa leaves was boiled with 300mL of water for 20 minutes at 80̊ C and cooled for 10 minutes. The extract was separated from the leaves using a sterile muslin cloth and filtered through a sterile Whatman filter paper [6]. The production of Moringa leaf extract is summarized in Scheme 2.

Scheme 2. Processing of Moringa leaf extract [6]

2.2.3  Processing of Lemon grass (Cymbopogon citratus) aqueous extract

Sixty grams of the leaves were washed with clean water, cut into pieces, boiled with 300mL of water for 30 minutes, cooled for 10 minutes and sieved using a sterile muslin cloth to obtain the Lemon grass aqueous extract [7]. The production of Lemon grass aqueous extract is summarized in scheme 3.

Scheme 3. Processing of lemon grass aqueous extract [8]         

2.3 Production and formulation of yoghurt

Seventy-five grams of milk was mixed with 0.6g of stabilizer, 6g of sugar and 215mL of water as an aqueous medium for mixing. Yoghurt mixes were homogenized to obtain a uniform product. It was pasteurized at 80 ̊C for 30 minutes to destroy unwanted microorganisms. The product was cooled to 43 ± 2 ̊C and starter culture was added and sample was incubated for 4 -6 hours. The leaf extracts were added separately at a proportion of 5, 10 and 15%. The product was kept refrigerated at 5 ± 1 ̊C for further analysis [8]. There were ten samples coded as RCAE1, RCAE2, RCAE3, MLAE1, MLAE2, MLAE3, LGAE1, LGAE2, LCAE3, YWNE. Sample YWNE served as the control sample (Table 1). The pictorial representation of the formulated yoghurt samples (Plate 1-4) is shown in Fig 1.

Table 1. Formulation of herbal yoghurt using different concentrations of Roselle calyx, Moringa leaves and Lemongrass leaves (aqueous extract).

Figure 1. Pictorial representation of the formulated yoghurt samples (Plate 1-4)

2.4 Proximate analysis of the herbal yoghurt samples

The following proximate analysis was carried out on the formulated samples of the Roselle, Moringa and lemon grass flavoured yoghurt and the control.

2.4.1  Determination of moisture content

The moisture content of the samples was determined using the hot air oven method described by Association of Official Analytical Chemists [9].

2.4.2  Determination of crude fat

The fat content of the samples was determined using the standard method described by Association of official Analytical Chemists [9].

2.4.3  Determination of crude protein

The crude protein determination was achieved using the standard method (Kjeldahl method) described by Association of Official Analytical Chemist [9].

2.4.4  Determination of ash content

The ash content of each yoghurt sample was determined according to Association of Official Analytical Chemists [9].

2.4.5  Determination of carbohydrate

Total carbohydrate content was calculated by difference as follows:

% carbohydrate = 100 – (% moisture + % fat + % protein + % ash + % crude fibre).

2.5 Determination of micronutrient composition

2.5.1  Determination of vitamin A content

Vitamin A content was determined according to Association of Official Analytical Chemists method [9].

2.5.2  Determination of vitamin C content

Vitamin C content was determined using the Association of Official Analytical Chemists method [9].

2.5.3  Determination of phosphorous content

Phosphorous content in the sample was determined according to Onwuka by molybdate method using hydroquinone as a reducing agent [10].

2.5.4  Determination of calcium content

The calcium content was determined by the titration method according to Kirk and Sawyer [11].

2.6 Determination of phytochemicals 

2.6.1  Determination of flavonoids

Total flavonoids content was determined by a colorimetric assay using the Kapoor’s method [12].

2.6.2  Determination of saponins

Total saponins was determined using Kapoor method [12].

2.6.3  Determination of tannins

Total tannins were determined using Kapoor method [12].

2.7 Microbial analysis

2.7.1  Determination of lactic acid bacteria (LAB)

The lactic acid bacteria in the formulated yoghurt was determined using deMan Rogosa Sharpe (MRS) Agar (CM 361) as described by Oxoid manual. Samples were serially diluted in duplicates using the surface pour plate method. The plates were incubated under anaerobic conditions at 37̊ C for 48 hours. After incubation, the number of colonies were counted using the colony counter and represented as colony forming unit per millilitre (cfu/ml) [13].

No of colonies (cfu/ml) = average count × dilution factor (D.F)

2.7.2  Determination of total viable count (TVC)

The total viable count test was carried out using the method described by Prescott [14]. Using sample and sterilized quarter strength ringer solution as diluents, one millilitre (1 mL) of the water sample was pipetted into a sterile test tube and 9 ml ringer solution pipetted into it and other test tubes arranged for serial dilutions (10^-3). The diluted sample was pipetted into a marked petri dish and sterile nutrient agar of 20 mL was poured into the same petri dish and swirled to mix and incubated (under conditions that permit microbial reproduction so that colonies develop could be seen without the aid of a microscope at a temperature of 37 °C for 24 hours. After incubation, the number of colonies were counted and represented as colony forming unit per millilitre (cfu/ mL).

No of colonies (cfu/mL) = average count × dilution factor (D.F)

2.7.3  Determination of mould count

The mould count test was carried out using the method described by Prescott [14] and Sabouraud dextrose agar (SDA) was used as the media. Fifteen milliliters of the media was added to one gram of sample in the Petri dish and mixed. The media was allowed to settle before incubating at 37 ⁰C for 48 hours. After incubation, the number of colonies were counted and represented as colony forming unit per millilitre (cfu/mL).

No of colonies (cfu/mL) = average count × dilution factor (D.F)

2.8 Titratable Acidity (TTA)

The titratable acidity was determined according to Association of Official Analytical Chemist Method [9].

2.9 pH

The pH of the samples was measured in a 10% (w/v) dispersion of the samples in distilled water. Each suspension was mixed thoroughly and a standard pH meter (Hanna meter model H196107) was used for pH determination. The pH electrode was dipped into mixture and reading taken [9].

2.10   Sensory evaluation

The sensory properties of samples were evaluated by a semi-trained panel consisting of 20 panelists (including students and staff in the Department of Food Science and Technology, University of Nigeria, Nsukka). A 9-point Hedonic scale was used to assess the formulated herbal yoghurt, 0 = like extremely and 1 = dislike extremely. The samples were served in plastic cups and randomly presented. A questionnaire comprising of the five sensory attributes namely, color, taste, aftertaste, flavor, texture, consistency and overall acceptability was given to each panelist [15].

2.11   Experimental design and data analysis

The experiment was laid on Completely Randomized Design (CRD). Data obtained was subjected to statistical analysis. The analysis of variance (ANOVA) tests were carried out by using the general linear model procedure of the SPSS (Version 23.0). Means were separated by Duncan’s new multiple range test (DNMRT). Significance was accepted at p<0.05 [26].

3.    Results and discussion

3.1 Proximate composition of the yoghurt samples formulated with Roselle calyx, Moringa leaf and Lemon grass (aqueous extract)

3.1.1 Moisture content

The moisture content of the flavored yoghurt samples ranged from 70.80 % for MLAE1 to 76.92 % for LGAE3. There showed a significant difference (p<0.05) between the flavored yoghurt samples and the sample YWNE which was plain and served as control sample. The difference in moisture increased as the concentration of the extracts increased. The increase in moisture could be attributed to the increasing water content of the aqueous extracts [16]

3.1.2 Ash content

The ash content of the samples ranged from 0.20 % for LGAE3 to 0.85 % to RCAE1. There showed a significant difference (p<0.05) between the flavoured samples and the control sample. The ash content of the flavoured yoghurts decreased as the concentration of the extract increased. This implies that increase in the concentration of the extracts did not contribute to the increase in the solid residues in the yoghurt samples. The ash content was similar to the report by Salisu [17].

3.1.3  Crude fat

The fat content ranged from 12.97 % for RCAE3 to 15.83 % for MLAE2. There showed no significant difference (p>0.05) between the flavoured yoghurt samples and sample YWNE. This could be attributed to the low fat content of Roselle calyx, Moringa and Lemon grass fresh leaves as with the report of Lakshmipriya [18].

3.1.4  Protein content

The protein content ranged from 5.11 % for YWNE to 5.59 % for MLAE2. There was no significant difference (p>0.05) between the flavoured yoghurt samples and sample YWNE. This could be attributed to the low protein content of Roselle calyx, Moringa and Lemon grass fresh leaves as to the report of Lakshmipriya [18].

3.1.5  Carbohydrate content

The carbohydrate content ranged from 3.84 % for LGAE2 to 7.51 % for MLAE1. There was no significant difference (p>0.05) between the flavoured yoghurt samples and sample YWNE. This could be due to the small amount (5, 10 and 15 ml) of Roselle calyx, Moringa and Lemon grass aqueous extracts added [16]. The proximate compositions are shown in Fig. 2.

Figure 2. Graphical representation of the proximate composition of the formulated yoghurt samples. (Values are means ± standard deviation of triplicate determinations. Means with different superscripts in the same column are significantly different at p˂ 0.05).

3.2 Selected micronutrient composition of the yoghurt samples formulated with Roselle calyx, Moringa leaf and Lemon grass (aqueous extract)

3.2.1  Vitamin C content

The vitamin C content of the yoghurt samples ranged from 1.06 mg/100g for YWNE to 3.60 mg/100g for LGAE3. There was a significant difference (p>0.05) between the control sample (YWNE) and the flavoured yoghurt samples. The increase in vitamin C content follows a simultaneous increase in the concentration of the aqueous extracts. This is due to Roselle calyx, Moringa and Lemon grass leaves already containing a high vitamin C content as reported by Martha [19].

3.2.2  Vitamin A content

The vitamin A content ranged from 0.19 mg/100g for MLAE2 to 0.26 mg/100g for RCAE3. There was no significant difference (p>0.05) between the control sample (YWNE) and the flavoured samples except for sample RCAE3 which increased the vitamin A content of the yoghurt at Roselle calyx extract concentration of 15 ml. This could be due to increase in the extract at 15 ml contributing to the increase in the vitamin A content of the yoghurt as reported by Fasoyiro [20].

3.2.3  Phosphorous content

The phosphorous content of the yoghurt samples ranged from 174.59 mg/100g for YWNE to 588.41 mg/100g. There was a significant difference (p<0.05) between the control sample and the flavoured yoghurts. The increase in the concentration of the aqueous extracts led to the increase in the phosphorous content of the flavoured yoghurt samples and this is due to the high phosphorus content of Roselle calyx, Moringa and Lemon grass leaves [18, 20].

3.2.4  Calcium content

The calcium content of the yoghurt samples ranged from 81.32 mg/100g for LGAE1 to 104.26 mg/100g for RCAE3. There was a significant difference (p<0.05) between the control sample and the flavoured yoghurts. The result shows that addition of aqueous extract with increasing concentration led to increase in the calcium content of the yoghurt samples. The values of the calcium content obtained agreed with the report of Tajidin [21]. The micronutrient composition of the yoghurt samples is shown in Fig. 3.

Figure 3. Graphical representation of the micronutrient of the formulated yoghurts. Values are means ± standard deviation of triplicate determinations. Means with different superscripts in the same column are significantly different at p ˂ 0.05)

3.3 Physicochemical composition of the yoghurt samples formulated with Roselle calyx, Moringa leaf and Lemon grass (aqueous extract)

3.3.1  Titratable acidity

The titratable acidity of the yoghurt samples ranged from 0.90 % for YWNE to 1.09 % for RCAE3. There was a significant difference (p<0.05) between the control sample and the flavoured yoghurt samples. The titratable acidity of the yoghurt samples was increasing with increase in the concentration of the aqueous extract [20].

3.3.2  pH

The pH of the yoghurt samples ranged from 3.75 for LGAE3 to 4.57 for YWNE. There was a significant difference (p<0.05) between the control sample and the flavoured yoghurt samples. The pH of the flavoured yoghurt samples decreased with increase in the concentration of the aqueous extract [19, 20]. The physicochemical composition of the yoghurt samples is shown in Fig. 4.

Figure 4. Graphical representation of the physicochemical composition of the formulated yoghurts. Values are means ± standard deviation of triplicate determinations. Means with different superscripts in the same column are significantly different at (p ˂ 0.05).

3.4 Physiochemical composition of the Roselle calyx, Moringa leaf and Lemon grass (aqueous extract)

3.4.1 Titratable acidity

The titratable acidity (Table 2) of Roselle calyx aqueous extract is 1.22 % showing the acidic level of the Roselle calyx, Moringa leaf is 0.64 % due to its alkaline nature and lemon grass is 0.41 % [17,19,20].

 3.4.2 pH

The pH (Table 2) of Roselle calyx aqueous extract is 3.88 due to the acidic level of Roselle calyx [20], Moringa leaf is 6.89 due to its alkaline nature and lemon grass is 5.92 [17, 19].

Table 2. Selected physiochemical composition of the Roselle calyx, Moringa leaf and Lemongrass leaf (aqueous extract).

3.5 Phytochemical composition of the yoghurt samples formulated with Roselle calyx, Moringa leaf and Lemon grass (aqueous extract)

3.5.1  Flavonoid content

The flavonoid content ranged from 0.17 mgQE/100g for YWNE to 8.76 mgQE/100g for RCAE3. There was no significant difference (p<0.05) between the control sample and flavoured yoghurt samples. This could be due to the amount (5, 10 and 15 ml) of Roselle calyx, Moringa and Lemon grass aqueous extracts added not been enough to cause a significant increase in the flavonoid content of the yoghurt samples [16].

 3.5.2 Tannin content

The tannin content ranged from 0.02 mgTA/100g for YWNE to 0.97 mgTA/100g for RCAE3. There was a significant difference (p<0.05) between the control sample and the flavoured yoghurt samples. The tannin content of the yoghurt formulated with Roselle calyx extract increased with increase in concentration of extract and this could be related to Roselle calyx containing non-hydrolyzable tannin, namely catechin [17]. Also, the tannin content of the yoghurt formulated with Moringa leaf and Lemon grass leaf aqueous extract was decreasing with increase in the concentration of the extracts and this could be due to Moringa and Lemon grass leaf containing hydrolysable tannin, namely tannic acid, which gets easily hydrolysed in water [17].

3.5.3  Saponin content

The saponin content ranged from 0.05 mg/g for YWNE to 4.83 mg/g for MLAE3. There was a significant difference (p<0.05) between the control sample and the flavoured yoghurt samples. The saponin content of the yoghurt samples was increasing with increase in concentration of the extracts and this is due to the saponin content of Roselle calyx, Moringa leaf and Lemon grass leaf [18].  The phytochemical composition of the yoghurt samples is shown in Fig. 5.

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