Comparative studies of yoghurt produced from animal and selected imitation vegetable milk

In this study, the possibility of producing yoghurt from both plant and animal sources was explored. Hence, a comparative study was carried out on the yoghurt samples that were produced from cow milk (CM), oat flakes (OM), and African yam beans (Sphenostylis stenocarpa) (AYB). The physicochemical and organoleptic properties, as well as the microbial stability of the yoghurt samples, were analyzed using standard methods. The proximate compositions (moisture, fat, protein, fibre, ash, and carbohydrate) of the samples were 85.50–86.45, 0.25–2.95, 5.42–6.10, 0.09–1.18, 0.94–0.88, and 3.56–7.43%, respectively. Meanwhile, their total solids ranged between 13.55 and 14.50 for the three yoghurt samples CM, OM, and AYB, respectively. The proximate results showed that all the chemical compositions, except their ash content, significantly (p < 0.05) differed in all the three samples. The ranges of the total viable bacteria count of the sample CM, OM, and AYB were 1.58–3.0 × 109, 1.95–4.0 × 108, and 5.50–1.1 × 109 cfu/mL during the 14 days of refrigeration storage, respectively. A decrease in the count was noticed on the third day thereby leading to the deterioration of the yoghurt samples and reduction in the composition with storage. The sensory evaluation results showed no significant differences (p ≤ 0.05) in the aroma, taste, and overall acceptability of the three yoghurt samples. This work has shown the potential of vegetable milk as an alternative source of milk. It is interesting to note that this plant-based yoghurt has longer shelf life and higher fiber content with improved compositional values and consumer’s acceptability than the animal-based yoghurt.


Introduction
Yoghurt is a coagulated or semi-solid milk product produced by souring or fermenting milk using pure starter cultures of Lactobacillus bulgaricus and Streptococcus thermophiles [3,15], Balogun, Arise, Kolawole and Mayowa, 2017). Yoghurt is among the most common dairy products consumed around the world [28], Rahmeh, Akbar, Kishk, Al-OnaiziAl-AzmiAl-Shatti, Shajan, Al-Mutairi and Akbar, 2019). This product now has a strong preference compared to fresh milk because of its better satisfying nature, in terms of its texture, flavour, and nutritional and health benefits [14]. For instance, cow milk is the largest source of animal milk in the world, due to its high availability and most commonly used milk for yoghurt production. Currently, alternatives are being considered from the plant source (vegetable milk) due to the cost and health issues associated with the increased consumption of cow milk or animal milk [9,14]. Vegetable milk, which is also known as analogous milk, could be obtained from seed or cereal soaked in water, ground, and filtered. Vegetable milk is at the forefront (as a substitute for cow milk) due to either the growing problem of lactose intolerance and allergies or to changes in food preference [11]. The alternatives for cow milk, which have increased the utilization of most of the underutilized legumes, were explored by most researchers to produce cheap and available milk and milk-like products to the teeming population. For instance, Bambara groundnut milk was used for the production of yoghurt either in isolation or mixed with milk from animal or other vegetable sources with resultant effect of improved nutritional contents and products of high consumers' acceptability (Bristone, BadauIgwebuike and Igwegbe, 2015, [9,14,29]. Oats (Avena sativa) is a cereal specially grown for its seeds utilization. The use of a fermentation process in combination with a new kind of tailored oat-based food rich in dietary fibre, to increase interest in oats as a base for new functional food products, could provide new food such as yoghurt-like products (Sumon Siri, Panjun, Naksuk, Boonmawat, Mukprasirt and Phasuthan, 2020). In the same vein, the acceptability of African Yam Bean (AYB) has been improved through fermentation by bacteria and fungi. The few fermented products developed from extracts of AYB included a cheese-like product, vegetable milk fermented with indigenous micro-organisms, and a yoghurt-like product [4]. A study revealed that probiotic drink produced from AYB exhibited in vivo probiotic effect on the experimental animals as evident in the prevention of the integrity of gastrointestinal tract (GIT) wall and the serum AST and ALT of the group of animals challenged with pathogenic E. coli [2]. Therefore, this study aimed to carry out a comparative study on different sources of milk (animal and plant sources), by looking for a better substitute/alternative to the animal source of milk for yoghurt production.

Source of materials
The fresh cow milk was obtained from the animal husbandry unit of the University of Ilorin, Ilorin, Nigeria. Other materials, such as the starter culture, African yam beans, oat flakes, stabilizer, and sweetener, were all purchased from Oja-tuntun, Ilorin, Kwara State, Nigeria.

Yoghurt production from cow milk
The yoghurt was produced according to the method previously described by Bristone et al. [12] with slight modification. Briefly, 100% fresh cow milk (1 L) was homogenized, pasteurized at 85 °C for 30 min and continuously mixed. The mixture was cooled and inoculated with starter cultures (Lactobacillus bulgaricus and Streptococcus thermophilus). 0.5 g of the culture was used to inoculate 1000 mL of milk to initiate fermentation and incubated at room temperature for 12 h. After incubation, sucrose was added to the yoghurt and stirred, after which the desired custard consistency was reached, packaged, and stored at 4 °C for 14 days (Fig. 1).

Production of imitated milk and yoghurt from oat flakes
Imitated milk was produced from oat flakes using method described by Mårtensson, Andersson, Andersson, Öste, and Holst [19]. Oats (100 g) were soaked in water for 15 min at room temperature. The soaking water was then discarded and the oat was blended in a Warring laboratory mill blender (HGBTWTS3, Torrington, CT, USA) with water in the ratio of 1:4 (oat to water) to form a smooth paste.
The mixture was strained with cheese cloth to obtain the imitated milk. The milk was cooled and inoculated with starter cultures (Lactobacillus bulgaricus and Streptococcus thermophilus). 0.5 g of the culture was used to inoculate 1000 mL of milk to initiate fermentation and incubated at room temperature for 12 h. After incubation, sucrose was added to the yoghurt and stirred, after which the desired custard consistency was reached, packaged, and stored at 4 °C for 14 days (Fig. 1).

Production of imitated milk and yoghurt from African yam beans
Imitated milk was produced from African yam beans using the method described by Adurotoye et al. [2] with slight modifications. The African yam bean seeds were sorted to remove extraneous materials. The seeds were soaked in water for 12 h, rinsed, and heated for 5 min at 100 °C and left to cool. The seeds were manually dehulled and blended in a Warring laboratory mill blender (HGBTWTS3, Torrington, CT, USA) with water in the ratio of 1:4 (seed:water) until a smooth slurry was obtained. The slurry was filtered through double folded cheesecloth to obtain the imitated milk. The imitated milk was cooled and inoculated with starter cultures (Lactobacillus bulgaricus and Streptococcus thermophilus). 0.5 g of the culture was used to inoculate 1000 mL of milk to initiate fermentation and incubated at room temperature for 12 h. After incubation, sucrose was added to the yoghurt and stirred, after which the desired custard consistency was reached, packaged, and stored at 4 °C for 14 days (Fig. 1).

Colour measurement
Colour measurement of the yoghurt samples was carried out using colour flex (A60-1014-593; Hunter Associates Laboratory, Reston, VA, USA) on the basis of lightness (L*), red-green (a*), and yellow-blue (b*) values. The instrument was calibrated against white and black colour tiles before colour measurement.

Determination of pH
A standard digital pH meter (Hanna instrument) was used. The pH meter was standardized using buffer solutions of pH 4.0 and 9.0. The pH electrode was dipped into the yoghurt, and after a few minutes of equilibration, the pH of the yoghurt sample was taken.

Determination of total solids
For each yoghurt sample, 20 mL was pipetted into already weighed Petri dishes. The samples plus the Petri dishes were then re-weighed. The samples were heated at 100 °C for 4 h in a hot air oven (S336RB, manufactured by carbolite parsons lane, Hope valley, England) until constant weights recorded. The dried samples with the Petri dishes were then cooled in a desiccator. The weight of the Petri dishes was then subtracted from the weight of the Petri dishes plus sample before and after drying and the total solid content expressed in percentage (AOAC 2012).

Determination of the viscosity
The viscosity of the yoghurt samples was determined using the method described by Salami et al. [29]. Briefly, 10% of each sample was suspended in distiled water and mechanically stirred for 20 min at room temperature (27 °C). Oswald type viscometer was used to measure the viscosity of the mixture.

Proximate composition of the yoghurt samples
The ash, fat, and moisture contents were determined using AOAC methods [6]. The protein content was determined by Kjeldahl method (N × 6.25). Total carbohydrate content was calculated by difference as expressed below: (1) Carbohydrate = 100 − (moisture + ash + fat + f ibre + protein)

Microbial analysis (total bacterial count and total fungal count)
Microbial analysis was carried out using the method of Balogun et al. [9]. Briefly, 1 mL of each sample was pipette aseptically into 9 ml of sterile distilled water in a test tube and shaken properly, a 10 -1 dilution. One milliliter was pipetted from the dilution 10 -1 into another test tube containing 9 ml of sterile distilled water to make a dilution of 10 -2 . This was repeated until a 10 -8 dilution was achieved. One milliliter each of 10 -8 of the different samples was dispensed into sterile Petri-dishes using sterile pipettes. The pour plate method was used for the microbial enumeration. Cooled molted sterile nutrient agar (for bacterial count) and potato dextrose agar (for fungi count) were added to cover the mixture in the Petri dishes and swirled. The plates were left for some minutes to solidify and later incubated at 37 °C for 24 h. Colonies were counted after incubation using a colony counter.

Sensory analysis
The sensory attributes (appearance, taste, aroma, crispiness, and overall acceptability) of the yoghurt samples were evaluated by a 50-member semi-trained panel (average age of 30 years, comprising 25 females and 25 males) drawn among the staff and students of University of Ilorin, Ilorin, Nigeria. The panelists were in good health and are familiar with the taste, flavour, and other attributes of yoghurt. The evaluation started at around 1 p.m. The coded yoghurt samples were prepared and served in sensory evaluation plates. White bulbs were fitted in the sensory room to detect the genuine colour of the sample presented to the panelists for assessment based on 9-point hedonic scale according to Ihekoronye and Ngoddy [17]. (1-dislike extremely, 2-dislike very much, 3-dislike moderately, 4-dislike slightly, 5-neither like nor dislike, 6-like slightly, 7-like moderately, 8-like very much, 9-like extremely). Potable water was provided for the panelists to rinse their mouth during taste assessment.

Statistical analysis
All experiments were conducted in triplicates except where it is stated otherwise. The data were subjected to one-way analysis of variance using Statistical Package for Social Sciences (SPSS, version 16.0) to obtain the mean values, which were separated using Duncan's multiple range rest (DMRT) at p < 0.05.

Colour quality of the yoghurt
Colour is considered one of the most important quality parameters in yoghurt production. The result presented in Table 1 revealed a significant difference (p < 0.05) in the lightness parameter of the samples, with sample from cow milk (CM) having the highest value (54.40 ± 041) followed by yoghurt from oat milk (OM) sample (50.98 ± 0.73) and African yam beans (AYB) sample having the least value (49.66 ± 0.34). Besides, a significant (p < 0.05) difference was also recorded in the redness parameter with sample OM having the highest value (3.71 ± 0.04) and sample AYB with the second value (3.53 ± 0.04) while sample CM had the least value of redness (2.90 ± 0.12). Unlike the first two parameters, there is no significant (p > 0.05) difference between sample OM (8.99 ± 0.69) and sample CM (8.51 ± 0.14) in the yellowness parameter whereas sample AYB (6.68 ± 0.18) is significantly lower when compared to samples CM and sample OM ( Table 1). The colour results obtained from this study showed that sample CM, which is produced from cow milk, had the typical yoghurt (light-white) colour. The implication of having a colour that is different from this could affect the marketability of the yoghurt. That is, when such yoghurt product deviated from the consumers' choice for yoghurt colours, which they were used to. Sample OM, which is the yoghurt produced from oat milk, had the closest value in terms of lightness, hence making oat seeds a close substitute to cow milk-based yoghurt when compared to sample AYB with least lightness value. The result of lightness obtained in this study is in line with the report of Salami et. al. (2020) in which yoghurt was produced from Bambara groundnut milk.

pH of the yoghurt
The pH is a measure of the hydrogen ion concentrations in any compound. Study [16] reported that the optimum pH of thick fermented milk coming into the market ranged from 3.27 to 4.59. It is interesting to note that the pH of the yoghurt samples (3.50-3.87) in this study, as presented in Table 2, fell into this optimum range. The pH values for the three samples are not significantly (p > 0.05) different from one another (Table 2). Notably, the lower pH affected the casein (milk protein), causing it to coagulate and precipitate, thereby forming the solid or thick curd that made up the yoghurt [14].

Total soluble solids of the yoghurt
The total soluble solids of the yoghurt samples from CM, OM, and AYB were in the range of 13.55 to 14.50% as presented in Table 2. Literature reported that the total soluble solid is the mass fraction of substances remaining after completion of the heating process [21]. It is important because it has effect on the viscosity of the yoghurt. Past finding had shown that an increase in the total solids led to an increase in viscosity, which was good for the rheological property of a yoghurt sample [14]. It is worthwhile to note that the current yoghurt samples produced from the vegetable sources (OM and AYB) had higher total solids than those produced from animal source (CM). The result obtained in this study is similar to the 12.4-14.50% previously reported for soy yoghurt [21].

Viscosity of the yoghurt
The viscosity of the yoghurt samples is shown in Table 2 and was between 11.45 and 18.40 mPa − ls. The yoghurt from CM had the highest value (18.40 mPa − ls), while AYB sample had the least value (11.45 mPa − ls). Obviously, viscosity is affected by the strength and number of bonds between casein micelles in yoghurt, as well as their structure and spatial distribution (Izadi et.al., 2015). The result obtained for sample CM (18.40 mPa − ls) was closely related to 19.40 mPa − s reported [29] for Bambara groundnut yoghurt. The viscosity of yoghurt has been found to be dependent on the lactic acid production. For instance, there existed a symbiotic or protocooperative relationship between Streptococcus thermophilus and Lactobacillus bulgaricus bacteria during yoghurt production. This is because the coagulation of milk proteins was induced by thermophilic bacteria (Streptococcus thermophilus and Lactobacillus bulgaricus), which propagated at high temperatures. Therefore, as the concentration of lactic acid increased, the proteins present in milk formed gel to give the end result as the viscous yogurt. Besides, the high protein content obtained for the CM sample (Table 3) might be responsible for its high viscosity ( Table 2).

Antioxidant activity of the yoghurt
Antioxidant is a molecule that inhibited the oxidation of other molecules caused by free radicals, for example, the DPPH radicals. Antioxidant activity of a dairy food is important both for the shelf life of the product as well as for protection from oxidative damage in the human body [7]. Free radicals are compounds that caused harm if their levels become too high in the body system. They have been linked to multiple illnesses, including diabetes, heart disease, and cancer. From Table 2, the DPPH radical scavenging activity of sample CM was significantly (p < 0.05) higher (27.99%) when compared to sample OM (23.25%) and sample AYB (20.69%), respectively. The hydrolysis of milk protein or organic acid production might have also contributed to the antioxidant activity of the yoghurt samples due to microbial metabolic activity during fermentation. For instance, a past study [13] reported a strong relationship between the high oxidative stability of yoghurt and the antioxidant peptides released during milk fermentation by Lactobacillus bulgaricus. Besides, oatmeal (OM) and African yam bean (AYB) have been widely known as good sources of phytochemicals, such as ß-glucan, flavonoids, anthocyanidins, and bioactive peptides, which have attributable antioxidative powers [18,24,27]. Hence, yoghurt from these two crops (OM and AYB) would contribute to fast, easy, and cheaper sources of antioxidants when compared to the expensive animal-based source, cow milk yoghurt.

Proximate composition of the yoghurt samples
The result of the proximate composition of the yoghurt samples is presented in  [27], respectively. Since typical yoghurt contained high moisture content, yoghurt samples from this study, therefore, complied with this standard. Although very high moisture content in yoghurt could affect the texture and mouth feel of the yoghurt but the high moisture content could also help to hydrate the body after consumption. Sample CM has the highest fat content (2.95%) while the result obtained for the present sample AYB was a bit higher than the 1.07% reported [1] for AYB produced yoghurt. The high fat content of the cow milk could be as a result of the cholesterol content of the milk compared to the vegetable milk yoghurts, but which was being a disadvantage since high cholesterol was not nutritionally good for the body. However, sample OM had the lowest fat content, which could not cause increase in the cholesterol level of the blood; hence, sample OM would be a perfect vegetable milk-yoghurt alternative for sample CM in order to help control the cholesterol level of the blood. In addition, past finding, Mårtensson et al. [19] reported that soluble fibre content and the beta-glucan in oats helped to lower the blood cholesterol level. 85.50 ± 0.03 b 1.25 ± 0.02 b 5.93 ± 0.00 a 0.09 ± 0.03 b 0.86 ± 0.00 b 6.37 ± 0.48 a Moreso, sample CM had the highest protein content, which was expected since CM is from animal source. However, the protein content contents for AYB and OM are in line with the ranges from previous findings [1,25], respectively. Sample AYB had the closest value to sample CM; hence, it could prove as a good alternative to animal source of milk for yoghurt production, most especially, to those who might be suffering from animal milk protein allergy or lactose intolerance. The sample AYB could also provide an easy source of protein to those suffering from malnutrition (kwashiorkor). The sample OM had the significant (p < 0.05) highest fiber content (1.18%) when compared to samples AYB and CM (with 0.09% and 0.02%), respectively. The high amount of fiber in sample OM could be as a result of its soluble fiber content (β-glucan), which helped in easy digestion and prevented colon cancer [25]. Moreover, the ß-glucans possessed the prebiotic function in the gastrointestinal tract by supporting the growth of beneficial microbial groups, whereby the slowly digestible fraction of oat starch moderated the glycemic response [5].
Ash is the inorganic residue remaining after the water and organic matter have been removed by heating in the presence of oxidizing agents, which provided a measure of the total amount of minerals within a food. The ash content of sample OM (0.88%) was not significantly different from sample CM (0.94%) and sample AYB (0.86%), respectively. The value obtained for sample OM in this study was a bit different from the 0.96% reported by Rashid et al. [27]. More so, the result recorded for sample AYB was closely related to 0.94% obtained by Aderinola and Olanrewaju [1]. However, the ash content (1.75%) reported by Bahareh and Mahastie (2008) was higher than the present values (0.86-0.94%) obtained from this current research work. Meanwhile, a high ash content implied that the inorganic (mineral) composition in the yoghurt was high as well. Though the highest ash content was found to be in sample CM (yoghurt made from cow milk), it was noticed that ash content value of sample OM was closed to that of sample CM; this simply meant that it could provide a good source of mineral component to the vegetarians.
There was a significant difference in the carbohydrate levels in each sample. For instance, the sample AYB was having the highest amount of carbohydrate (6.37%), while samples OM and CM had 6.12 and 3.56%, respectively. The result obtained for sample CM was a little different to the 4.56% reported by Bristone et al. [12], whereas the result recorded for sample AYB was different from the 9.09% reported by Aderinola and Olanrewaju [1]. The carbohydrate in plain yoghurt occurred mainly as a sugar called lactose (milk sugar) and galactose. However, the lactose content of yoghurt was lower than those in milk. This was because bacterial fermentation resulted in lactose breakdown. For instance, lactose broke down to form galactose and glucose; meanwhile, the glucose was mostly converted to lactic acid, the substance that contributed to the sour flavor of yoghurt and other fermented milk products (Sanders et al. 1998).

Total viable bacteria count
Total viable bacterial count was the most common microbiological test that gave a quantitative idea about the presence of microorganisms such as bacteria in a sample. Figure 2 shows the bacterial count results of the yoghurt samples stored at refrigeration temperature (4 °C). The result obtained on day zero of the storage showed that there was a significant difference (p < 0.05) between the yoghurt samples stored at 4 °C. For instance, the sample CM had the highest count 158 ± 0.30 cfu/ml, followed by sample AYB with 55 ± 0.60 cfu/ml and sample OM had the least count (19.50 ± 0.50 cfu/ml on the first day of storage). The high total viable count of the sample CM could be as a result of presence endogenous microorganism from the host cow. Meanwhile, the total viable bacteria count declined on day three (3) of the storage at 4 °C compared to the first day while a considerable increase was noticed on the seventh day of storage. A further decline in the total viable bacteria count also occurred on the fourteenth day of storage at 4 °C. The total bacterial count obtained from this study is similar to those reported by Aderinola and Olanrewaju [1], where decline was also experienced as the days of storage increased, which could be as a result of the low temperature storage. Panoff, Thamma, vongs, Guéguen, and Boutibonnes (1998) and Palova Charvat Masopust, Klapkova, and Kvapil (2007) have previously reported the probable cause of death of bacterial cells to be as a result of damage in cell membranes and DNA denaturation during low temperature storage. The result (Fig. 2) further showed that the sample OM, which was the yoghurt produced from oat milk, would have a longer shelf life compared with other yoghurt samples due to its least bacteria count after 14 days of refrigeration storage.

Total fungal count
The result of the total fungal count of the samples stored at 4 °C on day zero, day seven, and day fourteen is presented in Fig. 3. Sample CM was found to have the highest count of 78.50 ± 0.16 (cfu/ml). Meanwhile, a decline in the fungal count was also noticed as the storage days increased, which was similar to the result on the bacterial count (Fig. 4). The decrease in fungal population of the yoghurt could be as a result of the low storage temperature of the product. Past studies [23] had reported that the damage in the cell membranes and DNA denaturation were probable causes of bacterial cells death during low temperature storage. Contrarily, the implication of the high fungal count is short shelf life of the yoghurt. Hence, the sample OM has the tendency of longer shelf life (low fungal count) when compared to the other yoghurt samples.

Sensory attributes of the yoghurt samples
Interestingly, the sensory evaluation results revealed that the panelists generally accepted all the yoghurt samples as shown in Table 4. The current result is in line with the past reports that showed high consumers' acceptability of yoghurt produced from other vegetable sources such as soymilk [29] and Bambara groundnut milk [14], respectively.

Conclusion
This study revealed that acceptable yoghurt can be produced from vegetable milk, which is cheaper compared to animal milk. The use of underutilized legumes such as African yam beans in yoghurt production would increase its utilization and value addition to the people living in a low-income community. The present study further showcased the application of oats, a non-popular cereal in milk production, to be used in the production of health-beneficial yoghurt due to its beta-glucan and low-fat contents. It is noteworthy that the yoghurt sample OM, which is made from oats, was mostly preferred by the consumers. The high fiber content of the sample OM was found beneficial to the GIT for easy food digestion. The microbial/ storability analysis carried out in this study also showed that the yoghurt samples produced from vegetable sources had better shelf-life than the sample from animal source. Therefore, it was concluded that yoghurt could be prepared from vegetable milk sources, especially oat flakes and African yam bean milk.

Data availability
The data presented in the study are included in the article and additional material.

Competing interests
The authors declare no competing interests.

Ethics approval
The study proposal was presented within the Departmental Research and Ethical committee and was approved by the Ethical committee of the Department of Home Economics and Food Science, University of Ilorin, Ilorin, Nigeria.