Viability of Probiotic Strains in Moringa Aqueous Extract-Fortified Low-Fat Yogurt

INTRODUCTION

Progression within the food industry, coupled with an augmented consumer awareness concerning health-oriented and nutrient-laden dietary preferences, has engendered a marked escalation in the requisition for functional food items. In this context, dairy products hold a pronounced standing in the human diet, being acknowledged as all-encompassing sustenance replete with a comprehensive array of indispensable nutrients. The fermentation process, distinguished by its efficacy and economic feasibility, stands as a preeminent methodology for augmenting product functionality (Tamang, Shin, Jung, & Chae, 2016). There has been a tremendous increase in the production of fermented dairy products in the past few decades with their well-documented health benefits, which led the demand upwards (Gorlov et al., 2019; Lee, Cha, & Park, 2004). Prebiotics are the nonviable food components, mainly poly/oligosaccharides (inulin and its hydrolytic products, oligofructose, and galactooligosaccharides), which are selectively utilised by host microorganisms, thus, favoring/supporting the growth of probiotics in addition to other innate health effects (Davani-Davari et al., 2019; Gibson et al., 2017; Mohanty, Misra, Mohapatra, & Sahu, 2018). Various studies suggest the significance of synbiotic dairy foods incorporated with various probiotic bacteria and prebiotics (Li et al., 2020; Manoharan, Jayapratha, & Ashokkumar, 2020; Ranjitham & Poornakala, 2020). Bioactive chemicals are present in both plant- and animal-based origins. They have been investigated for use in creating functional synbiotic yogurt, including essential oils, honey, aloe vera, and moringa medicinal plant extracts (Ahmad et al., 2022).

Moringa (Moringa oleifera) is an ancient tree and one of 13 species in the Moringaceae family; it is the most well-known member of the genus. It is known as the drumstick tree or tree of life; the ability to utilize bark, pods, leaves, nuts, seeds, tubers, roots, and flowers of the plant makes it a multipurpose plant (Sacoto & Verlicchi, 2019). Moringa is known for its numerous nutritional and pharmacological activities, such as cardiac stimulants, antitumor agents, and may also have antioxidant, antihypertensive, anti-inflammatory, antibacterial and antifungal activities (Anwar, Latif, Ashraf, & Gilani, 2007). Moringa is a significant plant in the nutrition for people in many developing countries due to its substantial quantity of nutrients (Zhao et al., 2019). Moringa is rich in several nutrients, such as proteins, fiber and minerals (Jongrungruangchok, Bunrathep, & Songsak, 2010; Moyo, Oyedemi, Masika, & Muchenje, 2012), that play an important role in human nutrition. It contains numerous phytochemicals (such as neochlorogenic acid, rutin, chlorogenic acid, apigenin, quercetin, and Kaempferol), as secondary plant metabolites having health-promoting effects, thereby reducing the risk of noncommunicable diseases (Lako et al., 2007; Scalbert, Johnson, & Saltmarsh, 2005). Formulations in the food industry, as the bioactive compounds studied in this plant can be included in foodstuff development (Saucedo-Pompa et al., 2018).

The leaves of moringa are the most nutritious vegetative part of the plant and an important source of B and C vitamins, provitamin A as beta-carotene, vitamin K, manganese, and protein (Adepoju & Selezneva, 2020; Peter, 2008). In addition, it has been shown to exhibit high antioxidant properties due to its polyphenol content (Shih, Chang, Kang, & Tsai, 2011; Sreelatha & Padma, 2009). Moringa leaves are of special interest for both physiological and microbial food preservation (Saucedo-Pompa et al., 2018). However, there are limitations on its consumption due to its greenish appearance and it slightly bitter and unpleasant taste (Adepoju & Selezneva, 2020). Therefore, the aim of the present study is to utilize the benefits of moringa leaves and overcome its unpalatable taste by using its aquas extract.

Recent studies have suggested incorporating plants and their extracts with probiotics to produce more effective and functional products (Allam M.G. & S.M., 2019; Chand et al., 2021; M. Gomaa, Alneamah, & Ayad, 2018; Mukprasirt, Domrongpokkaphan, Kaewpanya, Khemkhao, & Sumonsiri, 2022; Yilmaz et al., 2022). Adding plant extracts can enhance the growth of lactic acid bacteria and thus increase the beneficial effects on the human gastrointestinal tract (Burgain, Gaiani, Linder, & Scher, 2011; M. A. Gomaa, Allam, Haridi, Eliwa, & Darwish, 2022; Huq, Khan, Khan, Riedl, & Lacroix, 2013). Moringa extract potentiated the LAB growth in the yogurt and attenuated the colon cancer by increasing the expression of antioxidant enzymes (Zhang [ea1] et al., 2019). In addition, it showed a protective effect against hepatic failure by enhancing the liver profile and lowering blood cholesterol levels (Cardiens [ea2] et al, 2018). 

Therefore, the aim of the present study is to produce a synbiotic yogurt that benefits from the lactic acid fermentation and phytochemistry of Moringa aqua extract. For this purpose, low-fat yogurt samples were prepared with different probiotic strains and enriched with different concentrations of Moringa aqua extract. The effect of the Moringa extract on the viability of the probiotics, the physicochemical properties of the yogurt, the sensory properties, and the shelf life of the product were evaluated.

MATERIALS AND METHODS[ea3] 

Milk preparation

Reconstituted low-fat bovine milk (0.5% fat,12% total solids, 3.37% protein, and 0.84% ash) was prepared from skimmed milk powder (RSM) for yogurt production.

Growth conditions of the starter culture and probiotic strains

pH-modified (4.58) MRS agar (Oxoid Ltd., Hampshire, England) (DeMan, Rogosa, & Sharpe, 1960) was used to count the commercial starter culture Yo-Mix® Real 300, containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus obtained from Danisco Denmark. Besides, the media were used to enumerate the four strains of probiotic LAB: Lactobacillus rhamnosus (Lr-32), Lactobacillus casie (Lc-11), Lactobacillus acidophilus (La-14), and Lactobacillus plantarum (Lp-115), which were obtained from the culture collection of Danisco (California Gold Nutrition). All bacteria were incubated at 42 °C for 72 h.

Moringa aqueous extract

Fresh Moringa leaves (Moringa oleifera) were obtained from the farm of the Faculty of Agriculture Saba Basha, Egypt. The leaves were soaked in cold water at 5 °C for three min to remove impurities. Subsequently, the leaves were dehydrated in an oven at 55°C for 16-18 h. The dried leaves were crushed into fine powder using a food processor (Tornado FP-1000SG, Egypt), sieved through a 60 mm mesh sieve, and stored at -20 °C until use.

The aqueous Moringa extract was prepared by mixing 40 g of the dried leaf powder in 100 mL of distilled water at 40°C for 24 h with shaking at 12 rpm. Afterwards, the extract was filtered twice with Whatman filter paper No. 1 and stored at -20°C until use (Arif et al., 2022).

Moringa aqueous extract effect on strains viable count

The number of viable bacteria was determined for each strain in a pH-modified (4.58) MRS broth supplemented with Moringa aqua extract at four concentrations (0.0, 0.5, 1.0, or 1.5%). The broth media were incubated with 0.1 mL of an overnight growth culture. Samples were collected in triplicate at seven time points (0, 4, 8, 12, 16, 20, and 24 h) of growth at 42 °C. Each sample was diluted up to 10-8 in MRS broth, then plated on MRS agar and counted under the same conditions for 48 hours. The number of viable bacteria was then recorded as Log CFU g^-1.

Yogurt manufacture

The yogurt was produced by lactic acid fermentation of reconstituted low-fat bovine milk heat-treated at 80°C for 10 minutes. Either the commercial starter culture Yo-Mix® Real 300 or one of the four strains of probiotic LAB was added: Lactobacillus rhamnosus (Lr-32), Lactobacillus casie (Lc-11), Lactobacillus acidophilus (La-14) and Lactobacillus plantarum (Lp-115) to reach 10⁸ cfu/mL in the final mixture at 42 ºC and mixed well. The inoculated milk was supplemented with Moringa aqua extract (0.5%, 1%, or 1.5%) in addition to the controls (0.0) (Table 1), then filled into 100 ml cups and incubated at 42ºC for about 3h, followed by cooling at 4°C. The synbiotic yogurt preparations were stored at 5 °C for 15 days for further experiments (Tamime & Robinson, 2007).

pH, acidity, and viscosity determination

The pH values of the yogurt samples were measured with a pH meter (Jenway 3505, England). The acidity of the yogurt samples was determined by direct titration with 0.11 molar NaOH, and the values were calculated as lactic acid (%) according to equation (1) (Llanos et al., 2019). The test was repeated four times over a period of 15 days at 1, 5, 10, and 15 days of storage. The viscosity of the yogurt treatments was measured at 15 °C using a viscometer (D.P. SELECTA, S.A. ST-2020R, Korea) at a speed of 60 to 200 rpm with spindle R5. The viscosity is expressed in mPa·s. and the temperature is automatically corrected (Kia, Ghasempour, Ghanbari, Pirmohammadi, & Ehsani, 2018).

Yogurt’s microbial viable counts during storage

One gram of low-fat yogurt was mixed in 9 mL of phosphate buffer, 1 mL of which was transferred to the appropriate agar medium and counted. Plates were incubated at 37°C for 48 hours (for all bacteria) and counts were expressed as log CFU g^-1. LAB were counted on MRS agar. Coliform bacteria were counted on Violet red bile agar (VRBA). Yeasts and molds were counted on potato dextrose agar for 7 days at 25°C (Kazama, 2020). The test was repeated four times during the 15-day storage period: on the 1^st, 5^th, 10^th, and 15^th day.

Probiotic count and specific growth rate

For counting LAB, the MRS agar (Biolife) was used as recommended by the Standard Methods for examination of dairy Products. The plates were incubated for 48 h at 37 ^oC.

Table 1: The formulation of twelve synbiotic low-fat yogurt trials and the control

Sensory evaluation of synbiotic yogurt

Nine experienced panelists, consisting of staff from the Faculty of Agriculture, Saba Basha, Alexandria University, aged 24–61 years, participated in the evaluation of the sensory attributes of fermented milk samples one day after production. Low fat yogurt samples (100 mL cups) were placed on white plates and randomly presented to the panelists. The panelists were asked to evaluate the sensory characteristics of the yogurt (color, flavour, appearance, and texture) on a 5-point scale (with 1 being the worst and 5 being the best).

First, the color was evaluated in contrast to the white paper in the background. Second, the flavour (smell and taste) was evaluated by swallowing ≈10 g (one spoon portion) of the sample. Appearance was assessed by visual observation, and finally, textural properties were assessed by breaking the yogurt gel and shaking the product. Overall acceptability was assessed at the end of the sensory evaluation of each sample(Soukoulis, Panagiotidis, Koureli, & Tzia, 2007).

Statistical analysis

Data were analyzed using one-way ANOVA [ea4] with IBM SPSS 25 (Armonk, New York, United States); the obtained data were expressed as mean ± standard deviation (SD). Differences between means from one-way ANOVA were compared using Duncan’s test at a 95% confidence level (p < 0.05).

RESULTS AND DISCUSSION

Probiotic strains viability [ea5] 

Viability of the four selected probiotic strains Lactobacillus rhamnosus (Lr-32), Lactobacillus casie (Lc-11), Lactobacillus acidophilus (La-14) and Lactobacillus plantarum (Lp-115) were assessed in MRS media supplemented with Moringa water extract at 0.5%, 1%, 1.5% v/v or 0.0% as control. At seven intervals of growth development (0, 4, 8, 12, 16, and 24 h), the number of viable microorganisms was presented as Log CFU g^-1 ± SD in Table 2 and as Δ Log CFU g^-1 in Figure 1.

All strains showed better growth in the presence of the aqueous Moringa extract, depending on the concentration. The highest value was recorded for Lactobacillus rhamnosus (Lr-32), which generally showed a higher growth rate in general and the trial MR3, 1290 ±0.01 log CFU g^-1 × 10⁶, followed by MR2 and MR1, and the lowest count for this strain was observed in MR0, where no aqueous Moringa extract was added (1170 ±0.02, 1020 ±0.02, and 296 ±0.02 log CFU g^-1 × 10⁶, respectively). The second-best performance was observed for Lactobacillus plantarum (Lp-115), with the trials containing aqueous moringa extract ranging from 430±0.03 to 300±0.02 Log CFU g^-1 × 10⁶, corresponding to an increase of 35-117% in viable count compared to the control MP0, which recorded 184 ±0.03 Log CFU g^-1 × 10⁶. Lactobacillus acidophilus (La-14) and Lactobacillus casei (Lc-11) probiotic strains showed a comparable viable count profile, with values ranging from 199 ±0.03 to 267 ±0.03 for the trials containing 0.5%, 1%, 1.5% v/v moringa aqueous extract (MA1, MA2, MA3, MC1, MC2, and MC3). While the controls (MA0, and MC0) recorded 174 ±0.03 and 181 ±0.01 Log CFU g^-1 × 10⁶, respectively.

The calculation of Δ Log CFU g^{-1 [ea6]} shows a higher contrast when comparing samples containing aqueous Moringa extract with controls and eliminates the inoculum effect, as shown in Figure 1. Lactobacillus plantarum (Lp-115), which showed the second highest growth among all strains, presented a limited difference between all trials (MR1, MR2, MR3) and the control MR0, which started to be noticeable after 12 hours of growth. Lactobacillus acidophilus (La-14) performed better in the higher concentrations of moringa aqueous extract of 1% and 1.5% (MA2 and MA3) since the first reading. A limited improvement was observed at 0.5% (MA1), which was close to the control MA0. The probiotic strains Lactobacillus rhamnosus (Lr-32) and Lactobacillus casei (Lc-11) shared a similar growth profile in which the addition of moringa aqueous extract at all concentrations posted the viable count upwards after 12 and 8 hours. The final Δ Log CFU g^-1 of MR3 was almost 10 times higher than the viable count of control (MR0). A similar trend was observed in the case of Lactobacillus casei (Lc-11); the highest moringa extract concentration in MC3 increased the viable count by 2.5 times the control MC0.

Table 2: Bacterial viable count of probiotic strains: Lactobacillus acidophilus (La-14), Lactobacillus plantarum (Lp-115), Lactobacillus casei (Lc-11), and Lactobacillus rhamnosus (Lr-32) in moringa aqueous extract-Fortified MRS media continuing 0.5%, 1%, 1.5% v/v or 0.0% as control

Figure 1: Bacterial viable counts of probiotic strains: Lactobacillus acidophilus (La-14), Lactobacillus plantarum (Lp-115), Lactobacillus casei (Lc-11), and Lactobacillus rhamnosus (Lr-32) in moringa aqueous extract-Fortified MRS media continuing 0.5%, 1%, 1.5% v/v or 0.0% as control all numbers are presented as Δ Log CFU g-1 × 10⁶.

Physio-Chemical properties of moringa Yogurt 

pH and acidity

Table 3 illustrates the pH values of the Yogurt samples. The highest pH value was observed in the control sample after one day of storage (4.64), which gradually decreased to 4.42 at the end of the 15-day of storage period. The lowest pH value was noticed in MC3 (1.5% moringa aqueous extract) with Lactobacillus casei (Lc-11), with 4.41, 4.30, 4.14, and 4.07 after 1, 5, 10 and 15 days of storage at 5°C, respectively. Overall, a concentration-dependent decrease in pH was observed in all treatments throughout the storage period. This decrease is attributed to the conversion of lactose into lactic acid, indicating higher activity in the presence of the aqueous extract. This pH decrease signifies the continuous growth of lactic acid culture during storage, which is enhanced by the presence of moringa aqueous extract. Similar results have been reported in other studies, where yogurt supplemented with moringa exhibited a significant reduction in pH compared to control samples (El-Gammal, Abdel-Aziz, & Darwish, 2017; Saeed, 2020).[AT8] 

Similar to the pH trend, a slight increase was also observed in the total titratable acidity. The highest values were recorded in trial MC3 with titratable acidity values of 0.79, 0.80, 0.81, and 0.82% after 1, 5, 10, and 15 days of storage at 5°C, respectively. This increase was dependent on the concentration of the aqueous Moringa extract. Conversely, the lowest titratable acidity was observed in the control sample with values of 0.60, 0.69%, 0.73, and 0.78% at the same storage intervals. The increase in acidity is due to bacterial activity converting lactose into organic acids, mainly lactic acid, resulting in a decrease in pH. This trend is consistent with previous reports Asfour and Anwer (2015)

Total solids

The addition of moringa to yogurt treatments increased the total solids content in a concentration-dependent manner. The highest total solids content was found in treatment MR3 (1.5% moringa aqueous extract) with Lactobacillus rhamnosus (Lr-32), measuring 12.39, 12.43, 12.46, and 12.51% ± 0.1 after 1, 5, 10 and 15 days of storage at 5 °C, respectively. The lowest value was observed in the fresh control sample at 12.22% ± 0.1. A slight increase in total solids was observed during storage in most trials, including the control (Table 3). The highest moisture content was observed in the fresh control sample at 87.78%, while the lowest content was observed in treatment MR3 (1.5% moringa aqueous extract). The slight variation in moisture content may be attributed to storage at low humidity conditions, which is consistent with the findings of I. Bakr, Mohamed, Tammam, and El-Gazzar (2015) and A. Bakr, Mousa, and EL-Shahawy (2020).

Viscosity

Viscosity significantly influences the quality of liquid and semi-solid foods (Karaman et al., 2014). The control's viscosity increased during the 15-day storage period. In all treatments, the addition of moringa aqueous extract led to an increase in viscosity, in contrast to the 0% trials (MA0, AP0, MC0, and MR0), and this increase was concentration-dependent. On the first day of storage, the highest viscosity value was 44.2 ± 2.57 mPa.s in the MR3 treatment with Lb. rhamnosus. The viscosity gradually decreased during storage to reach 40.8 ± 1.74, 33.4 ± 3.27, and 26 ± 1.66 after 5, 10, and 15 days of storage at 5°C, respectively. Similar trends were observed in all trials, with an overall decrease in viscosity during storage. All strains were in the same range as the commercial starter culture, aligning with the findings of Vital et al. (2015) and Zhang et al. (2019), who attributed increased yogurt viscosity with moringa extract to protein-polyphenol interactions. Moringa contains abundant polyphenolic compounds that can form complexes with milk proteins, such as casein.

Table 3: pH, acidity, and total solids (Ts) of 16 low fat yogurt trials 12 of which are fortified by moringa aquas extract 0.5, 1.0, and 1.5 % during storage for 15 days at 5°C presented as value ± SD.

Table 4: Viscosity averages mPa.s ± SDof 16 low fat yogurt trials 12 of which are fortified by moringa aquas extract 0.5, 1.0, and 1.5 % during storage for 15 days at 5 °C.

Microbial analysis of yogurt[ea11] 

Table 5 displays the probiotic viability values for the control and trial yogurt samples during the 15-day of storage period at 5 °C. The data show that increasing the level of moringa aqueous extract fortification in yogurt led to an increase in the viable probiotic counts. The highest count value was recorded in the MR3 treatment with Lb. rhamnosus, measuring 940 ± 5, 800 ± 5, 760 ± 10, and 480 ± 7 Log CFU g^-1 × 10⁶ on days 1, 5, 10, and 15 of storage, respectively. This indicates that the yogurt with the highest moringa extract concentration exhibited the maximum probiotic viability, in agreement with Zhang et al. (2019), who reported higher viable cell counts in moringa aqueous extract-supplemented yogurt compared to the control. They also suggested that the increased growth of lactic acid bacteria could be attributed to the polyphenolic components of moringa extract, as Moringa oleifera contains dietary polyphenolic substances (Siddhuraju & Becker, 2003), which may promote fermentation rates and enhance lactic acid bacteria metabolism and survival.

The yeast and mold count of control and treatments exhibited no significant difference that agreement with (Rupa & Vijay, 2022). The coliforms were found to be absent in both control and treatment yogurt samples, which confirms that the product has been produced under hygienic standards.

Table 5: Bacterial viable counts of probiotic strains: Lactobacillus acidophilus (La-14),Lactobacillus plantarum (Lp-115), Lactobacillus casei (Lc-11), and Lactobacillus rhamnosus (Lr-32) in moringa aqueous extract-Fortified low-fact yogurt continuing 0.5, 1, 1.5% (v/v) or 0.0% as control.

Sensory evaluation

The sensory evaluation results of the yogurt samples, in which attributes such as color, appearance, flavor, and texture, are presented in Figure 2. The radar charts depict the average sensory scores for sixteen different low-fat yogurt treatments fortified with moringa aqueous extract. Data revealed that there was a ±1 difference observed between all treatments and the control for all parameters, except for texture, where the control was the softest, with a score of 3.

All samples exhibited a normal white color similar to the control, indicating that the addition of moringa aqueous extract did not affect the natural color of the products. Samples containing Lactobacillus plantarum (MP0, MP1, MP2, MP3) were slightly preferred, possibly due to a starter culture effect, as one of the trials contained 0% moringa extract (MP0). Additionally, the flavor of samples containing Lactobacillus plantarum (MP0, MP1, MP2, MP3) and Lactobacillus rhamnosus (MR0, MR1, MR2, MR3) was similar to the control and received the highest scores.

Regarding appearance, all trials produced normal and homogeneous yogurt products. However, nine samples scored one point higher than the control. Among these, four contained Lactobacillus rhamnosus as a starter culture (MR0, MR1, MR2, MR3), three contained Lactobacillus casei (MC0, MC1, MC2), and the last two were fermented using Lactobacillus acidophilus.

In terms of texture, the control was softer than all trials. Nevertheless, samples containing Lactobacillus plantarum (MP0, MP1, MP2, MP3) and Lactobacillus rhamnosus (MR0, MR1, MR2, MR3) as a starter culture received the maximum score, while the other two strains scored four points.

Despite the light greenish color and slightly bitter taste of the moringa extract, all sensory scores for yogurt with moringa aqueous extract were very positive. The addition did not affect the color, smooth appearance, flavor, or texture of the products. These findings contrast with prior studies by Saeed (2020) and  Rupa and Vijay (2022), which reported a reduction in body and texture when moringa leaf powder was introduced into functional Greek yogurt. Moreover, the incorporation of moringa extract in our study did not result such negative attributes.

 Figure 2: Sensory evaluation of sixteen different moringa aqueous extract-fortified low-fat Yogurt treatments containing probiotic strain as a starter culture: Lactobacillus acidophilus (La-14), Lactobacillus plantarum (Lp-115) (MP0, MP1, MP2, MP3), Lactobacillus casei (Lc-11) (MC0, MC1, MC2), and Lactobacillus rhamnosus (Lr-32) (MR0, MR1, MR2, MR3) in four levels each (0.0, 0.5%, 1%, or 1.5% v/v) in addition to the control.