Moringa oleifera: A Prebiotic Material for Better Health ^†

Abstract

Probiotics are live beneficial microbes in the gut microbiota that exert health benefits on the host. Maintaining the balance of probiotics is key to preserving the health of the host. Prebiotics are the primary energy source utilized by these microbes to survive. Various non-digestible dietary fibers serve as prebiotics. In this study, plant-based materials were screened for their prebiotic potential on Lactobacillus paracasei and Lactobacillus rhamnosus, with E. coli as the enteric microorganism. The prebiotic efficiency ranged from 1.6 to 1.9, and the prebiotic index exceeded 1.0 for both probiotic strains. These findings suggest that Moringa is a promising prebiotic material with potential applications in the development of health-promoting functional foods.

1. Introduction

The human gut microbiota plays a significant role in preserving overall health. The microbiota comprises the entire population of microorganisms present and colonizes at different sites. Among the microorganisms, Bacteroidetes and Firmicutes are the major bacterial phyla present in the human gut microbiota [1]. The gut microbiota protects human health by involving itself in various functions such as nutrient metabolism, modulating immunological responses, and also protecting against pathogenic microorganisms by acids and bacteriocins produced by Lactobacillus and Bifidobacterium genera [2,3]. However, the imbalance of the number and diversity of beneficial microorganisms present in the gut results in the development of many diseases [4]. Various factors can alter the microbial balance, including mucin secretions and the human diet. Microbes utilized the human diet as a source of energy, and thus it is highly sensitive to maintaining the survival of the gut microbial community. The non-digestible carbohydrates can significantly alter the composition and function of the gut microorganisms [5]. As the gut microorganisms utilize non-digestible dietary substances known as prebiotics and derived energy for their survival from hydrolysing indigestible prebiotics [6]. Thus, it is essential to maintain the balance of beneficial microorganisms in the gut to preserve overall health. Recently, interest has rapidly increased in dietary supplementation and strategies that help a healthy gut microbiota.

Prebiotics are indigestible dietary fibers and carbohydrates that promote the growth and activity of beneficial microbes in the gut, including probiotic microorganisms. Several compounds have been reported for their prebiotic activity, such as inulin and different types of oligosaccharides [6]. The gut microorganisms ferment these prebiotics to produce various acids that promote health through various mechanisms. Different plant parts have been tested for their prebiotic potential, which selectively enhances the growth and activity of probiotic microorganisms [7].

Moringa is a ‘miracle tree’ found in nearly in worldwide regions. It comprises almost 13 species, among which Moringa oleifera is well known for its nutritional properties. It is considered one of the cheapest alternatives for fulfilling nutritional requirements, as it is rich in protein, potassium, calcium, minerals, vitamins and dietary fiber [8,9]. Interestingly, nearly every part of the Moringa tree has been used for various applications. The pods and leaves are commonly used as vegetables in Asian countries and are consumed as nutritious food. Considering the highly nutritious and medicinal properties of the Moringa tree, the present study focused on its prebiotic activity on beneficial gut bacteria.

2. Materials and Methods

2.1. Plant Material

Gum acacia, Moringa bark extract, Moringa pod without seed crush extract, Moringa leaves extract, Moringa gum, and Moringa whole pod extract were used for evaluating their prebiotic activity.

2.2. Probiotic and Enteric Bacteria

Two probiotic bacteria, including Lactobacillus paracasei and Lactobacillus rhamnosus, and the enteric bacterium E. coli were used to determine the prebiotic effect of the plant materials. The probiotic bacteria were grown on MRS medium (de-Man, Rogosa and Sharpe) and E. coli on nutrient agar (NA).

2.3. Sample Preparation for Prebiotic Assay

The Moringa leaves, Moringa gum and Moringa pods were collected and cleaned by surface swabbing with 70% ethanol. The samples were then air-dried and cut into small pieces then dried at 50 °C in a hot air oven for 2 h or upto complete dry of materials. They were then ground into a fine powder using a grinder and sieved through a 0.4 mm sieve. These powders were used as carbon sources in media to observe the growth of their prebiotic potential. Different test materials were used as carbon sources in different probiotic growth media to evaluate their prebiotic efficiency. MRS with glucose was used as a complete medium and served as the positive control, and MRS with the substrate replacing glucose was used as the test and MRS without glucose was used as the negative control.

2.4. Evaluation of Prebiotic Activity

The prebiotic activity of plant materials was evaluated by the assay previously reported by Anprung and Sangthawan (2012) [10], with necessary modifications. Lactobacillus paracaise and Lactobacillus rhamnosus were used as probiotic bacteria, and E. coli was used as a representative of enteric bacteria. The experiment was performed in replicates. A purified single colony of each bacterium was inoculated into 10 mL of medium (MRS for probiotics and nutrient broth for E. coli) and incubated overnight. After that, 1 mL of inoculum was transferred into freshly prepared 100 mL of medium supplemented with 1% prebiotic material (Moringa plant parts) and glucose. For E. coli, we used minimal medium plus prebiotics and glucose. All flasks were incubated for 24 h. The CFU/mL was determined at 0 h and after 24 h, and the PAS was calculated using the following equation:

$$\begin{array}{c}\mathit{Prebiotic} \mathit{activity}=\left[{\displaystyle \frac{(Probiotic Log CFU/\mathrm{m}\mathrm{L} on prebiotic at 24 \mathrm{h} - Probiotic Log CFU/\mathrm{m}\mathrm{L} on prebiotic at 0 \mathrm{h})}{(Probiotic Log CFU/\mathrm{m}\mathrm{L} on glucose at 24 \mathrm{h} - Probiotic Log CFU/\mathrm{m}\mathrm{L} on glucose at 0 \mathrm{h})}}\right]-\hfill \\ \left[{\displaystyle \frac{(Enteric Log CFU/\mathrm{m}\mathrm{L} on prebiotic at 24 \mathrm{h} - Enteric Log CFU/\mathrm{m}\mathrm{L} on prebiotic at 0 \mathrm{h})}{(Enteric Log CFU/\mathrm{m}\mathrm{L} on glucose at 24 \mathrm{h} - Enteric Log CFU/\mathrm{m}\mathrm{L} on glucose at 0 \mathrm{h})}}\right]\hfill \end{array}$$

Similarly, the prebiotic index (PI) was calculated by dividing the log CFU on the prebiotic by the log CFU on glucose.

2.5. Statistical Analysis

The results of the prebiotic activity assay were presented as the mean ± standard deviation of three replicates. The data were analyzed by one-way ANOVA, with statistical significance set at p < 0.05.

3. Results and Discussion

Prebiotic Activity Score

Various plant-based materials have been evaluated for their prebiotic activity based on the prebiotic activity score (PAS) and in vivo effect on animal models. In this study, we tested different parts of the Moringa plant for their prebiotic activity scores using probiotic and enteric bacteria by in vitro assay. A total of six plant materials were used, among which Moringa whole pod extract and Moringa gum showed the highest prebiotic activity scores of 1.90 and 1.22, respectively, followed by gum acacia, with a score of 1.15 against Lactobacillus rhamnosus. Similarly, for Lactobacillus paracasei, the PAS values were 1.38, 1.07, and 1.12, respectively. The other tested Moringa plant parts showed non-significant PAS values (<1) (Table 1).

Table 1. Prebiotic activity score of Moringa plant parts on probiotic bacteria.

Bacteria	Lactobacillus paracasei	Lactobacillus rhamnosus
Prebiotic
Gum acacia	1.07 ± 0.041	1.15 ± 0.047
Moringa bark extract	0.30 ± 0.031	0.19 ± 0.017
Moringa pod without seed crush extract	0.35 ± 0.037	0.16 ± 0.035
Moringa leaves extract	0.42 ± 0.027	0.40 ± 0.054
Moringa gum	1.15 ± 0.036	1.22 ± 0.034
Moringa whole pod extract	1.68 ± 0.012	1.90 ± 0.012

The values presented are the mean ± SD of three replicates.

The statistical significance of the prebiotic effect of each material on the tested bacteria was tested using one-way ANOVA. The ANOVA revealed a significant effect of the prebiotic treatment of Moringa plant parts on the response of L. rhamnosus, F(5, 12) = 1088.16, p < 0.05, and L. paracasei, F(5, 12) = 243.53, p < 0.05. Overall, these results indicated that the Moringa plant parts exhibited significant prebiotic activity, especially the Moringa whole pod extract and Moringa gum. The prebiotic index for all six plant parts on three probiotics ranged from 0.99 to 1.13 (Table 2).

Table 2. Prebiotic index of Moringa plant parts on probiotic bacteria.

Bacteria	Lactobacillus paracasei	Lactobacillus rhamnosus
Prebiotic
Gum acacia	1.13	1.04
Moringa bark extract	0.99	1.00
Moringa pod without seed crush extract	1.01	1.01
Moringa leaves extract	1.02	1.02
Moringa gum	1.10	1.07
Moringa whole pod extract	1.17	1.08

The present study is in accordance with previous enzyme pectinase-based extracts of Longan with prebiotic activity scores of 1.44 and 1.69 when tested against L. acidophilus and B. lactis [11]. Similarly, Wang et al. (2019) reported on the Moringa oleifera-derived polysaccharide for its beneficial effects on the gut microecology in mice [12].

4. Conclusions

The present study highlighted the prebiotic activity of the Moringa plant parts, especially the Moringa whole pod extract and Moringa gum, which have the highest effects on the growth of the beneficial bacteria. As it indicates that the Moringa not only provides nutrition to the host but also an energy source for the survival of the beneficial microbes presented in the host gut microbiota. However, the present study has limitations, such as it being a purely in vitro study, and no detailed phytochemical analysis was performed to analyze which components contribute to selectively supporting the growth of the tested probiotic bacteria. As the present study evaluated specific plant materials, the results may not be representative of all varieties. Thus, further studies are required to analyze the phytochemicals and conduct animal studies to evaluate their effects under real-life conditions.