INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue X October 2025

Application of Lactobacillus Plantarum in Fermentation of Moringa

Oleifera Leaves and How It Effects on Nutritional Quality

Dalugoda O.S. and Amarakoon R

Department of Microbiology, Faculty of Science, University of Kelaniya, Sri Lanka

DOI: https://dx.doi.org/10.51244/IJRSI.2025.1210000331

Received: 02 November 2025; Accepted: 08 November 2025; Published: 21 November 2025

ABSTRACT

Moringa oleifera leaves are highly nutritious but underutilized and have a very low shelf life. The major aim of

this study is to examine the effect of nutritional quality of Moringa oleifera leaves by fermenting with

Lactobacillus plantarum.

Sundried Moringa oleifera leaf powder followed with fermentation by Lactobacillus plantarum were

investigated for % moisture, ash, crude protein, crude fat, crude fiber and total carbohydrates, based on AOAC

methods. The antioxidant potential was analyzed via DPPH assay.

In different incubation periods, moisture content was significantly (p<0.05) increased from 10.24±0.04%

(unfermented) to 11.46±0.07% (24h), 12.52±0.07% (48h), 27.48±0.20% (72h). Ash content was significantly

(p<0.05) reduced from 8.25±0.05% (unfermented) to 7.33±0.03% (24h), 6.25±0.1% (48h), 5.33±0.13% (72h).

Crude protein content was significantly (p<0.05) increased from 24.63±0.18% to (unfermented), 25.81±0.04%

(24h), 28.43±0.11% (48h), 30.24±0.04% (72h). Crude fat content was significantly (p<0.05) reduced from

5.09±0.07% (unfermented), to 4.17±0.08% (24h), 4.17±0.08% (48h), 2.83±0.10% (72h). Crude fiber content

was significantly (p<0.05) reduced from to 8.33±0.06% (unfermented) to 7.29±0.05% (24h), 6.12±0.03% (48h),

5.82±0.08% (72h). Total carbohydrate content was significantly (p<0.05) reduced from 41.45±0.19%

(unfermented), to 39.63±0.17% (24h), 36.6±0.21% (48h), 30.49±0.17% (72h). The IC 50 values recorded for

Moringa oleifera leaves fermented by Lactobacillus plantarum was 136.3 (unfermented), 115.6 (24h), 65.3

(48h), 50.2 (72h), indicating the highest antioxidant potential in 72h incubation period.

Therefore, 72 h fermented Moringa oleifera leaf powder is the best incubation period than unfermented, 24h and

48h.

Key words: Moringa oleifera leaves, Lactobacillus plantarum, Brine preservation, Proximate composition

analysis, Antioxidant properties

INTRODUCTION

Moringa oleifera tree is a very versatile tree which is indigenous to tropical and sub-tropical countries. In Sri

Lanka it is especially grown in dry zones. Fresh leaves, fruits, flowers and immature pods of Moringa oleifera

are edible and are very delicious diets. Moringa oleifera leaves are highly nutritious and contain enormous health

benefits. Due to the importance of Moringa oleifera tree it is known as “tree of life”, “miracle tree” and “God’s

gift”(Martín Ortega & Segura Campos, 2018). The tree is also known by such regional names as “Drumstick

tree” and “Horseradish tree” (Fahey, 2016).

The increasing population of many tropical countries led to awareness of the importance of Moringa oleifera

leaves as a source of essential nutrients which may not be available in other food sources (Iheanacho & Udebuani,

2010). Recently, many researches have done to identify nutritional components of Moringa oleifera leaves and

to develop it as a main food item or as a food supplement (Moyo et al., 2011). Since, Moringa oleifera leaves

are rich in proteins and calcium it can be used as an alternative food source for children and even for pregnant

mothers and feeding mothers (Gopalakrishnan, Doriya and Kumar, 2016). Moringa oleifera leaves can be used

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INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

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to fight against malnutrition and mineral deficiencies, because it contain significant amounts of phenolic

compounds, dietary fiber, proteins and minerals.

Moringa oleifera leaves have brilliant antibacterial and fungicidal activities. Today many resistant bacterial

strains have been developed against antibiotics. Due the low toxicity and low cost Moringa oleifera leaves extract

can be used to fight against resistant bacterial strains and parasitic worms. Therefore, it can be used as

alternatives or adjuvants for antibiotics. Methanol and ethyl acetate extracts of Moringa oleifera leaves prompt

a great fungicidal activity. It can especially degrade the chitin in the cell walls of fungi. Since Moringa oleifera

leaves have an extraordinary fungicidal activity it is used to cure skin diseases including skin lesions (DAAM,

2020). Moringa oleifera leaves can also be used as a cosmetic agent.

Today many researches have been undergone by using Moringa oleifera leaves. As a result researchers have

been able to produce many productions such as Moringa tea, Moringa powder, Moringa capsules, Moringa soap,

Moringa face wash, Moringa tablets, and Moringa beverages. Generally, a wide range of beneficial

microorganisms are used for the preservation of Moringa oleifera leaves, in order to enhance the nutritional

qualities and to provide numerous health benefits for the consumer.

Though there are many studies have been carried out in other countries such as India and China related to the

preservation of Moringa oleifera leaves, there is a lack of studies on preservation of Moringa oleifera leaves by

using microorganisms, in Sri Lanka. Therefore, the major objective of this study is to investigate the possibilities

of using food grade microorganisms in fermentation of Moringa oleifera leaves.

METHODOLOGY

Collection of samples

Fresh Moringa oleifera leaves were plucked from a Moringa oleifera tree growing at Kalapaluwawa, Rajagiriya,

Sri Lanka (I.F, Offor et al., 2014).

Preparation of samples

Drying of fresh Moringa oleifera leaves

Plucked Moringa oleifera leaves (200g) were weighed by using an electrical balance. Then leaves were dried

under sunlight for 24 hours and were grounded into a fine powder.

Packaging and storage

Sundried Moringa oleifera leaf powder was immediately packed in air tight bottles which were sterilized by

using alcohol (70%). Then bottles were stored in the room temperature (28°C ± 2°C).

Preparation of starter culture of Lactobacillus plantarum

Stock cultures were grown in MRS broth (10.0 mL) at 37° C for 48 hours were inoculated into sterile MRS broth

(10% w/v, 50.0 mL) with Moringa oleifera leaf powder (2.5 g), skim milk (1.0 g) and refined sugar (1.5 g). This

was incubated at 37° C for 24 hours. Preparation of Lactobacillus plantarum starter cultures were done according

to the method described in (Ouwehand et al., 2001) with slight modifications.

Preparation of fermented Moringa oleifera leaf powder

Sun dried Moringa oleifera leaf powder (50.0 g) were added separately into four conical flasks with sterile

distilled water (500.0 mL). Flasks were sterilized in an autoclave at 121°C for 5 minutes. Prepared starter cultures

of Lactobacillus plantarum were transferred into each flask in order to carry out the fermentation process in

different time durations such as 0h, 24 h, 48 h, and 72 h at 37° C. Then the fermented samples were pasteurized

in order to stop the fermentation.

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INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

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Analysis of proximate composition unfermented and fermented Moringa oleifera leaf powder by

Lactobacillus plantarum

Determination of moisture content

Moisture content was determined using AOAC, 2012 with slight modifications. Sample (2g) was weighed into

a previously dried and weighed Petri dishes. Then it was dried in the oven (Philip Harris, UK) at 105 ±1°C for

4 hours. Finally, Petri dishes were cooled and weighed until a constant weight was obtained.

Determination of total Ash content

Total ash content was determined using AOAC, 2012 with slight modifications. Sample (2 g) was measured to

a dry crucible and was incinerated in the muffle furnace (Wise Therm, South Korea) at 550°C for 6 hours.

Finally, crucibles were cooled and weighed until a constant weight was obtained.

Determination of crude protein content. (Kjeldahl method)

In the digestion process the protein content was determined using the micro-Kjeldahl method (AOAC, 2012)

with slight modifications. Sample (2 g) was weighed and was placed on digestion tubes. The catalyst [3.5 g

K₂SO₄+ 0.4 g CuSO₄. 5 H₂O (Sigma Aldrich)] and conc. H₂SO₄(95-98%, 5.00 mL) were added. Digestion was

carried out in the digester for 60 minutes at 410°C. Tube rack was then taken out of the block digester and was

placed on a rack holder, and was allowed to cool for 1 hour and distilled water (20 mL) was added to tubes.

In the distillation process H₃BO₃solution (20 mL) and few drops of indicator were added to an Erlenmeyer flask.

Flask was located under the tip of the condenser. Then a clean tube was placed in the distillation unit. Water (20

mL) and NaOH (20 mL) were added to it and the Distillation process was carried out.

Finally, in titration process the solution in the Erlenmeyer flask was titrated with H₂SO₄to the end point of the

indicator. Crude protein content of the sample was calculated by multiplying the obtained Nitrogen content from

the conversion factor of 6.25.

Determination of crude fat content

Fat content was determined using the method of AOAC, 2012 with slight modifications. Finely chopped sample

(2 g) and anhydrous sodium sulphate (4.00g) were placed in an extraction thimble. Then the mouth of the thimble

was plugged with a piece of cotton wool. Extraction thimble with the sample was kept in the Soxhlet apparatus.

Petroleum ether (200.00 mL) and pumic chips were added into the cleaned and weighed round bottom flask.

Flask was connected to the Soxhlet extractor and the condenser was fixed. Refluxing was carried out for 5 hours.

After refluxing is over, the solvent was distilled off and flask and the contents were placed in an oven at 105°C

for two hours. Finally, the flask and the contents were allowed to cool for 30 minutes and were reweighed.

Determination of crude fiber content

Crude fiber content was determined using the method described by AOAC, 2012 with slight modifications. 2g

of defatted samples were transferred into a 400.0 mL beaker and H₂SO₄(Sigma Aldrich) (5%, 50 mL) was added

and the volume was made to 200 mL mark with distilled water. The content was brought to the boiling point and

kept boiling for 30 minutes while stirring with a glass rod. Hot water was added time to time to keep the volume

constant. It was then filtered through a 15.00 cm #4 Whatman filter paper on a Buchner funnel attached to filter

pump. The residue was transferred to the funnel with a jet of hot water and was washed it with hot water until

the filtrate was free from acid. This was checked by using a litmus paper. The residue was scraped off from the

filter paper, and was placed in the same beaker and remaining last traces with a jet of hot water.

NaOH (5%, 50 mL) and distilled water (200 mL) was added. Then it was brought to the boiling point and was

kept boiling for 30 min. It was filtered instantaneously through the same piece of filter paper and the residue was

moved to the filter by means of a jet of hot water. Then the residue was washed with hot water and with HCl

(Sigma Aldrich) (1%) and again with hot water until it was free from acid. After that it was washed twice with

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small amounts of alcohol (95%) and diethyl ether (Sigma Aldrich) and residue was transferred into a porcelain

dish. Remaining liquid was evaporated in an oven (Philip Harris, UK) at 100° C until the residue comes to a

constant weight. It was then allowed to cool and was weighed. Finally, it was kept in a muffle furnace (Wise

Therm, South Korea) at 500° C and it was allowed to cool and the final weight was taken.

Determination of total carbohydrate content

Total carbohydrate content of fermented Moringa oleifera leaf powder by Lactobacillus plantarum was

calculated from following equation. Total carbohydrate content = 100-(moisture content% + ash content% +

crude fat content% + crude fiber content % + crude protein content).

Determination of antioxidant potential

The antioxidant potential of fermented Moringa oleifera leaf samples was determined by using 2, 2-diphenyl-1-

picrylhydrazyl (DPPH) assay by using a 96 well microtiter plate according to the method described by (Wasana

& Amarakoon, 2021) with slight modifications.

Determination of chemical quality (pH)

The chemical quality was determined by measuring the pH according to the method described by (Menaka et

al., 2011) with slight modifications.

Statistical analysis

Minitab 16 statistical package was used. One-way ANOVA technique and Tukey’s multiple comparison tests

were used to determine the significant differences of mean values of fermented Moringa oleifera leaf powder by

Lactobacillus plantarum. (p˂0.05 was considered as significant.)

RESULTS AND DISCUSSION

Determination of Moisture content

Turkey’s comparison in ANOVA shows that the means of moisture contents are significantly (p<0.05) different

from each incubation periods suggesting that there is an effect of fermentation by Lactobacillus plantarum. The

percentage of moisture content increases with increase of incubation period (unfermented, 10.24^D±0.04% < 24h,

11.46^C±0.07% < 48h, 12.52^B±0.07% < 72h, 14.28^A±0.08). The highest moisture content was observed in 72h

fermented Moringa oleifera leaves by Lactobacillus plantarum. The percentage moisture content of fermented

Moringa oleifera leaf powder shows a higher percentage of (>10%). Therefore, it confirms the fact that Moringa

oleifera leaves are rich in moisture.

Determination of Ash content

Turkey’s comparison in ANOVA shows that there is a significant (p<0.05) reduction in the ash content between

unfermented (8.25^A±0.05) and 72h fermented Moringa oleifera leaf powder (5.33^D±0.13%) by Lactobacillus

plantarum. This may be because minerals present in Moringa oleifera leaf powder has been used by

Lactobacillus plantarum for its metabolic purposes. (Scheuer et al., 2021). As a result percentage of ash content

has been decreased with the increase of incubation period (unfermented, 8.25^A±0.05% < 24h, 7.33^B±0.03 < 48h,

6.25^C±0.1 < 72h, 5.33^D±0.13).

Determination of Crude protein content

Turkey’s comparison in ANOVA shows that there is a significant (p<0.05) increment in the crude protein content

between unfermented (24.63^D±0.18) and 72h fermented Moringa oleifera leaf powder (30.24±0.04^A) by

Lactobacillus plantarum indicating the highest protein content. This increment may be due to the extracellular

enzymes secreted by Lactobacillus plantarum in order to degrade cellulolytic materials during the time of

fermentation (Munishamanna et al., 2017).

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INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)

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Determination of crude fat content

Turkey’s comparison in ANOVA shows that the means of crude fat contents are significantly (p<0.05) different

from each other suggesting that there is an effect of fermentation by Lactobacillus plantarum. A significant

(p<0.05) reduction in crude fat content can be observed at 72h incubation period (2.83^D±0.10%). There is no

significant (p<0.05) difference between the crude fat contents of 24h (4.17^B±0.08%) and 48h fermented Moringa

oleifera leaf powder (4.17^B±0.08%) by Lactobacillus plantarum.

According to Scheuer et al. (2021) extensive breakdown of large molecules of fat into simply fatty acids causes

the enhancement of crude fat during fermentation. However, the results obtained in this study does not agree

with that. This is due to the activity of Lactobacillus plantarum in Moringa oleifera leaf powder.

Determination of crude fiber content

A significant (p<0.05) reduction in crude fiber content can be observed at 72 h incubation period (5.82^D±0.08%).

This is a result of unstiffening of fibrous tissues at the time of fermentation. Bioconversion of dietary fiber and

lignocelluloses in to protein also reduces the fiber content (Scheuer et al., 2021).

Fiber is an essential component in food which proliferates the digestibility and reduces constipation. This study

shows a higher fiber content in fermented Moringa oleifera leaf powder Therefore, it could function as a good

source of dietary fiber providing more health benefits.

Determination of total carbohydrate content

Based on Turkey’s comparison in ANOVA, it can be concluded that there is a significant (p<0.05) difference

between the means obtained for the total carbohydrate content in different incubation periods indicating an effect

of fermentation by Lactobacillus plantarum. The amount decreases in the order of unfermented (41.45^A±0.19%>

24h (39.63^B±0.17%) > 48h (36.6^C±0.21%) > 72h (30.49^D±0.17%). Statistical analysis revealed that there is a

significant (p<0.05) reduction in the total carbohydrate content in 72h incubation period. Reduction arises as a

result of utilizing carbohydrates by Lactobacillus plantarum as a source of energy for metabolic functions and

growth. During the period of fermentation oligosaccharides are converted into simple sugars. This reaction also

reduces the total carbohydrate content (Scheuer et al., 2021).

Determination of Antioxidant activity

Moringa oleifera leaves are rich in phenols, flavonoids, and para-anthocyanin making it a good source of

antioxidants. (Iqbal & Bhanger, 2006).

IC50 value is the concentration of antioxidant required to give 50% inhibition of the probe in the antioxidant

assay. Hence, lower IC50 value denotes high antioxidant activity of a given food source. According to the results,

IC50 values decrease in the order of unfermented (136.3) > 24h (115.6)> 48h (65.3)> 72h (50.2).

More importantly, a significant (p<0.05) increase in the antioxidant potential was observed in 72 h fermented

Moringa oleifera leaf powder by Lactobacillus plantarum. Therefore, 72 h fermented Moringa oleifera leaf

powder has the highest antioxidant potential. Antioxidant properties are mostly due to the phytochemicals

present in plants. These phytochemicals are important for the human health making them functionally vital for

consumption. As a result of bioactive peptide synthesis by Lactobacillus plantarum via hydrolysis of proteins,

antioxidant properties are enhanced during the fermentation (Nkhata et al., 2018).

Analysis of chemical quality (pH)

According to the statistical analysis there is a significant (p<0.05) difference between the means obtained for pH

in fermented Moringa oleifera leaf powder by Lactobacillus plantarum. There is a gradual reduction of pH with

increasing incubation time in the order of unfermented (6.10^A±0.04) >24h (5.50^B±0.06) >48h (4.80^C±0.05> 72h

(4.20^D±0.03). This result agrees with almost all the lactic acid bacteria mediated fermentation and natural

fermentation reported by (Obadina et al., 2013). This may be due to the production of lactic acid by Lactobacillus

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plantarum during fermentation period in the process of energy synthesis for their metabolism (Obadina et al.,

2013).

CONCLUSION

Proximate composition analysis of sundried Moringa oleifera leaf powder followed with fermentation by

Lactobacillus plantarum in different incubation periods, moisture content 10.24±0.04% (unfermented)

11.46±0.07% (24h), 12.52±0.07% (48h), 27.48±0.20% (72h). Ash content 8.25±0.05% (unfermented)

7.33±0.03% (24h), 6.25±0.1% (48h), 5.33±0.13% (72h). Crude protein content 24.63±0.18% (unfermented),

25.81±0.04% (24h), 28.43±0.11% (48h), 30.24±0.04% (72h). Crude fat content 5.09±0.07% (unfermented),

4.17±0.08% (24h), 4.17±0.08% (48h), 2.83±0.10% (72h). Crude fiber content 8.33±0.06% (unfermented),

7.29±0.05% (24h), 6.12±0.03% (48h), 5.82±0.08% (72h). Total carbohydrate content 41.45±0.19%

(unfermented), 39.63±0.17% (24h), 36.6±0.21% (48h), 30.49±0.17% (72h) were recorded respectively. The IC

50 values recorded for Moringa oleifera leaves fermented by Lactobacillus plantarum were 136.3

(unfermented), 115.6 (24h), 65.3 (48h), 50.2 (72h). The highest antioxidant potential was recorded in 72h

fermented Moringa oleifera leaf powder by Lactobacillus plantarum.

Fermentation by using Lactobacillus plantarum had significantly (p<0.05) affected on ash content, crude protein

content, crude fiber content, crude fat and total carbohydrate content at the maximum incubation period. Based

on the above results, it can be concluded that the 72 h fermented Moringa oleifera leaf powder by Lactobacillus

plantarum is the best preservation method in terms of nutritional quality to increase its’ shelf life.

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of-Lactobacillus-plantarum-in-fermentation-of-jackfruit-Artocarpus-heterophyllus-seeds-and-

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Table 1: Nutrient content of unfermented and fermented Moringa oleifera leaf powder by Lactobacillus

plantarum

Treatment

Moisture *

Ash *

Crude protein Crude fat *

*

Crude fiber Total

*

carbohydrate *

0h (unfermented) 10.24^D±0.04 8.25^A±0.05 24.63^D±0.18 5.09^A±0.07

8.33^A±0.06

7.29^B±0.05

6.12^C±0.03

41.45^A±0.19

39.63^B±0.17

36.6^C±0.21

24 h

48 h

72 h

11.46^C±0.07 7.33^B±0.03 25.81^C±0.04 4.17^B±0.08

12.52^B±0.07 6.25^C±0.1 28.43^B±0.11 4.17^B±0.08

14.28^A±0.08 5.33^D±0.13 30.24^A±0.04 2.83^C±0.10

5.82^D±0.08 30.49^D±0.17

*The values are mean ±standard deviation of the replicates. The values with common superscript letters in each

column are not significantly different (p<0.05).

The graph of nutrient conytent vs incubation period

45

40

35

30

25

20

15

10

5

0

Moisture *

Ash *

Crude protein *

Crude fat *

Crude fiber *

Total carbohydrate *

Incubation period (hours)

0 h 24 h 48 h 72 h

Figure 1: Bar chart representing the nutrient content of unfermented and fermented Moringa oleifera leaf powder

by Lactobacillus plantarum

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Table 2: Representing % inhibition of unfermented and fermented Moringa oleifera leaf powder by

Lactobacillus plantarum

Sample concentration 0 h (unfermented)

24 h

48.0

40.1

31.9

30.8

28.7

48 h

50.8

39.2

33.4

19.8

16.1

72 h

60.6

45.2

36.2

25.9

15.3

1500 *

250 *

125 *

62.5 *

125 *

66.7

56.4

42.3

35.4

32.1

*The values are mean ±standard deviation of the replicates. The values with common superscript letters in each

column are not significantly different (p<0.05).

Graph of % inhibition vs concentration (μg/mL)

80

70

60

50

40

30

20

10

0

200

400

600

0 h

800

24 h

1000

72 h

1200

1400

1600

48 h

Figure 2: Variation of antioxidant potential in unfermented and fermented Moringa oleifera leaf powder by

Lactobacillus plantarum

Graph of pH vs incubation period (hours)

7

6

5

4

3

2

1

0

0 h

24 h

48 h

72 h

Incubation period (hours)

Figure 3: Bar chart representing the pH of unfermented and fermented Moringa oleifera leaf powder by

Lactobacillus plantarum

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