Journal of Ayurveda and Integrative Medicine 16 (2025) 101037

Contents lists available at ScienceDirect

Journal of Ayurveda and Integrative Medicine

journal homepage: elsevier.com/locate/jaim

Acute oral toxicity evaluation of synbiotic mixture containing Streptococcus

salivarius K12 and Musa acuminata aqueous peel extract in
Sprague-Dawley rats
Aalina Sakiinah Mohd Fuad a , Mohd Hafiz Arzmi a,b,c , Muhammad Ekmal Bakar d,
Izatus Shima Taib d , Fazle Khuda e,f , Nurrul Shaqinah Nasruddin e,*
a
Cluster of Cancer Research Initiative IIUM (COCRII), International Islamic University Malaysia, Kuantan Campus, Pahang, Malaysia
b
Department of Fundamental Dental and Medical Sciences, Kulliyyah of Dentistry, International Islamic University Malaysia, Kuantan Campus, Pahang, Malaysia
c
Melbourne Dental School, The University of Melbourne, Swanston Street, 3053, Victoria, Australia
d
Centre of Diagnostics, Therapeutics and Investigative Studies, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
e
Department of Craniofacial Diagnostics and Biosciences, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
f
Department of Restorative Dentistry, Faculty of Dentistry, Lincoln University College Malaysia, Petaling Jaya, Selangor, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: Background: The combination of S. salivarius K12 and M. acuminata are being used as synbiotic, but its safety
Acute oral toxicity evaulation is required.
LD50 Objective: This study aimed to determine the LD50 of synbiotic containing probiotic Streptococcus salivarius K12
Synbiotic mixture
and prebiotic Musa acuminata peel extract.
Streptococcus salivarius
Musa acuminata
Materials and Methods: The determination of LD50 is done according to the Acute Oral Toxicity test No. 425
(AOT425). For limit test, five female Sprague Dawley rats were given a limit dose of 2000 mg/kg of the synbiotic
mixture once orally, and observed for 12 days. For subacute toxicity test, twenty female Sprague Dawley rats
were randomised into 4 groups (n = 5). Control group received saline, others received synbiotic mixture at doses
175 mg/kg, 550 mg/kg, and 2000 mg/kg, respectively, and observed for 14 days. Animals were euthanised on
day-15, blood was collected, and subjected to haematological and biochemical analyses. Kidney and liver were
preserved for histopathological examination.
Result: No significant changes on the average body weight of the animals throughout the study. Haematological
parameters and biochemical analysis do not depict any changes related to acute toxicity. Histopathology analysis
depicted mild changes on kidney and liver.
Conclusion: Based on the data, the LD50 of the synbiotic formulation is higher than 2000 mg/kb, with no sign of
acute toxicity observed on all parameters.

1. Introduction prebiotics are fructans, galacto-oligosaccharides, starch and

glucose-derived oligosaccharides, pectin oligosaccharides, and
Probiotics are described as living microorganisms that, when taken non-carbohydrate oligosaccharides [3]. The synergistic combination of
in sufficient amounts, have beneficial effects on the host’s health [1]. It prebiotics and probiotics is known as a synbiotic. It is developed to
has been long used to maintain gastrointestinal health through the overcome the survival of the probiotic upon travelling through the upper
maintenance of the intestinal microbiome. To date, the most common intestinal tracts. Other studies demonstrate a better probiotic estab­
strains of probiotics are in the genera Lactobacillus, Bifidobacterium, lishment in the colon, thus contributing to the maintenance of intestinal
Saccharomyces, Streptococcus, Enterococcus, Escherichia, and Bacillus [2]. homeostasis [4].
Prebiotic is defined as a substrate that is selectively utilized by host Streptococcus salivarius K12 is a Gram-positive cocci bacterium, and it
microorganisms and that results in a health benefit. Common types of is part of the normal flora of the oral region. It was originally isolated

Peer review under responsibility of Transdisciplinary University, Bangalore.

* Corresponding author. Department of Craniofacial Diagnostics and Biosciences, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
E-mail address: shaqinah@ukm.edu.my (N.S. Nasruddin).

https://doi.org/10.1016/j.jaim.2024.101037
Received 17 September 2023; Received in revised form 13 December 2023; Accepted 5 July 2024
Available online 15 December 2024
0975-9476/© 2024 The Authors. Published by Elsevier B.V. on behalf of Institute of Transdisciplinary Health Sciences and Technology and World Ayurveda
Foundation This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A.S.M. Fuad et al. Journal of Ayurveda and Integrative Medicine 16 (2025) 101037

from the oral cavity of healthy children that had sustainably raised a biomass of the tested Candida spp. The study also showed that the
sizable native K12 strain oral cavity population for over a year, during S. salivarius K12 is able to remain viable when mixed with M. acuminata,
which no new S. pyogenes infections occurred [5]. Mainly inhabiting the with the optimized concentration of M. acuminata at 200 mg/mL.
tongue, this bacterium is among the pioneer colonisers of the oral sur­ Comparing total cell counts in prebiotic and synbiotic-treated biofilms
faces. It aids by preserving the healthy oral state of an individual by revealed that the synbiotic had a greater inhibitory effect on Candida
providing auxiliary defence against oral pathogens [6]. Some spp. total cell count in biofilms and hyphal formation than the prebiotic
commensal strains of this bacteria have been utilized as probiotics for alone. These studies also suggested that the presence of probiotics can
oral and upper respiratory health [5,7]. S. salivarius plays a very boost the anti-biofilm and anti-hyphae activity of the prebiotic,
important role in maintaining oral and gastrointestinal ecology by sus­ demonstrating a synergistic impact between the probiotic and prebiotic
taining the stability of microbiota composition, bacterial interference, [26]. To date, no acute toxicity study has been done to determine the
and interaction with the host [8]. Several studies have demonstrated the safety of the formulation. Therefore, this study is proposed.
antagonistic effect of S. salivarius against common oral pathogens.
During early oral colonization, S. salivarius has been described in 2. Methodology
competition with Streptococcus mutans and Streptococcus sobrinus for
adhesion sites in the oral cavity [9]. It also competes for adhesion site 2.1. Preparation of Streptococcus salivarius K12
against S. pyogenes on the human pharyngeal cell layer and inhibits
epithelial colonization by the periodontal pathogen Aggregatibacter Streptococcus salivarius K12 DSM-13084 was grown on Brain Heart
actinomycetemcomitans [7,10]. Infusion (BHI) agar and incubated at 37 ◦ C under anaerobic conditions.
The development of S. salivarius K12 as a probiotic is based on its S. salivarius K12 was sub-cultured on BHI liquid medium in an anaerobic
broad inhibitory activity against S. pyogenes. Further research explored jar supplied with an anaerobic gas generator sachet (Thermo Scientific
its diverse health benefits, ranging from treating halitosis to stimulating Oxoid AnaeroGen, USA). A 50% (v/v) glycerol stock was prepared by
immune defence against viral infection [11]. Probiotic therapy from mixing 500 μL of the inoculum with 500 μL of sterile glycerol in 1.5 mL
S. salivarius is used as an alternative treatment for pharyngotonsillitis of an Eppendorf tube and stored at − 80 ◦ C freezer (Thermo Scientific,
through several mechanisms, such as locally inhibiting the adhesion and USA).
growth of pathogenic bacteria and modulation of immune responses
[12,13]. A study by Taverniti et al. illustrates that S. salivarius can 2.2. Preparation of Musa acuminata aqueous peel extract
activate the macrophages by stimulating the pro-inflammatory cytokine
tumour necrosis factor-alpha (TNF-α) [12]. Musa acuminata, also known The peel of Musa acuminata was collected from local supplier. The
as the banana, is one of the most widely consumed fruits worldwide, due peel was cleaned, and the wet weight was recorded. Next, 100g of the
to its texture, flavour, high nutritional value, widely available and easy peel was blended to produce a soluble powder and added to a beaker of
to consume [14]. M. acuminata belongs to the Kingdom Plantae, Order of 500 mL distilled water. The decoction was filtered into another 500 mL
Zingiberales, and family of Musaceae. Originating from Southeast Asia. beaker. The concentrated filtrate undergoes a freeze-drying process until
One possible source of prebiotics is bananas [15]. This is contributed it produces a greenish, powder-like product in the jar. The M. acuminata
by several attributes, such as it contains a small amount of inulin that extract powder was kept in a dry environment with 30% relative
supports the growth of bacteria, mainly Bifidobacteria, related to humidity.
improving bowel health and general health [16,17]. Unripe banana has
a high amount of type 2 resistant starch, which can act as prebiotics 2.3. Preparation of synbiotic mixture
upon consumption [18]. This owes to the fact that resistant starch resists
digestion in the small intestine and undergoes fermentation in the large The concentration of MASE used in the biofilm study was optimized
intestine, hence feeding the growth of good bacteria in the colon [19]. A to 200 mg/mL, which has been shown to allow the probiotic to remain
study by Vu et al. illustrated that banana peel contains mixtures of viable under static biofilm formation methodology [26]. The formula­
phenolic compounds, although their composition may differ according tion is prepared through serial dilution, starting with a stock solution of
to the cultivation methods, pre-treatments, varieties, and maturity [20]. 1000 mg/mL by adding 1000 mg of freeze-dried extract of Musa acu­
Bioactive compounds such as phytosterols, biogenic amines and carot­ minata skin to 1 mL of sterile distilled water. The stock is then diluted to
enoids are also found in banana peels, suggestive of high antioxidant produce a working dilution of 800 mg/mL and 80 mg/mL. This is fol­
properties, and nutritional and gastrointestinal benefits of the peels lowed by the addition of 0.1 mL of 1 x 106 cells/mL of Streptococcus
[21]. Fructo-oligosaccharides (FOS) is another polysaccharides-based salivarius K12 into each working dilution. Working stock is prepared
prebiotic that is commonly extracted from banana peels. It acts as an daily (within 2 h before the oral gavage procedure) to ensure the
additive by enriching the taste and enhancing the texture. Another freshness and viability of the probiotic.
function of FOS is improving the nutritional prominence by acting as
prebiotics to support the viability of probiotics, boost the immune sys­ 2.4. Approval of animal ethics committee
tem, as well as reducing the risk of diabetes and cardiovascular disease
without altering the overall sensorial characteristic of the food [22,23]. All experiments involving an animal model were performed
A cellulose-based prebiotic fibre was successfully developed through following the guidelines for the care and use of an animal model
enzymatic hydrolysis by Phirom-on and colleagues. The water-soluble approved by the Universiti Kebangsaan Malaysia Animal Ethics Com­
cellulose did not support the growth of pathogenic bacteria (E. coli), munity (UKMAEC approval number: FPG/2021/NURRUL SHAQINAH/
but it significantly enhanced the growth of L. plantarum TISTR2075, 13-JAN./1148-JAN.-2021-DEC-2022).
which met the criteria for prebiotic fibre. Furthermore, the
water-soluble cellulose had a higher prebiotic activity index than the 2.5. Acute toxicity assay
commercial prebiotic [24].
This synbiotic mixture is developed as an oral mouthwash that is The procedure was performed according to OECD test Guidelines no
based on the combination of S. salivarius K12 and M. acuminata as an 425 (Up and Down Procedure) [27]. The Limit Test precedes the Main
intervention against oral candidiasis. This patented formulation has Test. Nulliparous and non-pregnant female Sprague-Dawley rats with an
been previously described to inhibit co-aggregation and biofilm forma­ average weight of 250g were randomly selected. This study complies
tion of oral pathogens, including C. albicans, in vitro [25]. A recent with the ARRIVE 2.0 reporting guidelines [28]. All animals were sub­
in-vitro study revealed that the synbiotic treatment reduced the biofilm jected to the standard husbandry setting, with a 5-day acclimatisation

2
A.S.M. Fuad et al. Journal of Ayurveda and Integrative Medicine 16 (2025) 101037

period. Commercialized rat pellets and water were supplied ad libitum. performed using SPSS software version 25.0 (IBM, USA), and the results
Photoperiods were adjusted to 12 h of light and 12 h of darkness daily, are regarded as statistically significant when p < 0.05.
and the environmental temperature was maintained at 21 ◦ C throughout
the study. Oral gavage procedure was done by trained competent 3. Results
handler and the ratswere checked for signs of aspiration pneumonia to
ensure the procedure was correct. The syringe was checked for leftovers 3.1. Body weight and behavioural pattern
to ensure that all substance entered the gut. Dosing concentrations
follow the OECD no. 425 guidelines for both Limit Test and Main Test The Body weight of the animals was measured daily from the
[27]. beginning of the study until euthanasia day. The result is tabulated as
the average weight of the animals according to the week (Fig. 1). Group
2.5.1. Limit test B (175 mg/kg) and Group D (2000 mg/kg) depicted the same pattern of
Limit test was carried out as a single dose of 2000 mg/kg p.o. given increase in average body weight in the control group. Group C (550 mg/
according to the body weight. The rats were kept without food 3–4 h kg) shows a slight increase in the average body weight. Statistical
before dosing, and 1–2 h after dosing. Following the survival of the analysis resulted in insignificant weight changes in all groups.
treated rat, the same dose was delivered to four additional rats in Post-treatment behavioural observation of the animals does not show
identical conditions. The animals were observed for 14 days for any any changes in respiration rate, sleepiness or drowsiness. Throughout
behavioural or toxic changes. the study period, the animals remained active with no signs of stress or
sickness.
2.5.2. Main Test
Main test was performed by administering a single-ordered dose 3.2. Biochemical analysis
progression in which the animal was dosed one at a time. 20 SD rats
were randomised into four groups (group A; treated with saline, group B; Serum was tested for kidney and liver biomarkers’ level. Important
treated with 175 mg/kg synbiotic formulation, group C; treated with biomarker for the kidney is creatinine and urea, while alanine trans­
550 mg/kg synbiotic formulation, group D; treated with 2000 mg/kg aminase (ALT), alkaline phosphatase (ALP), and aspartate transaminase
synbiotic formulation). All doses were given through oral route, with 48 (AST) are the biomarkers for the liver.
h interval of dosing, from 2000 mg/kg bw, down to 550 kg/bw and 175 A significant decrease in the creatinine level is observed in Group B
mg/kg, respectively. The dosing follows the 3.2 factor outlined in the (175 mg/kg) and Group D (2000 mg/kg), compared to the control
OECD Guidelines [27]. The animals were observed for 14 days for any group. The level of urea also decreased significantly in all treated
behavioural or toxic changes. groups, when compared to the control group. No significant changes are
Blood samples were collected in plain tubes for biochemical analysis observed in the level of liver biomarkers. All results are shown in
and EDTA tubes for haematological parameters analysis through cardiac Table 1.
puncture. The rats were euthanised using intramuscular injection of an
overdosed ketamine-xylazine cocktail (ketamine at 200mg/kg bw, 3.3. Haematological analysis
xylazine at 20 mg/kg bw). The liver and kidney were resected for his­
topathological analysis. Effects of the synbiotic formulation on haematological indices were
examined at the end of the observation period (Day 14). The formulation
2.6. Biochemical and haematological analysis does not have any significant effects on all haematological parameters
except for the Mean Corpuscular Haemoglobin Concentration (MCHC),
Blood was collected in plain tube (red-cap) to separate the serum (BD between the treatment group 550 mg/mL and 2000 mg/mL, when
Vacutainer, USA) for analysis of liver panel (alanine aminotransferase compared to the control group. The level of white blood cells and im­
(ALT), aspartate aminotransferase (AST), alkaline phosphate (ALP)) and mune cells remains within the normal range. All results are shown in
kidney panel (urea and creatinine). Other blood samples were collected Table 2 and Table 3.
in EDTA-containing tubes for haematological study (BD Vacutainer,
USA). CBC parameters, haemoglobin (Hb), total RBC, packed cell vol­
ume (PVC), mean corpuscular volume (MCV), mean corpuscular hae­
moglobin (MCH), mean corpuscular haemoglobin concentration
(MCHC), platelet count, white blood cells (WBC) count, neutrophils (N),
lymphocytes (L), monocytes (M), and eosinophils (E) were determined.
The samples were sent to the Veterinary Laboratory Service Unit,
Faculty of Veterinary Medicine, Universiti Putra Malaysia for analysis of
all parameters.

2.7. Histopathology

Kidney and liver were resected from all animals, The specimens were
fixed in 10% neutral-buffered formalin at room temperature, fixed,
embedded, and microtomed in 5.0 μm [29] thick slices along the lon­
gitudinal centre line. This was followed by deparaffinisation, rehydra­
tion and staining with Haematoxylin and Eosin (H&E). The slides were
examined under a light microscope and magnified images of tissue
structure were captured for further study.

2.8. Statistical analysis Fig. 1. The average body weight of all groups of animals during the study
period. All groups show an increase of body weight throughout the study
A descriptive analysis was achieved via mean (standard deviation) period. (*p values < 0.05 were considered as significant using one way ANOVA
for continuous data using one-way ANOVA. All statistical analyses were followed by Bonferroni post-hoc tests.

3
A.S.M. Fuad et al. Journal of Ayurveda and Integrative Medicine 16 (2025) 101037

Table 1
Values are expressed as mean ± SD (n = 5 for each group). *P values < 0.05 were considered as significant using one-way ANOVA followed by Bonferroni post-hoc
tests. Asterisks denote significant differences compared to the control.
ALP AST ALT TP ALB DBil TBil Creat Urea

U/L U/L U/L g/L g/L umol/L umol/L umol/L mmol/L

Control 228.332 232.428 77.584 71.86 30.606 0.1236 0.813 47.716 7.6694
175mg/kg 200.068 196.466 78.152 78.5 35.286 0.0366 0.902 36.59* 5.898*
550 mg/kg 173.374 213.608 81.122 76.94 29.442 0.0872 1.452 44.584 5.889*
2000 mg/kg 156.86 173.616 81.838 77.98 33.556 0.172 1.651 39.484* 5.734*

Table 2
Values are expressed as mean ± SD (n = 5 for each group). *P values < 0.05 were considered as significant using one-way ANOVA followed by Bonferroni post-hoc
tests. Asterisks denote significant differences compared to the control.
RBC Hb PCV MCV MCHC MCV MCHC WBC
12
x10 /L g/L L/L fL g/L fL g/L x109/L

Control 7.34 137.80 0.42 57.92 343.97 57.92 324.96 9.12

175mg/kg 7.31 140.20 0.42 57.23 335.56 57.23 335.56* 7.60
550 mg/kg 7.13 141.40 0.41 56.94 348.46 56.94 348.46 9.52
2000 mg/kg 7.52 143.20 0.41 54.77 347.75 54.77 347.75* 6.96

M. acuminata skin extract shows no evidence of acute toxicity [30,31].

Table 3
Blood indices from the sub-acute toxicity study depicted a significant
Values are expressed as mean ± SD (n = 5 for each group). *P values < 0.05 were
change in the Mean Corpuscular Haemoglobin Concentration (MCHC)
considered as significant using one-way ANOVA followed by Bonferroni post-
hoc tests. Asterisks denote significant differences compared to the control.
from group C (550 mg/kg) and group D (2000 mg/kg), compared to the
control group. The MCHC test is one of the RBC indices, which is a group
Band Neutro Lymp Mono Eosin Baso PLT
of tests that offer information regarding RBC parameters such as size,
N
shape, and quality. An increase in MCHC is noted in individual animals
x109/ x109/ x109/ x109/ x109/ x109/ x109/L
from both groups. This can be attributed to technical error as the sam­
L L L L L L
ples were kept standing for more than an hour. Samples that have been
Control 0.09 1.45 6.84 0.56 0.178 0 816.4 sitting for more than an hour should be properly mixed by inverting 5 to
175mg/ 0.08 1.42 5.23 0.57 0.30 0.00 848.40
10 times before testing. Automation’s 45-degree mixing of samples may
kg
550 mg/ 0.10 2.51 6.09 0.62 0.20 0.00 769.80 not be enough to disperse aggregated red cells, resulting in an errone­
kg ously low red cell count and a high MCHC [32]. In the current study, no
2000 mg/ 0.07 1.44 4.69 0.62 0.14 0.00 830.00 significant changes were seen in the level of immune cells, attesting the
kg
formulation does not alter the immune level, nor have an impact on the
immune pathways that may cause unwarranted physiological changes.
3.4. Histopathology analysis This result is in line with a randomized, placebo-controlled, double-­
blind study on the safety of S. salivarius K12 [33]. A previous study on
3.4.1. Kidney chicken fed with banana peel also exhibited no changes in the immune
Light microscopy examination of kidney histology depicted normal cell level, thus supporting this result [34].
histology in the control group Figure A. Several glomerular shrinkages The liver is a susceptible organ to hepatotoxicity since it is the pri­
are seen in group Figure B and C. Congestions of blood vessels are also mary organ for xenobiotic processing and detoxification. The AST and
observed in group C and D. All results are shown in Fig. 2. ALT are important enzymes in the conversion of amino acids into -keto
acids, which are then transported via the Krebs cycle and electron
3.4.2. Liver transport chain for full metabolism. They are thought to be accurate
Light microscopy examination of kidney histology depicted normal indicators of liver injury. When the liver is damaged, these marker en­
histology in the control group (A). Vacuolation and blood vessel zymes are released into the system over a specific threshold [35]. Liver
congestion are seen in group B. Empty nucleus is observed in group C. parameters (ALT, AST, and ALP) present no significant changes, estab­
Vacuolation is also observed in Group D. All results are shown in Fig. 3. lishing that the formulation does not have a significant impact on bodily
metabolism and detoxification. Previous studies on the sub-acute
4. Discussion toxicity of S. salivarius K12 also demonstrated similar results [30]. Pre­
vious studies on the acute toxicity of M. acuminata peel extract also
In the current study, acute toxicity assays were conducted using limit exhibit matching results [31]. Thus, this establishes that this synbiotic
tests, which were carried out by administering various dosages orally in formulation is not hepatotoxic. On the other hand, liver histology de­
accordance with OECD guidelines. Acute toxicity study resulted in the picts several abnormalities such as vacuolation, loss of nucleus, and
determination of the LD50 of the synbiotic formulation of M. acuminata blood congestion. The levels of ALT and AST, which are the main in­
peel extract and S. salivarius K12 to be greater than 2000 mg/kg. Ac­ dications of liver injury, were not considerably altered in the liver. As a
cording to the findings, although Group B and Group D showed a similar result, these changes may not be toxicologically important, given that
trend of growth in average body weight with the control group. The other biochemical analysis findings contradicted them.
average body weight does, however, slightly increase in Group C. The Serum creatinine and urea are common indicators for predicting
animals exhibited a uniform increase in the body weight throughout the renal dysfunction because they are significantly raised when the kidney
observation period, with no signs of clinical distress. It is supported by is dysfunctional. Serum creatinine levels normally do not rise until at
previous individual studies of acute toxicity for S. salivarius K12 and least half of the kidney’s nephrons are destroyed or damaged. As
simultaneous detection of blood creatinine and urea is usual practice for

4
A.S.M. Fuad et al. Journal of Ayurveda and Integrative Medicine 16 (2025) 101037

Fig. 2. The histology of kidneys from rats treated with the synbiotic formulation. The green arrow depicts the normal glomerulus. Glomerular shrinkage is shown by
the black arrow, while blood vessel congestion is shown by the blue arrow. (A; control, B; 175 mg/kg, C; 550 mg/kg, D; 2000 mg/kg).

Fig. 3. The histology of liver from rats treated with the synbiotic formulation. vacuolation is shown by green arrow, while empty nucleus is shown by yellow arrows.
(A; control, B; 175 mg/kg, C; 550 mg/kg, D; 2000 mg/kg).

differential diagnosis, serum urea was also calculated [36]. Kidney S. salivarius K12.
parameter show significant changes in the level of creatinine and urea.
The level of creatinine illustrated a significant decrease in Group B (175 5. Conclusion
mg/kg) and Group D (2000 mg/kg). Urea levels also depicted a signif­
icant decrease in all groups, compared to the normal control. This Based on this study, the LD 50 of the oral synbiotic formulation is
finding contradicts a study on the effect of banana peel extract on kidney greater than 2000 mg/kgbw. No changes are observed in the haema­
indices resulted in an increase in both urea and creatinine levels [31]. tology indices and biochemical parameters of the kidney and liver. The
This decrease can be attributed to the anti-inflammatory effect of weight of the rats increases throughout the study period. Histology of

5
A.S.M. Fuad et al. Journal of Ayurveda and Integrative Medicine 16 (2025) 101037

the liver and kidney depicted minimal changes. Thus, this oral synbiotic [10] Teughels W, Haake SK, Sliepen I, Pauwels M, Eldere JV, Cassiman JJ, Quirynen M.
Bacteria interfere with A. actinomycetemcomitans colonization. J Dent Res 2007;86
mouthwash is generally safe. A study on the sub-chronic toxicity can be
(7):611–7.
done to test the effect of the synbiotic upon long-term exposure. Further [11] Wescombe PA, Hale JD, Heng NCK, Tagg JR. Developing oral probiotics from
study on the minimal therapeutic effective dose can be done to identify Streptococcus salivarius. Future Microbiol 2012;7(12):1355–71. https://doi.org/
the dose that, on average, produces a therapeutic effect on the disease. 10.2217/fmb.12.113.
[12] Taverniti V, Minuzzo M, Arioli S, Junttila I, et al. In Vitro functional and
Efficacy study with a larger population sample or bigger animal is also immunomodulatory properties of the Lactobacillus helveticus MIMLh5-Streptococcus
suggested to improve the evaluation of the safety and provide better salivarius ST3 association that are relevant to the development of a pharyngeal
guidelines for prescribed dose levels. probiotic product. Appl Environ Microbiol 2012;78(12):4209–16. https://doi.org/
10.1128/AEM.00325-12 [Article].
[13] Tapiovaara L, Pitkaranta, Korpela R. Probiotics and the upper respiratory tract—a
Funding sources review. Pediatr Infect Dis Open Access 2016;1:19.
[14] Miranda-Castro SP. Chapter 3 - application of chitosan in fresh and minimally
processed fruits and vegetables. In: Bautista-Baños S, Romanazzi G, Jiménez-
This work was supported by the Fundamental Research Grant Aparicio A, editors. Chitosan in the preservation of agricultural commodities.
Scheme, Ministry of Higher Education Malaysia, (FRGS/1/2022/SKK0/ Academic Press; 2016. p. 67–113. https://doi.org/10.1016/B978-0-12-802735-
UIAM/02/2). 6.00003-3.
[15] Powthong P, Jantrapanukorn B, Suntornthiticharoen P, Laohaphatanalert K. Study
of prebiotic properties of selected banana species in Thailand. Journal of Food
Author contributions Science Technology 2020;57(7):2490–500. https://doi.org/10.1007/s13197-02
0-04284-x.
[16] Moshfegh AJ, Friday JE, Goldman JP, Ahuja JKC. Presence of inulin and
Original Idea from Mohd Hafiz Arzmi. Experimental design and data
oligofructose in the diets of Americans. J Nutr 1999;129(7):1407S–11S. https://
collection by Nurrul Shaqinah Nasruddin, Aalina Sakiinah Mohd Fuad doi.org/10.1093/jn/129.7.1407S.
and Muhammad Ekmal Bakar. Data analysis and confirmation by Nurrul [17] Kelly G. Inulin-type prebiotics–a review: part 1. Alternative Med Rev 2008;13(4):
Shaqinah Nasruddin, Fazle Khuda and Izatus Shima Taib. Manuscript 315–29.
[18] Dibakoane SR, Du Plessis B, Da Silva LS, Anyasi TA, Emmambux MN, Mlambo V,
writing by Aalina Sakiinah Mohd Fuad. All authors thoroughly revised et al. Nutraceutical Properties of Unripe Banana Flour Resistant Starch: A Review.
the manuscript. Starch –Stärke 2023;75(9–10):2200041. https://doi.org/10.1002/star.202200041.
[19] Guo J, Tan L, Kong L. Impact of dietary intake of resistant starch on obesity and
associated metabolic profiles in human: a systematic review of the literature.
Declaration of generative AI in scientific writing Critical Reviews in Food Science and Nutrition 2020;61(6):889–905. https://doi.
org/10.1080/10408398.2020.1747391.
The authors declare that this article did not use generative AI or AI- [20] Vu HT, Scarlett CJ, Vuong QV. Phenolic compounds within banana peel and their
potential uses: a review. J Funct Foods 2018;40:238–48. https://doi.org/10.1016/
assisted technologies. j.jff.2017.11.006.
[21] Singh B, Sing JP, Kaur A, Sing N. Bioactive compounds in banana and their
Conflict of interest associated health benefits – a review. Food Chem 2016;206:1–11. https://doi.org/
10.1016/j.foodchem.2016.03.033.
[22] Pereira GA, Arruda HS, Molina G, Pastore GM. Extraction optimization and profile
The authors declare that they have no known competing financial analysis of oligosaccharides in banana pulp and peel. J Food Process Preserv 2018;
interests or personal relationships that could have appeared to influence 42(1):e13408. https://doi.org/10.1111/jfpp.13408.
[23] Azam M, Saeed M, Pasha I, Shahid. A prebiotic-based biopolymeric encapsulation
the work reported in this paper.
system for improved survival of Lactobacillus rhamnosus. Food Biosci 2020;37:
100679. https://doi.org/10.1016/j.fbio.2020.100679.
Acknowledgement [24] Phirom-on K, Apiraksakorn. Development of cellulose-based prebiotic fiber from
banana peel by enzymatic hydrolysis. Food Biosci 2021;41:101083. https://doi.
org/10.1016/j.fbio.2021.101083.
We acknowledge the Ministry of Higher Education Malaysia for [25] Rismayuddin NAR, et al. The effect of synbiotic Streptococcus salivarius K12 and
providing research funds (FRGS/1/2022/SKK0/UIAM/02/2). We also Musa acuminata on Candida albicans biofilm formation. Malaysian J Med Health Sci
acknowledge International Islamic Unversity Malaysia and Universiti 2019;15(108).
[26] Rismayuddin NAR, Badri EAM, Ismail AF, Othman N, Bandara HMHN, Arzmi MH.
Kebangsaan Malaysia for providing the laboratory facilities. Synbiotic Musa acuminata skin extract and Streptococcus salivarius K12 inhibit
Candida species biofilm formation. Biofouling 2022:1–14. https://doi.org/
References 10.1080/08927014.2022.2105142.
[27] OECD. Test No. 425: acute oral toxicity: up-and-down procedure. https://doi.
org/10.1787/9789264071049-en; 2008.
[1] Zendeboodi F, Khorshidian N, Mortazavian AM, da Cruz AG. Probiotic:
[28] Percie SN, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The Arrive
conceptualization from a new approach. Current Opinion in Food Science 2020;32:
guidelines 2.0: updated guidelines for reporting animal research. PLoS Biol 2020;
103–23. https://doi.org/10.1016/j.cofs.2020.03.009.
18:e3000410. https://doi.org/10.1186/s12917-020-02451-y.
[2] Sánchez B, Delgado S, Miguez AB, Lourenco A, Gueimonde M. Probiotics, gut
[29] Feldman A, Wolfe D. Tissue processing and hematoxylin and Eosin staining.
microbiota, and their influence on host health and disease. Mol Nutr Food Res
Methods Mol Biol 2014;1180:31–43. https://doi.org/10.1007/978-1-4939-1050-
2017;61(1):1600240. https://doi.org/10.1002/mnfr.201600240.
2_3.
[3] Saleem U, Amin S, Ahmad B, Azeem H, Anwar F, Mary S. Acute oral toxicity
[30] Burton JP, et al. Extended safety data for the oral cavity probiotic Streptococcus
evaluation of aqueous ethanolic extract of Saccharum munja Roxb. roots in albino
salivarius K12. Probiotic Antimicrobial Proteins 2010;2(3):135–44. https://doi.
mice as per OECD 425 TG. Toxicol Rep 2017;4:580–5. https://doi.org/10.1016/j.
org/10.1007/s12602-010-9045-4.
toxrep.2017.10.005.
[31] Edenta C, Okoduwa SIR, Okpe O. Effects of aqueous extract of three cultivars of
[4] Tester RF, Al-Ghazzewi FH. Role of prebiotics and probiotics in oral health. Nutr
banana (Musa acuminata) fruit peel on kidney and liver function indices in wistar
Food Sci 2018;48(1):16–29. https://doi.org/10.1108/nfs-03-2017-0056.
rats. Medicines 2017;4(4):77. https://doi.org/10.3390/medicines4040077.
[5] Wescombe PA, Burton JP, Cadieux PA, Klesse NA, Hyink O, Heng NCK, et al.
[32] Constantino BT, Cogionis B. High mean corpuscular hemoglobin concentration:its
Megaplasmids encode differing combinations of lantibiotics in Streptococcus
causes and effects on automated CBC results. Can J Med Lab Sci 2007;69(3):
salivarius. Antonie Leeuwenhoek 2006;90(3):269–80. https://doi.org/10.1007/
113–26.
s10482-006-9081-y.
[33] Burton JP, Chilott CN, Wescombe PA, Tagg JR. Evaluation of safety and human
[6] Masdea L, Kulik EM, Gerspach IH, Ramseier AM, Filippi A, Waltimo. Antimicrobial
tolerance of the oral probiotic Streptococcus salivarius K12: a randomized, placebo-
activity of Streptococcus salivarius K12 on bacteria involved in oral malodour. Arch
controlled, double-blind study. Food Chem Toxicol 2011;49(9):2356–64. https://
Oral Biol 2012;57(8):1041–7. https://doi.org/10.1016/j.archoralbio.2012.02.011.
doi.org/10.1016/j.fct.2011.06.038.
[7] Guglielmetti S, Taverniti V, Minuzzo M, Arioli S, Stuknyte M, Karp M, Mora D. Oral
[34] Duwa H, Saleh B, Lamido M, Saidu A. Growth, haematological and serum
bacteria as potential probiotics for the pharyngeal mucosa. Appl Environ Microbiol
biochemical indices of broiler chickens fed banana peel meal as replacement for
2010;76(12):3948. https://doi.org/10.1128/AEM.00109-10.
maize in the semi-arid zone of Nigeria. Online J Anim Feed Res 2014;4(5):121–6.
[8] Delorme C, Abraham AL, Renault P, Guedon E. Genomics of Streptococcus salivarius,
[35] Casotti V, D’Antiga L. Basic Principles of Liver Physiology. In: D’Antiga L, editor.
a major human commensal. Infect Genet Evol 2015;33:381–92. https://doi.org/
Pediatric Hepatology and Liver Transplantation. Cham: Springer International
10.1016/j.meegid.2014.10.001.
Publishing; 2019. p. 21–39. https://doi.org/10.1007/978-3-319-96400-3_2.
[9] Tanzer J, Kurasz AB, Clive J. Inhibition of ecological emergence of mutans
streptococci naturally transmitted between rats and consequent caries inhibition by
Streptococcus salivarius TOVE-R infection. Infect Immun 1985;49(1):76–83.