Introduction
Prebiotic
is non-digestible food ingredients that beneficially affect the host by
selectively stimulating the growth of bacteria in the colon and stimulating
native microflora in the gut (Ashaolu et al.
2021; El-Sayed et al. 2021; Tawfick et al. 2022). The prebiotic also
could confer a health benefit (Slizewska and
Chlebicz-Wójcik 2020; Zabihollahi et al. 2020; El-Sayed et al.
2021). Prebiotics not only have protective effects on the
gastrointestinal system but also on other parts of the body, such as the
central nervous system, immune system, and cardiovascular system (Davani-Davari et
al. 2019). Prebiotic-based substances are derived from the sources
natural such as inulin, oligofructose, fructo-oligosaccharide,
galacto-oligosaccharide, mannooligosaccharide, etc. (Zhang et al. 2022; Parsana et al.
2023). Inulin is a natural and polydisperse β-(2-1) fructans (Zhang
et al. 2015). Inulin is type
of prebiotics which can be added food to stimulate the proliferation of lactic
acid bacteria (Valero-Cases and Frutos 2017;
Davani-Davari et al. 2019). Inulins are carbohydrates and act like dietary
fiber (Zhang et
al. 2015). Inulin-type fructans are dietary fiber that cannot be
digested by human digestive enzymes (Sun et
al. 2020). According to a recent study in 2021, mangrove apples were
revealed to be soluble dietary fibers that significantly contain inulin, which
can be a source to natural prebiotics (Wibawanti
et al. 2021).
Fermentation of prebiotics by gut microbiota
produces short-chain fatty acids (SCFAs), such as propionic acid, butyric acid,
and acetic acid. They add to SCFAs role in human body, and maintaining
intestinal health (Boger et al. 2018; Davani-Davari et
al. 2019; Sun et al. 2020).
There are abundant studies to determine the prebiotics activity by employing
probiotics bacteria Lactobacillus is
a strain of lactic acid bacteria that can utilize prebiotics of inulin (Paula et al.
2020). Prebiotics from inulin and fructo-oligosaccharide support the growth of L. acidophilus bacteria (Masroor et al. 2019). Prebiotics have been investigated in several
studies to support the growth of lactic acid bacteria. Lesser yam inulin was
stimulated on the growth of Lactobacillus
and Bifidobacterium (Winarti et al.
2013). Carrot and orange juice fortified with inulin was supports the
survival of L. plantarum (Valero-Cases and Frutos 2017). Various
concentrations of prebiotic inulin was stimulated on the growth of L. bulgaricus, L. acidophilus, and Streptococcus
thermophilus (Setiarto et al. 2017).
L. bulgaricus as gram
positive are used for dairy products in yogurt (Wibawanti et al. 2022; Wibawanti et al. 2023).
To date, the information about the use of inulin
from mangrove apples as a source of prebiotics is still limited. The aim of this study was to investigate the efficacy of inulin extracts
from mangrove apples as type a
prebiotic source for L. bulgaricus growth using In
vitro fermentation experiments.
Materials and Methods
Bacteria treatment
The
probiotic bacteria used in this research was L. bulgaricus (FNCC 0041) containing 108 CFU/mL. The
bacteria were obtained from Food and Nutrition Culture Collection, Center for the
Study of Food and Nutrition, Universitas Gadjah Mada, Yogyakarta, Indonesia.
The purified colonies were introduced to de Man Rogosa and Sharpe (MRS) broth
and incubated at 37°С for 24 h.
Inulin of mangrove apple treatment
The
mangrove apple inulin with the extraction method was carried out according to
the method used by Wibawanti et al. (2022) method. The
mangrove apple was sliced thin and heated in hot water 90°C for 60 mins with 1:
4 ratios. The filtrate was precipitated with 40% ethanol and kept at-18°C for 6
h. The filtrate was defrosted at room temperature. The filtrate was centrifuge
for 5 min at 5000 rpm and supernatant was collected.
The experiment was principally set up based on a
completely randomized design. The research was randomly divided into five
treatment groups with four replicates. The different concentrations of inulin
of mangrove apples (IMA) were 0, 2.5, 5, 7.5 and 10% (w/v) in the Man Rogosa
Sharpe (MRS) Broth of media culture.
Assessment of prebiotic activity
The
prebiotic activity of inulin from mangrove apple was determined In vitro by counting the bacterial
population of Lactobacillus bulgaricus (Shubha et al.
2022). MRS agar was used to count lactic acid bacteria (LAB) and the
plates were anaerobically incubated at 37°C for 24 h. Total number of colonies
was recorded as CFU/mL. It was calculated in plates containing 25–250 colonies.
Determination of pH values
The
pH values of the inulin from mangrove apple were determined using a pH meter
that had previously been calibrated with pH 7.0 and 4.0 standard buffers (Rezende et al.
2022). All analyses were performed in duplicate at 20°C.
Determination of titratable acidity
(TA)
The
TA value of inulin of mangrove apple was determined using the method described
by Zhao et al. (2022). A few drops of
phenolphthalein were added as the colored indicator solution and titrated with
0.1 M NaOH solution.
SCFAs analysis
The
SCFAs were measured following the method of Ashwar
et al. (2021). The
High-Performance Liquid Chromatography (HPLC; Shimadzu GC-2010, Japan) system
was modified to examine the short chain fatty acids (SCFA). Together with
vigorous shaking, 20 mL of 0.4%. HCl and about 1 mL of the fermentation broth
were combined. Then it underwent the centrifugation process (3000 rpm, 4°C) for
10 mins. Syringe filters with a 0.2 m pore size were used to filter the
supernatant. Acetonitrile and 0.1% H3PO3 were used as the
mobile phase and at a flow rate of 1 mL/min during in chromatographic
separation on an Agilent Zoffax Eclipse Plus C18 column (4.6 100 mmol/L, 3.5
m).
Statistical analysis
All
experimental data were presented in mean values with ± standard deviation (SD).
Statistical differences among the results were analyzed using one way analysis
of variance (ANOVA) using SPSS software. Differences were considered
statistically significant at P < 0.05.
Results
The effect of IMA on the growth of L. bulgaricus is presented in Fig. 1. Based on the results, total lactic acid with
different concentration of IMA had significant (P<0.05) effect on the
viability of L. bulgaricus. The amount of probiotic L. bulgaricus varied following
the incubation for 24 h. The highest growth rate of L. bulgaricus the MRS
Broth media added with 10% IMA (P<0.05) with value 9.63±0.61 log CFU/mL. However, concentrations of 5 and 7% of IMA did
not show significant (P>0.05) difference in the viability of L. bulgaricus with a
value of 8.96±0.67 and 9.09±0.61 log CFU/mL, respectively. Total population of L. bulgaricus
with 0 and 2.5% was 8.28±0.5 and 8.77±0.47 log CFU/mL, respectively
(P>0.05).
The pH values of IMA are represented in Fig. 2.
Based on the results, L. bulgaricus
exhibited trends of pH reduction at higher concentrations of IMA. The pH value
of IMA with a concentration of 10% was the lowest compared with another concentration
3.46±0.01 (P<0.05). The pH value of 7.5% IMA was also lower (P<0.05)
compared to other treatments. However,
concentrations 0, 2.5 and 5.0% of IMA did not show distinct differences
(P>0.05) in the pH value, where 3.6±0.01; 3.58±0.01, and 3.55±0.03 were
marked, respectively.
There was an increase in lactic acid
levels during the inulin fermentation by L.
bulgaricus for 24 h in the control %20339-343_files/image002.png)
Fig. 1:
The addition of IMA on the
growth of L. bulgaricus
%20339-343_files/image004.png)
Fig. 2: The addition of IMA on the pH value
%20339-343_files/image006.png)
Fig. 3: The addition of IMA on the titratable acidity
and concentration IMA of 2.5, 5.0, 7.5, and 10%.
Fig. 3 shows the titratable acidity of inulin from mangrove apples. The highest
concentrations of lactic acid were observed when the L. bulgaricus was grown in the presence of 10% of IMA with an
average amount of 1.55±0.02% (P<0.05). However, the titratable acidity value
of the IMA concentration of 7.5% (1.50±0.01%) was not significantly different
(P>0.05) with the value obtained by the IMA concentration of 10%. The IMA
concentrations 0, 2.5 and 5% of IMA were not different (P>0.05) in the
titratable acidity value, 1.48±0.01, 1.47±0.07 and 1.48±0.03%, respectively.
The content of acetic acid during fermentation of
IMA for 24 h incubation is shown in Fig. 4. The result of acetic acid
fermentation from L. bulgaricus with
the different IMA concentrations of 0, 2.5, 5.0 7.5 and 10% as much as 30.79,
30.22, 29.47, 27.53 and 29.58 mM, respectively. The content of propionic acid during fermentation
at incubation time 24 h of IMA are shown in Fig. 5. The result of propionic
acid fermentation from L. bulgaricus
with the diverse IMA proportions of 0, 2.5, 5.0, 7.5 and 10% as much as 0.07,
0.58, 0.39, 0.21 and 0.24 mM, respectively. The content of butyric acid during fermentation at incubation time 24 h
of IMA are shown in Fig. 6. The result of acetic acid fermentation from L. bulgaricus with the addition of IMA
with the various IMA amounts of 0, 2.5, 5, 7.5 and 10% as much as 0.34, 0.73,
0.85, 0.18 and 0.38 mM, respectively.
Discussion
In
this study, increasing total viability of L.
bulgaricus was associated with
the concentration of the IMA as a prebiotic (Fig. 1). This means that L. bulgaricus growth could be stimulated by IMA, which could
thus be used as an energy source in correlation with decrease of pH and the
increase of lactic acid content. Kanjan and
Hongpattarakere (2017) reported that inulin could support the
growth of L. paracasei. Further, Setiarto et al. (2017) claimed that the
addition of inulin could increase the growth of L. bulgaricus bacteria. Valero-Cases
and Frutos (2017) also claimed that the addition of inulin could support
on the viability of L. plantarum. Palacio et al.
(2014) reported that the activity of prebiotics which could act as a
carbon source so as to stimulate the growth of probiotic. In the literature Kanjan and Hongpattarakere (2017) observed a
significant grow of Lactobacilli and Bifidobacteria along with an increase in
the amount of lactic acid the decrease of pH, and SCFAs. The improving of
probiotic stability by addition of inulin can be explained the formation of
interactions (hydrogen bonds) between inulin and polar head groups of membranes
phospho-lipids of probiotic bacteria (Zabihollahi
et al. 2020).
The presence of L. bulgaricus resulted in a decrease in the pH values of the
culture media. In this research, a decrease
in pH affected the lactic acid produced by L. bulgaricus (Fig. 2).
Reduction in pH, which results in a higher level of organic acid produced, may
indicate greater fermentability. Oluwatosin et al. (2022) reported that the
pH reduction of L. plantarum
cultivated in 2% (m/v) glucose-free MRS media of inulin with value of 4.78. Rezende et al.
(2022) reported that the decrease in pH values indicates metabolic
activity of probiotic microorganisms and production of organic acid. Napisah et al.
(2022) reported that L. brevis
was also found to reduce pH in media containing inulin and fibersol-2.
An increase in the titratable acidity value may
be due to the decrease in pH value (Fig. 3). Lactic acid is also used as
a carbon source by L. bulgaricus in
improving growth by increasing metabolism. Napisah
et al. (2022) reported that %20339-343_files/image008.png)
Fig. 4: The
addition of IMA on the acetic acid
%20339-343_files/image010.png)
Fig. 5: The addition of IMA on the propionic acid
%20339-343_files/image012.png)
Fig. 6: The addition of IMA on the butyric acid
lactic acid is the main end
product of LAB’s use of carbohydrates during fermentation. Winarti et al.
(2013) reported that lesser yam inulin was used L. acidophilus for produces lactic acid production during
fermentation.
Different amounts of acetic
acid, propionic acid and butyric acid were produced by the fermentation of L. bulgaricus with the addition of IMA
(Figs. 4, 5 and 6). IMA is one of the substrates, which is fermented in the
intestine and is known to stimulate the production of SCFA. Johnson et al.
(2015) reported that inulin is one of the substrates that is fermented
in the intestine and is known to stimulate the production of SCFAs. Ashwar et al. (2021) reported
that varying propionic acid, acetic acid and butyric acid were produced from the
fermentation of resistant starch by Lactobacilli.
Buruiana et
al. (2017) investigated the prebiotic of xylo-oligosaccharides from corn
stover giving effect to the amount of SCFA produced by Lactobacillus, Enterococcus
and Bacteroides genera.
Conclusion
The IMA at 10% concentration stimulated the growth
of L. bulgaricus with a value of
9.63±0.61 log CFU/mL, pH value 3.46±0.01 and total lactic acid with a value of
1.55±0.02%. Fermentation by L. bulgaricus
with IMA produced different amounts of acetic acid, propionic acid and butyric
acid. The IMA may serve as a potential source of prebiotics that may help L. bulgaricus survive.
Acknowledgements
This study was supported
by Majelis Dikti-Litbang
PP Muhammadiyah through Hibah Riset
Muhammadiyah Batch VI. We would like to express our gratitude for the
collaboration between Muhammadiyah University Purworejo (UMPWR), Universiti
Teknologi Malaysia (UTM), and Mangrove
Institut Komango Foundation.
Author Contributions
JMWW planned the experiments, conducted lab works,
statistically analyzed the data, and interpreted the results, Z interpreted the
results, LMS and HK reviewed and revised the manuscript and SP conducted field
work.
Conflicts of Interest
All authors declare no conflict of interest.
Data Availability
Data presented in this study will be available on a fair
request to the corresponding author.
Ethics Approval
Not applicable to this paper
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