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Studies on Growth Variables, Metabolites and Safety Effect of
Lactobacillus Acidophilus, Streptococcus Salivarious Subsp.
Thermophilus and Lactobacillus Delbrueckii Subsp. Bulgaricus (As
Starter Culture) on Cocos Typical Based Extract During Time-
Monitoring Bioprocessing (Fermentation)
Esther Omobola Areo
*
Department of Microbiology, Faculty of Natural and Applied Sciences, Hallmark University, Ijebu Itele,
Ogun
*Corresponding Author
DOI:
https://doi.org/10.51583/IJLTEMAS.2026.150300042
Received: 21 March 2026; Accepted: 28 March 2026; Published: 09 April 2026
ABSTRACT
The role of Lactic Acid Bacteria (LAB) is vital during bio-processing (fermentation) which could result to
desirable nutritional, sensory and safety quality product. This study investigated on growth kinetics variables,
metabolites and safety effect of Lactobacillus acidophilus, Streptococcus salivarious subsp. thermophilus and
Lactobacillus delbrueckii subsp. bulgaricus (as starter culture) on Cocos typical based extract during time-
monitoring bio-processing. This was done anaerobically in the bioreactor with optimized timing of 0-72 h at
37 °C, but samples were drawn every 12 h intervals. Specific LAB growth rate was calculated using Monod
equation while specific consumption rate of substrates and generation rate of metabolite in the fermenting
samples were modeled using Luedeking-Piret equation. The total LAB count (6.36 to 10.54 log10 cfu/ml) and
lactic acid concentration (0.07 to 1.74%) increased but pH (6.48 to 4.03), total sugar (20.96 to 10.88%), total
soluble sugar (3.01 to 0.17%), total solid (5.98 to 2.81%) and fructo-oligosaccharide (100.05 to 34.31 mg/kg)
decreased with increasing fermentation period. At 12 h, higher specific growth rate with the lowest doubling
time of 0.22 h
-1
and 3.15 h respectively. Both specific fructose and sucrose rate consumed by the lactic acid
bacteria were higher at 72 h. Similarly, at 72 h, more lactic acid was produced and the least concentration was
observed at 36 h of fermetation period. Zone of inhibition as antibacterial effect of each samples against
Escherichia coli, Salmonella typhi and Staphylococcus aureus ranged from 10.11-19.33 mm, 9.13-19.83 mm
and 9.89-21.17 mm respectively. It was deduced accordingly that the Cocos typical extracts (at 24 h and 36 h)
served as good carbon sources for Lactobacillus acidophilus, Streptococcus salivarious subsp. thermophilus and
Lactobacillus delbrueckii subsp. bulgaricus) and the production of useful metabolites that could guarantee the
prevention of pathogens was enhanced.
Keywords: Cocos typical extract; bio-processing (fermentation); growth kinetics; antibacterial activity, Lactic
Acid Bacteria (LAB)
INTRODUCTION
Plant milk are becoming more demanding because they aid consumers’ health and they are used to replace
animal-based milk (with exorbitant cost in Nigeria) (Amapu et al., 2024; Alma and Judith, 2024; Adebo and
Sobowale, 2025). As a result, focus has been on milk extract from plants such as fruits, nuts and legumes (Akusu
and Emelike, 2018; Fatemeh, 2014; Okoroafor et al., 2025). Within the last few decade, more researchers have
interest to study the growth kinetics variables of Lactic Acid Bacteria (LAB) in fermented plant-sourced foods
(Kpikpi et al., 2009). Most of them focused on the enhancement of flavor, texture, composition and safety
qualities that Cocos typical extract that LAB could impart (Zipori et al., 2024; Pua et al., 2022). However, Lactic
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Acid Bacteria ingested (via this food) must be in right proportion, before they can render health benefits beyond
inherent general nutrition (Coşansu et al., 2021; Domínguez-Murillo and Urías-Silvas, 2024).
Optimum desirable quality of LAB fermented foods (in terms of nutritional, sensory and safety values are
exhibited at specific fermentation time and temperature (Undugoda and Nilmini, 2019). Their activity and
growth vary under various proportions, temperature, fermentation time, pH and water activity. According to
Shetty et al., (2017). these parameters could be determined or illustrated mathematically. This study focused
more on the optimization of fermentation time and specific temperature for LAB-processed Cocos typical and
how these affect other advantageous parameters. This could limit cost of industrial plant mass and ensure correct
conditions as well as consistent outputs (Barao et al., 2018). This further can predict the amount of desirable
metabolites produced at specific condition or the quantity of consumed carbon sources during fermentation
(Zhang et. al., 2018). The production of lactic acid is time and temperature dependent. The microorganisms
involved are known to have optimum growth at 37
o
C, according to Olusola (2014).This work centered on several
fermentation period, in order to establish the optimum time the vital metabolites or improved properties could
widely be generated in the food.
Growth of LAB is stimulated by prebiotics, which are indigestible food ingredients that stimulate the growth
and maintain LAB microbiota. Prebiotics in Cocos typical extract are in form of fruto-oligosaccharide. Fructo-
oligosaccharide in Cocos typical extract are thereby significant part of the food nutrient that selectively stimulate
the growth and activity of LAB (Panitantum, 2004). Meanwhile, above normal body required level, non-
digestible fruto-oligosaccharide in Cocos typical causes Small Intestinal Bacterial Overgrowth (SIBO) (Leena,
2007). This challenge disrupts digestion, causes intense physical discomfort and damages the small intestine
(Leena, 2007). The effect of bioprocessing on non-digestible fruto-oligosaccharides need to be studied and linked
with growth rate of LAB, with the fact to enhance safety and quality of Cocos typical extract (Leena, 2007).
LITERATURE REVIEW
Existence of traditional bioprocessed (fermented) dairy and plant milk analogues are significant in Nigeria
(Yuliana et al., 2010). Such food products could be inform of yogurt, cheese and others (Ladokun and Oni, 2014).
Probiotic LAB are known to exert medical and nutritional quality, which could be delivered to humans via
various plant extracts and related products (Han et al., 2021). Certain minimum number of probiotics needed
daily in the guts, for an average individual, is 10
6
–10
7
CFU/g (Han et al., 2022). According to Leena, (2007),
twelve gramme of LAB per day or less is usually well tolerated. These beneficial LAB that persist in the gut
ecosystem could decrease intestinal pH and suppress proteolytic bacteria. They likewise slow aging process in
consumers (Vernazza et al., 2006; Gordon, 2008).
More so, proportions of LAB in foods are time and temperature dependent (Amapu et al., 2024). Prebiotics act
as lubricant stimulant to bowels and have effect on LAB growth, gastrointestinal flora, stool characteristics and
mineral (like calcium, magnessium and iron) absorption in adolescents and postmenopausal women. They act as
biomarkers of immune function and increase feacal flora. They ensure exhibition of inhibitory effect on
precancerous colon and reduce fasting glucose and apolipoprotein B levels in type-2 diabetic patients (Sharon et
al., 2009).
Aside from the nutritional and health benefits, Anyogu et al. (2021) and Ajibola et al. (2023) emphasized on the
safety value of fermentation. Most life-threatening diseases are consequences of unsafe food consumed.
Enhancing the quality and safety of food consumed is a major concern in the world today. As highlighted by
Coşansu et al. (2021), LAB could cure issues like food allergies, bowel disorder or gastrointestinal infections,
lactose intolerance, genitourinary infections, food allergies, in humans, most especially among the children.
Moreover, one of the important metabolites that contribute to antibacterial properties of foods, during LAB
fermentation is lactic acid (Barão et al., 2019). This could prevent cancer of the large intestine (Mrudula et al.,
2024). Pathogens that cause bacteria infections could be rendered inactive in the presence of this metabolite,
during production and storage (Asli, 2011). Nevertheless, small intestinal LAB overgrowth resulting from over
produced number of LAB leads to the production of toxins, enzymes, and intestinal gases (H
2
, CH
4
and CO
2
),
which interrupt with the health of consumers (Thammarutwasik, 2009).
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The current study was directed towards examining the growth, substrates and metabolites kinetics of
Lactobacillus acidophilus, Streptococcus salivarious subsp. thermophilus and Lactobacillus delbrueckii subsp.
bulgaricus starter cultures during the time-monitoring bioprocessing (fermentation) of Cocos typical based
extract. The effect of these LAB on non-digestible fructo-oligosaccharides was assessed and the antagonistic
behavior of the extract was screened against common pathogens (Escherichia coli, Salmonella typhimurium and
Staphylococcus aureus).
MATERIALS AND METHODS
Microorganisms utiized
Matured 12 month old western tall Cocos typical was purchased from the farm from Sapo, Badagry, in Lagos
State. The commercial starter cultures, Lactobacillus acidophilus, Streptococcus salivarious subsp. thermophilus
and Lactobacillus delbrueckii subsp. bulgaricus of Direct Vat type (DVS) Yo mix, were gotten from Kuto market,
Ogun State, Nigeria.
Laboratory fermentation of Cocos typical extract
Immediately after the drilling out of water in broken-shelled, de-husked Cocos typical, homogenizing of 4 kg of
comminute meat with 4500 ml of distilled water was done, according to modified method of Olusola (2014).
Thermal treatment of the mixture was then employed at 65 ºC for 30 min and cooled at 4 ºC in ice bath. Viability
of LAB from starter cultures (Lactobacillus acidophilus, Streptococcus salivarious subsp. thermophilus and
Lactobacillus delbrueckii subsp. bulgaricus) from the Direct Vat type (DVS) Yo mix was verified by sub-
culturing on De Mann Rogosa Sharoe (MRS) Agar at 37
o
C for 24 h of
incubation. Identification was by the
method of Ayodeji et al. (2017) at 1500 bp of 16S RNA with primer 27F (3’-GAGTTTGATCCTGGCTCAG-
5’) and 1492R (3’-TACCTTGTTACGACTT-5’). Probiotics test of the cultures was confirmed using Tambekar
and Bhutada (2010). Thereafter, six flasks containing the extract were inoculated with a milliliter of 10
5
MacFarland of the mixture
of 3% (v/v) of L. acidophilus, S. salivarious subsp. thermophilus and L. delbrueckii subsp. bulgaricus at 37
o
C for 72 h. Fermented
samples from each flask were drawn and refrigerated at 12 h interval, one after the other (with label A, B, C, D, E, F and G).
Measurement of pH were executed, using AOAC (2000).
Total lactic acid bacteria count
This was done using MRS agar with 0.01% sodium azide prepared according to the manufacturer’s specification.
This medium was sterilized in an autoclave at 121
o
C for 15 min. Each sample (1 ml) was dissolved in sterile
de-ionized peptone water and serially diluted. An appropriate dilution of 1 ml was inoculated on nutrient and
MRS agar plates and the plates were incubated anaerobically using anaerobic jars for 48 h at 37 °C. The total
Lactic Acid Bacteria count was done for each sample in log10 cfu/ml (AOAC, 2000).
Total lactic acid concentration
This was determined through AOAC (2005) by titrating 1.0 ml of sample against 0.1N NaOH using
phenolphthalein as the indicator. The appearance of a pink colour marked the end point of titration. The
measurements were done in duplicate. The titratable acidity (expressed as percent lactic acid) was determined
using:
1 ml 0.1N NaOH = 0.009008 ml = lactic acid (Acid Factor);
TA= [Normality of Base x Volume of Base x Acid Factor x100]/Volume of Sample.
Estimation of total solids, total soluble solids and total sugar
This is the amount of solids remaining after heating the sample at 105 °C to constant weight. Conversely, the
moisture content is a measure of the amount of water (and other components volatilized at 105 °C) present in
the sample. This was determined in gravimeter using the method described by Bradley Jr (2003). A measured
weight of each test sample was put in a previously weighed dish and evaporated to dryness over a steam bath. It
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was then dried in an oven at 105 °C for an hour. It was cooled in desiccators and then reweighed by difference.
The dry weight of the samples was obtained and expressed as a percentage of the sample weight.
The total soluble solids were determined using a digital hand held refractometer and the total soluble solid
content was expressed as Brix at 25 °C. The amount of residue remaining from a 0.2 μm filtered liquor sample
after heating the sample at 105 °C to constant weight was calculated, according to by Bradley Jr (2003).
Total soluble solids (%)= {(Weight-dry pan plus dry sample – Weight-dry pan)/ Weight sample as received} x
100
Moisture (%) = 100 – [{(Weight-dry pan plus dry sample – Weight-dry pan)/ Weight sample as received} x 100]
The total sugar contents indicator was determined as described by AOAC (1999), using the principle of ethanolic
extraction of sugar from samples by the organic solvent - petroleum ether using absorbance method. One gram
of each sample was weighed into 200 ml volumetric flask. Cotton wool was placed over the sample in the
volumetric flask to prevent splashing. Fifteen milliliters of 85% ethanol was added to each flask. The solutions
were voltexed and filtered through Whatman’s No. 1 filter paper. Four separating funnels were set up.
Chloroform solution of 10 ml was added to each filtrate. Further extraction was carried out in the separating
funnels. Each filtrate formed a layer on the chloroform.
The chloroform layer was discarded and 10 ml of petroleum either was added to each filtrate to remove the lipid
content present. The petroleum ether layer below was used to determine the total sugar contents. Five milliliters
of petroleum ether was weighed out inside a boiling flask. Distilled water of 0.5 ml was added to obtain a diluted
extract. Phenol (0.5 ml of 5% (w/v)) was added to each of the content in each flask, along with 2.5 ml of
concentrated sulphuric acid (Conc. H
2
S04), for colour development. Each of the flask content was then heated
and allowed to cool after which the absorbance was read off at 490 nm wavelength using Spectronic 20D –
Spectrophotometer.
Estimation of glucose, fructose and sucrose concentration
This was determined using the phenol-sulphuric acid method (Shetty et al., 2017). Each sample of 0.1 g was
weighed and homogenized with 600 ml of distilled water, in a beaker. Filtrate of the mixture was transferred
into a 1 L volumetric flask. Two millimeters of aliquot of carbohydrate solution was pipetted with the addition
of 1 ml of 5% aqueous phenol solution and 5 ml of concentrated H
2
S04, in a test tube. The solution was cool for
10 min, vortexed for 30 s and placed in a water-bath at room temperature for 20 min for color development. At
absorbance of 490 nm, the solution was quantified using a UV visible spectrophotometer. It was blanked with 2
ml distilled water, 1 ml aqueous phenol (5%) and 5 ml conc. H
2
S04. Then, extrapolation was done to get the
concentration of the carbohydrate standard graph. A standard graph of absorbance against the known
concentrations of the standards for all the carbohydrate was plotted. Finally, the concentration of the sugar was
calculated using:
Sucrose/glucose/fructose (mg/100g) = conc. (mg/l) x volume of sample x dilution factor
Sample weight x 1000
Estimation of fructo-oligosaccharide
Each sample of 10 g was weighed into a beaker and 30 ml of boiled deionised water (hot) were added, stirred on
hot plate at 80 degree Celsius for 10 min and allowed to cool. Carrez reagent 1 of 5 ml were added with 5 ml of
carrez reagent 11, mixed and filtered into a 50 ml standard flask and made up to mark of 50 ml using distilled
water. Absorbance at 480 nm was read using equal volume of carrez 1 and carrez 11 reagents as blank. Also
standard fructose standard graph was prepared or from an existing fructose standard graph, extrapolated to obtain
the concentration of fructo-oligosaccharide in each sample (Petkova et al., 2011).
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Modelling study of growth kinetics of Lactobacillus acidophilus, Streptococcus salivarious subsp.
thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in Cocos typical extract biomass
The specific LAB growth rate was calculated using Monod equation (Monod, 1942) while specific consumption
rate of glucose, fructose, sucrose and generation rate of lactic acids in samples were modeled using Luedeking-
Piret equation. During the log phase when cell utilizes nutrients and grow to increase biomass, the growth
behaves similar to autocatalytic reaction. At this phase, growth rate is proportional to cell mass (x) of that period.
At this time, t, the rate of cell mass increase (dx/dt) is equal to the specific growth rate (μ) and the cell
concentration.
td = 0.693/ μ……………………………………………………………………….(i)
μ = 2.303(log
10
X – log
10
X
0
)/t – t
0
………………………………………………… (ii)
Monod equation for Exponential growth (Shuler, 2003): Cells + Substrate → More cells + Products.
From Luedeking- Piret type kinetics, the specific consumption rate of glucose (v; g glucose/g cell per h) and the
specific production rate of lactic acid (π; g lactic acid/g cell per h or percentage) can be calculated from the
differentials of glucose concentration (ΔS; g glucose/l) and lactic acid concentration (ΔP; g lactic acid/l) at each
time as the following equations:
V = -(1/x)(ΔS/Δt)……………………………………………………………….(iii)
Π = (1/x)(ΔP/Δt)………………………………………………………………...(vii)
Lactobacillus acidophilus, Streptococcus salivarious subsp. thermophilus and Lactobacillus delbrueckii
subsp. bulgaricus antimicrobial Assay
Salmonella typhi, Escherichia coli 0157:H7 and Staphylococcus aureus were isolated from spoilt fruits in the
microbiology laboratory of the Department of Food Science and Technology, Federal University of Agriculture
Abeokuta, Ogun State Nigeria. These pathogens were isolated from spoilt Cocos typical fruits streaked on
Salmonella-Shigella Agar, MacConkey Agar and Mannitol Salt. Pure culture of the isolate were subjected to
series of biochemical tests and identified genotypically with 16S RNA primer. The sequence and condition of
the PCR were described in Appendix 1.
Then, 24 h incubated culture of Salmonella typhi, Escherichia coli 0157:H7, and Staphylococcus aureus each
was prepared and inoculated in 10 ml of sterile water, homogenized and standardized by 10
5
MacFarland-
standardized. These were spread on Muellen Hinton Agar plates. Wells of 7 mm in diameter were cut into these
agar plates using sterile cork borer and 50 μl of the bioprocessed Cocos typical extract were placed into each
well. One well was filled with fermented Cocos typical extract which serve as negative control. The culture
plates were incubated at 37°C for 24 h and the zones of inhibition was measured in diameter (mm). Meanwhile,
paper discs were impregnated with streptomycin and imipenem antibiotics were used as control according to
Olateru et al. (2020). Antimicrobial tests were done in triplicate.
Other Statistical Analysis
Triplicate values of data were subjected to one-way analysis of variance (ANOVA SPSS 21.0 version) and means
were separated using Duncan multiple range test at 95% confidence level.
RESULTS AND DISCUSSION
Results of the total Lactic Acid Bacteria count, pH, lactic acid concentration produced, as well as total solid,
soluble solid and sugar of the fermented Cocos typical extract were presented in Table 1. There was significant
difference (p<0.05) in all the parameters evaluated. A log phase was observed in total LAB count and the lactic
acid produced ranged from 6.36 to 10.54 log10 cfu/ml and 0.07% to 1.74% respectively. The pH, and total solid,
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soluble solid with sugar decreased from 6.48 to 4.03, 20.96 to 10.88%, 5.98 to 2.81%, 3.01 to 0.17% respectively
as fermentation time increased.
Metabolic activities of L. acidophilus, S. salivarious subsp. thermophilus and L. delbrueckii subsp. bulgaricus
during the processing accounted for increase in total LAB count and lactic acid produced. This also is responsible
for the decrease observed in the pH, total solid, total soluble solid and total sugar of the samples.
This is in support to Pato et al. (2021), who reported L. acidophilus to have imparted better taste and aroma in
Cocos spp extract from Indonesia, due to higher pH and lactic acid values (Yuliana et al., 2010). The pH values
of samples were observed to be in right amount that consumers’ gastrointestinal tracts could tolerate. In this
regard, results from this study fall at best favourable pH even at 72 h of fermentation, so they could deliver
desired beneficial probiotic LAB (Guler-Akin and Akın, 2007; Obi and Ikenebomeh, 2007).
During fermentation, there was gradual release of soluble mineral element leached out through the soft texture
of samples and dissolution of the bound sugars into the fermented milky extract. This, however, were utilized
by the LAB. Trend in this outcome is seen with the study of Shetty et al., (2017). So, reduction or depletion of
sugars occurs, as viable LAB count increase with fermentation time.
From Table 2, monosaccharides found in the fermented Cocos typical extract with LAB were glucose and
fructose while the dissaccharide was sucrose. It was observed that as fermentation period and LAB growth
increased, there was reduced trend in the glucose, fructose and sucrose concentration from 1.69 to 0.79 g/100g,
2.17 to 1.02 g/100g and 1.08 to 0.51 g/100g respectively.
The amount of fructo-oligosaccharide (prebiotics) progressively reduced as fermentation period increased. It
falls rapidly from 100.05 to 34.31 mg/kg between 0-72 h of fermentation. The results showed no significant
difference (p<0.05) existed in the samples’ fructo-oligosaccharide. As growth of the fermenting organisms upto
72 h, there was tremendous reduction in fructo-oligosaccharide content. This was also presented in Table 2.
Reduction of glucose, fructose and sucrose occurred due to hydrolysed breaking down of complex carbohydrates
into simpler form by the metabolic action of LAB. Ngoc et al., 2013) described the action of enzyme invertase
on the sugar during bioprocessing.
Combination of three LAB could also be factor that speedy the rate of decomposition process of complex sugars
giving higher rate of reduction of glucose, fructose and sucrose, as LAB population increase with time. This
result corroborate with the earlier report of Tuitemwong and Tuitemwong (2003) and Adelodun and Abiodun
(2012). The relationship of fruto-oligosacharide (prebiotics) amount per fermentation time and Total LAB count
showed inversely proportion
Table 1: Growth Attributes of Lactobacillus acidophilus, Streptococcus salivarious subsp. thermophilus
and Lactobacillus delbrueckii subsp. Bulgaricus in the Fermentation of Cocos typical Milk.
Sample
Fermentation
Period (h)
Total LAB
Count (log₁₀
cfu/ml)
pH
Lactic Acid
Concentration
(%)
Total Solid
(%)
Total Soluble
Solid (%)
Total Sugar (%)
A
0
6.36 ± 0.52ᵇ
6.48 ± 0.02ᶜ
0.07 ± 0.02ᵃ
20.96 ± 0.35ᶠ
5.98 ± 0.03ᶠ
3.01 ± 0.26ᶠ
B
12
7.49 ± 0.32ᵇᶜ
4.56 ± 0.23ᵇ
0.31 ± 0.06ᵇ
19.09 ± 0.11ᵉ
6.33 ± 0.03ᵍ
2.45 ± 0.05ᵉ
C
24
7.79 ± 0.27ᵈ
4.31 ± 0.05ᵃᵇ
0.59 ± 0.05ᶜ
16.20 ± 0.47ᵈ
3.82 ± 0.04ᵉ
2.01 ± 0.01ᵈ
D
36
8.08 ± 0.15ᵃ
4.24 ± 0.05ᵃ
0.62 ± 0.00ᶜ
15.62 ± 0.02ᵈ
3.39 ± 0.00ᵈ
1.06 ± 0.00ᶜ
E
48
8.87 ± 0.30ᵉ
4.17 ± 0.03ᵃ
0.74 ± 0.00ᵈ
14.21 ± 0.02ᶜ
3.11 ± 0.01ᶜ
0.52 ± 0.04ᵇ
F
60
9.90 ± 0.11ᵉ
4.09 ± 0.02ᵃ
0.89 ± 0.00ᵉ
12.37 ± 0.08ᵇ
2.66 ± 0.03ᵃ
0.37 ± 0.01ᵃᵇ
G
72
10.54 ± 0.59ᶜ
4.03 ± 0.01ᵃ
1.74 ± 0.01ᶠ
10.88 ± 0.59ᵃ
2.81 ± 0.02ᵇ
0.17 ± 0.01ᵃ
Mean of samples with different superscript letter are significantly different (p<0.05); Mean of triplicate
values±standard error
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Table 2: Relationship between Lactobacillus acidophilus, Streptococcus salivarious Subsp. thermophilus
and Lactobacillus delbrueckii Subsp. Bulgaricus with reducing sugar and fructo-oligosaccharide of the
Cocos typical milky extract during time-monitoring fermentation
Mean of samples with different superscript letter are significantly different (p<0.05); Mean of triplicate
values±standard error to one another. The beneficial LAB whose growth is affected by the prebiotics in bio-
processed product, which must be present in adequate amount at the time of consumption in order to render it
being effective as (Siró, 2011). Therefore, the fermented Cocos typical extracts can efficiently deliver probiotic
bacteria due to the proportion of fruto-oligosacharide in them and prevent the risk of Small Intestinal Bacterial
Overgrowth (SIBO). Reduction rate of fruto-oligosaccharide as fermentation time increased with higher LAB
count, rendered bioprocessed Cocos typical extract to be safe for individual, including the infants.
Growth rate with residual doubling time, glucose, fructose and sucrose at a specific fermentation period (from
Monod parameters) for L. acidophilus, S. salivarious subsp. thermophilus and L. delbrueckii subsp. bulgaricus,
was given in Table 3. The fermentation period of 12 h had higher specific growth rate with lowest doubling time
of 0.22 h
-1
and 3.15 h respectively. Fermentation period at 24 h and 36 h gave the least values of 12.00 and
12.59 respectively, and more doubling time of 0.06 h
-1
. From this study, evidences showed that specific growth
rate increased but reduction in doubling time of LAB was observed. This was in line with the reports of Phuc
(2011) and Dubey (2012). The lowest specific glucose rate consumed by the lactic acid bacteria was at 48 h of
bio-processing with -0.4 gh
-1
, meanwhile, more consumption was observed at 60 h of fermentation period with
218.28 gh
-1
. This reflection of the utilization of the extracts’ nutrients by the LAB with accumulation of lactic
acid concentration was observed in the reports of Van-Neil et al., (2002) and Otles and Cagindi (2003). The
speedy up by environmental factors such as pH, medium composition, aeration and so on may also contribute to
this (Fadela et al., 2009). Both specific fructose and sucrose rate consumed by LAB were at 24 h of fermentation
was at least value but highest outcome was seen at 72 h. Similarly, at 72 h, more lactic acid was produced and
few amount was observed at 36 h.
The growth kinetics reflect that Cocos typical extract support the growth of L. acidophilus, S. salivarious subsp.
thermophilus and L. delbrueckii subsp. bulgaricus. This is based on material balance, limit substrates (specific
glucose, fructose and sucrose consumed) and metabolite (specific lactic acid produced), distributed between
cells, at different fermentation time. This vitality and number of probiotic bacteria are critical factors for their
beneficial functions in the host, according to (Coşansu et al., 2021). Temperature and fermentation time played
vital role in the proliferation of the LAB, and impacted direct desirable characteristics of the fermented Cocos
typical milky extract. This is in agreement with the research of Barão et al. (2019). After 36 h, it was observed
that the tread of growth rates, doubling time, specific glucose, fructose and sucrose rate, and lactic acid rate
produced, begin to change, unfavourably. This revealed these samples could render the best quality composition
to consumers, at 36 h of bio-processing. This is in support with the outcome of study of Yuliana et al. (2010).
Zone of inhibition quantified was shown in Table 4. Ranged results were seen in L. bulgaricus (10.11-19.33
mm), S. thermophilus (9.13-19.83 mm) and L. acidophilus (9.89-21.17 mm). Within the zone of inhibition,
streptomycin and imipenem had values ranging from 13.49-16.41 mm, with significant difference. Present of
ntimicrobial properties of LAB (in all samples) against commonly found pathogens (E. coli, S. typhi and S.
Sample
Fermentation
Period (h)
Total LAB
Count
(log10cfu/ml)
Glucose
(g/100g)
Fructose
(g/100g)
Sucrose
(g/100g)
Fructo-
oligosaccharide,
FOS (mg/kg)
A
0
6.36±0.52
b
1.69±0.34
a
2.17±0.08
a
1.08±0.22
ab
100.05±0.02
f
B
12
7.49±0.32
bc
1.52±0.11
b
1.97±0.23
b
0.98±0.15
cd
85.43±0.12
e
C
24
7.79±0.27
d
1.46±0.14
b
1.88±0.51
bc
0.93±0.30
c
60.3±0.06
d
D
36
8.08±0.15
a
1.27±0.07
c
1.71±0.01
c
0.85±0.16
d
60.05±0.05
d
E
48
8.87±0.30
e
1.28±0.10
d
1.63±0.26
d
0.83±0.55
d
59.26±0.03
c
F
60
9.90±0.11
e
0.81±0.12
e
1.40±0.44
e
0.66±0.13
e
34.61±0.20
b
G
72
10.54±0.59
c
0.79±0.04
f
1.02±0.02
f
0.51±0.49
f
34.31±0.02
a
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aureus), might be due to production of organic acids (lactic acid), peptides (bacteriocins), carbon dioxide,
hydrogen peroxide, ethanol and diacety (Farnworth, 2005; Sarkar, 2007). Although, antimicrobial properties of
LAB is said to be strain specific but collective activities of more than one strain ensure more efficient
antimicrobial action. The increase in the medium chain fatty acid as supported by Jay (1982) and this could exert
antimicrobial activities (Akinpelu et al., 2015). The mechanism(s) of antimicrobial activity in probiotic LAB
strains appears to be multifactorial, due to bacteriocins and/or lactic acid produced (Gauri et al., 2013). This was
reflected in the result of mixed cultured strains of LAB used in this study. It was observed that short-chain acids
(lactic) produce during fermentation which increase with time had inhibitory effect. Combination of three
different probiotic strains in this study, achieve stronger inhibitory effect on the growth of the pathogenic bacteria.
This is in line with the report of Denkova (2013). Other inhibitory agents found in LAB that could control major
pathogenic bacteria in Nigerian bioprocessed food are acidocin (in L .acidophilus) and bacteriocin (Amin, 2011).
These agents including lactic acid, weaken the outer plasma membrane and sequestrate magnesium ions by
chelating effect, causing inhibition of pathogens. Some antimicrobial activities in LAB such as those
Table 3: Growth Kinetics of Lactobacillus acidophilus, Streptococcus salivarious Subsp. thermophilus
and Lactobacillus delbrueckii Subsp. bulgaricus during fermentation of Cocos typical milky extract using
Monod parameters
Sample
Fermentation
Period (h)
Specific
Growth
Rate, μ
(h⁻¹)
Doubling
Time, td
(h)
Specific
Glucose Rate
Consumed, V
(g/h)
Specific
Fructose Rate
Consumed, P
(g/h)
Specific
Sucrose Rate
Consumed, Y
(g/h)
Specific Lactic
Acid Rate
Produced, π
(g/h)
A
0
–
–
–
–
–
–
B
12
0.22 ±
0.01ᶜ
3.15 ±
0.15ᵃ
0.32 ± 0.03ᶜ
0.37 ± 0.01ᵃ
0.19 ± 0.012ᵇ
6.86 ± 0.08ᵃ
C
24
0.06 ±
0.01ᵃ
12.00 ±
0.78ᶜ
0.12 ± 0.03ᵇ
0.18 ± 0.01ᵇ
0.10 ± 0.01ᵃ
14.14 ± 0.60ᵇ
D
36
0.06 ±
0.01ᵃ
12.59 ±
0.32ᶜ
0.72 ± 0.01ᵈ
0.64 ± 0.01ᶜ
0.29 ± 0.01ᶜ
28.11 ± 0.01ᶜ
E
48
0.15 ±
0.05ᵇᶜ
4.57 ±
0.17ᵇ
-0.40 ± 0.06ᵃ
3.10 ± 0.12ᵈ
0.97 ± 0.02ᶜ
357.50 ± 0.80ᵈ
F
60
0.20 ±
0.02ᶜ
3.51 ±
0.20ᵃ
218.22 ± 0.058ᵉ
129.54 ± 0.02ᵉ
69.18 ± 0.01ᵈ
4964.26 ± 0.02ᵉ
G
72
0.12 ±
0.01ᵃᵇ
5.59 ±
0.01ᵇ
22.85 ± 0.02ᶠ
593.98 ± 0.02ᶠ
304.02 ± 0.01ᵉ
36735.12 ±
0.01ᶠ
Table 4: Lactobacillus acidophilus, Streptococcus salivarious subsp. thermophilus and Lactobacillus
delbrueckii subsp. bulgaricus antimicrobial Assay of the fermented Cocos typical milky extract on
different indicators
Antimicrobial in Cocos typical
extract and antibiotics as
control
Zone of Inhibition (mm) of the Tested Pathogens
Escherichia coli
Salmonella typhimurium
Staphylococcus aureus
A
10.11±0.17
c
9.13±0.17
a
9.89±0.17
b
B
13.33±0.44
b
13.50±0.76
b
12.67±0.88
b
C
14.67±0.60
c
15.17±0.60
c
15.00±0.00
c
D
16.50±0.29
d
16.67±0.17
d
17.00±0.29
d
E
18.00±0.29
e
17.50±0.29
de
17.67±0.17
d
F
18.67±0.17
ef
18.33±0.17
e
19.67±0.44
e
G
19.33±0.17
f
19.83±0.60
f
21.17±0.44
f
Streptomycin
15.67±0.34
e
13.49±0.29
d
16.25±0.55
c
Imipenem
15.98±0.29
d
15.34±0.18
e
16.41±0.28
d
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Mean of samples with different superscript letter are significantly different (p<0.05); Mean of triplicate
values±standard error associated with molecules frequently exported by bacteria, (the hemolysins or hydrolytic
enzymes), have effectiveness in diarrhoea caused by Salmonella among children (Amit and Vipan, 2013).
Many researchers had confirmed that the component of fatty acid (such as capric, lauric, miristic, oleic, palmitic
and miristic acids) had effective activity against Candida spp, Aspergillus niger, Bacillus cereus, Bacillus subtilis,
Micrococcus lysodeikticus, Penicilium citrinum, Peudomonas aeroginosa, Streptococcus pneumonia (also
Palmitic and Myristic Acid), Saccharomyces cerevisiae, Streptococcus group A and others (Bergsson et al, 2001;
Sheehan et al, 1999; Ogbolu et al, 2007).
CONCLUSIONS
Cocos typical extract served as good carbon source for Lactobacillus acidophilus, Streptococcus salivarious
subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. As they feed on the macromolecules, there
was increase in their growth and in produced metabolites. Therefore, this study established that fermented Cocos
typical can render probiotic LAB, which exert health benefit above nutritional value. In addition, this study
proved that as at 72 h of fermentation, viability was still enhanced in Cocos typical extract, exhibiting log phase
of microbial growth. Meanwhile, stability of desirable or nutritious compositions and the safety of this product
was be maintained at 36 h of fermentation.
It could also be inferred that L. acidophilus, S. salivarious subsp. thermophilus and L. delbrueckii subsp.
bulgaricus impart inhibitory effect on pathogens (E. coli, S. typhimurium and S. aureus) and greatly reduced the
non-digestible frutooligosaccharide, in the samples.
It was deduced accordingly that the bioprocessed Cocos typical extracts (at 24 h and 36 h of fermentation with
L. acidophilus, S. salivarious subsp. thermophilus and L. delbrueckii subsp. bulgaricus) were safe for
consumption and they could be recommended for the vulnerable group, the pregnant women, vegetarian, infants
and children.
ACKNOWLEDGEMENT
Molecular Analysis Research Group in Microbial Safety, Quality and Diseases Control (MARG-MSQDC),
Department of Microbiology, Hallmark University, Km 65, Sagamu-Ore Expressway, Ijebu-Itele, Ogun State
122101, Nigeria are more appreciated by the author, for their support to make sure the original draft preparation
is done successfully. The author also appreciated and declared that the study was also supported by Hansataj
Nigeria Limited, Riocharistos Engineering Limited and Shenkeve Engineering and Procurement Limited.and
appreciate
Author and Contributors
Areo E.O. (Methodology, Conceptualization, Software, Validation, Formal Analysis, Investigation, Resources,
Administration, Data Curation, Project Funding Acquisition, Editing, Supervision, Writing – Original Draft
Preparation, Visualization)
Non-Ethical Approval
Author declares that this research work did not involve the use of human data or human tissue and It is thereby
affirmed that during the course of researching, no harm nor discomfort to the participant occurred.
Conflict of Interest Disclosure
Not available
Funding: There was no funding received for this research work.
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Appendix 1 Supporting information
Supplementary data associated with this article can be found in the online version at
DOI:10.1016/j.microb.2025.100286.
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