Microorganisms play a crucial role in
fulfilling human life needs. The use of probiotics as a therapeutic tool can be
developed, among other things, as an immunomodulator and antimicrobial agent.
The diverse functions of probiotic bacteria are considered an alternative
solution to various issues related to bacterial antibiotic resistance,
irrational antibiotic use, and its increasing use in recent decades (1). The potential of probiotics as an alternative to antibiotics is
because research by Halder (2017) states that probiotics have the potential to
act against various infectious agents, one of which is Lactobacillus (2). Lactobacillus can fight pathogens by producing different
antimicrobial components, lowering pH, competing with pathogens for adhesion
and nutrients in the intestine, and suppressing the growth of pathogenic
bacteria through direct coaggregation with bacteria (3). Sekendar (2017), states that Lactobacillus produces bacteriocins,
which have strong antibacterial properties and act bactericidally or
bacteriostatically against various food pathogens (4). Bacteriocin synthesis may also contribute to the probiotic
activity of intestinal Lactobacillus and, in some cases, may be directly
responsible for beneficially altering the intestinal microbiota or suppressing
some gastrointestinal pathogenic bacteria (5). Many benefits of using probiotics come from their ability to
combat pathogenic organisms through various mechanisms, including their
immunomodulatory effects and their ability to restore the balance of normal gut
flora (6).
Probiotics are beneficial live bacteria
that promote digestive health and protect against pathogenic bacteria.
Typically, probiotics are found in foods and beverages. Probiotics are live
microorganisms that can provide positive health effects when consumed in
sufficient amounts. Probiotic products available in the market today use
bacteria from the Lactobacillus and Bifidobacterium species. The development of
drinks and supplements containing probiotics is in accordance with research by
Shabbir (2023), who carried out the development of Fermented Skim Milk drinks
equipped with Probiotics with a combination of Lactobacillus casei and Lactobacillus rhamnosus, concluding that Probiotics can be used in milk
fermentation to make fermented milk drinks and beverages. Yakult can be used to
market innovative beverages to specific consumer groups, manufacturers and
industries, as well as large-scale retail distribution (). Researcher Badgeley (2021) said that in recent years, researchers
have investigated various health benefits associated with probiotic
supplements, which primarily contain live microorganisms from Bifidobacterium
and Lactobacillus species. The main areas of focus of his research are the
effects of probiotic supplements on gut microbiota and cancer prevention, as
well as on the efficacy and toxicity of chemotherapy (). Commonly used Lactobacillus species include (), , , , , and ,
while Bifidobacterium species include , , , and (). Both the Lactobacillus and Bifidobacterium have different roles in
human digestion. aids
in intestinal lactose digestion, stimulates the immune response against
unwanted microorganisms, and helps control blood cholesterol levels. is one of the lactic acid bacteria that can be utilized as a
probiotic microbe and is currently one of the most widely developed probiotic
strains ().
Declarations
Conflict of Interest
The authors declare no conflicting interest.
Data Availability
The unpublished data is available upon request to the corresponding author.
Lactic acid bacteria (LAB) have long been
used in the food industry for their ability to convert sugars, including
lactose and other carbohydrates, into lactic acid (10). L. acidophilus bacteria can impart the characteristic sour
taste to fermented dairy products such as yogurt and also produce a low pH,
which can inhibit the growth of other microbes and survive in the stomach
alongside thousands of other bacteria (11). For the fermentation process, the raw material used (milk) must
undergo initial heating (pasteurization), cooling, adding lactic acid bacteria
and an incubation process. After the incubation process, the process of storing
the fermented yogurt product is by storing it in the refrigerator (11). The role of lactic acid bacteria in making yoghurt and similar
products is in producing acids and flavor compounds such as acetaldehyde.
According to Abedi (2020), lactic acid produced by the metabolism of L.
acidophilus can lower the pH thereby inhibiting the growth and reproduction
of pathogenic bacteria (12).
Given the importance of L. acidophilus
bacteria, one way to utilize them is by granulating the bacteria into granules.
In a study using Lactobacillus casei (13), it was found that drying at 45°C for 2 hours was the optimal
condition to achieve good viability and quality of probiotic granules, with a
viability result of 7.43 cfu/gram (meeting the requirements). According to the
FAO and WHO, probiotic products should contain at least 106 – 107
cfu of live bacteria per gram of the probiotic product (14).
In the study conducted by Gantini (2016),
probiotic products were made into instant granules using the wet granulation
method. In this research, probiotic products will be formulated into
effervescent granules using the wet granulation method. In addition to the
active ingredient, the formulation of effervescent granulation also includes
several additives, such as carbonate additives to enhance taste and acid source
additives (15). The acid sources referred to here are citric acid and tartaric
acid. Citric acid and tartaric acid have their respective properties that, when
combined, will affect the physical properties of the resulting granules (16). The wet granulation method is a process of transforming fine
powder into granules using a suitable binder solution. The advantages of the
wet granulation method include achieving good flowability, improving
compressibility, controlling release, preventing component separation during
the process, ensuring even granule distribution, and increasing dissolution
rate (13).
Based on the above, we formulated
effervescent granules containing L. acidophilus bacteria using the wet
granulation method as a probiotics supplement. Therefore, physical properties
of effervescent granules were evaluated including the organoleptic, flow rate,
angle of repose, bulk density, tapped density, compressibility index, water
content, dissolution time, particle size distribution, sedimentation, foam
height, and viability of lactic acid bacteria using the Total Plate Count (TPC)
method.
Methods
Materials
Lactobacillus acidophilus was obtained from Agritama Sinergi Inovasi (AGAVI) Co., Ltd
(Indonesia). Excipients used were polyvinylpyrrolidone (Anhui
Sunhere Pharmaceutical Excipients Co., Ltd, China), lactose
(DMV-Fonterra Excipients GmbH & Co., KG, Germany), tartaric acid (Justus
Kimiaraya Co., Ltd, Indonesia), citric acid (Shandong Ensign Industry Co. Ltd,
China), sodium bicarbonate
from (AGC Inc. Chemical Co., Ltd, Japan), De Man Rogosa
and Sharpe Broth (Merck KGaA, Darmstadt, Germany).
Tools
The tools used in this research include
sieves No. 12 and 18, glassware (Pyrex), mortar and stamper, micropipettes,
test tubes, 10 mL centrifuge tubes, tube racks, stopwatches, analytical scales
(Mettler Toledo), hot plates (IKA®C-MAG HS-4), autoclave (ALL AMERICAN®), incubator
(Memmert®), centrifuge (DLAB®), oven, granule flow tester, tap density tester
(TDT-3-H), moisture balance (Radwag®), pH meter (Mettler Toledo), orbital shaker
(IKA® KS 4000-i), microscope, colony counter (Funke Gerber®), vortex mixer
H-VM-300 (HEALTH®).
Morphological Identification
of Lactobacillus Acidophilus Isolate
Confirmation Test for Bacterial Growth
The deMan
Rose Sharp Agar (MRSA) media and test
tubes underwent sterilization by autoclaving at 121°C for 15 min. The working
table was sanitized using a cloth/tissue dampened with 70% alcohol. Subsequently, 15 mL of sterile MRSA media was dispensed into each test tube
and allowed to solidify with the tube positioned at an angle between two Bunsen
burners. Once solid, a single dose of pure L. acidophilus bacteria was
streaked onto the solidified media in a zig-zag pattern. Incubation was carried
out for 24 h at 37°C (17).
Bacterial Gram Staining Test
The bacterial suspension
was streaked onto the slide using a loop and fixed to the bottom of the slide.
Following this, 1-2 drops of methylene blue were
dripped, allowed to stand for 1 min, then cleaned with distilled water and
air-dried. Subsequently, 1-2 drops of iodine were
added, left for 1 min, rinsed with distilled water, and dried. Once fully
dried, 70% alcohol was applied, left for 30 min, followed by rinsing with
distilled water and drying. After complete drying, 1-2 drops of fuchsin were
added, left for 1 min, then rinsed using distilled water and dried.
Observations were conducted using a microscope at a magnification of 100x.
Preparation of Lactobacillus acidophilus Effervescent Granules
Sucrose was initially dissolved in 300 mL of distilled water and subsequently
sterilized through autoclaving at 121°C for 15 min. A 10% v/v of L. acidophilus bacterial culture suspension (inoculum) was introduced into 30 mL of
sterile MRSB medium within a 100 mL Erlenmeyer flask. The mixture was incubated
at room temperature with agitation at 125 rpm for a duration of 48 h. To
achieve biomass production of L. acidophilus bacteria, the inoculum was
integrated into the cultivation medium (sucrose medium) and subjected to
incubation for 24 h. This incubation occurred with continuous agitation at 125
rpm at room temperature. Harvesting
and subsequent separation of the biomass were achieved through centrifugation
at 5000 rpm for 20 min. The obtained mass of L.
acidophilus were subsequently
subjected to a drying process within an oven set at 40°C for a duration of 24
h.
Determination of the concentration of bacteria used is in accordance
with research by Gantini (2016) which formulated Lactobacillus casei bacteria
which were made into instant granules. Where the concentration of bacteria used
is 3% (13). When designing the formula to be
used, formula optimization is first carried out to obtain the optimum formula.
In this optimization, Design Expert Software was used to optimize the
ingredients, citric acid and tartaric acid were chosen as independent variables
by setting citric acid with a lower limit value of 0.3 and upper limit 2.0,
while tartaric acid had a lower limit value of 0.5 and an upper limit value of
50. PVP binder is used in the form of an alcohol solution with a concentration
of 3-15%. Granulation using this material will produce good granules, dry
quickly, and have good compressive strength (18). So the presentation used is a
minimum PVP presentation of 3%.
The formulation of effervescent granules was achieved using the wet
granulation technique (19). Initially, the base portion comprising Sodium
Bicarbonate, lactose, and PVP K30 solution was blended in a mortar until
achieving a smooth dough-like consistency, followed by sieving through a mesh
with a pore size of 12, and subsequently subjected to drying at 40°C for a
duration of 15 minutes, resulting in Mass 1. Concurrently, the mixture of
citric acid, tartaric acid, lactose, and PVP K30 solution was similarly
combined in a mortar to form a homogenous dough, sieved through mesh 12, and
subjected to drying at 40°C for 15 minutes, yielding Mass 2. Subsequently, Mass
1, Mass 2, and L. acidophilus bacteria were combined, sieved through
mesh 12, and subjected to a 3-hour drying process, followed by dry sieving
through mesh 18. Finally, the granules were evaluated for their
characteristics.
Physical Characterization of Lactobacillus acidophilus Effervescent Granules
Examinations for the physical characteristics of effervescent granules
containing L. acidophilus bacteria include organoleptic evaluation, flow
rate examination, angle of repose analysis, compressibility assessment, loss on
drying measurement, dissolving time evaluation, pH testing, and foam height
examination
Organoleptics
An ample quantity of granules was collected,
followed by a direct observation where parameters such as shape, color, taste,
and aroma were taken into consideration (18).
Flow Rate
The flow rate test was carried out by
inserting 100 g of granule into the funnel. The bottom cover of the funnel was
opened and the fall time of the granules was calculated using a stopwatch (20). The flow rate is said to be good if it has a flow rate of less
than 10 g/sec (21).
Angle of Repose
As much as 50 g of granules was put into
the funnel or into the Granule Flow Tester tool which was stored on a flat
surface. The funnel valve was then opened until all the granules fall down form
the cone. The diameter and height of the granule cone formed were measured (22). Requirements for a good angle of repose range from 25o
– 40o (23).
Compressibility
As much as 50 g of granules was put into a
volumenometer measuring cup, then the initial height of the granules was
recorded. The measuring cup was tapped for 250 beats at a constant speed after
which the granule weight was obtained. It was compressed and the real specific
gravity and incompressed specific gravity were measured (24). Good compressibility value is less than 20% (25).
Loss on Drying
As much as 2 g of dry granules was weighed
and put into the moisture balance tool. After 5 minutes, the water content was
measured (26). The water content requirements for effervescent granules range
from 0.4% - 0.7% (27).
Dissolving Time, Foam Height, and pH
Ten grams of granules were placed into a
beaker glass and dissolved with 250 mL of water. The dissolution process was
observed using a stopwatch, and the results obtained were recorded. The time
taken for the granules to completely dissolve was determined (28). The test was conducted to assess the dispersion time of the
granules in water. The prepared granules met the criteria for a good granule,
with a dissolution time ranging between 1-2 minutes, as per the effervescent
granule dissolving time requirement (20). During the granule dispersion process, foam height was measured to
assess the granules' dispersion capability (28). pH of the solution was then measured.
Viability Test
A total of 68.2 grams of MRSA media was dissolved in 1000 milliliters of distilled water and heated until homogeneous. The media was then sterilized in an autoclave at 121 degrees Celsius for 15 minutes to ensure complete sterilization. Subsequently, a total of 15 milliliters of sterile MRSA media was dispensed into a petri dish and allowed to solidify. Once solidified, effervescent granules were sprinkled onto the surface of the MRSA media and evenly spread. The prepared petri dish was then incubated for 24 hours with the lid inverted to prevent condensation. Following the incubation period, the colonies that grew on the media were manually counted using a colony counter, and the total plate count was calculated based on these observations.
Result
Morphological Identification
of Lactobacillus acidophilus Isolate
The confirmation test for bacterial growth was conducted using MRSA selective media, with the goal of enhancing bacterial proliferation at 37°C during a 24-hour incubation period. The growth of L. acidophilus isolates was conspicuously manifested through the development of turbidity on the agar's surface, indicating robust growth. The outcomes of macroscopic observations of LAB are depicted in Figure 1, illustrating the distinct characteristics observed. Additionally, the results of microscopic observations with Gram staining can be seen in Figure 1, providing a detailed view of the bacterial morphology and arrangement.
Figure 1. Morphological identification of L. acidophilus isolate. (A) L. acidophilus bacteria (a) and MRSA media (b), (B) Gram staining in 100x magnification, and (C) Effervescent granules of L. acidophilus.
Figure 2. Flow rate of Lactobacillus acidophilus effervescent granules.
Figure 3. Angle of repose of Lactobacillus acidophilus effervescent granules.
Figure 4. Compressibility of Lactobacillus acidophilus effervescent granules.
Figure 5. Lost on drying of Lactobacillus acidophilus effervescent granules.
Organoleptic Test
The result
of organoleptic assessment, the manufactured effervescent granule formulations
underwent evaluations concerning their shape, color, and aroma.
Organoleptically, all three formulas yielded a white appearance, presented a
slightly sour taste, and exhibited a relatively mild scent. The inclusion of
the active ingredient L. acidophilus bacteria in the formula did not
noticeably influence the color, resulting in the observed white coloration as
depicted in Figure 1.
Flow Rate of Effervescent Granules
The granules flow rate
test aims to determine the quality of the effervescent granules because, in the
tablet printing process, the flow rate of the powder/granules is greatly
affected by the flow rate test (29). The flow rate is said to be good if it has a
flow rate of not less than 10 g/sec
(21). The results of the flow rate test
showed that formula 1 had a better granule flow rate than formulas 2 and 3 (see
Figure 2).
Angel of Repose
The angle of repose test is intended to
determine the flow properties of granules. The angle of repose can be
determined by pouring the granule into the funnel. Then, a repose angle will be
formed. The angle of repose requirement for granules ranges
from 25o– 40o
(23). The results of the angle of repose test show
that formula 1 has a better granule angle of repose or equal to 40o
so that formula 1 is closer to the requirements than formulas 2 and 3 (see
Figure 3).
Compressibility Test
The compressibility test
is intended to describe the stable and compact nature of the powder when
pressed in the tablet molding process. The better the ability of the granules
to flow, the lower the compressibility value. The
compressibility test requirement for granules of not less than 20% (21). The compressibility test results
show that all formulas meet the requirements. Formula 3 has a smaller
compressibility value compared to formulas 1 and 2 (see Figure 4).
Loss on Drying
When testing the water content, which was
carried out by weighing several granules and putting them into the moisture
balance, the result of formula I was 0.62%, formula II was 1.54%, and formula
III was 1.26% (see Figure 5). From the three results of the water content of
formulations II and III, it was said that they did not meet the requirements
because the water content requirements for effervescent granules, according to
Fausett (2000), ranged from 0.4% - 0.7% (27). This is due to the formulation's
concentration of acids and bases, and the room temperature where the
preparation is made has high enough humidity.
Figure 6. Dissolving time of Lactobacillus acidophilus effervescent granules.
Figure 7. pH of Lactobacillus acidophilus effervescent granules.
Figure 8. Foam height of Lactobacillus acidophilus effervescent granules.
Figure 9. Viability of Lactobacillus acidophilus in effervescent granules.
Dissolving Time
Testing the dissolving time of
effervescent granules, the tool used is a stopwatch. The dissolving time test
is characterized by the granules completely dissolving in water until an acid
and base reaction is formed which can produce carbon dioxide gas (CO2).
Between 1-2 min is the effervescent granule dissolving time. effervescent
granules dissolve well (dispersed) within = 5 min can be categorized as meeting
the dissolution time requirements (20). From the results of the dissolving
time test, formula 1 has faster dissolving time test results than formulas 2
and 3 (see Figure 6).
pH Test
The acidity level was measured using a
pH meter in an effervescent solution that had been dissolved as much as 1 g in
9 mL. The test was carried out 2 times for each formulation. The results showed that all formulas met the
requirements because the pH test value was <5 (see Figure 7).
Foam Height
Measured based on the
height of the foam produced by a sample of 1 g of sample for each formulation
that has been dissolved, it can be seen that the highest foam was produced by formula I (see Figure 8).
In Vitro Viability
Testing the viability of probiotic
granules is a test that needs to be considered so that the probiotic bacteria
contained in the granules can provide a therapeutic effect.
The Lactic Acid Bacteria viability test of probiotic products used the spread method. The spread method is a technique for growing microorganisms in agar media by spreading the preparations on agar media that have solidified (30). World Health Organization (WHO) requirements Lactic Acid Bacteria Viability are 107 cfu/g (14). The viability of the lactic acid bacteria for all formulas can be seen in Figure 9.
Discussion
In addition to the MRSA
selective media confirmation test for bacterial growth (depicted in Figure 1),
morphological identification was performed through Gram staining of bacteria
obtained from pure L. acidophilus isolates. The purpose of Gram staining
is to microscopically visualize colony morphology and distinguish between
Gram-negative and Gram-positive bacteria. Bacteria that retain a purple hue
after alcohol dissolution, following Methylene Blue absorption, are termed
Gram-positive Bacteria. Conversely, bacteria that lose their purple color when
washed with alcohol subsequently absorb Fuchsin dye, resulting in a pink hue;
these are referred to as Gram-negative Bacteria. The response to staining is
also influenced by the cell wall structure. Gram-positive bacteria, primarily
featuring peptidoglycan in their cell walls, retain the violet color (31).
As indicated by the
results of Gram staining, the bacteria exhibit a rod-like shape and belong to
the Gram-positive category. This observation aligns with the findings of Putri
(2018), who noted that Lactic Acid Bacteria generally assume the form of
Gram-positive rods and are generally non-spore-forming members of the LAB group (31). Abedi and Hashemi (2020) also affirmed that
Lactic Acid Bacteria are Gram-positive, non-spore-forming microorganisms
capable of thriving in oxygen-rich environments and producing lactic acid
through carbohydrate fermentation (e.g., glucose, lactose) (12).
In the lactic acid fermentation
process, this process begins with the change of one glucose molecule into 2
pyruvic acid molecules. This process is a stage of glycolysis so that 2 ATP
molecules are produced. After the glycolysis process does not proceed to the
oxidative decarboxylation stage unless 2 pyruvic acids are converted into 2
molecules of lactic acid.
During the organoleptic
assessment, the manufactured effervescent granule formulations underwent
evaluations concerning their shape, color, and aroma. Organoleptically, all
three formulas yielded a white appearance, presented a slightly sour taste, and
exhibited a relatively mild scent. The inclusion of the active ingredient L.
acidophilus bacteria in the formula did not noticeably influence the color,
resulting in the observed white coloration as depicted in Figure 1.
The outcomes of the
effervescent granules' flow rate evaluation are presented in Figure 2, revealing distinct flow rates for Formulas I,
II, and III. The table's data indicate the influence of varying concentrations
of acidic ingredients on the flow rate of the effervescent granules.
Specifically, Formula I exhibited a more favorable flow rate compared to
Formulas II and III. As per the available literature, tartaric acid boasts a
higher density, implying that granules containing greater tartaric acid
concentrations exhibit increased density. Consequently, higher density
corresponds to larger molecular weight, thus leading to enhanced ease of flow
due to heightened gravitational forces (32).
Zuraidah's research
(2018) aligns with these findings, demonstrating that increased tartaric acid
concentration corresponds to an improved flow rate (33). Furthermore, in line with Hermanto's findings
(2019), variations in granule shape and size also contribute to the attainment
of a desirable flow rate (34). Additionally, Syahrina's work (2021) emphasizes the role of the binder in
influencing flow rate. Notably, the present study employs PVP K30 as the
binder, akin to Syahrina's study (2021). Syahrina's research findings highlight
that PVP K30 can diminish cohesion forces, thereby augmenting particle size and
leading to favorable flow rates (21).
Figure 3 illustrates the outcomes
of the angle of repose measurements, all of which are equal to or exceed 40
degrees. These angle of repose data collectively indicate deficient flow
properties across Formulas I, II, and III. Drawing insights from both Figure 2 dan Figure 3, the flow rate and
angle of repose results in Formula I are notably smaller when compared to
Formulas II and III. This correspondence is consistent with the findings of
Zuraidah (2018), who emphasized the impact of citric acid and tartaric acid
concentrations on variations in angle of repose across the three formulas (33).
Within Formula I, a
smaller angle of repose is observed compared to Formulas II and III, an effect
attributed to the substantial molecular weight of tartaric acid and its
consequent influence on particle size. The presence of larger particles reduces
particle cohesiveness, leading to a swifter flow rate and subsequently yielding
a smaller angle of repose (33). Correspondingly, Salmatuzzahro's study (2022) indicated that the angle of
repose is subject to the influences of granule shape, size, and moisture
content. Variations in granule shape might stem from divergent emphases during
the granulation process, potentially resulting in distinct angle of repose
measurements (35).
In accordance with the
study, the results of the compressibility test indicated that Formula I
exhibited a value of 11.45%, Formula II measured 14.03%, and Formula III
recorded 7.27%. The data presented in Figure 4 demonstrate that all
attained values conform to the prescribed criteria for satisfactory
compressibility, as denoted by values falling below 20% (25). Notably,
the highest compressibility value is observed in Formula II. This outcome is
attributed to the influence of moisture content within the powder; granules
possessing elevated water content, when subjected to pressure or vibration,
tend to undergo substantial volume reduction, consequently leading to
heightened compressibility. Moreover,
the configuration and dimensions of granule particles significantly impact
compressibility values. Optimal compressibility values are associated with
granules that feature uniform shapes and sizes, facilitating a smoother tablet
compression process (34).
The assessment outcomes,
as presented in Figure
5, demonstrate that Formula I aligns
with the specified requirements. This alignment can be attributed to the
hygroscopic nature of citric acid, enabling it to readily absorb atmospheric
moisture. This observation resonates with the findings of Romantika (2017), who
reported that augmenting citric acid content leads to increased water content
in effervescent baby java oranges (36). This trend is further supported by
Widyaningrum's research (2015), which highlighted that citric acid addition in
pandan leaf effervescent granules correlates with heightened water content
(37). Moreover, according to Audrey (2022) quoted in Lieberman et al. (1994) emphasized that citric
acid exhibits marked hygroscopic characteristics, rendering effervescent
granules with higher citric acid content exceptionally prone to water
absorption during the manufacturing process, consequently resulting in elevated
water content (38).
Figure 6 displays that the dissolving time for Formula I
is notably swifter when contrasted with Formula II and Formula III. This
divergence underscores the influence of distinct citric acid and tartaric acid
concentrations on the solubility testing of effervescent granules. This aligns
with the findings of Zuraidah's study (2018), indicating that the pronounced
hygroscopic nature of higher-concentration tartaric acid fosters enhanced water
absorption and, consequently, quicker reactivity in effervescent granules (33). Correspondingly, Salmatuzzahro's research (2022)
expounded on the faster dissolution time of the citric acid formula
characterized by the lowest concentration (35). Consistently, the current study's findings
parallel these trends. Formula I boasts the lowest citric acid concentration
coupled with the highest tartaric acid concentration, leading to the most rapid
dissolution time compared to Formula II and Formula III. The rapid carbonation
reaction between sodium bicarbonate, tartaric acid, and citric acid is
facilitated by the hygroscopic nature of both citric and tartaric acids.
Notably, Figure 6 illustrates that all three formulations exhibit
dissolution times ≤ 5 min or within the range of 1 to 2 min. Thus, all three
formulas satisfy the requirements of the solubility test.
In Figure 7, the pH value of Formula I exceeds that of
Formula II and Formula III. This divergence stems from the variations in citric
acid and tartaric acid concentrations, particularly the elevated tartaric acid
concentration in Formula I, which significantly impacts the pH value.
Kusnadhi's study (2003) highlighted that the emergence of carbon dioxide (CO2)
during the interaction of two effervescent components in water, leading to the
partial formation of carbonic acid, results in a reduction of H+ ions within the
solution. This reduction contributes to increased solution alkalinity and
consequently elevates the pH value
(39). Furthermore, the properties of L. acidophilus
bacteria exert an influence on the preparation's pH. Given that the optimal
growth pH for L. acidophilus bacteria is ≤ 5, the presence of these
bacteria in the effervescent granule formulation maintains the solution's
acidity even after granulation. Based on the outcomes for the three formulas,
all are classified as compliant with the pH test requisites, as their pH values
are ≤ 5.
The mean froth height
measurement extracted from L. acidophilus bacteria effervescent granules
(Figure 8) ranged from 2 to 3 cm. Formula I exhibited a
froth height of 3.1 cm, Formula II recorded 2.55 cm, and Formula III showed
2.25 cm. The optimal froth height outcome aligns with the effervescent market
standard, indicating a minimal deviation of approximately 3 cm. As articulated
by Bryant (1970), foam comprises numerous min bubbles originating from liquid
and arising due to either chemical reactions (acidulant and carbonate) or
mechanical manipulation (stirring). As these bubbles swiftly accumulate and
expand on the liquid's surface, foam emerges. The substantial froth formation observed in this study can be attributed to
the nature of tartaric acid. In comparison to other acids, tartaric acid
exhibited the most robust outcomes in terms of carbon dioxide generation,
albeit with a longer disintegration period.
In Gantini's
investigation (2016), the optimal conditions involving a temperature of 45°C
and a drying duration of 2 h yielded a Lactic Acid Bacteria viability of 7.43
cfu/g in instant probiotic granules. In this current study, the temperature
applied was 40°C for a duration of 3 h (13). As demonstrated in Figure 9, the average Lactic Acid Bacteria viability for effervescent granules was
412.5 x 101 cfu/g in Formula I, 422.7 x 101 cfu/g in
Formula II, and 544.7 x 101 cfu/g in Formula III. These findings
signify the continued viability of L. acidophilus bacteria within the
effervescent granule preparations. However,
all formulated formulas fell short of meeting the WHO criteria for Lactic Acid
Bacteria viability in probiotic products. This discrepancy could potentially be
attributed to the drying duration, indicating an influence on Lactic Acid
Bacteria viability. Gantini's research (2016) underscores that prolonged
granule drying periods correlate with reduced bacterial viability. This
underscores the critical role of drying time in determining the survival or
demise of bacteria (13).
Conclusion
Based on the undertaken
research focusing on the formulation and evaluation of L. acidophilus
bacteria effervescent granules, the conclusion drawn is that among the three
formulations, Formula I emerges as the most favorable (L. acidophilus 3%; Polyvinylpyrrolidone
3%; Citric Acid 0.3 %; Tartaric Acid 49.7%; Sodium Bicarbonate 25% and Lactose
Ad). Formula I demonstrates the
closest alignment with the physical requisites stipulated for effervescent
granule preparations, as corroborated by the outcomes of the comprehensive
tests, including the organoleptic assessment. Specifically, the granules are in
the appropriate shape, white in color, and devoid of odor. The flow rate test
yielded a value of 1.497 g/sec, the angle of repose was measured at 40.75°, the
compressibility test resulted in 11.45%, loss on drying test showed 0.62%, and foam height measured
3.075 cm.
All three formulations,
i.e., Formulas I, II, and III, effectively incorporate L. acidophilus
bacteria after being converted into effervescent granules, as substantiated by
the viability of Lactic Acid Bacteria. Specifically, Formula I exhibited a
Lactic Acid Bacteria viability of 412.5 x 101 cfu/g, Formula II
registered 422.7 x 101 cfu/g, and Formula III recorded 544.7 x 101
cfu/g, as evidenced by the results of the Lactic Acid Bacteria viability test.
Lactobacillus acidophilus, a strain of lactic acid bacteria widely used as a probiotic microorganism, has been extensively employed in developing probiotic products including in effervescent granules. This study's objectives were to ensure that the resultant granules possessed the desired physical attributes and retained the requisite viability of lactic acid bacteria. A wet granulation method was used. The formula was physically evaluated and analyzed using Design Expert software, followed by gram staining and bacterial harvesting. Gram staining verification demonstrated the gram-positive nature of the pure L. acidophilus bacterial isolates, as evidenced by their consistent purple coloration and characteristic basil shape. Evaluation of physical properties revealed organoleptic attributes such as granular shape, white coloration devoid of odor, flow rate of 1.497 g/sec, an angle of repose at 40.75o, compressibility of 11.45%, drying loss of 0.62%, and a foam height of 3.075 cm, consistently meeting the stipulated criteria. Furthermore, formula I, II, and III preserved L. acidophilus bacteria after being converted into effervescent granules, as evidenced by viable lactic acid bacteria counts, with formula I (412.5 x 101 cfu/g), formula II (422.7 x 101 cfu/g), and formula III (highest at 522.7 x 101 cfu/g). The formula I emerges as the most favorable effervescent granules containing L. acidophilus 3%; polyvinylpyrrolidone 3%; citric acid 0.3%; tartaric acid 49.7%; sodium bicarbonate 25% and lactose as filler. Based on the finding, this probiotic effervescent granules has the potential to be developed as a daily supplement, especially for flora normal stabilization.
Tanujaya D, Riniwasih L. Formulasi dan Uji Stabilitas Fisik
Tablet Effervescent yang Mengandung Bakteri Probiotik Lactobacillus Bulgariscus
dengan Metode Granulasi Basah. Indoensia Nat Res Pharm J. 2019;4(2):pp.101-112.
Halder D, Mandal M, Chatterjee SS, Pal NK, Mandal S.
Indigenous probiotic Lactobacillus isolates presenting antibiotic like activity
against human pathogenic bacteria. Biomedicines. 2017;5(2):1–11.
Surendran Nair M, Amalaradjou MA, Venkitanarayanan K.
Antivirulence Properties of Probiotics in Combating Microbial Pathogenesis
[Internet]. Vol. 98, Advances in Applied Microbiology. Elsevier Ltd; 2017. 1–29
p. Available from: http://dx.doi.org/10.1016/bs.aambs.2016.12.001
Ali MS, Lee EB, Hsu WH, Suk K, Sayem SAJ, Ullah HMA, et al.
Probiotics and Postbiotics as an Alternative to Antibiotics: An Emphasis on
Pigs. Pathogens. 2023;12(7).
Cotter PD, Ross RP, Hill C. Bacteriocins-a viable alternative
to antibiotics? Nat Rev Microbiol [Internet]. 2013;11(2):95–105. Available
from: http://dx.doi.org/10.1038/nrmicro2937
Rusli R, Amalia F, Dwyana Z. POTENSI BAKTERI Lactobacillus
acidophilus SEBAGAI ANTIDIARE DAN IMUNOMODULATOR. Bioma J Biol Makassar. 2018;3(2):25–30.
Shabbir I, Al-Asmari F, Saima H, Nadeem MT, Ambreen S,
Kasankala LM, et al. The Biochemical, Microbiological, Antioxidant and Sensory
Characterization of Fermented Skimmed Milk Drinks Supplemented with Probiotics
Lacticaseibacillus casei and Lacticaseibacillus rhamnosus. Microorganisms.
2023;11(10).
Badgeley A, Anwar H, Modi K, Murphy P, Lakshmikuttyamma A.
Effect of probiotics and gut microbiota on anti-cancer drugs: Mechanistic
perspectives. Biochim Biophys Acta - Rev Cancer [Internet].
2021;1875(1):188494. Available from: https://doi.org/10.1016/j.bbcan.2020.188494
Aini M, Rahayuni S, Mardina V, Quranayati Q, Asiah N. BAKTERI
Lactobacillus spp DAN PERANANNYA BAGI KEHIDUPAN. J Jeumpa. 2021;8(2):614–24.
Leoanggraini U, Muhadi BI. Fermentasi Mikroaerofilik Lactobacillus
acidophilus untuk Produksi Probiotik. Ind Res Work Natl Semin. 2011;188–92.
Hidayati H, Afifi Z, Triandini HR, Permata I. Pembuatan
Yogurt Sebagai Minuman Probiotik untuk Menjaga Kesehatan Usus. Pros SEMNAS BIO
2021 Univ Negeri Padang. 2021;1265–70.
Abedi E, Hashemi SMB. Lactic acid production – producing
microorganisms and substrates sources-state of art. Heliyon [Internet].
2020;6(10):e04974. Available from:
https://doi.org/10.1016/j.heliyon.2020.e04974
Gantini SN, Widayanti A. ANALISA DATA RESPONSE SURFACE
METHODOLOGY PADA PENGEMBANGAN GRANUL PROBIOTIK LACTOBACILLUS CASEI. Penelit
Pengenbangan Iptek. 2016;109.
The World Health Organization. Health and Nutritional
Properties of Probiotics in Food including Powder Milk with Live Lactic Acid
Bacteria. Fao Who [Internet]. 2001;(October):1–34. Available from: Cordoba,
Argentina
Mindawarnis, Hasanah D. Formulasi Sediaan Tablet Ekstrak Daun
Nangka (Artocarpus heterophyllus L) dengan variasi Polivinil Pirolidon (PVP )
sebagai Pengikat dan Evaluasi Sifat Fisiknya. Univ Nusant PGRI Kediri
[Internet]. 2017;01(1):1–7. Available from: http://www.albayan.ae
Anam C, Kawiji, Setiawan RD. Study of physical and sensory
characteristics and antioxidant activity of beet fruit effervescent granules
(beta vulgaris) with different granulation methods and combination of acid
sources. J Teknosains Pangan. 2013;2(2):21–8.
Andarilla W, Sari R, Apridamayanti P. OPTIMASI AKTIVITAS
BAKTERIOSIN YANG DIHASILKAN OLEH Lactobacillus casei DARI SOTONG KERING. J
Pendidik Inform dan Sains. 2018;7(2):187.
Tungadi R. Teknologi sediaan solid. Team WADE Publish,
editor. Ponorogo; 2018. 136 p.
Sidoretno WM. Formulasi Dan Evaluasi Granul Effervescent
Kombinasi Ekstrak Kering Rimpang Jahe Merah, Temulawak Dan Kayu Manis. JOPS
(Journal Pharm Sci. 2022;5(2):21–35.
Santosa L, Yamlean PVY, Supriati HS. Formulasi Granul
Effervescent Sari Buah Jambu Mete (Annacardium Ocidentale L.). Pharmacon.
2017;6(3):56–64.
Syahrina D, Noval N. Optimasi Kombinasi Asam Sitrat dan Asam
Tartrat sebagai Zat Pengasam pada Tablet Effervescent Ekstrak Ubi Jalar Ungu
(Ipomoea batatas L). J Surya Med. 2021;7(1):156–72.
Maulida Yuniar Widya Putri, Wilda Amananti HP. UJI SIFAT
FISIK GRANUL EFFERVESCENT EKSTRAK DAUN SALAM ( Syzygium polyanthum ) DENGAN
PEMANIS DAUN STEVIA ( Stevia Rebaudiana. Politek tegal. 2020;3(1):1–10.
Yenrina R, Sayuti K, Anggraini T. Effect of natural colorants
on color and antioxidant activity of “kolang kaling” (Sugar palm fruit) jam.
Pakistan J Nutr. 2016;15(12):1061–6.
Pratama R, Roni A, Fajarwati K, Keilmuan K, Farmasi F, Bhakti
U, et al. UJI SIFAT FISIK GRANUL INSTAN EKSTRAK PEGAGAN ( Centella asiatica ).
2022;52:299–304.
Noval N, Kuncahyo I, Pratama AFS, Nabillah S, Hatmayana R.
Formulasi Sediaan Tablet Effervescent dari Ekstrak Etanol Tanaman Bundung
(Actionoscirpus grossus) sebagai Antioksidan. J Surya Med. 2021;7(1):128–39.
Sudarsono APP, Nur M, Febrianto Y. Pengaruh Perbedaan Suhu
Pengeringan Granul (40°C,50°C,60°C) Terhadap Sifat Fisik Tablet Paracetamol. J
Farm Sains Indones. 2021;4(1):44–51.
Rani KC, Parfati N, Muarofah D, Sacharia SN. Formulasi Granul
Effervescent Herba Meniran (Phyllanthus niruri L.) dengan Variasi Suspending
Agent Xanthan Gum, CMC-Na, dan Kombinasi CMC-Na-Mikrokristalin Selulosa RC-
591. J Sains Farm Klin. 2020;7(1):39.
Putra. D., Antari. N., Putri. N., Arisanti. C. SP. Penggunaan
Polivinill Pirolidon (PVP) Sebagai Bahan Pengikat Pada Formulasi Tablet Ekstrak
Daun Sirih (Piper betle L.). J Farm Udayana. 2019;8(1):14–21.
Damayanti NWE, Abadi MF, Bintari NWD. Perbedaan Jumlah
Bakteriuri Pada Wanita Lanjut Usia Berdasarkan Kultur Mikrobiologi Menggunakan
Teknik Cawan Tuang Dan Cawan Sebar. Meditory
J Med Lab. 2020;8(1):1–4.
Putri AA, Erina, Fakhrurraz. Isolasi Bakteri Asam Laktat
Genus Lactobacillus Dari Feses Rusa Sambar ( Cervus unicolor ). Jimvet E-Issn :
2540-9492. 2018;2(1):170.
Egeten KR, Yamlean PVY, Supriati HS. Formulasi dan Pengujian
Sediaan Granul Effervescent Sari Buah Nanas (Ananas comosus L. (Merr.).
Pharmacon J Ilm Farm. 2016;5(3):116–21.
Zuraidah N, Ayu WD, Ardana M. Pengaruh Variasi Konsentrasi
Asam Sitrat dan Asam Tartrat terhadap Sifat Fisik Granul Effervescent dari
Ekstrak Daun Nangka (Artocarpus heterophyllus L.). Proceeding Mulawarman Pharm
Conf. 2018;8(November 2018):48–56.
HERMANTO. Pengaruh PVP Dan HPMC Sebagai Bahan Pengikat
Terhadap Sifat Fisik Tablet Effervescent. 2019;
Salmatuzzahro A, Nawangsari D, Fitriana AS. Uji Sifat Fisik
Tablet Effervescent dari Ekstrak Kulit Buah Pisang Raja ( Musa X Paradisiaca L
) dengan Perbandingan Asam Sitrat dan Natrium Bikarbonat. skripsi. 2022;
Romantika RC, Wijana S, Perdani CG. Formulasi dan
Karakteristik Tablet Effervescent Jeruk Baby Java (Cytrus sinensis L. Osbeck)
Kajian Proporsi Asam Sitrat Formulation and Characterization of Baby Java
Orange (Cytrus Sinensis L. Osbeck) Effervescent Tablets Study on Cytric Acid
Proportion. J Teknol dan Manaj Agroindustri [Internet]. 2017;6(1):15–21.
Available from: http://www.industria.ub.ac.id
Widyaningrum A, Lutfi M, Argo BD. Karakterisasi Serbuk
Effervescent dari Daun Pandan (Pandanus amaryllifolius Roxb) dengan Variasi
Komposisi Jenis Asam. J Bioproses Komod Trop. 2015;3(2):1–8.
Perbandingan Konsentrasi Asam Sitrat Dan Asam Malat Terhadap
Karakteristik Granul Effervescent Bunga Telang P, Sophia Rachma Putri A, Luh
Ari Yusasrini N, Timur Ina P, Studi Teknologi Pangan P, Teknologi Pertanian F,
et al. The Effect of Comparative Concentration of Citric Acid and Malic Acid on
the Characteristics of Effervescent Granules of Butterfly Pea Flower (Clitoria
ternatea L.). Online) Audrey Sophia RP dkk / Itepa. 2022;11(4):2022–788.
Astuti RD, Wahyu AW. Formulasi dan Uji Kestabilan Fisik
Granul Effervescent Infusa Kulit Putih Semangka. J Kesehat. 2016;11(1):162–71.
