Online - 2455-3891

Vol 11, Issue 12, 2018 Print - 0974-2441

Research Article

THE EFFECT OF STORAGE CONDITION ON ANTIOXIDANT ACTIVITY OF PROBIOTICS IN

YOGURT DRINKS

JANSEN SILALAHI1*, DARSHIENY NADARASON1, YOSY C E SILALAHI2

1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Nanomedicine Center Universitas Sumatera Utara, Indonesia.
2
Department of Pharmacy, Faculty of Pharmacy and Health Sciences, Universitas Sari Mutiara, Indonesia. Email: [email protected]
Received: 27 June 2018, Revised and Accepted: 02 August 2018

ABSTRACT

Objective: The aim of this study was to investigate the effect of temperature and time of storage on antioxidant activity of probiotics in yogurt drinks.

Methods: The samples used were Yakult, Cimory, YoFit, and BioKul marketed in Medan city. The samples were stored at room and refrigerate
temperatures for 2 weeks. Various volumes of samples were withdrawn and mixed with 1 ml of 2,2-diphenyl-1-picrylhydrazil solution and
homogenized. Then, it was incubated for 30 min and centrifuged at 5000 rpm for 15 min. The supernatant was collected, and the absorbance was
measured at 516 nm by spectrophotometer. The absorbance of different volume of supernatant of each sample was used to construct linear regression
equation. Then, the IC50 value was determined.

Results: The present study shows that the higher the temperature and the longer the time of storage decreased the antioxidant activity of yogurt
drinks. Yakult exhibited the highest antioxidant activity among the analyzed samples. Before storage, the IC50 value of Yakult was 0.2763 ml, and after
stored for 2 weeks, the IC50 value of Yakult was 0.3867 ml at refrigerated and 0.5393 ml at room temperature.

Conclusions: Probiotic in yogurt drinks stored at refrigerated temperature has higher antioxidant activity than stored at room temperature. The IC50
values of yogurt drinks decreased with storage time.

Keywords: Antioxidant, 2,2-diphenyl-1-picrylhydrazil, Probiotics, Storage condition, IC50 value.

© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2018.v11i12.28156

INTRODUCTION and time of storage on antioxidant activity of probiotics from four plain
products of different types/brands of yogurt drinks.
Oxidation is an essential process for many living organisms for the
production of energy to fuel biological processes. Physiological MATERIALS AND METHODS
metabolites such as free radicals and reactive oxygen species (ROS)
are formed as a result of respiration in the aerobic organism [1-3]. Materials
Formation of free radicals such as superoxide anion radical and hydroxyl Reagents and materials were 2,2-diphenyl-1-picrylhydrazil (DPPH),
radical is unavoidable during respiration, and excessive production can methanol, Vitamin C, and distilled water. The samples used were plain
cause damage on lipid, protein, carbohydrate, and nucleic acids [4,5]. yogurt products purchased from market Pondok Indah Pasar Buah in
Fortunately, human body has developed a number of enzymatic and Jalan Setia Budi, Medan, Indonesia. The samples (plain products) did
non-enzymatic defense mechanism which can neutralize the free not contain flavoring agents of four different type/brand yogurt drinks
radicals and oxidant molecules [5,6]. In certain circumstances such as included Yakult, Cimory, YoFit, and BioKul.
chronic hyperglycemia, this antioxidant defense system fails and not
effective enough to totally neutralize free radicals which are known as Instruments
oxidative stress [2,7]. Therefore, to prevent the damage by oxidative Refrigerator, incubator, centrifugation, vortex, analytical balance, and
stress, antioxidant compounds contained in fruits, vegetables, and UV-visible Spectrophotometer (Shimadzu UV-1800) and glasswares
food supplement containing antioxidant is required to reduce oxidative were used.
damage [4,6,7].
Preparation of DPPH solution
Probiotic is defined as live microorganisms when administered in 50 mg of DPPH was transferred into 50 ml of brown volumetric flask and
adequate amounts, confer a health benefit on the host by improving dissolved in methanol and calibrated up to the marked line. From this
microbial balance. Probiotic bacteria, especially such as Lactobacillus solution, 2 ml was transferred into 50 ml volumetric flask and added
and Bifidobacterium, are found to be beneficial in preventing the methanol to volume to get a solution of 40 µg/ml (0.5 mM) [1,16].
growth of pathogenic bacteria, reducing inflammation, and inhibiting
glycation of protein in hyperglycemia. Some bioactive components are Preparation of Vitamin C solution
produced by probiotic including antioxidants [7-12]. The antioxidant 50 mg of Vitamin C powder was dissolved with methanol in 25 ml brown
mechanisms of probiotics could be assigned to scavenge ROS by volumetric flask. From this solution, 2.5 ml was transferred into a 25 ml
non-enzymatic and enzymatic antioxidants contained in yogurt volumetric flask and added methanol up to the marked line of volumetric
drinks [6,13]. Factors such as pH, redox potential, and acidity, buffering flask to make Vitamin C solution with concentration of 200 µg/ml. [1,16].
capacity, packaging, molecular oxygen, yogurt composition, and storage
time and temperature affect the viability of probiotic bacteria during Procedure
storage and hence antioxidant activity of yogurt drinks [14,15]. The The samples were divided into two groups. Group A samples were
objective of this study was to examine the effect of the temperature stored at cold storage by placing them inside the refrigerator. Group B
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samples were stored at the ambient or room temperature. The samples percentage of the all samples decreased with the storage time. The
were stored for 2 weeks, and antioxidant activity was determined prominent decreased in scavenging percentage can be observed in
before storage, after 1 week, and 2 weeks of storage. storage at room temperature compared to the refrigerate temperature.

Determination of absorbance curve of DPPH solution Figs. 2-6 show that the lower the absorbance value of DPPH solution
The DPPH solution with a concentration of 40 μg/ml was transferred mixed with sample the higher antioxidant activity of the analyzed
into the cuvette and placed into the spectrophotometer. The absorbance samples. This is due to the ability of Vitamin C and bioactive components
was measured within the wavelength between 400 to 800 nm to produced by probiotic to chemically reduce DPPH solution which is
get absorbance curve. From this absorbance curve, the maximum used to evaluate the antioxidant activity. Interaction of antioxidant
absorbance of DHHP solution was found to be 516 nm and used in this compounds with DPPH is based on the transfer of hydrogen atom to
study. DPPH radical and converts it into DPPH-H. The result of the reduction
of DPPH radicals results in color change from a purple color to yellow
DPPH free radical scavenging assay of Vitamin C and tested pale color which indicates the antioxidant activity of the sample [7,18].
samples
1 ml of methanolic DPPH solution (40 μg/ml) was transferred into Antioxidant activity of Vitamin C and yogurt drinks
a volumetric flask of 5 ml and added 0.0625 ml Vitamin C solution The IC50value of the samples and Vitamin C was determined using linear
of 200 μg/ml; the same procedure was carried out with 0.1250 ml, regression equation obtained by plotting the volume of test samples as
0.2500 ml, and 0.500 ml of Vitamin C solution. This procedure also X-axis and the scavenging percentage of tested samples as Y-axis. The IC50
applied to each yogurt drink with a series of volumes, namely 0.25 ml, value was determined by calculating the volume that required to scavenge
0.50 ml, 0.75 ml, and 1.0 ml of Yakult, Cimory, YoFit, and BioKul, 50% of DPPH radicals by the samples. The result of IC50 value can be seen in
respectively, then methanol was added to each mixture to the volume of Fig. 7. From Fig. 7 can be seen that the smaller the volume of tested sample
5 ml (to the marked line). It was well vortexed, and then incubated for the higher the antioxidant activity indicate that the lower volume needed to
30 min at room temperature. The samples were centrifuged for 15 min scavenge DPPH solution at 50% and stated or called as IC50 and vice versa.
at 5000 rpm. The supernatant was collected, and the absorbance of the
sample was measured at 516 nm using the spectrophotometer. Negative
control was prepared using 1.0 ml of DPPH solution transferred into 5 ml
volumetric flask and added methanol to volume without addition of the
sample. It was allowed to stand for 30 min at room temperature then the
absorbance (A) was measured. The readings were recorded in triplicates.
The percentage of radical scavenging activity (RSA) was calculated:

A(control) − A(sample)
RSA (%) = × 100
A(control) ,

Graphs for the effect of storage condition of samples and Vitamin C

on DPPH scavenging activity (%) were plotted, and the regression
equation determined [1,17].
Fig. 1: Absorbance curve of 2,2-diphenyl-1-picrylhydrazil solution
Determination of IC50 value in methanol (40 µg/ml)
The effective volume of Vitamin C and samples required to scavenge DPPH
radical by 50% (IC50 value) was obtained from linear regression analysis of
dose-response curve obtained by plotting between percentage inhibition
and volume (ml). The results obtained from the DPPH scavenging activity
were substituted into the regression equation, where the X-axis was the
volume of vitamin C, and Y-axis was the percentage of DPPH scavenging
activity [17].

Determination of IC50 from regression equation; Y=aX+b

Notes: Y=Percentage of DPPH scavenging activity (IC50, dependent

variable); a=Slope/gradient; x=Volume of vitamin C (independent
variable); b=intercept. Fig. 2: The effect of storage condition of Yakult on 2,2-diphenyl-
1-picrylhydrazil scavenging activity (%). Rt: Room temperature;
RESULTS AND DISCUSSION Ret: Refrigerator temperature

Absorbance curve of DPPH solution

The absorbance measurement of the DPPH solution in methanol using
a UV-visible spectrophotometer and the absorbance curve is presented
in Fig. 1.

From Fig. 1, the maximum absorbance of the DPPH solution is at the

wavelength of 516 nm and used to determine IC50.

DPPH scavenging activity (%) of yogurt drinks

The effect of storage condition of each sample on DPPH scavenging
activity (%) can be seen in Figs. 2-5. The scavenging activity percentage
of Vitamin C is shown in Fig. 6.
Fig. 3: The effect of storage condition of Cimory on 2,2-diphenyl-
Regression equation from Figs. 2-6 was used to calculate the IC50 1-picrylhydrazil scavenging activity (%). Rt: Room temperature;
value of each sample. From Figs. 2-5 can be seen that the scavenging Ret: Refrigerator temperature

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Fig. 4: The effect of storage condition of YoFit on 2,2-diphenyl-1-

Fig. 6: 2,2-diphenyl-1-picrylhydrazil scavenging activity (%) of
picrylhydrazil scavenging activity (%). Rt: Room temperature;
Vitamin C 2.5 µg/ml
Ret: Refrigerator temperature

Fig. 5: The effect of storage condition of BioKul on 2,2-diphenyl-

Fig. 7: Antioxidant activity (IC50) of samples and Vitamin C.
1-picrylhydrazil scavenging activity (%). Rt: Room temperature;
RT: Room temperature; ReT: Refrigerator temperature
Ret: Refrigerator temperature

The other samples contain more than one probiotic bacteria as written
The IC50 value decreased with storage time for both room and refrigerates
on the label. Cimory contains two probiotic bacteria, Streptococcus
temperature. Before storage, IC50 value of Yakult (0.2763 ml) is the highest
thermophylus and Lactobacillus delbrueckii subsp. bulgaricus. BioKul
value followed by YoFit, Cimory, and BioKul to scavenge the DPPH radicals
by 50%. Even after storage at room and refrigerate temperature, Yakult contains four probiotics, Streptococcus thermophylus and Lactobacillus
has the highest IC50 value compared to other samples. Vitamin C with bulgaricus, Lactobacillus acidophilus and Bifidobacterium, and Yofit
concentration of 2.5 ug/ml as positive control of antioxidant was found to contains L. acidohpilus LA-5, Bifidobacterium BB12, and Streptococcus
be the strongest antioxidant with IC50 value of 0.1742 ml which is much thermophilus.
higher than Yakult [19,20].
The antioxidant activity of probiotic bacteria in yogurt drinks is inversely
Probiotics demonstrate antioxidant activity by secreting enzymes related to the storage temperature. Food that contain probiotics
such as superoxide dismutase (SOD) and promote the production preferably stored at a temperature of 2–8°C [22,23]. This is because
of the major non-enzymatic antioxidant and free radical scavenger storage above 20°C for prolong duration of time decreases the viability
glutathione (GSH). SOD catalyzes the breakdown of superoxide into of certain probiotic bacteria such as L. acidophilus and Bifidobacterium
hydrogen peroxide and water. Thus, it acts as a central regulator of lactis. Thus, the decrease in the viability of bacteria could decrease the
ROS levels. GSH eliminates radicals such as hydrogen peroxides, and antioxidant activity of probiotic bacteria that present in yogurt drinks
hydroxyl radicals, by collaborating with selenium-dependent GSH at room temperature as can be seen from the data that the scavenging
peroxidase [16,21]. Probiotics also promote the production of certain percentage decrease for all the samples at 1st and 2nd week of storage.
antioxidant biomolecules, such as the exopolysaccharides [11]. In The common probiotics that present in all the samples is L. acidophilus
addition, antioxidant activity could also be due to metal chelating and B. lactis. Hence, the most suitable temperature for the viability of
activity, but the mechanism is still unclear [16,21]. L. acidophilus is 2°C and B. lactis is 4°C [23].

Although Yakult has the shortest expiry date compared to others, Yakult has the shortest expiry followed by Cimory, YoFit, and BioKul
Yakult exerted the highest scavenging percentage followed by YoFit, with the longest expiry. Addition of growth promotors such as glucose,
Cimory, and BioKul in both room and refrigerate temperature for vitamins, minerals, casein, whey protein, yeast extract, and antioxidants
2 weeks. For Yakult, only small volume was withdrawn to react with are fortified in dairy products to increase the viability of the probiotics.
DPPH solution because Yakult expressed high scavenging percentage Oxygen content and redox potential are harmful to the probiotic
in very small volume (0.0625 ml) before and after storage in both room survival during storage because most of the species are anaerobic
and refrigerate temperature for 2 weeks compared to other samples and saccharolactic [22]. Free radicals produced from the oxidation of
(Cimory, YoFit, and BioKul). Several factors affecting survival of the components (e.g., fat and sugar) can be hazardous to the probiotics.
probiotic bacteria and thus the antioxidant activities include storage Therefore, the level of oxygen during packaging should be as low as
temperature, composition of food, type of packaging materials, moisture possible, because all the tested samples in this study contain sucrose
content of powders, oxygen content, and exposure to light. [22]. The and skim milk powder in their composition, which may produce free
species and number of probiotic bacteria present in the products radicals during storage; thus, the scavenging activity also decreases on
could also affect the viability of bacteria during storage and hence the storage [22,23].
the ability to produce the bioactive components including antioxidant
peptides [1]. In this study, Lactobacillus casei (L. casei shirota) is the Packaging aspects such as type and thickness of the materials, gas
only probiotic present in Yakult products as mentioned on the label. (oxygen, carbon dioxide, and water vapor) and light permeability

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