J Food Sci Technol (June 2015) 52(6):3711–3718

DOI 10.1007/s13197-014-1412-9

ORIGINAL ARTICLE

Characterization of pomegranate juice and whey based novel

beverage fermented by kefir grains
Nayereh Sabokbar & Faramarz Khodaiyan

Revised: 8 June 2013 / Accepted: 19 May 2014 / Published online: 13 June 2014
# Association of Food Scientists & Technologists (India) 2014

Abstract Mixture of pomegranate juice and whey was eval- which are recognized as functional food. Probiotics can be
uated as a potential substrate for production of a novel probi- described as foods containing live microorganisms that when
otic beverage by kefir grains. Different fermentation condi- are ingested in adequate amounts, confer a health benefit on the
tions were used as viz: two fermentation temperature (19ºC host (Vasiljevic and Shah 2008). Fruit juices are a rich source
and 25ºC) and two levels of kefir grains inoculum (5 % and of calcium and vitamin and could serve as suitable media for
8%w/v). pH, acidity, lactose consumption as well as organic cultivating probiotic bacteria. Use lactic acid bacteria in food
acids formation were determined during 32 hours of fermen- industry, has been utilized for production of functional foods in
tation. Results showed that kefir grains were able to utilize recent years. Kefir which is made with kefir grains is a refresh-
lactose and decrease pH, increase acidity, produce lactic acid ing, naturally carbonated fermented dairy beverage that has a
and acetic acid, while the level of citric acid decreased. It was slightly acidic taste, yeasty flavor and creamy consistency
observed these change depended on temperature and level of (Powell et al. 2007). The traditional kefir is produced by the
kefir grains with the highest changes at the temperature of addition of small kefir grains to fresh milk. Kefir grains are like
25ºC and kefir grains inoculum of 8%w/v. Pomegranate juice small cauliflower florets, 1–3 cm in length, lobed, irregularly
and whey mixture therefore may serve as a suitable substrate shaped, white to yellow-white in color, and have a slimy but
for the production of novel probiotic dairy-fruit juice beverage firm texture (La Riviére et al. 1967). Kefir grains contain
by kefir grains and the sensory characteristics of this beverage proteins, polysaccharides and complex mixture of microorgan-
were shown desirable results. isms. Lactic acid bacteria (LAB) and yeasts have a complex
symbiotic relationship in kefir grains and responsible for alco-
Keywords Kefir . Fermentation . Pomegranate juice . Whey holic and lactic acid fermentation, respectively. Some of dis-
tinctive features of lactic acid fermentation are growth of lactic
acid bacteria, production of different organic acids, degrada-
Introduction tion of some anti-nutritional factor such as metal chelating
agents in raw plant materials like phytate, oxalate and tanins
In recent years, there has been an increased interest in the and decrease in pH (Reddy and Pierson 1994).
production of fermented beverages specially probiotics due to Whey is a rich source of proteins with high biological
their health beneficial effects and nutritional properties. This value. Whey proteins include α-lactalbumin (α-La), β-
has led to an increase in production and consumption of food lactoglobulin (β-Lg), bovine serum albumin and
immunoglubolins (BSA). β-Lg is a small and soluble globular
N. Sabokbar (*) protein which has monomer mass of 18 KDa. It has a lot of
Department of Food Science and Technology, School of Agriculture,
useful nutritional and functional food characteristics that have
Shiraz University, Shiraz, Islamic Republic of Iran
e-mail: nayereh.sabokbar@yahoo.com made it a choice as an ingredient for formulation of novel
foods and beverages. β-Lg shows many biological activity
F. Khodaiyan such as anti-hypertensive, anti-cancer, hypo-cholesterolemic
Bioprocessing and Biodetection Laboratory, Department of Food
and anti-microbial (Chatterton et al. 2006). α-La is another
Science, Engeneering and Technology, Faculty of Agricultural
Engineering and Technology, Campus of Agriculture and Natural major protein in whey that makes up about of 25 % of total
Resource, University of Tehran, Karaj, Islamic Republic of Iran whey proteins. It enhances calcium absorption because it is a
3712 J Food Sci Technol (June 2015) 52(6):3711–3718

calcium binding protein. Amino acids such as lysine, leucine, determined by titrating the samples with 0.1 N NaOH to
threonine, tryptophan and cysteine are present in large pH 8.1 and expressed as %w/w lactic acid. Quantitative anal-
amounts in the whey (Permyakov and Berliner 2000). ysis of organic acids (lactic, citric and acetic acids) was carried
Pomegranate (Punica granatum, Punicaceae) is one of the out by HPLC (Knauer, Germany) apparatus equipped with a
important table fruit and is known to have considerable health- K- 2600 UV-visible detector at 210 nm. A separation column
promoting properties such as antimicrobial, antiviral, antiox- (Ultrasep ES-FS special 250 × 3 mm) set with 0.001 N
idant and anti-mutagenic effects (Negi et al. 2003). sulphuric acid as the mobile phase with 0.2 ml/min flow rate.
Considerable amount of total soluble solids, total sugars, Room temperature was used for operation and the injection
reducing sugars, anthocyanins, phenolics, ascorbic acid, pro- volume was 20 μl. Organic acids content was determined
teins and antioxidants are present in fresh juice. Level of using external standards (Mousavi et al. 2011). Lactose
antioxidants in pomegranate juice was shown to be higher as amount was also determined by HPLC (Knauer, Germany)
compared to green tea and red wine (Gil et al. 2000) and these equipped with a K-2301 refractive index detector. A 10 μm
antioxidant are more potent than many other antioxidants such separation column (Eurokat H 300×8 mm) was employed and
as vitamin C and E (Aviram et al. 2002). the mobile phase was sulphuric acid 0.01 N with a flow rate of
The aim of this research was to prepare a fermented dairy- 0.5 ml/min. The injection volume was 20 μl and temperature
fruit juice beverage and study the suitability of mixture of of 30ºC was used for this analysis. Lactose content was
whey and pomegranate juice as a media for the growth of reported using external standards (Mousavi et al. 2011).
lactic acid bacteria and yeasts, sugar consumption and organic Lactobacilli counts (Log CFU/ml) were performed on
acids formation during kefir grains fermentation. Pomegranate MRS medium at an incubation temperature of 30ºC for 2 days.
juice was used in this study because of its antioxidant activi- PDA medium was used for counting yeast at incubation
ties and nutrient factor. Also it may make better the sensory temperature of 25ºC for 3 days and expressed as log CFU/
quality of beverage and enhances the acceptability of it. ml (Liu and Lin 2000).

Sensory evaluation
Materials and methods
Each sample was tested for smell, flavor, consistency, color
Kefir grains were collected from a household in Tehran, Iran. and overall acceptability by 10 panelists. Each sensory char-
The grains were kept in pasteurized milk at room temperature. acteristic was scored on an increasing scale from 1 (bad) to 5
Milk was exchanged every 2 days to maintain the grains (great) (Watts et al. 1989).
viability. The commercial concentrated pomegranate juice
and whey used in this study were supplied from Alifard Statistical analysis
(Sunich, Iran, Saveh) and Safadasht (Iran, Karaj) cheese mak-
ing company, respectively. All experiments were carried out in triplicate and each sample
was analyzed in duplicate. The results are expressed as mean ±
Preparation of fermented beverage SD. Duncan's multiple range tests were used to compare the
difference among mean values of beverage's properties at the
Whey was diluted with distilled water in a portion of 1:1 and level of 0.05 and SAS software (version 9.1; statistical anal-
then mixed with pomegranate juice concentrate to 14° brix. ysis system institute Inc., Cary, NC, USA) was used for
Mixture was pasteurized at 60ºC for 30 min. Kefir grains were analysis.
removed from milk, washed with distilled water and then
inoculated into the prepared beverage at two levels (5 % and
8 % w/v). The beverage was incubated at two temperatures
(19 and 25ºC) for 32 hours. The fermentation runs were Results and discussion
assessed through periodic sampling in order to determine
characteristics of beverages. After completion of fermentation Some of the initial characterizations of whey and pomegranate
the kefir grains were removed, washed with distilled water and juice concentrate are given in Table 1a and b, respectively.
returned to milk and beverage was kept at suitable temperature
for future analysis. pH and acidity

Chemical and microbial analysis Changes in pH value during cultivation of different levels of
kefir grains in beverage are depicted in Fig. 1. As can be
The pH of the samples was measured by a digital pH meter observed pH value decreased from an initial value of 4.23±
(Metrohm 744, Netherland). Total titratable acidity was 0.03 to lower value depended on type of fermentation. Hence
J Food Sci Technol (June 2015) 52(6):3711–3718 3713

Table 1 a: some of initial characteristics of pomegranate juice concentrate, b: some of initial characteristics of whey

a
pH Acidity (%w/w) Brix Turbidity Density (g/ml) Vitamin C Reducing sugar Sucrose Citric acid Acetic acid
(mg/100 ml) (g/100 ml) (g/100 ml) (g/l) (g/l)
3.84±0.02 2.1±0.03 64 0.84±0.011 1.33±0.21 129.23±3.1 41.32±1.5 0.75±0.04 20±1.1 0.15±0.01

b
pH Density (g/ml) Ash (%w/w) Dry matter (%w/w) Lactose (g/100 m) Protein (%w/w) Fat (%w/w)
4.9±.03 1.01±0.17 0.47±0.015 5.9±0.034 5.01±0.06 0.83±0.41 0.55±0.03

Data are means ± standard deviation

total acidity of beverages increased. The highest decrease in could affect more on pH value change as compared to 19ºC
pH value was observed during the second and third 8 hours of (Fig. 1). This might be due to the optimum temperature for
fermentation. pH value in the examined beverages was the enzymes or metabolic activities of microorganisms in kefir
lowest in beverage fermented with 8 % w/v kefir grains in grains. In our previous experiment (data have not been
temperature of 25ºC, 3.40±0.02. Higher pH value was ob- published) three levels of fermentation temperature were
tained for fermented beverages in temperature of 19ºC and used for preparation of a novel fermented beverage by kefir
25ºC with 8 %w/v and 5 % w/v kefir grains, respectively grains (20ºC, 25ºC and 30ºC). We found that temperature
(3.48±0.022 and 3.50±0.017, respectively) and the highest in of 25ºC was more suitable than others. Also temperature of
beverage fermented with 5 % w/v kefir grains in 19ºC (3.61± 20ºC was better than 30ºC for metabolic activity of kefir
0.02). Bensmira and Jiang (2011) reported a drop in pH value grains.
of peanut-milk kefir during fermentation. They attributed this
drop to a decrease in lactose amount and a consequent Lactose consumption during the fermentation process
increase in lactic acid content. On the other hand Magalhães
et al. (2010) reported production of lactic acid during fermen- Kefir microorganisms metabolized lactose as a carbon source,
tation of kefir is of great importance due to its inhibitory effect but in different beverages amount of lactose consumption
on both spoilage and pathogenic microorganisms. So it can be were different as shown in Fig. 2. It can be observed that most
said reduction in pH value moreover leads to increase in total of lactose present in beverage was metabolized within
acidity, causes an inhibition of spoilage and pathogenic mi- 32 hours of fermentation. On the other hand, among all
croorganism growths, too. It was shown temperature of 25ºC beverages the highest consumption of lactose occurred in

Fig. 1 Changes in pH and acidity

of different beverages during
fermentation process
Fermentation temperature of
19ºC: open symbol, Fermentation
temperature of 25ºC: closed
symbol, kefir inoculation of
5 % w/v: circle, kefir inoculation
of 8 % w/v: square. Filled lines:
pH value and Dashed lines:
titratable acidity. Bars represent
the standard deviation
3714 J Food Sci Technol (June 2015) 52(6):3711–3718

Fig. 2 Kinetic of lactose

consumption during fermentation
process Fermentation temperature
of 19ºC: open symbol,
Fermentation temperature of
25ºC: closed symbol, kefir
inoculation of 5 % w/v: circle,
kefir inoculation of 8 % w/v:
square. Bars represent the
standard deviation

fermented beverage with 8 %w/v kefir grains in temperature time evolution of organic acids (Lactic acid, citric acid and
of 25ºC which decreased from the initial amount of 2.4± acetic acid) during the fermentation process. As depicted in
0.06 g/100 ml to 0.5±0.042 g/100 ml with 79.17 % reduction. Fig. 3, lactic acid is recognized as the main metabolic produce
It was shown earlier this sample had the lowest pH and highest by kefir microorganisms during fermentation in all samples.
acidity, too. The lower lactose metabolize was observed for Lactic acid which is a common end product of bacterial
fermented beverages with 8 % w/v kefir grains in temperature fermentation increased significantly (P<0.05) from initial
of 19ºC and 5 %w/v kefir grains in 25ºC, while the lowest was value of 0.9±0.1 g/l to 5.9±0.15 g/l, 5.3±0.11 g/l, 5.2±
found in beverage fermented with 5 %w/v kefir grains in 0.13 g/l and 4.6±0.1 g/l in beverage fermented with 8 % w/
temperature of 19ºC with decrease from 2.4±0.06 g/100 ml v kefir grains in temperature of 25ºC, with 5 % w/v kefir
to 1.1±0.068 g/100 ml (reduction of 54.17 %). It was earlier grains in 25ºC, with 8 % w/v kefir grains in 19ºC and in
reported by Magalhães et al. (2011) that the use of whey as temperature of 19ºC with 5 % w/v kefir grains, respectively.
substrate for the production of whey-based beverage resulted This is according to Güzel-Seydim et al. (2000) who reported
in lower lactose metabolize than that was found during milk kefir has lower lactic acid content as compared to yogurt (8.8-
fermentation with kefir grains. They suggested it could be 14.6 g/l) and it is probably due to the preferential use of the
probably due to the characteristics of milk which is richer in heterofermentative pathway rather than homoferementative
nutrients than whey. In present study, it was observed that pathway. Production of lactic acid during fermentation has
fermentation temperature and level of kefir grains might have been attributed to lactic acid bacteria metabolisms and is
affected the lactose metabolized in beverage during important due to inhibitory effect on both spoilage and path-
fermentation process, while Magalhães et al. (2011) have ogenic microorganisms in kefir (Magalhães et al. 2010). In
reported that type of substrate is another factor which could case of citric acid significant decrease (P<0.05) was observed
have affected on this parameter. after 32 hours of fermentation (Fig. 3). This decrease in
beverage fermented with 8 % w/v kefir grains in temperature
Organic acid changes during fermentation of 25ºC was significantly (P<0.05) more than others with a
reduction from initial amount of 6.1±0.2 g/l to 0.9±0.1 g/l.
Volatile compounds are important in flavor of beverage as According to Güzel-Seydim et al. (2000) citric acid is the
they determine different desirable sensory characteristics preferred substrate for formation of diacetyl and acetoin by
(Arrizon et al. 2006). Formation of organic acids in dairy some lactic acid bacteria.
products may occur as a result of hydrolysis of butterfat (fatty The initial mean value of acetic acid was 0.1±0.014 g/l
acids), biochemical metabolic processes and bacterial metab- which increased during 32 hours of fermentation process. The
olisms (Güzel-Seydim et al. 2000). Figures 3 and 4 show the highest final concentration for acetic acid was observed in
J Food Sci Technol (June 2015) 52(6):3711–3718 3715

Fig. 3 Kinetics of changes in

lactic acid and citric acid during
fermentation process
Fermentation temperature of
19ºC: open symbol, Fermentation
temperature of 25ºC: closed
symbol, kefir inoculation of
5 % w/v: circle, kefir inoculation
of 8 % w/v: square. Filled lines:
citric acid and Dashed lines: lactic
acid. Bars represent the standard
deviation

beverage fermented with 8 % w/v kefir grains in temperature of present in beverage before fermentation and increased from
25 ºC, 0.7 ± 0.016 g/l. Bensmira and Jiang (2011) and the first hour of fermentation. This difference may be due to
Magalhães et al. (2011) reported that during first 18 and the presence of pomegranate juice in formulation. The increase
24 hours of peanut milk kefir fermentation and whey or milk of acetic acid could be attributed to the heterofermentative
kefir fermentation, respectively mean concentration of acetic acetic acid and lactic acid cultures present in kefir grains mi-
acid was practically zero, while we found acetic acid was croflora (Magalhães et al. 2010). According to the results

Fig. 4 Kinetic of changes in

acetic acid during fermentation
process Fermentation temperature
of 19ºC: open symbol,
Fermentation temperature of
25ºC: closed symbol, kefir
inoculation of 5 % w/v: circle,
kefir inoculation of 8 % w/v:
square. Bars represent the
standard deviation
3716 J Food Sci Technol (June 2015) 52(6):3711–3718

Fig. 5 Bacterial growth kinetic

during 32 hours fermentation of
beverages Fermentation
temperature of 19ºC: open
symbol, Fermentation
temperature of 25ºC: closed
symbol, kefir inoculation of
5 % w/v: circle, kefir inoculation
of 8 % w/v: square. Bars represent
the standard deviation

obtained, it was revealed that temperature of 25ºC was more Microbial analysis
suitable for metabolic activity of kefir grains microflora as
compared to 19ºC, since changes in organic acid as well as Figure 5 shows the changes in lactobacilli population during
pH, acidity and lactose amount were higher at this temperature. 32 hours of fermentation. Immediately after the addition of
On the other hand with increase in kefir grains inoculation from 8 % w/v kefir grains the population of lactobacilli bacteria
5 % w/v to 8 % w/v these changes were occurred more. were 5±0.1 log CFU/ml, while in the case of 5 % w/v kefir

Fig. 6 Yeast growth kinetic

during 32 hours fermentation of
beverages Fermentation
temperature of 19ºC: open
symbol, Fermentation
temperature of 25ºC: closed
symbol, kefir inoculation of
5 % w/v: circle, kefir inoculation
of 8 % w/v: square. Bars represent
the standard deviation
J Food Sci Technol (June 2015) 52(6):3711–3718 3717

Table 2 Final characteristics of different beverages

Samples pH Acidity (%) Lactose (g/100 ml) Lactic acid (g/l)

Fermentation Temperature (ºC) Kefir grains inoculation (%w/v)

19 5 3.61±0.02a 0.95±0.036c 1.1±0.068a 4.6±0.1c
b b b
19 8 3.48±0.022 1.21±0.028 0.8±0.08 5.2±0.13b
b b b
25 5 3.50±0.017 1.19±0.03 0.86±0.079 5.3±0.11b
c a c
25 8 3.40±0.02 1.44±0.035 0.5±0.042 5.9±0.15a
Citric acid (g/l) Acetic acid (g/l) Lactobacilli bacteria (log CFU/ml) Yeast (log CFU/ml)
19 5 2.5±0.13a 0.5±0.018c 7±0.13d 5.25±0.09a
19 8 1.7±0.14b 0.61±0.02b 7.3±0.13c 5.3±0.08a
25 5 1.9±0.17b 0.62±0.019b 7.6±0.2b 5.28±0.1a
25 8 0.9±0.1c 0.7±0.016a 8.2±0.15a 5.4±0.13a

Means within the same column with different letters are significantly (P<0.05) different. Data are means ± standard deviation

grains it was 4.7±0.12 log CFU/ml. This shows part of kefir Results obtained for lactobacilli bacteria and yeasts were in
grains microflora was transferred to beverage after inocula- the range reported by other researchers (Liu and Lin 2000;
tion. These results are according to Liu and Lin (2000) who Irigoyen et al. 2005; Fontán et al. 2006).
reported after addition of kefir grains to soymilk, bacteria were At table 2 the final characteristic including pH, acidity,
transferred to beverage. After 32 hours of incubation at 25ºC lactose amount, organic acids, lactobacilli and yeast popula-
the lactobacilli counts increased by 3.2 log cycles from 5±0.1 tion of different beverages, are given.
log CFU/ml to 8.2±0.15 log CFU/ml in beverages fermented
with 8 % w/v kefir grains, but by only 2.3 log cycles from 5± Sensory evaluation
0.1 log CFU/ml to 7.3 ± 0.13 log CFU/ml in beverage
fermented with 8 % w/v kefir grains but in temperature of Results of smell, flavor, consistency, color and overall
19ºC. This indicates a temperature of 25ºC is more suitable acceptance evaluation are shown in table 3. As can be
than 19ºC for lactobacilli bacteria growth. Similar result was observed smell and flavor of fermented beverage by
obtained for when 5 % w/v kefir grains was used for 5%w/v kefir grains in temperature of 25ºC have gotten
fermentation. the highest degree as compared to others (scores of 3.92±
The initial counts of yeasts in beverages fermented with 0.27 and 4.02±0.24, respectively). Between consistency of
8 % w/v and 5 % w/v kefir grains were 2.8±0.1 log CFU/ml this sample and beverage prepared with 5%w/v kefir grains
and 2.53±0.08 log CFU/ml, respectively (Fig. 6). After in temperature of 19ºC, no significant difference (P<0.05)
32 hours of fermentation, no significant difference (P<0.05) was observed. Also there weren’t any significant difference
was observed in yeast counts in different beverages with a (P<0.05) between various beverage in parameter of color.
final concentration of 5.4±0.13 log CFU/ml, 5.3±0.08 log The overall acceptability of beverage fermented by 5%w/v
CFU/ml, 5.28±0.1 log CFU/ml and 5.25±0.09 log CFU/ml kefir grains in temperature of 25ºC was the highest with
for fermented beverages with 8 % w/v kefir grains in temper- score of 4.32±0.32 and it wasn’t significantly different (P<
ature of 25ºC, with 8 % w/v kefir grains in 19ºC, with 5 % w/v 0.05) with overall acceptability of beverage by the same
kefir grains in 25ºC and 5 % w/v kefir grains in 19ºC, level of kefir gains but was fermented at temperature of
respectively. 19ºC.

Table 3 Sensory characteristics of beverages

Samples Smell Flavor Consistency Color Overall acceptability

Fermentation temperature (ºC) Kefir grains inoculation (%w/v)

19 5 3.3±0.17b 3.31±0.2b 4.02±0.24ab 4.12±0.18a 4.01±0.26ab
19 8 2.9±0.2c 3.14±0.11b 3.5±0.19c 4.2±0.18a 3.4±0.16c
25 5 3.92±0.27a 4.02±0.24a 4.41±0.3a 4.43±0.22a 4.32±0.3a
25 8 3.03±0.12bc 3.04±0.22b 3.61±0.22bc 4.1±0.2a 3.6±0.14bc
3718 J Food Sci Technol (June 2015) 52(6):3711–3718

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department of University of Tehran throughout the research project. the production of novel whey-based kefir beverages and traditional
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