World Applied Sciences Journal 19 (10): 1367-1380, 2012

ISSN 1818-4952
IDOSI Publications, 2012
DOI: 10.5829/idosi.wasj.2012.19.10.2797

Production of Cereal-Based Probiotic Beverages

1

Amal A. Hassan, 2Mona M.A. Aly and 2Soher T. El-Hadidie

Deptartment of Food Science, Faculty of Agriculture,

Ain Shams Univesity, Shoubra El-Kema, Egypt
2
Food Technology Research Institute, Agricultural Research Center, Giza, Egypt
1

Abstract: In the present study, rice and millet grains were fermented with ABT-2 starter culture (S. thermophilus
L. acidophilus and Bifidobacterium BB-12) to obtain probiotic beverages, fortified with pumpkin and sesame
seed milk in order to combine the high nutritive value of seeds and the health benefits of the probiotic culture.
The levels of several factors, i.e. starter culture concentration, rice and millet level and the amount of honey,
affecting the fermentation process, were established for completing a controlled fermentation for 16 hours.
The viable cell counts reached at the end of fermentation time about 4.3 x 10 9 cfu/ml. Changes were observed
in acidity and counts of probiotic bacteria as a result of fortification with pumpkin and sesame milk at 10%.
Sensory properties were also evaluated. Fermentation with ABT-2 starter culture improved the color, flavor,
texture and overall acceptability of the beverages. Slight changes in the count of probiotic, pH and acidity
during storage of the beverages at 4C were observed. The shelf-life of the rice and millet fermented beverages
was estimated to be 15 days under refrigerated storage.
Key words: Rice Millet ABT-2 starter culture
properties Shelf-life

Pumpkin seed

INTRODUCTION
Most probiotic foods at the markets worldwide are
milk based and very few attempts are made for
development of probiotic foods using other fermentation
substrates such as cereals. Their large distribution and
important nutritive value have focused the attention on
their use as raw materials for the development of new
fermented functional foods [1]. The interest in the
development of non-dairy probiotic products is increasing
due to consumer interest for exotic and different tastes,
emerging number of vegan consumers and also because
some consumers are lactose intolerant or have milk
protein allergies [2]. Cereal products often ferment
spontaneously, resulting in improved shelf-life and better
nutritional properties compared with the raw material.
Single or mixed cereals are used as a substrate in the
production of fermented foods and the final product can
vary according to the microbial population and
fermentation conditions. Fermentation procedures have
been used to develop new foods with enhanced health
properties [3].
Corresponding Author:

Sesame

Probiotic

Acidity

Sensory

Rice (Oryza sativa L.) competes closely with wheat

as the world most important crop. It is grown in more than
100 countries including Egypt, when compared with other
cereals; it has the highest food yield. Regarding to wheat
and maize, rice supplies more than half of all the calories
for human consumption. Rice is consumed as milled rice
after dehulling process and whitening (removal of
pericarp, bran layer and embryo from brown rice). After
whitening parts of rice were removed which has very
low starch content but has high percentages of oil,
protein, vitamins and minerals, however, the remained part
(the endosperm of the milled rice) mainly composed of
carbohydrates and proteins [4]. Millet (Pennisetum
typoides) is one of the oldest foods known to human and
possibly the first cereal grain to be used for making bread.
Today millet ranks as the sixth most important grain in the
world. Millet is equal or superior to grain of wheat, corn,
sorghum and rice in protein and oil content, it contains
similar amount of calcium (Ca) and phosphorus (P),
more iron (Fe) than cereals. In Egypt, millet is mainly used
in feeding rations as bird seeds and relatively small
amount of total crop is utilized directly in foods [5].

Mona M.A. Aly, Food Technology Research Institute, Agricultural Research Center, Giza, Egypt.

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Pumpkin (Cucurbita pepo) seeds were found to be rich in

oil (about 50%), protein (38%) and alpha-tocopherols
(3g/100g). The seed oil contains an appreciable amount of
unsaturated fatty acids (78%) and found to be a rich of
linoleic (47%) [6]. Sesame (Sesamum indicum L.) is one of
the most important oil seed crops worldwide and has
been cultivated since ancient Egyptian and used as
traditional health food. Its a good source of protein,
minerals and oil. Sesame seeds are used in the making of
Tahin (sesame butter) and halawa, for the preparations of
rolls, crackers, cakes and pastry products. They are used
for their nutritive, preventive and curative properties.
Sesame oil seeds are sources of some phytonutrients
such as minerals, vitamins, omega-6 fatty acids, flavonoid,
phenolic anti-oxidant and dietary fibers with potent
anti-cancer as well as health promoting properties [7].
Probiotic microorganisms have been studied as
successful ingredients in the functional food segment.
The popularity of probiotic foods is due to the numerous
health benefits attributes to its intake [8]. A number of
health benefits of probiotic products have been proposed
including: antimicrobial, antimutagenic, anticarcinogenic,
antihypertensive properties, reduction in serum
cholesterol, alleviation of lactose intolerance, reduction of
allergic symptoms, reduction of diarrhea and stimulation
of the immune system [9, 10, 11, 12].
To achieve the benefits from their ingestion, the
probiotic microorganisms must reach the intestine alive
and in sufficient concentration, surviving to harsh
conditions found during the flow through the
gastrointestinal tract [13]. A minimum daily intake of
8.9 log of colony forming unit (CFU) or 6-7 log cfu/g or
1ml of food is generally recommended [14]. The survival
of probiotic microorganisms in food products is strongly
influenced by pH and post-acidification, which may occur
during refrigerated storage of fermented products.
Other factors such as production of hydrogen peroxide,
oxygen level, temperature and food matrix also affect the
microbial viability [15].
The aim of this study was to develop rice and millet
probiotic beverage products using commercial probiotic
starter culture (ABT-2). As well as to study the effect of
pumpkin and sesame seeds addition on some
physicochemical, microbiological and sensory properties
during refrigerated storage.

Research Center, Ministry of Agriculture Giza, Egypt.

Sesame and pumpkin seeds were purchased from local
market. A commercial probiotic starter culture (ABT-2)
was used (Peyma - Chr. Hansen, Badr city, Cairo)
containing S. thermophilus ST-20Y, L. acidophilus LA-5
and Bifidoacterium BB-12.
Preparation of Cereal-based Probiotic beverages: was
Carried out on three stages:
Stage 1: Preparation of Rice and Millet Milk as Follows:

Soaking
(12 hr)

Soaking
(2hr)

Draining
Draining
Wet milling
Cooking
(1:3)/30 min

Addition of water (1:1)

Filtration

Blending with water

Settling and sedimenting (over night)

Rice milk
Removing the top layer
Adding water to make millet milk

Fig. 1: Preparation of rice and millet milk

Stage 2: Preparation of Pumpkin and Sesame Seed Milk
as Follows:
Pumpkin seed

Sesame seeds

Dehulling

Soaking (8 hr)

Wet milling (1:1)

Sieving
Pumpkin seed milk

Heat treatment (90C/5min)

Cooling

MATERIALS AND METHODS

Dried millet (Shandewel 1), brown rice (Giza 181)
were obtained from Crops Research Institute, Agricultural

Millet

Rice

Draining
Wet milling (1:1)

Sieving

Sesame seed milk

Heat treatment (90C/5min)

Cooling

Fig. 2: Preparation of pumpkin and sesame seeds milk

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Stage 3: Preparation of Cereal Based Probiotic

Beverage as Follows:

Microbiological Analysis: Viable counts were determined

in fermented beverages (1 ml), after 3, 5, 7, 9, 12 and 15
days of storage. Differential media used for enumeration
of S. thermophilus, L. acidophilus and Bifidobacterium
BB-12 where those previously described by Martin-Diana
et al. [19].

Rice or Millet milk

Heat treatment (90 C/20 min)

Cooling (37 C)

Sensory Properties: Ten tasters trained are usually used

in the Food Technology Research Institute assessed the
produced eight beverage samples. The eight beverage
samples were scored for color, taste, texture, flavor and
overall acceptability. Score were based on a hedonic
scale of 1 to 9 where: 1 = dislike very much (very bad) and
9 = like very much (excellent) [20].

5% honey

Inculcation with 5% ABT-2 starter culture

Incubation (37C/16hr)

Plain beverage

(10% Pumpkin seed ) milk)

(10% sesame seed milk)

Estimation of Shelf-Life: The self life of the fermented rice

and millet beverages was defined as the period of
refrigerated storage (4C) during which pH remained
above 4.0 and the number of viable cell counts was
above 106 cfu/ml. Refrigerated storage was carried out for
15 days with periodical observation of pH, total acidity
and viability of starter culture.

Refrigerated storage (4 C/15 days)

Fig. 3: Preparation of probiotic cereal - based beverage

Formula of beverage:

Amount of serving
(100 ml)
Fermented rice or millet beverage: 85
Sesame or pumpkin milk:
10
Honey or sugar:
5
*Vanilla or fruit flavor
----------100
Chemical Analysis: Total solid were determined by
drying the sample at 1022C to constant weight. Protein,
ash, fat content was determined according to A.O.A.C
[16]. Nitrogen free extract (total carbohydrates) was
calculated by difference. Na, Zn, Fe, K and Ca were
determined using the flame photometer (Galienkamp,
EGA 330, England) and Perkin Elmer atomic absorption
spectrophotometer (model 80, England) as described in
A.O.A.C [16]. Total phosphorus was determined by
spectrophotometer at 650 nm according to the method
described in A.O.A.C [16]. Total phenols were determined
as described by Singleton and Rossi [17]. Gallic acid was
chosen as a standard to prepare the standard curve.
Phenols were expressed as mg/100g sample.
Physical Analysis
Measurement of pH, Acidity and Viscosity During
Fermentation: The pH of the fermented samples was
monitored during fermentation by measures of pH-meter.
The viscosity was measured by a dynamic viscometer
(Brookfield Model RVDI, USA). Titratable acidity was
determined as lactic acid percentage by titrating with
0.1 NaOH, using phenolphthalein as an indicator [18].

Statistical Analysis: The experimental data were analyzed

using analysis of variance (ANOVA) followed by
Duncans multiple range tests (P < 0.05) to determine a
significant difference among samples. The data were
analyzed according to users guide of statistical analysis
system [21].
RESULTS AND DISCUSSION
Chemical Composition of Raw Materials: Moisture,
crude
fat, crude protein, ash, crude fiber and
carbohydrate of brown rice, millet, pumpkin seeds and
sesame seeds flour were determined. The results are
presented in Table 1 . Moisture, crude fat, crude
protein, ash, fiber and carbohydrate of brown rice were
12.23, 1.60, 8.38, 1.38, 1.14 and 87.17%, respectively.
These results are in agreement with the values reported
by Asad [4, 22].
Results of the proximate composition of millet flour
are presented in same (Table 1). Millet flour contained
8.40, 12.0, 7.20, 1.95, 3.0 and 71.46% moisture, protein,
fat, ash, fiber and carbohydrate respectively. The revealed
results are in agreement with Ali et al. [23], Belton and
Taylor [24]. Data presented in Table 1 indicated also
that dehulled pumpkin seeds contained 8.05 moisture,
33.44 crude protein, 46.50 crude fat, 3.02 ash, 3.12 crude
fiber and 13.42% total carbohydrates, respectively.

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Table 1: Chemical composition (% dry basis) of raw materials
Parameter

Brown rice

Millet

Pumpkin seed

Sesame seed

L.S.D

Moisture

12.2 3a

8.40b

8.05c

6.55d

0.9525

Protein

8.38d

12.00c

33.44a

29.76b

0.8639

Fat

1.60d

7.20c

46.50b

51.17a

0.6917

Ash

1.38d

1.95c

3.02b

4.19a

0.0490

Crude fiber

1.14c

3.00b

3.12b

4.07a

0.3626

Total carbohydrate

87.17a

71.46b

13.42 c

10.41d

0.8273

Data are averages from three independent trials

Data in the same row with different small letters are significantly different (p<0.05)
Table 2: Physical properties of different beverage samples
Sample

pH

(1)

7.83a

Titratable acidity (%)

0.08e

Dry matter %
9.92g

(2)

4.55

0.54

(3)

5.29

0.31

(4)

5.30

0.32

(5)

7.11

0.16

9.18

(6)

4.34

0.59

11.06

(7)

5.26d

(8)
L.S.D

Viscosity CP
601b

10.41

772a

12.24

240g

14.17

119e
500c

h
e

600b

0.45b

13.44c

390f

5.20d

0.41b

14.20a

440d

0.03

0.03

0.02

3.78

Data are averages from three independent trials

Data in the same column with different small letters are significantly different (p<0.05)
1= unfermented rice beverage 2= fermented plain rice beverage 3= fermented rice beverage + 10% pumpkin seed milk 4= fermented rice beverage + 10% sesame
seed milk 5= unfermented millet beverage 6= fermented plain millet beverage 7= fermented millet beverage + 10% pumpkin seed milk 8= fermented millet
beverage + 10% sesame seed milk

These results are in agreement with those obtained by

Alfawaz [25]. Data in Table 1 also indicated that the
proximate composition of sesame seeds which contained
6.55, 29.76, 51.17, 4.19, 4.07 and 10.41% moisture, protein,
fat, ash, crude fiber and carbohydrates, respectively. From
the above mentioned data indicated that cereal grains
have high carbohydrate, meanwhile pumpkin and sesame
seeds have high fat, protein and ash content.
Physical Properties: The physical properties of the
beverage samples studied in this work are shown in
Table 2. These quality parameters (pH, titratable acidity,
dry matter and viscosity) are related to stability of
bioactive components in plant derived products.
Adding probiotic starter culture caused decreasing in pH
value of the beverage relative to control, at the same time
titratable acidity increased. Fortification with pumpkin
and sesame seed milk increased the beverage pH value.
This implies that pumpkin and sesame seed milks are less
acidic. Thus an addition of pumpkin and sesame seed milk
will increase pH value of the beverages. This shows that
these probiotic beverage blends will acceptable or
suitable for people with ulcers and other related problems
than probiotic beverage alone since it is less acidic.

Regarding to viscosity, addition of probiotic bacteria

caused increasing in viscosity of the produced
beverages; meanwhile, adding pumpkin and sesame milk
caused decrement in viscosity. Sesame seed milk fortified
beverages had the lowest viscosity. The viscosity
behavior was related to the pH, yielding maximum values
at the lowest pH. So, the increase in viscosity takes place
at acidic pH values [26]. In the same Table, dry matter
increased in the produced beverages as probiotic starter
added and the fortification with sesame and pumpkin seed
milk. Dry matter ranged between 9.92 and 14.17 % for
sample 1 and 4, respectively.
Chemical Composition of Different Beverages: Chemical
composition of different produced beverages is presented
in Table 3. Percentage of protein content was 1.27 and
1.35% for control rice and millet beverages (sample 1 and
5), respectively. Protein content was observed to increase
about two times from that for control as a result of
fortification with pumpkin and sesame seed milk. Protein
content were 3.11 and 3.63% for fermented rice beverage
+ 10% pumpkin seed milk and fermented rice beverage +
10% sesame seeds milk, respectively. Fat content was
significantly (p<0.05) increased as pumpkin and sesame

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Table 3: Chemical composition (% dry basis) of different beverages
Sample
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
LSD 0.05

Energy Kcal.

Protein

Ash

Fat

36.49
44.40
70.50
72.07
38.39
46.38
64.04
72.81
--

1.270f
2.752d
3.119c
3.635a
1.350f
2.073e
3.453b
3.510b
0.163

0.089c
0.257d
0.416b
0.312c
0.095e
0.234d
0.475a
0.389b
0.040

0.187f
0.440e
2.700b
3.070a
0.476e
0.918d
2.365c
2.750b
0.058

Total carbohydrate

Total phenols mg/100g

9.16a
6.96ef
6.91ef
6.77f
8.09b
7.83bc
7.25de
7.55cd
0.38

23.61b
36.13a
35.19a
25.50c
30.98b
37.83a
35.72a
31.40b
2.68

Data are averages from three independent trials

Data in the same column with different small letters are significantly different (p<0.05)
1= unfermented rice beverage 2= fermented plain rice beverage 3= fermented rice beverage + 10% pumpkin seed milk 4= fermented rice beverage + 10% sesame
seed milk 5= unfermented millet beverage 6= fermented plain millet beverage 7= fermented millet beverage + 10% pumpkin seed milk 8= fermented millet
beverage + 10% sesame seed milk

seed milk fortification. Fat content of the control rice and

millet beverages were 0.18 and 0.47%, respectively.
Fermented rice beverage + 10% sesame and pumpkin seed
milk were 2.70 and 3.07%, respectively. Similar findings
were observed for fermented millet beverages. The
increment in fat content of produced beverages attributed
to high fat content of oily pumpkin and sesame seeds.
Ash content of produced fermented probiotic
beverages increased about two times relative to controls.
Fermented rice and millet beverages + 10% sesame milk
had ash content amounted in 0.31 and 0.38%,
respectively. Ash content increased significantly may be
attributed to fermentation process and fortification with
pumpkin and sesame seed milk. Hughes and Hoover [27]
reported that bifidobacteria produce nutrients and
improve lactose tolerance, calcium absorbability and
digestibility. Total carbohydrate content of fermented rice
and millet beverages decreased relative to control
beverages. Carbohydrate content ranged from 6.77 to
9.16%, respectively. These differences could be generally
attributed to fermentation process, since lactic acid
bacteria form exoplyaccharides which have been of
interest for their technological potential as thickening
agent or as a source of ropines and for their potential as
dietary fiber [28]. On the other hand, the energy values of
produced beverages were found between 36.49 and
72.81 kcal/100ml. caloric values of different beverages
were increased by pumpkin and sesame seed fortification.
The caloric value was increased to reach 70.50 and
72.07%, for fermented rice beverages + 10% pumpkin milk
and sesame milk, respectively. Meanwhile, caloric values
were increased to reach 64.04 and 72.81% for fermented
millet beverages + 10% pumpkin milk and sesame seed
milk, respectively. The caloric value of commercial
available probiotic products, which are dairy based
products, falls in the range from 60 to 120 kcal/g,

depending on the kind of milk used in its preparation

(defatted, low fat or whole milk). According to the result,
the fermentation of rice or millet resulted in a non-dairy
probiotic beverage with lower caloric value compared to
commercial available fermented dairy products. As
noticed in the same Table, total phenols content of the
produced probiotics beverages were higher than that of
control (without fermentation). Total phenols increased
to reach 36.13 and 37.83 mg/100g for plain probiotic rice
and millet beverages, respectively. These results
supported by Tseng et al. [29] who reported that total
phenolic content of fermented rice beverage was higher
than that using rice solely. They also mentioned that total
phenolics were the major naturally occurring antioxidant
component.
Minerals Content: Table 4 shows minerals i.e. sodium
(Na), potassium (K), phosphorus (P), calcium (Ca), iron
(Fe) and zinc (Zn) content of different probiotic
beverages. Results indicated that minerals content
increased as a result of pumpkin and sesame seed milk
added. Samples 4 and 8 contained the highest value of Na
(190.6 mg/L, 114.0 mg/L) followed by sample (6) 76.1 mg/L.
The lowest value was 30.5 mg/L for sample (5) followed by
32.0 mg/L for sample (1). Potassium content ranged 114.7
to 284.1 mg/L. The highest value for sample (8). Notable
increase in Ca content of produced beverages as a result
of sesame milk added, this means sesame seeds are rich
source of calcium. The highest value 457 mg/L for sample
(4) followed by 270 for sample (8), increasing percentage
for that samples were 124.08 and 174%, respectively.
Regarding to iron content data showed that iron content
of sample (8) and (4) were 18.9 and 18.2 mg/L, followed by
(3) and (7) which were 17.5 and 16.1 mg/L, respectively.
Meanwhile, the obtained values of iron content in (2) and
(6) were 15.4 and 14.3 mg/L, respectively.

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Table 4: Mineral profile of different produced beverage samples (mg/L)
Sample

Na

Fe

Zn

Ca

Na/K

Ca/P

(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)

32.0
63.3
68.0
190.6
30.5
76.1
75.9
114.0

114.7
117.5
172.3
258.8
205.4
210.0
275.8
284.1

14.7
15.4
17.5
18.2
12.4
14.3
16.1
18.9

2.7
2.7
6.1
2.9
2.0
2.05
4.4
3.2

130
204
208
457
50
100
110
270

180
200
241
300
100
160
200
230

0.28
0.53
0.39
0.73
0.20
0.36
0.30
0.31

0.73
1.02
0.99
1.52
0.60
0.62
0.55
1.19

1= unfermented rice beverage 2= fermented plain rice beverage 3= fermented rice beverage + 10% pumpkin seed milk 4= fermented rice beverage + 10% sesame
seed milk 5= unfermented millet beverage 6= fermented plain millet beverage 7= fermented millet beverage + 10% pumpkin seed 8= fermented millet beverage
+ 10% sesame seed milk.
Table 5: Sensory evaluation of different beverages
Sample

Color

Taste

Flavor

Texture

Acceptance

(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
LSD 0.05

7.90b 0.66
8.61a 0.31
8.20ab 0.34
7.15c 0.34
6.20d 0.26
8.00b 0.75
7.50c 0.61
6.20d 0.88
0.50

7.55bc 0.98
8.65a 0.24
8.10ab 0.32
6.83de 0.59
6.35e 0.71
7.45ab 1.04
7.75bc 0.83
6.50e 0.48
0.52

7.75bc 0.82
8.60a 0.32
8.02b 0.68
7.25c 0.49
7.25c 0.49
8.260b 0.68
7.75bc 0.83
6.20d 0.43
0.56

7.87bc 0.80
8.70a 0.47
7.26c 0.47
8.35ab 0.41
7.45cd 0.49
8.54a 0.45
7.05d 1.27
7.70bd 0.52
0.60

8.07b 0.61
8.64a 0.51
8.25b 0.41
7.42c 0.29
6.20e 0.34
7.86d 0.47
6.9f 0.16
6.15e 0.34
0.38

Data in the same column with different small letters are significantly different (p<0.05).
1= unfermented rice beverage 2= fermented plain rice beverage 3= fermented rice beverage + 10% pumpkin seed milk 4= fermented rice beverage + 10% sesame
seed milk 5= unfermented millet beverage 6= fermented plain millet beverage 7= fermented millet beverage + 10% pumpkin seed milk 8= fermented millet
beverage + 10% sesame seed milk

Zinc contents in samples (3) and (7) increased with

pumpkin seed milk added amounted in 6.1 and 4.4 mg/L.
This means pumpkin seeds are good source of zinc.
Moderate amount of zinc was observed for samples
fortified with sesame milk. Antony and Chandra [30]
reported that mineral contents of finger millet were (Ca)
313.1, (P) 467.2, (Fe) 6.53, (Cu) 1.01, (Zn) 2.02 and (Mn)
9.86 mg/100g on dry weight. Arora et al. [31] reported that
content of pearl millet seeds were 123.67, 15.90, 2.13, 0.84,
0.50 and 0.35 mg/100g, on dry matter basis for
phosphorous, calcium, iron, zinc, copper and manganese,
respectively.
Sensory Evaluation: This study evaluated the
acceptability for the color, taste flavor and texture of
probiotic rice and millet beverages. Results of the sensory
evaluation of fermented beverages on a hedonic scale
from 1 (very bad) to 9 (excellent) are shown in Table 5.
Data revealed that there are generally significantly
different (p < 0.05), which indicates that the different
beverages have distinctively different tastes. Addition of
probiotic bacteria (commercial ABT-2 starter) at 5 % level,
have more acceptable than that of control beverage, while

the sensory properties of beverages when sesame seed

milk fortification decreased. Fortification with pumpkin
seed milk had more acceptable than sesame seed milk; this
may be because the beany taste of the later. The probiotic
juices were found to contain aromas flavor (sour)
characteristic of functional ingredients [32].
In general, the presence of pumpkin and sesame seed
milk increased the nutritional quality of beverages.
Sesame seed milk had a negative influence on the
fermented product taste and flavor but at the same time it
had positive effect on the beverage texture. These defects
can overcome by adding different flavor components
vanilla, chocolate or fruit juice or jam i.e. straw berries and
peaches. Molin [33] reported that to make the fermented
cereal gruel more acceptable to consumers in the
industrialized world, it mixed into a fruit drink.
Fermentation of Rice and Millet by the Commercial
Starter Culture ABT-2:
Changes in pH and Titratable Acidity: The chemical
quality attributes (pH and titratable acidity) of the
fermented cereal- based beverage during production
process are shown in Figs. 4 and 5. Significant differences

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Fig. 4: Changes in pH values of probiotic non - diary

beverages samples during fermentation process

Fig. 5: Changes in titratable acidity (% lactic acid) of

probiotic non-diary beverages samples during
fermentation process
were noticed between the two fermented substrates
employed for their performances during the fermentation
process. The pH decreased markedly at 4 to 8h until about
12 h and further decreased afterward. The maximum
acidification (pH) ranged from 4.34 to 4.55 according to
the fermented substrate employed. The time to reach the
desired pH (4.5) of the fermented rice beverage samples
was 1.6 times higher than with the fermented millet
beverage samples. The results demonstrated a slow
acidification profile. The time required to reach pH 4.5 for
fermented plain rice beverage samples was 16 h while it
was reduced to 10h for fermented plain millet beverage
samples. This implies that rice substrate is less acidic
than millet substrate, thus the use of millet as a substrate
for fermentation will encourages greater capability of
producing acid and decrease the pH value of fermented
millet beverage. For all fermented non-dairy beverage

samples, pH values significantly reduced (p>0.05)

during the fermentation process (16h) from 5.320.02 to
4.55 0.01 and from 5.300.02 to 4.340.01 for fermented
plain rice and millet beverage samples, respectively,
which could be due to the presence of the three bacterial
strains of ABT-2 starter culture, S. thermophilus ST-20Y,
L. acidophilus LA-5 and Bifidobacterium BB-12. When
all data were analyzed together using ANOVA (Fig. 4),
it could be observed that a significant effect of fermented
substrate employed on final pH (p>0.05) with lower values
observed in the fermented millet beverage samples and
higher values in the fermented rice beverage samples after
fermentation time (16h). The different fermentation rates
could be attributed to strain specificities and differences
of cereal media. These results are in harmony with
those obtaine by Afaneh et al. [34] and Mrtensson et al.
[35, 36] who reported that after much longer fermentation
(16h) of an oat base with commercial mixed dairy cultures,
pH reached 3.9-4.5, while Marklinder and Lonner [37]
achieved pH 4.3-4.5 for 6 h when fermenting oat meal
soups with L. plantarum strains. Angelov et al. [1]
applied oat as substrate for lactic acid fermentation with
dairy starter cultures and found that 8h was an
appropriate fermentation time to reach the desired pH
levels of 4.0-4.5. Coda et al. [38] achieved pH 3.72 to 4.03
for 8h when fermenting cereals (rice, barely, emmer, oat
or soy flour) and concentrated red grape must with
L. plantarum strains for making vegetable yogurt-like
beverages. The results showed that the titratable acidity
values of the two types of fermented beverage under
study were increased during the fermentation process
(16 h) (Fig. 5). Total acidity content ( as percent lactic
acid) significantly increased (p>0.05) from 0.32% (0h)
to 0.54% (16h) and from 0.35% (0h) to 0.59% (16h) at the
end of fermentation process for fermented plain rice and
millet beverage samples, respectively. However, fermented
plain millet beverage samples had significantly higher
(p>0.05) acidity than that of fermented plain rice beverage
samples. These results may be related to fermented
substrate employed. Generally, the production of acidity
was low at the early culture period but increased
considerably from 4 h fermentation until 16h.These results
are in agreement with those reported by Tangular and
Erten [39], Ramos et al.[40] and Colakoglu and Gursoy
[41]. Wongkhalaung et al. [42] reported that the titratable
acidity of fermented hydrolyzed rice with L. casei and
L. acidophilus was produced up to 0.6% lactic acid, while
Wongkhalaung and Boonyaratanakornkit [43] achieved
0.86 % acidity as lactic and pH 3.48 for 24 h when
fermenting rice milk with mixed cultures of L. acidophilus

1373

World Appl. Sci. J., 19 (10): 1367-1380, 2012

and L. casei subsp. rhamnosus. The increase in acidity

with decrease in pH as fermentation proceed may
eliminate or discourage the growth of most spoilage and
pathogenic microorganisms that cannot withstand
such condition hence making the probiotic non-dairy,
cereal-based beverages safer for consumption and also
help to increase the shelf- life.
Assessment of the Two Types of Probiotic Beverages
During Refrigerated Storage: In this study rice and millet
was used as the main substrates for the commercial starter
culture ABT-2 and pumpkin and sesame seed milks were
added after fermentation to investigate their effects on the
survival and acidifying activities of probiotic cultures
during refrigerated storage at 41C for 15 days.
Changes in pH and Titratable Acidity: The effects of
pumpkin and sesame seed milk addition on pH and
titratable acidity values of fermented rice and millet
beverages during refrigerated storage for up to 15 days
are shown Figs. 6-9. As expected, the storage time
significantly affected the acidity level in the three types
of rice beverages; titratable acidity increased, whilst the
pH was decreased. Differences between pH values of
fermented plain rice beverage samples at the first nine
days of storage were insignificant (p>0.05), thereafter,
the pH values significantly decreased at the twelfth
and fifteenth day of storage reaching 4.42 and 4.32,
respectively (Fig. 6). In parallel with pH values, the
titratable acidity values of fermented plain rice beverage
samples at the first two days of storage were insignificant
(p>0.05). After that, the acidity values of the beverage
samples significantly increased (p>0.05) from 0.54% to
0.57% after ninth day of storage until the fifteenth day
of storage (Fig. 8).
The addition of pumpkin and sesame seed milk to
the plain rice beverage affected the initial pH value from
(4.55 to 5.29 and 5.30) and the titratable acidity from
(0.54% to 0.31% and 0.32%), respectively. The mean pH
values of the fermented rice beverage samples containing
10% pumpkin seed milk slightly decreased at the third day
of storage but this was statistically insignificant (p>0.05).
On day six, pH values of fermented pumpkin-containing
rice beverage samples showing a 0.06 pH unit drops
compared to the beginning of storage period. The mean
pH values of the fermented pumpkin - containing rice
beverage samples significantly declined (p>0.05) at the
end of storage reaching 5.15 compared to the mean pH
values at the first day of storage (Fig. 6). In parallel with
pH values, differences between titratable acidity values of

Fig. 6: Changes in pH values of probiotic rice beverage

samples during refrigerated storage (4C)

Fig. 7: Changes in pH values of probiotic millet beverage

samples during refrigerated storage (4C)

Fig. 8: Changes in titratable acidity (% lactic acid) of

probiotic rice fermented rice beverage during
storage

1374

World Appl. Sci. J., 19 (10): 1367-1380, 2012

Fig. 9: Change in titratable acidity ( % lactic acid) of

probiotic millet beverage samples during
refrigerated storage (4C)
the rice beverage samples containing 10% pumpkin seed
milk at the first six days of storage were significant
(p>0.05) reaching 0.32%. At the end of storage the acidity
values of fermented pumpkin containing rice beverage
samples significantly increased (p>0.05) reaching 0.35%
compared to the first day of storage (Fig. 8).
There were no significant differences (p>0.05) in
pH values of fermented sesame containing rice beverage
samples throughout storage period, while differences
between titratable acidity values of fermented sesamecontaining rice beverage samples at the first six days of
storage were significant (p<0.05), thereafter the titratable
acidity values of samples remained unchanged until the
last day of storage reaching 0.34% (Fig. 8). In general,
fermented plain rice beverage samples had the lowest pH
and the highest titratable acidity values at the first and
last day of storage, respectively, compared to other types
of rice beverages (Figs. 6&8). The same trend was also
observed in fermented millet beverage samples. Figs. 7&9
show variation in pH and titratable acidity values of
fermented millet beverage during refrigerated storage for
up to 15 days. Data indicated that storage time
significantly affected the acidity level in the three types
of millet beverage; titratable acidity increased, whilst
the pH was decreased. Probiotic fermented beverage
prepared from millet only without additives had the
lowest pH values ranged between 4.34 and 4.4 and the
highest titratable acidity values ranged between 0.59%
and 0.63%, respectively, throughout storage period as
compared with the two other types of millet beverages
(Figs. 7&9). Shah [10,44] observed similar decreases in pH
values during storage of commercial yogurts containing

L. acidophilus and B. bifidium without any effect on cell

counts, while the increase in acidification in the probiotic
yogurt was mainly due to the growth of L. debrueckii
spp. bulgaricus, S. thermophilus during refrigerated
storage [45].
The pH level above 4 is generally required for a
fermented oat beverage throughout storage [46].
Results in Figs. 6&7 showed that the pH remained above
4.0 for fifteen days of refrigerated storage for fermented
three types of rice or millet beverages. These results are
in line with those reported by Colakoglu and Gursoy [41]
and Paseephol and Sherkat [47]. Guo et al. [48] reported
that the pH of the fermented buffalo milk containing
L. casei decreased from 5.02 to 4.00 after 30 days of
refrigerated storage. Akalin et al. [49] found that probiotic
microorganisms reduced pH values of different types of
yoghurts from 4.51 to 4.40, after 28 days of refrigerated
storage at 4C.
From data reported above it was observed that pH
values of all beverage samples decreased while titratable
acidity values were increased during refrigerated storage
and these differences were found to be significant
(p>0.05) statistically. This is can be explained by further
metabolic activity of starter culture during refrigerated
storage [50].
Survival of Lactic Acid Bacteria in the Probiotic Rice
Beverage During Refrigerated Storage: The probiotic
rice beverage was produced in the follow conditions:
initial pH of 5.32, fermentation temperature of 37C,
with inoculums level of 5% of commercial starter culture
(ABT-2) and 16h of fermentation. After fermentation
process, pumpkin and sesame seed milk in a ratio of 10%
(v/v) were added separately to the fermented plain rice
beverage and all the three types of probiotic rice beverage
(fermented plain rice beverage, fermented plain rice
beverage containing 10% pumpkin seed milk and
fermented plain rice containing 10% sesame seed milk)
were stored for up to 15 days at 41C. The results of the
bacterial counts after the production and during
refrigerated storage of the fermented rice beverage
samples were illustrated in Figs. 10-12. On the
production day (zero time), the addition of pumpkin
and sesame seed milk to fermented plain rice
beverage significantly increased (p<0.05) the count of
L. acidophilus from the initial level 9.66 log cfu/ml to 9.73
and 9.80 log cfu/ml, respectively, while the count of
Bifidobacterium BB-12 was significantly decreased
(p<0.05) from 9.87 log cfu/ml to 9.66 and 9.74 log cfu/ml,
respectively. However, there were no significant (p>0.05)

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World Appl. Sci. J., 19 (10): 1367-1380, 2012

Fig. 10: Viability of S. thermophilus in probiotic rice beverages with ABT-2 starter culture during refrigerated storage
at 4C

Fig. 11: Viability of L. acidophilus in probiotic rice beverages with ABT-2 starter culture during refrigerated storage at
4C

Fig. 12: Viability of Bifidobacterium BB-12 in probiotic rice beverages with ABT-2 starter culture during refrigerated
storage at 4C
1376

World Appl. Sci. J., 19 (10): 1367-1380, 2012

differences between the fermented plain rice beverage and

the rice beverage containing pumpkin or sesame seed milk
in terms of the viable cell counts of S. thermophilus.
Generally, the viability of S.thermophilus decreased
gradually about 1 log cycle and significantly (p<0.05)
in the three types of fermented rice beverages, during cold
storage (Fig. 10). During storage, the viable cell counts of
the bacteria ranged from (9.68, 9.78 and 9.82 log cfu/ml)
at the beginning of storage to (8.01, 8.04 and 8.02 log
cfu/ml) at the last day of storage for the fermented plain
rice beverage, rice beverage containing pumpkin seed
milk and rice beverage containing sesame seed milk,
respectively. The same trend was also observed in the
viable cell counts of L. acidophilus and Bifdobacterium
BB-12 (Figs. 11 & 12). However, at the last day of storage
there were no significant (p>0.05) differences between
the three types of fermented rice beverages in terms of
the viable cell counts of S. thermophilus and
Bifidobacterium BB-12, while significant (p<0.05)
differences were found between fermented plain rice
beverage and fermented rice beverage containing
pumpkin or sesame seed milk in terms of the viable cell
counts of L. acidophilus. Similar pattern was also
observed for probiotic millet beverage samples (data not
shown). Bakirci and Kavaz [51] mentioned that the total
viable counts of S. thermophilus, L. acidophilus and
Bifidobacterium spp. decreased slightly during the
refrigerated storage, but remained at sufficient levels
(>6 log cfu/g) for up to 14 days. In addition, Al-Otaibi
[52] found that the viable counts of L. acidophilus of
the commercial probiotic fermented milk products
reduced about 1-2 log cycles, during refrigerated
storage period. Speck [53] reported that 108 to 109 viable
cells of L. acidophilus should be ingested daily to
ensure that consumers receive health benefits. All the
three types of rice beverages under study fulfilled this
requirement for viability of L. acidophilus at the end
of the storage. Moreover, several studies have shown
that yogurt bacteria (S. thermophilus and L. debrueckii
spp. bulgaricus) survive well in yogurt throughout
the shelf life [49, 54]. Olson and Aryana [55] found that
the L. acidophilus counts in plain yogurt tended to
decrease from 6.84 to 4.43 log cycles during 8 weeks of
storage time. The present results were better than those
obtained by Mousavi et al. [56] for probiotic pomegranate
juice. The authors reported the reduction of 3 log cycles
for L. plantarum and L. debrueckii after 14 days of storage
at 4C. High survival rates of L. casei in non-dairy
fermented products stored refrigerated was reported by
Pereira et al. [57], who studied L. casei fermentation and
survival in cashew apple juice.

The reduction in the count of L. acidophilus during

the cold storage may be due to the production of
antimicrobials such as bacteriocins, H2O2or organic acids.
Interestingly, Al-Otaibi [52] mentioned that the
populations of bifidobacteria decreased significantly
from the production day to the end of refrigerated storage
period for three weeks at 51C in the tested eight
commercial probiotic fermented milk products. The author
also reported that only two of the tested products
maintained 106 cfu/ml viable count of bifidobacteria tills
the end of cold storage period. According to Kurman and
Rasic [58], the viable level of bifidobacteria must be above
106cfu/ml to provide therapeutic benefits. All the resultant
three types of rice beverages fulfilled this requirement
for viability of bifidobacteria till the end of cold storage
period. The viability losses or the reduction of
bifidobacterial count may be due to the decrease of pH
values, post process acid production [59], sensitivity to
oxygen [60] and metabolites such as hydrogen peroxide
and ethanol and to bacteriocins produced by lactic acid
bacteria [61]. In the present study, good cell viability was
kept throughout storage. These results are in agreement
with those obtained by Medina and Jordano [61] and
Lankaputhra et al. [62] who found poor viability of
bifidobacteria in yoghurt during storage. The coliform
and pathogenic bacteria and molds and yeasts counts
were not detected in all the three tested rice beverages on
the production day and during the refrigerated storage at
41C for 15 days.
CONCLUSION
Shelf-life study revealed that during 15 days storage
at 4C, pH and acidity of rice or millet-based beverage
remained above 4 and lower than 1%, respectively,
while numbers of S. thermophilus, L. acidophilus and
Bifidobacterium BB-12 remained above 8 log cfu ml 1.
This study shows a new possibility to make an acceptable
fermented product based mainly on rice or millet which are
suitable substrates thats can support high cell viability
during cold storage for 15 days for different probiotic
strains. In addition these produced beverages combines,
therefore interesting nutritional qualities and probiotic
characteristics. This kind of study can facilitate the
development of new, fermented, non-dairy, nutritionally
well-balanced food products with unique physical
properties.
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