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ro

Journal of Agroalimentary Processes and
Technologies 2014, 20(2), 128-132



Journal of
Agroalimentary Processes and
Technologies



___________________________________________
Corresponding author: e-mail: alina.leescu@ugal.ro

Influence of refrigerated storage on viability of
microorganisms in fermented vegetables juice

Alina G. Profir
*
, Camelia V. Neagu, Daniela Istrati, Camelia Vizireanu

Food Science, Food Engineering and Applied Biotechnology Department, Faculty of Food Science and
Engineering,Dunrea de Jos University of Galai, RO-800008, Domneasc Street, 47, Galai, Romania
Received: 12 May 2014; Accepted: 10 June 2014
.____________________________________________________________________________________
Abstract
The suitability of vegetables juice as food matrix for the production of functional beverage with probiotic
strains (Lactobacillus acidophilus, Lactobacillus casei and Saccharomyces boulardii) was investigated.
The tested microorganisms were capable of a good stability on vegetables juice with nutrient
supplementation, honey and lyophilized vegetables pulp, in different proportion. Using statistical analysis
the proper combination of factors with influence on the probiotic viability was selected for optimization
of stability of microorganisms during storage for 21 days at 4 2C. The samples were evaluated in 7
days interval for viable cell count and pH.
Cell viability of 7.048.5 log cfu/mL was achieved at the end of storage period. The pH value has
decreased from 4.6 to 4.2. The results show that the increase of stability is correlated with availability of
nutrients necessary for probiotic growth. This study indicated that probiotic microorganisms can be use
by food producers for development of functional beverage based on vegetables juice.
Keywords: Fermentation, vegetables juice, probiotic, viability
______________________________________________________________________________________

1. Introduction
The use of vegetables has been widely discussed as
an alternative to obtain products that contain
probiotic strains. These products can be use by all
type of consumers, including vegetarians and
individuals with cholesterol restricted diets, lactose
intolerance and milk protein allergies. Vegetables
have nutrients which promote the growth of
probiotic microorganisms and do not have high
amount of compounds which may limit their
consumption by some population groups [1].
Lactic acid bacteria tailored to the various intrinsic
and extrinsic environmental conditions completely
exploit the potential of vegetables juice which
enhances the hygiene, sensory, nutritional and
shelf life properties [2]. The lactic acid
fermentation of vegetable products, applied as a
preservation method for the production of finished
and half-finished products, is considered as an
important technology and it is further investigated
because of the growing amount of raw materials
processed in this way in the food industry [3].
Previous studies show that fermentation of beetroot
and carrot juices, with addition of brewers yeast
autolysate, can be carried out in order to improve
their nutritive values. Brewers yeast autolysate
contributes to the increase of the number of viable
cells of lactic-acid bacteria during the fermentation
and to better production of lactic acid [4].
Bifidobacterium strains were capable of having
biochemical activities in carrot juice without any
nutrient supplementation [5]. Fiber addition could




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markedly improve the Bifidobacterium strains
tolerance to organic acids and the stability in low
pH juice [6].
The FAO/WHO expert committee has defined
probiotics as microorganisms that exhibit
beneficial effects on hosts health when ingested in
a sufficient amount. Therefore is very important
to assure the viability of probiotics during the
entire shelf life of product. Combined skills of the
microbiologist, food technologist and clinician are
necessary to sustain effect of probiotics [7].
The ability of probiotic cells to survive and
maintain their functional characteristics during
production and storage are key issues in the
development of effective production processes [8].
The aim of our study is to increase the stability of
three probiotic strains, Lactobacillus acidophilus,
Lactobacillus casei and Saccharomyces boulardii,
in fermented vegetables juice. Amount of
necessary nutrients in the development of probiotic
microorganism can be optimized by the addition of
honey and lyophilized pulp obtained after juicing
of vegetables.
2. Materials and methods
2.1. Raw materials. The vegetables were
purchased from a local market. After the
preliminary operations the vegetables were juiced
with a centrifugal juicer provided by Philips. The
proportion of each vegetable in the blend juice was
55% beetroot, 25% carrot and 20% celery.
2.2. Starter culture and fermentation conditions.
Freeze dried Lactobacillus acidophilus LA-5 and
Lactobacillus casei 431 were obtained from Chr.
Hansen SRL (Braov, Romania) and directly used
as starter culture. Saccharomyces boulardii was
obtained from Biocodex Laboratories (Montrouge,
France). Honey and lyophilized vegetables pulp
was added in different proportion to increase the
stability of microorganisms during the refrigerated
storage. Samples were inoculated with bacteria
and yeast and incubated at 37 C. The
fermentations were carried until the value of 4.6
was reached (after ca. 8h) and stopped by quick
cooling. The samples were stored for 21 days at 4
2C and monitored.

2.3. Analytical method.
2.3.1.Viable cell enumeration. Suspensions of
fermented beverages were decimally diluted in sterile
physiologically normal saline serum. Using a
calibrated 0.11 mL micropipette, 1 mL drops of the
appropriate dilution were placed on plate. Mann
Rogosa Sharpe agar medium was used for the
selective enumeration of Lactobacillus acidophilus
LA-5 and Lactobacillus casei 431. Plates were
incubated at 37C for 48 h, in anaerobic conditions,
following which the colonies were enumerated.
Enumeration of viable cells of Saccharomyces
boulardii was performed by determining the number
of colony-forming units on Yeast Extract Glucose
Chloramphenicol Agar plate after incubation at 25C
for 72 h using pour plate technique.
Microbial analysis and enumerations were performed
in technical triplicate to adjust for intra-experimental
errors.
2.3.2. pH. The pH of the samples was monitored
using a digital pH meter (S20K Mettler Toledo Seven
Easy, Switzerland). In preparation for pH monitoring,
the sensor was calibrated in buffer solutions with two
distinct pH values, 4.0 and 7.0, chosen by the
manufacturer of the equipment.
2.4. Statistical analyses and experimental design.
The Unscrambler X (CAMO Software) was used to
generate the experimental design. The results of the
experiment were analyzed using XLSTAT 2014.
Principal component analysis is a statistical
procedure that can reduce the number of
variables. It can be use to evaluate the results of
the experiment.
3. Results and Discussion
A design with 2 factors: amount of honey and
amount of lyophilized vegetables pulp were used in
11 runs to obtain a good viability of probiotic in
vegetables juice. Table 1 presents the experimental
design used for optimization.
A good viability of probiotic bacteria (9.2110.52
log cfu/mL) was achieved in juice after fermentation.
After the storage period a loss of 0.982.06 log
cfu/mL was reached, the decrease in viability was
between 0.09 and 0.99 log units at each monitoring
interval (Fig.1).




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This survival is better than the survival of
Lactobacillus acidophilus and Bifidobacterium
animalis ssp. lactis in stirred fruit yogurts [9]. In
the study of Buriti et al. (2010) the probiotic
populations were below 6 log cfu/g on day 28.
Inulin did not improve the probiotic viability in
refrigerated mousses and the degradation of this
polysaccharide by L. acidophilus during the
storage was undesirable, reducing prebiotic
potential. They have concluded that freezing may
represent a more stable condition to preserve
microorganisms, when compared to refrigeration
[10].
Table 1. Experimental design

7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
Day 1 Day 7 Day 14 Day 21
V
i
a
b
i
l
i
t
y

(
l
o
g

c
f
u
/
m
L
)



)
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11

Figure 1. Evolution of bacteria viability during storage
The initial viability of yeast was lower than the
viability of bacteria (8.8410.23 log cfu/mL). The
yeast population was above 7 log cfu/mL on day
28 (Fig.2). The decrease found at each monitoring
interval was between 0.55 and 2.56 log cfu/mL.
The difference between the viability of yeast after
fermentation and the final of storage period was
between 1.083.33 log cfu/mL. Wilson et
al.(2013) suggest that some vegetal compounds
can have antifungal properties and may reduce the
viability of Saccharomyces boulardii [11].
An insignificant decrease of pH was found in all
samples during storage (Fig.3). From the initial
value 4.6 the pH has decrease to a value of
4.24.35. The result is similar who that obtained by
Bulatovi et al. (2014) [12]. Ongol et al. (2007)
conclude that L. delbrueckii subsp. bulgaricus did not
greatly reduce the pH of fermented milk during
storage at 4C [13].
7.00
7.50
8.00
8.50
9.00
9.50
10.00
10.50
Day 1 Day 7 Day 14 Day 21
V
i
a
b
i
l
i
t
y

(
l
o
g

c
f
u
/
m
L
)


)






































Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11

Figure 2. Evolution of yeast viability during storage
4.2
4.25
4.3
4.35
4.4
4.45
4.5
4.55
4.6
Day 1 Day 7 Day 14 Day 21
p
H
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11

Figure 3. Evolution of pH during storage
A medium correlation was obtained between
variation of yeast viability and pH (Table 2). A low
negative correlation was found between variations of
bacteria viability and of pH. This result is different
than that obtained by Kailasapathy et al. (2008) who
found significant relationships between pH and
viable probiotic counts [9]. A low correlation was
obtained also between variation of yeast and bacteria
viability. These results suggest the analyzed factors
(amount of honey and amount of lyophilized
vegetables pulp) have the higher influence on the
viability of probiotic microorganisms.
These variations of viability and pH were subjected
to principal component analysis (PCA) and the first
two principal components explained 85.34% of the
variance as shown in Fig. 4. These results suggest
that the use of variables is adequate for viability
evaluation of fermented juices. Principal component
PC1 had high loadings on all variation of analyzed
factors. PC2 had positive loading for variation of
bacteria and yeast viability. The mean scores for all
samples on the two components were also projected.
The samples 3, 7 and 11 were the samples with a
good stability of viability during storage period.




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Table 2. Correlation matrix (Pearson (r))
Variables
Variation of yeast
viability
Variation of bacteria
viability
Variation of pH
Variation of yeast viability 1 0.234 0.450
Variation of bacteria viability 0.234 1 -0.116
Variation of pH 0.450 -0.116 1



Figure 4. PCA-Bi-plot showing sample mean scores in
relation to the variation of viability and pH loadings on
the first two principal components
The counts in all samples declined with time. The
counts were significantly with the increase of
nutrient supplements. These results are similar
with those obtained by Aryana and McGrew
(2007) [14].
Varga (2006) demonstrated that the presence of
honey at 1.0% to 5.0% (w/v) did not significantly
influence (p>0.05) the survival of characteristic
microorganisms in yogurt during a 6-week storage
period at 4 C. In addition, at a concentration of
approximately 3.0% (w/v), it highly improves the
sensory characteristics of yogurt without having an
inhibitory effect on starter bacteria. [15].
4.Conclusion
It is very important to understand the relationship
between the stability of probiotic strains and the
medium characteristics, such as the temperature,
composition, pH, time, during storage period. The
ability of probiotic cells to survive and maintain
their functional characteristics during storage are key
issues in the development of new products.
The identification of factors positively affecting the
survival of probiotics may lead to the development of
food processing optimization. The results of the
present study illustrated that the potential probiotic
strains used as starters in vegetables juice were able
to complete a normal fermentation and survive at
proper numbers at the end of the fermentation.
Acknowledgment. This study has been founded by the
Sectorial Operational Programme Human Resources
Development of the Romanian Ministry of Education,
Research, Youth and Sports trough the Financial
Agreement POSDRU/159/1.5/S/132397 ExcelDOC. The
authors are grateful to Bioaliment Research Platform,
Dunarea de Jos University of Galati, Romania for
scientific support.
Compliance with Ethics Requirements: Authors declare
that they respect the journals ethics requirements. Authors
declare that they have no conflict of interest and all
procedures involving human and/or animal subjects (if
exists) respect the specific regulations and standards.
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