International Food Research Journal 24(2): 787-794 (April 2017)

Journal homepage: http://www.ifrj.upm.edu.my

Role of protective agents on the viability of probiotic Lactobacillus plantarum

during freeze drying and subsequent storage
1*
Savedboworn, W., 1Kerdwan, N., 1Sakorn, A., 2Charoen, R., 2Tipkanon, S. and
3
Pattayakorn, K.

Department of Agro-Industry Technology and Management, Faculty of Agro-Industry, King

1

Mongkut’s University of Technology North Bangkok, Prachinburi, Thailand 25230

2
Department of Innovation and Product Development Technology, Faculty of Agro-Industry, King
Mongkut’s University of Technology North Bangkok, Prachinburi, Thailand 25230
3
Department of Food Technology and Nutritional, Faculty of Natural Resources and Agro-
Industry, Kasetsart University, Chalemphrakiat Sakon Nakhon Provinces campus, Sakon Nakhon,
Thailand 47000

Article history Abstract

Received: 17 December 2015 The aim of this study was to determine the effect of various protective agents on the survival
Received in revised form: of probiotic Lactobacillus plantarum TISTR 2075 grown in Plai Ngahm Prachin Buri rice
4 April 2016
Accepted: 8 April 2016
extract during freeze drying and subsequent storage. A combination of protein-trehalose (Prot
+ Tre) and protein-maltodextrin (Prot + MD) significant (P<0.05) improved the viability of the
strain after freeze drying with the survival rate of 98.13 and 97.58%, respectively. Among all
protective agents tested, Prot + Tre was found to maintain high degrees of viable cell number
Keywords with the lowest specific rate of cell death (k) of 7.45 x 10-4 and 1.79 x 10-2 day-1 after storage at
4oC for 168 days and 30oC for 84 days, respectively. Additionally, the accelerated storage tests
Lactobacillus plantarum
using accelerated temperatures of 40, 50, 60 and 70oC were used to develop a model system in
Probiotic
Freeze drying
order to estimate the viability of freeze-dried probiotic L. plantarum TISTR 2075 in different
Protective agents protective agents for long-term storage. It was concluded that accelerated storage testing is a
Storage stability useful technique with certain predictability in this study.

© All Rights Reserved

Introduction the time of consumption to provide health benefit

(Kosin and Rakshit, 2006). In order to preserve
Food products containing probiotic probiotic bacteria for long-term viability and
microorganisms are gaining interest and available functionality, dehydration process which involved
in market worldwide (Vasudha and Mishra, 2013; the transition of microorganisms from a liquid to a
Ashraf and Smith, 2015). The global market for solid medium is commonly used for production of
probiotic food is expected to reach US$52.34 billion dried powder of probiotics (Iaconelli et al., 2015).
by 2020 (James, 2014). Over the last few years, the Freeze drying is considered as a suitable method for
development of non-dairy probiotic products is a stabilizing microorganisms that are greatly sensitive
challenge for the food industry due to the ongoing to high temperature (Goderska, 2012; Fonseca
trend of vegetarianism and a high prevalence of et al., 2015). However, freezing and subsequent
lactose intolerance in many people around the world sublimation of frozen water could be attributed to
(Nualkaekul et al., 2012). Probiotics are available cellular injuries including damage to cell membrane
for consumers in an increasing variety of non-dairy and DNA (Tripathi and Giri, 2014). Additionally,
applications such as fruit and vegetable beverages, the changes in the physical state of membrane lipids
dessert products, cereal products, meat products and during storage may result in severe loss of bacterial
health supplements for direct consumption (Yeo and viability during storage (Fonseca et al., 2015). A
Liong, 2010; Behboudi-Jobbehdar et al., 2013). number of cryoprotective agents such as proteins,
The viability of probiotics should be maintained sugars and carbohydrates have been used to minimize
during processing, storage and delivery to target the bacterial inactivation after freeze drying and
site in gastrointestinal tract (Ying et al., 2010). The subsequent storage (Carvalho et al., 2004). According
minimum concentration of viable probiotic bacteria to Jofré et al. (2015), survival rate of L. rhamnosus
at least 106-107 CFU/mL was typically proposed at CTC1679, L. casei/paracasei CTC1677 and L. casei/

*Corresponding author.
Email: wanticha@yahoo.com
Tel: +66 37217300
788 Savedboworn et al./IFRJ 24(2): 787-794

paracasei CTC1678 was ≥ 94% after freeze drying supplemented with prebiotic maltodextrin, fibersol-2,
with glucose, lactose and skim milk. Furthermore, trehalose and inulin were used as protective media
soy protein isolate mixed with maltodextrin provided for L. plantarum TISTR 2075 in freeze drying. The
protective capability of >80% survival on the protective ability of these materials to enhance the
viability of freeze-dried Bifidobacterium longum stability of the strain during subsequent storage
1941 (Dianawati et al., 2013). Prebiotics are non- was also determined. An accelerated storage testing
digestible carbohydrates that resist hydrolysis and based on the Arrhenius equation was applied so as to
absorption in the upper parts of the gastrointestinal develop a model system to predict storage stability of
tract and affect the host by selectively stimulating freeze-dried probiotic for long-term storage.
the growth and/or activity of colonic microflora
(Roberfroid, 1998). Prebiotics were also applied as Materials and Methods
protective agents during freeze drying process. Inulin
is a natural polysaccharide composed of a chain Microorganisms
of fructose units with a terminal glucose unit. The The probiotic Lactobacillus plantarum TISTR
application of the inulin in food industry is related to 2075 isolated from fermented vegetables was obtained
its capability of substituting sugar and fat with a low from Microbiological Resource Center, Thailand
count of calories (Toneli et al., 2010). The presence of Institute of Scientific and Technological Research
inulin as a stabilizing agent in freeze drying process (TISTR), Thailand. The strain was preserved in de
was reported. Nualkaekul et al. (2014) suggested a Man-Rogosa-Sharpe (MRS) broth (Difco, Detroit,
reduction in viable cell number of 0.92 log CFU/g MI, USA) with 20% (v/v) glycerol content at -20oC.
when inulin was used as protectant of freeze-dried For routine analysis, the strain was subcultured twice
L. plantarum NCIMB 8826 in pomegranate powder in MRS broth and was incubated at 37oC for 24 h
after storage at room temperature for 12 months. under microaerobic-static conditions to maintain
Also, trehalose as stabilizer is widely used to stabilize freshness and then used as inoculum.
protein during drying (Hinrichs et al., 2001). It has
been demonstrated that trehalose is an effective Preparation of cereal extracts fermentation
cryoprotectant during freeze drying of L. rhamnosus Fermented Plai Ngahm Prachin Buri rice
GG and L. plantarum IFA No. 278 (Pehkonen et al., extract was prepared according to the procedures
2008; Strasser et al., 2009). This is probably due to described by Savedboworn and Wanchaitanawong
the remarkably high glass transition temperature (Tg) (2015). Plai Ngahm Prachin Buri rice was washed
of trehalose and the strong ion-dipole interactions and soaked in distilled water. The soaked rice was
and hydrogen bonding between trehalose and the mixed with distilled water (rice:water = 1:10 w/v).
biomolecule (Meng et al., 2008). Additionally, After decanting the soaking water, the soaked rice
maltodextrin as bulking agent and stabilizing agent was mixed with distilled water and then comminuted
in drying process was reported. The survival rate of in a blender for 3 min. The resultant slurry was
79.9% with the viable cell number of approximately filtered through double-layered cheesecloth 2 times
10 log CFU/g was achieved after freeze drying of to yield cereal extracts. Rice extract was dispensed
L. plantarum G2/25 (Yao et al., 2009). Besides, an into containers and sterilized by heating at 121oC for
accelerated storage testing is a useful method for the 15 min. Sterilized rice extract was inoculated with
prediction of storage stability and for the estimation overnight culture of 1% (v/v) L. plantarum TISTR
of shelf-life (Tsen et al., 2007). Several studies 2075. The fermentations were performed under no
have proposed a model to extrapolate the shelf life pH control in Duran screwcapped glass bottles at
of probiotics in powdered form during storage. A 37oC for 24 h. Viable cell counts were determined by
successful prediction of storage stability of freeze- the standard plate count method with MRS medium
dried L. acidophilus BCRC 10695, L. acidophilus supplemented with 0.5% CaCO3 at 37oC for 24 h. pH
CCRC 10695 and L. brevis ATCC 8287 (Desmond was measured with a pH meter.
et al., 1998; King et al., 1998; Tsen et al., 2007)
using the accelerated storage testing method based Freeze-drying of probiotic L. plantarum TISTR 2075
on Arrhenius theory has been proposed. Moreover, Prior to freeze drying, the 24-h incubated culture
no information regarding accelerated storage testing of probiotic L. plantarum TISTR 2075 grown in Plai
of freeze-dried L. plantarum has been reported. In Ngahm Prachin Buri rice extract was mixed with
order to find the efficient protective media which 15% (w/v) protein (Prot; All plant protein, Nutrilite,
have a great capability to stabilize probiotic cells Amway, USA) and 5% (w/v) of each protective agent
during freeze drying and storage, protein and protein used as follows: trehalose (Tre; Hayashibara, Japan),
 Savedboworn et al./IFRJ 24(2): 787-794 789

fibersol-2 (Fib; Matsutani, Japan), maltodextrin

DE 10 (MD; Du Zhi Xue, China) and inulin (Inul;
Nutrition Sc Co., Ltd, Thailand) for 30 min by a
magnetic stirrer. The suspensions were transferred
into lyophilized flask under aseptic conditions and
frozen at -18oC for 17 h. A freeze-drier was operated
at 0.110 mbar and -50oC for 18 h. Freeze-dried
samples were analyzed immediately for the viability.

Storage of freeze-dried probiotic L. plantarum TISTR

2075
Figure 1. Viable cell number, survival rate and moisture
The freeze-dried powders were kept in sealed content of probiotic L. plantarum TISTR 2075 after freeze
aluminum foil bags (7.5 x 12 cm) and stored at 4 drying with various protective agents.
and 30oC. The viability was determined every month Values with different lowercase letters (a-c) are significant
at 4oC and every 2 weeks at 30oC. The specific rate differences by Duncan’s multiple range test (P < 0.05).
of cell death (k, day-1) of freeze-dried L. plantarum Prot: Protein; MD: Maltodextrin; Fib: Fibersol-2;Tre:Trehalose;
Inul: Inulin
TISTR 2075 was calculated as a first-order reaction
from k = ln (N0/N)/t, where N refers to the bacterial Water activity and moisture content
cell count at a particular storage period (CFU/g), N0 Water activity was measured after freeze drying
represents the bacterial cell count at the beginning of using an Aqualab water activity instrument (Aqualab,
the storage (CFU/g) and t is the storage time (day) Model Series 3TE, USA). The residual water content
(Tsen et al., 2007). of the freeze-dried powders was evaluated in a drying
oven at 105oC until a constant weight was attained.
Accelerated storage test
Accelerated storage test was determined according Statistical Analysis
to the procedures described by Tsen et al. (2007) with Each result was expressed as the mean ± S.D
minor modifications. The freeze-dried samples were of two determinations. The data were assessed
incubated at 40, 50, 60 and 70oC. The residual viable using analysis of variance (ANOVA) with a level
cell number was evaluated on each sample collected of significance at P < 0.05. Significant divergences
at constant time intervals to calculate the specific rate among mean values were determined with Duncan’s
of cell death (k). Samples were taken every 24 h for multiple range tests. All statistical analyses were
6 days at 40oC, every 12 h for 3 days at 50oC, every 6 performed using SPSS Software, version 12 (SPSS,
h for 1.5 days at 60oC and every 1 h for 6 h at 70oC. White Plains, NY, USA).
Scanning electron microscopy (SEM) Results and Discussion
The freeze-dried powders were attached to a
brass stub with double-sided adhesive tape and Viability of L. plantarum TISTR 2075 after freeze
sputter coated with a layer of gold. Digital images drying with different protective agents
were recorded with a scanning electron microscope After freeze drying, the strain survival rate
(JSM 6400, JEOL, Tokyo, Japan) and captured at the of 23.45% was achieved when Prot was used as
required magnification. protective agent. The addition of protective agents
were found to significantly (P < 0.05) improve the
Enumeration of viable cell number survival rate of the strain with different degrees of
Freeze-dried powder (1 g) was resuspended in protection. Among all protectants tested, Tre, MD
9 mL of sterile 0.85% NaCl solution for 30 min at and Fib enhanced the viability of L. plantarum
room temperature. The appropriate serial dilutions TISTR 2075 with the survival rates of 98.13, 97.58
were prepared before pour plating on MRS agar and 75.71%, respectively. However, no significant
(added with 0.5% CaCO3) and incubated at 37oC for difference in survival rate was observed when Inul
24 h. The percentage of cell survival was defined as was added comparing with Prot alone (Figure 1).
follows : survival rate (%) = (N/N0) x 100, where N Freeze-drying might cause cell membrane damage,
represents the number of viable cell count after freeze protein and DNA denaturation resulting in the loss
drying (CFU/g) and N0 denotes the viable cell count of cellular viability and activity (Meng et al., 2008;
before freeze drying (CFU/g). Tripathi and Giri, 2014; Fonseca et al., 2015).
Protective agents play an important role in the
790 Savedboworn et al./IFRJ 24(2): 787-794

Table 1. Experimental k values of freeze-dried L. plantarum TISTR 2075 during storage at

4oC for 168 days and 30oC for 84 days and predicted k values of freeze-dried L. plantarum
TISTR 2075 during storage at 4 and 30oC

Prot: Protein; MD: Maltodextrin; Fib: Fibersol-2; Tre: Trehalose; Inul: Inulin

conservation of viability. The protective capability and fusing with each other (Ghandi et al., 2012).
of trehalose could be due to the stabilization of cell Moisture contents of the strain after freeze drying
membranes by replacing the water between lipid with various protective agents were in the range of
headgroups and the prevention of unfolding and 1.38-3.83%. Zayed and Roos (2004) revealed that a
aggregation of protein by hydrogen bonding with certain amount of water must remain in dehydrated
polar group of protein (Crowe et al., 2001). The state for a satisfactory survival rate. The residual
greater flexibility in the glycosidic bond between moisture in freeze-dried materials is directly related
the two D-glucose molecules, as compared to other to the type of freeze-drying medium. Moreover,
disaccharides, may allow trehalose to conform to the the morphology of freeze-dried L. plantarum
irregular polar groups of macromolecule (França et TISTR 2075 with different protective agents was
al., 2007). illustrated in SEM micrographs. It was observed
Also, maltodextrin exhibited high protective that L. plantarum TISTR 2075 were entrapped and
capability after freeze drying process. Many covered by protective matrices (Figure 2). All freeze-
researchers suggested that maltodextrin has the dried powders exhibited a similar particle shape
ability to retain water, stabilize enzyme, prevent with a porous-sheet-like structure. This result is in
cellular injuries, provide good oxidative stability concordance with Xu et al. (2016) that freeze drying
and overcome the stickiness (Bhandari et al., 1993). process created a porous structure. Additionally,
Incorporation of maltodextrin could be beneficial Poddar et al. (2014) suggested that freeze-dried
due to their relative high Tg values (Semyonov et material has connected porosity giving internal
al., 2010) and amorphous form are able to prevent surface area for water absorption.
protein unfolding during drying (DePaz et al.,
2002). Furthermore, protein as carrier was found Effect of protective agents on the viability of freeze-
to have a great protective effect on the survival dried L. plantarum TISTR 2075 during storage
of probiotic in this study. Protein is capable of The stability of freeze-dried L. plantarum TISTR
preventing cellular injury by forming a protective 2075 in various protective agents was evaluated during
coating on the cell wall (Gharsallaoui et al., 2007). storage temperature of 4oC for 168 days and 30oC for
Protein macromolecules may not pass through the 84 days. It was obvious that storage temperature was
structure of the peptidoglycan layer that covers the a crucial parameter affecting the survival of freeze-
plasma membrane of lactic acid bacteria. It is only dried cells. The viability of the strain was quite stable
capable of acting as inactive bulking agents, forming during storage at 4oC. A high storage temperature led
a protective coating around the cells and lowering the to a great decrease in the number of viable probiotic
probability of a large number of cells coming closer cell for all protective agents. The viability loss of
 Savedboworn et al./IFRJ 24(2): 787-794 791

Table 2. Moisture contents of freeze-dried L. plantarum TISTR 2075 with various

protective agents during storage at 4oC for 168 days and 30oC for 84 days

Prot: Protein; MD: Maltodextrin; Fib: Fibersol-2; Tre: Trehalose; Inul: Inulin

protectants could be considered in terms of a specific

rate of cell death (k value). The k values were
various depending on storage conditions and types
of protectants. As shown in Table 1, Prot + Tre as
protective agent was found to be relatively effective
with the lowest k values at both storage temperatures.
The k values of 7.45 x 10-4 day-1 with the final viable
cell count of 9.22 log CFU/g and 1.79 x 10-2 day-1
with the final viable cell number of 8.79 log CFU/g
were achieved at storage temperature of 4 and 30oC,
respectively. Unfortunately, Prot + Inul (k4oC = 3.48
x 10-3 day-1 and k30oC = 5.77 x 10-2 day-1) were found
to be less effective at 4 and 30oC, respectively,
however the viable cell numbers were still > 8 log
CFU/g higher than recommended effective dosage
of probiotic products. As shown in Table 2, moisture
contents of freeze-dried cells were 2.13-4.74% and
1.82-4.55% after storage at 4oC for 168 days and
30oC for 84 days, respectively. Consistent with the
report of Zayed and Roos (2004) that the optimum
moisture content for the storage of L. salivarius
subsp. Salivarius (UCC 500) ranged from about 2.8
to 5.6%. The inactivation of freeze-dried lactic acid
bacteria during storage almost resulted from chemical
Figure 2. Scanning electron micrographs of freeze-dried L. reaction such as oxidation and protein denaturation
plantarum TISTR 2075 powders with different protective (Passot et al., 2012). Among all possible degradation
agents; Prot (a), Prot + MD (b), Prot + Fib (c), Prot + Tre events, lipid oxidation of membrane fatty acid was
(d) and Prot + Inul (e)
mainly deemed responsible for cell death during
Prot: Protein; MD: Maltodextrin; Fib: Fibersol-2; Tre:
Trehalose; Inul: Inulin storage. Lipid oxidation is also accompanied by the
formation of free radicals which mainly damage
0.05-0.24 and 0.70-2.86 log CFU/g was detected DNA and cell membrane during long-term storage
during storage at 4 and 30oC, respectively. The rising (Albadran et al., 2015).
temperature was not only increasing the metabolic
activity in the cells, but also modified the molecular Prediction of the viability of freeze-dried L. plantarum
mobility of water as the environmental temperature TISTR 2075 by accelerated storage test
approached Tg (the T-Tg gradient). As the results, The accelerated storage testing was used to
the matrix will move closer to the rubbery state and develop a model system in order to predict the
water molecular mobility will increase (Behboudi- long-term preservation of freeze-dried probiotic L.
Jobbehdar et al., 2013). plantarum TISTR 2075. The specific rate of cell
From the results, the protective capability of death (k) of freeze-dried microorganism in various
792 Savedboworn et al./IFRJ 24(2): 787-794

protective agents kept under accelerated temperatures

at 40, 50, 60 and 70oC could be determined from
Equation 1.

ln N = ln N0 - kt [1]

where N0 is the initial viable cell number (CFU/g), N

is the viable cell number at any time (CFU/g), k is the
specific rate of cell death (day-1) and t is the storage
time (day).
The correlation between temperature and k value
could be described by the Arrhenius equation as
shown in Equation 2.

[2]

where k is the specific rate of cell death (day-1), Ea is

the energy of activation (J.mol-1), R is the gas constant
(8.32 J.mol-1.K-1), and T is the absolute temperature
(K). When taking the natural logarithm of both sides
of Equation 2, the Equation 3 is achieved.

[3]

The Arrhenius graph was plotted from the determined

k values in terms of natural logarithms versus the
reciprocals of their absolute temperatures (Figure
3). Consequently, k4oC and k30oC were estimated.
The predicted k values of the strain freeze-dried
with various protective agents during long-term
preservation at 4 and 30oC were shown in Table 1.
From the results, predicted k value of freeze-dried
L. plantarum TISTR 2075 in different protective Figure 3. Arrhenius plots of the specific rate of cell death
agents was verified by the experimental k value. The (k) of freeze-dried L. plantarum TISTR 2075 at various
ratio of predicted and experimental k values were temperatures in different protective agents; Prot (a), Prot
approximately 0.24-0.30 and 1.31-2.05 at 4 and + MD (b), Prot + Fib (c), Prot + Tre (d) and Prot + Inul (e)
30oC, respectively. Roos (1995) suggested that the Prot: Protein; MD: Maltodextrin; Fib: Fibersol-2; Tre:
phase transition is important causes for the observed Trehalose; Inul: Inulin
deviations from Arrhenius equations. A change in
the physical state of freeze-dried powders during dried L. acidophilus CCRC 10695 during storage.
storage may change activation energy. Additionally, Consistent with Hamsupo et al. (2005) that there
nonenzymatic browning reaction is also responsible was no significant difference in viability between
for cell death during storage depended on the physical prediction and experimental survival rates of spray-
state (Passot et al., 2012). Rate of browning was low dried L. reuteri KUB-AC5 at 4 and 30oC for 4 months.
below a critical temperature, above which the rate This indicated that the accelerated storage testing is
of the reaction increased substantially (Roos, 2001). the potential extrapolation tool for estimation of the
Nonenzymatic browning is not always prevented bacterial shelf-life with certain degree of correctness
in the glassy state. The reaction rates were lower at and predictability (Lapsiri et al., 2012).
temperature below Tg comparing with temperature
above Tg (Kawai et al., 2005). Several studies Conclusion
have been successfully predicted the viability of
microorganism during storage. According to Tsen In this study, probiotic L. plantarum TISTR
et al. (2007), there was no significant difference 2075 has the capability to survive after freeze drying
between predicted and actual results of freeze- process depending on the type of protective agents.
 Savedboworn et al./IFRJ 24(2): 787-794 793

Prot + Tre, Prot + MD and Prot + Fib were found Biochemistry and Biotechnology 70-72:513-526.
to be the most effective on probiotic in retaining Dianawati, D., Mishra, V. and Shah, N. P. 2013. Survival
the viability after freeze drying, especially Prot + of Bifidobacterium longum 1941 microencapsulated
Tre which exhibited significant impact on probiotic with proteins and sugars after freezing and freeze
drying. Food Research International 51(2):503-509.
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Fonseca, F., Cenard, S. and Passot, S. 2015. Freeze-
analysis of accelerated storage test data induced the drying of lactic acid bacteria. In Wolkers, W.F., and
equation indicating the prediction model of probiotic Oldenhof, H. (Eds). Cryopreservation and Freeze-
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extrapolated the strain survival stored at 4 and 30oC França, M. B., Panek, A. D. and Eleutherio, E. C. A. 2007.
with certain predictability. The correction factor Oxidative stress and its effects during dehydration.
would require rectifying the models. These predictive Comparative Biochemistry and Physiology - Part A:
equations will be useful for probiotic manufacturers Molecular Integrative Physiology 146:621-631.
to design and expect the probiotic shelf-life. Ghandi, A., Powell, I. B., Chen, X. D. and Adhikari,
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Acknowledgements
of Lactococcus lactis during spray drying. Drying
Technology: An International Journal 30:1649-1657.
This research was funded by King Mongkut’s Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A.
University of Technology North Bangkok. Contract and Saurel, R. 2007. Applications of spray-drying in
no. KMUTNB-GOV-58-33.1. microencapsulation of food ingredients: An overview.
Food Research International 40:1107-1121.
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