Agriculture and Natural Resources 50 (2016) 1e7

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Original article

Survival and shelf life of Lactobacillus lactis 1464 in shrimp feed pellet
after fluidized bed drying
Maneerat Wirunpan,a Wanticha Savedboworn,b Penkhae Wanchaitanawonga, *
a
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand
b
Department of Agro-Industry Technology and Management, Faculty of Agro-Industry, King Mongkut's University of Technology North Bangkok,
Prachinburi Campus, Prachinburi Province 25230, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: In the present study, Lactobacillus lactis 1464 was attempted to be incorporated in shrimp feed pellets.
Received 10 June 2014 The fresh culture (25% volume per weight) with and without pH adjustment was mixed into feed in-
Accepted 5 January 2015 gredients prior to the pelleting process at ambient temperature. The wet pellets were dried using a
Available online 14 January 2016
fluidized bed dryer at 50
C, 60
C, 70
C and 80
C to achieve a moisture content below 11%. The results
indicated that the strain viability depended on the drying temperature with a viable cell number of
Keywords:
approximately 106108 CFU/g and the pH of the culture was found to affect the strain viability during
Fluidized bed drying
drying. At all drying temperatures, the strain survival after drying ranged from 75.94% to 92.28% at pH 3.8
Lactobacillus lactis
Prediction model
and from 89.54% to 96.87% at pH 7.0. Moreover, the addition of protectants was found to enhance the
Probiotic strain survival during drying. In particular, milk powder and monosodium glutamate (MSG) exhibited
Protective agents significant (p < 0.05) protective effect on the viability at a high temperature of 80
C. During storage at
30
C, a high survival rate was found for the strain with MSG and acacia gum. Furthermore, the prediction
model for long-term storage stability of the strain was found to validate only at a low temperature of 4
C,
in comparison to a high temperature of 30
C.
Copyright © 2016, Kasetsart University. Production and hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction susceptible to the high temperature of the pelleting and drying

process. According to Biourge et al. (1998), Bacillus CIP5832 spores
Nowadays, probiotics offer a promising alternative approach for in dog diet were found to have in excess of 99% loss after the
controlling shrimp diseases and improving shrimp health through extrusion, expansion and drying processes. Furthermore, the viable
their potential to control pathogens, to stimulate the immune count of yeast in shrimp feed pellet decreased by 105 fold after
response, to improve water quality and to enhance nutrition extruding through a meat grinder at 72
C for 31 s followed by
through the production of digestive enzymes (Verschuere et al., drying at 65
C for 6 h (Aguirre-Guzma n et al., 2002).
2000; Gullian et al., 2004; Wang, 2007). To achieve health bene- Fluidized bed drying is extensively used for drying wet particu-
fits, probiotic bacteria must be viable and available at high con- late and granular materials. In a fluidized bed dryer, the probiotic cell
centration, typically 106e107 CFU/g of product (Kosin and Rakshit, suspension is mixed with a vibrating bed of absorbers or matrix
2006). Furthermore, incorporation into feed pellets is more effec- molecules which helps to form capsules by adherence (Nag and Das,
tive in conveying probiotics into animals compared to direct 2013). This process is comparatively economical. It involves low
application into rearing systems. It is also applicable for intensive energy consumption, high throughput and imparts moderate heat
aquaculture and requires no additional labor or shrimp handling stress to the bacterial cells (Beker and Rapoport, 1987; Nag and Das,
(Gomez et al., 2007). The viability and stability of probiotics have 2013). Furthermore, this process was successfully used for the
been a technological challenge in feed manufacturing because preparation of dried granules or powders containing lactic acid
probiotics, including Lactobacillus, Bacillus and yeast, are bacteria (Santivarangkna et al., 2007; Nag and Das, 2013). According
to Nag and Das (2013), fluidized bed drying was able to retain
viability of Lactobacillus casei CRL 431 of more than 7.7 log CFU/g
during storage at 25
C for 12 wk. Mille et al. (2004) revealed that the
* Corresponding author. Lactobacillus plantarum viability in casein powder was up to 80% after
E-mail address: [email protected] (P. Wanchaitanawong). fluidized bed drying at 35
C for 30 min. Correspondingly, the

http://dx.doi.org/10.1016/j.anres.2016.01.001
2452-316X/Copyright © 2016, Kasetsart University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
2 M. Wirunpan et al. / Agriculture and Natural Resources 50 (2016) 1e7

survival of Lactobacillus brevis in fish feed was 99% (108e109 CFU/g) calculated as described by first-order kinetics as shown in Equation
after fluidized bed drying at 40
C for 40e60 min with a moisture (1) (Desmond et al., 1998).
content of 5% (Toledo et al., 2010). Several studies have reported that
the viability of lactic acid bacteria during drying and storage was log N ¼ log N0  kt (1)
enhanced by the addition of protective agents such as trehalose, skim
milk, whey protein, soy protein isolate, monosodium glutamate, where N0 is the number of initial viable cells and N is the number of
sucrose, lactose, sorbitol and polymers such as carboxymethyl cel- viable cells at any time both expressed in colony forming units
lulose, dextran and acacia gum (Morgan et al., 2006; Santivarangkna (CFU) per gram, k is the specific rate of degradation per minute and
et al., 2007; Golowczyc et al., 2011; Lapsiri et al., 2013). In the present t is the drying time in minutes. A plot of the term of log N versus
study, an overnight culture of Lactobacillus lactis 1464 was incorpo- time (t) yields the estimate of k from the slope.
rated into shrimp feed pellets prior to the pelleting process at
ambient temperature and dried in a fluidized bed dryer to achieve a Effect of protective agents on viability of L. lactis 1464 after fluidized
moisture content lower than 11%. The effect of the drying tempera- bed drying
ture, culture pH and protectants on the strain survival during drying
was determined. Additionally, the storage stability of the strain in the Each protective agent (5% w/v) including, monosodium gluta-
pellets at 4
C and 30
C was also evaluated. mate (MSG) (Ajinomoto; Bangkok, Thailand), milk powder
(Dumex; Samut Prakan, Thailand), acacia gum (MT Instrument;
Materials and methods Bangkok, Thailand), maltodextrin (Du Zhi Xue, China) was added
into the overnight cultures of L. lactis 1464 prior to mix with the
Preparation of soymilk medium feed mixture. The shrimp feed pellets were prepared by the same
manner as described in the preparation of the shrimp feed pellets.
Soymilk medium was prepared as described by Wang et al. The wet pellets with an initial moisture content of approximately
(2002). Soybeans were washed and soaked overnight in distilled 26.5% were dried at various temperatures (50
C, 60
C, 70
C and
water. The soaked soybeans were blended with distilled water 80
C) until the moisture content was below 11%.
(soybean:water ¼ 1:10 w/v) for 3 min and then filtered through a
double-layer cheesecloth to obtain soymilk. 1% (w/v) glucose (Ajax Storage of dried shrimp feed pellets
Finechem; Taren Point, NSW, Australia) was added into the soymilk
before sterilization at 121
C for 15 min. Dried shrimp feed pellets (5 g) were placed into plastic zip bags
and kept at 4
C and 30
C for 6 mth. The viable cell counts were
Microorganism determined every month. Each treatment was duplicated.

L. lactis 1464 isolated from the sediment of a Nile tilapia fish Accelerated storage test
pond with antimicrobial activity against shrimp pathogen was
obtained from the Department of Biotechnology, Kasetsart Uni- Dried shrimp feed pellets were incubated in a hot air oven at
versity, Thailand. After two successive transfers of the strain in de 50
C, 60
C, 70
C and 80
C. At 50
C, samples were taken after
Man, Rogosa and Sharpe (MRS) broth (Merck; Darmstadt, Ger- 24 h, 48 h, 72 h, 96 h and 120 h of exposure; at 60
C, after 3 h, 6 h,
many) at 37
C for 24 h, the activated culture was again inoculated 9 h, 12 h, 15 h and 18 h; and at 70
C and 80
C after 1 h, 2 h, 3 h, 4 h,
into MRS broth at 37
C for 24 h which served as the inoculum 5 h and 6 h to determine the residue viable counts. The specific rate
(Lapsiri et al., 2011). The overnight cultures were conducted in of degradation (k) was calculated.
250 mL Erlenmeyer flasks containing 120 mL of sterile soymilk and
inoculated with 5 mL of the inoculum (approximately 109 CFU/mL). Enumeration viable counts
The sample was incubated at 37
C for 24 h.
Viable counts were enumerated using a pour plate technique.
Preparation of shrimp feed pellets The sample of feed pellets (5 g) was rehydrated with 45 mL of sterile
0.85% NaCl (Ajax Finechem; Taren Point, NSW, Australia) to obtain
The formulated shrimp feed consisted of 40% fish meal, 8% 1:10 dilution and mixed in a stomacher (Seward Laboratory Systems
shrimp head meal, 20% rice bran, 10% wheat flour, 5% sago flour, 10% Inc; Davie, FL, USA) for 1 min. Serial dilutions were made for each
horse tamarind leaves powder, 5% soybean oil and 2% premix by sample and plated on MRS agar containing 0.03% bromocresol
weight. The feed mixture was sterilized at 121
C for 30 min and purple (Ajax Finechem; Taren Point, NSW, Australia). Plates were
dried overnight in a hot air oven (Memmert GmbH; Memmert, incubated at 37
C for 24 h and enumerated for colony forming unit
Germany) at 55
C. Gelatin (3% w/v) as a feed binder was mixed per gram (CFU/g). Each treatment was duplicated. The survival rates
with the dried mixture in a stand mixer. The overnight culture (25% were calculated as: Survival rate (%) ¼ (log N/log N0)  100, where N0
v/w) of L. lactis 1464 with and without pH adjustment to 7.0 with is the number of initial viable cells and N is the number of viable cells
5 M NaOH (Ajax Finechem; Taren Point, NSW, Australia) was then at any time both expressed in CFU per gram (Reddy et al., 2009).
added. After mixing for 3 min, soybean oil was then added and
mixed for 3 min again. The feed mixture was pressed into pellets at Moisture content
ambient temperature using a laboratory pellet mill with a 2 mm
diameter (California Pellet Mill Co.; Crawfordsville, IN, USA). The The moisture content of the feed pellets was determined ac-
wet pellets (250 g) were dried in a laboratory fluidized bed dryer cording to the relevant international standard 6496 (International
(Sherwood Scientific; Cambridge, UK) with a 5 L stainless chamber Standard Organization, 1999).
at various air inlet temperaturesd50
C, 60
C, 70
C and 80
C. The
fluidizing air flow velocity was held constant at 3.10 m/s. Samples Statistical analysis
were collected during drying to determine the viability of L. lactis
1464 and the moisture content. The specific rate of degradation (k) All experiments were carried out in duplicate. The data were
of L. lactis 1464 during drying at constant temperature was statistically analyzed for analysis of variance in a completely
 M. Wirunpan et al. / Agriculture and Natural Resources 50 (2016) 1e7 3

randomized design. Significant divergences among mean values fluidized bed drying of shrimp feed pellets at 50
C, 60
C, 70
C and
were established using Duncan's multiple range tests at the 95% 80
C, respectively.
confidence interval. All statistical analyses were performed using At pH 7.0 (Fig. 1B), a similar trend was also observed; however,
the SPSS Software version 12 (SPSS Inc., White Plains, NY, USA). the viability of the strain was more stable than at pH 3.8. The sur-
vival rate of the strain after drying ranged from 89.54% to 96.87% at
Results and discussion all drying temperatures. As shown in Table 1, when heating at low
pH, the reduction of the strain decreased faster than at high pH. The
Effect of temperatures and pH on the viability of L. lactis 1464 specific degradation rate (k) of the strain at pH 3.8 was higher than
during fluidized bed drying at pH 7.0 for every drying temperature. Many studies have reported
that microorganisms have their maximum heat resistance at a pH
The fresh culture of L. lactis 1464 with and without pH adjust- value close to neutral (Ocio et al., 1994; Lopez et al., 1996). Corre-
ment was added into shrimp feed pellets and subjected to drying spondingly, Juneja and Eblen (1999) found that the heat tolerance
using a fluidized bed dryer to prevent deterioration over long term of bacteria decreased with decreasing pH and decreased even more
storage. The viability loss of the strain at pH 3.8 and pH 7.0 in when presented in high temperatures. Therefore, the strain with
shrimp feed pellets and the moisture content during the fluidized the pH adjustment to 7.0 was chosen for further study.
bed drying at various temperatures are shown in Fig. 1A and B. It
was observed that the moisture content decreased gradually from Effect of protective agents on survival of L. lactis 1464 in shrimp feed
approximately 26.5% to below 11% after drying at 50
C for 15 min, pellets
60
C for 10 min, 70
C for 5 min and 80
C for 5 min. It was clear
that higher temperatures resulted in a shorter time to reach the In order to enhance the viability of the strain during fluidized
equilibrium moisture content. However, it affected the viability of bed drying, four protective agentsdMSG, acacia gum, milk powder
the strain which decreased as the temperature increased. At pH 3.8, and maltodextrindwere tested for their protective effect against
a cell reduction of only 0.57 log CFU/g and 0.94 log CFU/g with various drying temperatures. As shown in Table 2, the survival rate
survival rates of 92.28% and 87.38%, respectively, (Table 1) were of the strain was related to the type of protective agents. In all
obtained during drying at 50
C and 60
C, respectively, while a treatments, the strain exhibited greater high survival rates of
high cell reduction of 1.63 log CFU/g and 1.80 log CFU/g with sur- approximately 84%e99% (viable cell number of 106e108 CFU/g)
vival rates of 78.16% and 75.94%, respectively, were observed after after the drying process. There was no significant difference in
drying at 70
C and 80
C, respectively. It is known that temperature survival after drying at 50
C. In contrast, a significant (p < 0.05)
is the important factor affecting the viability of probiotics during effect of protectants was observed during drying at 80
C. Among
drying process (Cha vez and Ledeboer, 2007). The viability loss was the four protective agents, MSG showed the highest protective ef-
mainly due to the damage to the cell membrane and proteins fect on the viability of the strain, followed by milk powder. This was
(Ananta et al., 2005). Wang et al. (2004) reported that the numbers consistent with the report of Sunny-Roberts and Knorr (2009),
of bifidobacteria and other lactic acid bacteria decreased with where the survival of spray-dried Lactobacillus rhamnosus
increasing outlet air temperature in spray drying. A similar finding increased approximately by 1 log cycle when MSG was added in the
was reported by Bayrock and Ingledew (1997) for fluidized bed drying medium. MSG was able to stabilize the cell membrane via
dried Saccharomyces cerevisiae. Additionally, the decrease in viable the reaction between its amino group and the carboxyl group of the
cell depends not only on the drying temperature but also on the microorganism proteins (Carvalho et al., 2003). The results also
time of exposure to heat (Cha vez and Ledeboer, 2007). Hence, the showed that milk powder exhibited a protective effect against high
drying time for biomaterial preservation should be as short as temperatures. This was probably because milk protein prevented
possible. In the current study, the wet pellets of probiotic shrimp cell injury by coating the cell wall proteins (Gharsallaoui et al.,
feed were dried to achieve a moisture content below 11% and to 2007; Ghandi et al., 2012) and stabilizing the cell membrane con-
maintain a high viable cell count (above 106 CFU/g) which was stituents (Reddy et al., 2009). Moreover, Silva et al. (2011) suggested
recommended for animal feed (Uppal et al., 2008). Therefore, that protein may create a structure which is easy to rehydrate after
drying times of 15 min, 10 min, 5 min and 5 min was sufficient for drying. The macromolecules of protein, such as sodium caseinate,

Fig. 1. Viability loss of L. lactis 1464 at (A) pH 3.8 and (B) pH 7.0 in shrimp feed pellets and moisture content during fluidized bed drying at various temperatures. Viability is shown
with solid lines, while short dashed lines show the moisture content.
4 M. Wirunpan et al. / Agriculture and Natural Resources 50 (2016) 1e7

Table 1
Specific rate of degradation (k) and survival of L. lactis 1464 in shrimp feed pellets after fluidized bed drying at different temperatures.

Drying temperature (
C) pH 3.8 pH 7.0

ka (per min) Survival (%) ka (per min) Survival (%)

50 0.013 92.28 ± 0.92 0.005 96.87 ± 0.58

60 0.021 87.38 ± 0.2 0.006 94.38 ± 0.76
70 0.066 78.16 ± 0.8 0.025 90.73 ± 1.74
80 0.084 75.94 ± 0.18 0.034 89.54 ± 0.48
a
k ¼ Specific rate of degradation.

are not capable of passing through the structure of the peptido- dried at 70
C and 80
C was observed. The viability loss during
glycan layer that covers the cell membrane of lactic acid bacteria storage was mainly due to the lipid oxidation of the cell membrane
(Ghandi et al., 2012). It is able to form non-interacting osmotically (Teixeira et al., 1996; Santivarangkna et al., 2007). Acacia gum and
inactive bulking compounds causing spacing amongst cells and not MSG exhibited protective coating on the cell wall and had antiox-
allowing their cell walls to come closer and fuse (Oldenhof et al., idant properties which could prevent cell damage due to an
2005; Joshi and Thorat, 2011). oxidation reaction (Desmond et al., 2002; Sunny-Roberts and
Knorr, 2009).
From the results, cell injury and inactivation occur not only
Viability of L. lactis 1464 in shrimp feed pellets during storage during feed processing, but also during storage. The viability of
probiotic bacteria during storage is inversely related to the storage
In order to determine the shelf-life of L. lactis 1464 in shrimp temperature (Gardiner et al., 2000). It is clear that pellets stored at
feed pellets, the dried pellets were kept in plastic zip bags and 4
C showed the highest stability over 6 mth. However, refrigera-
stored at 4
C and 30
C for 6 mth. As shown in Fig. 2, the decline in tion is inconvenient for the supplier and retailer due to its high cost.
viable cells is represented by the logarithmic value of the N/N0 for Thus, it is a major challenge to produce probiotic products that are
different storage periods. After storage for 6 mth at 4
C, there was stable at ambient temperature. In this study, the viability of the
no significant (p > 0.05) loss of initial viability of fluidized bed dried strain dried at 50
C and 60
C with the addition of acacia gum and
L. lactis 1464 either with or without protective agents. On the other MSG was maintained at 107 CFU/g over 3 mth which could still
hand, high mortality was observed in all treatments at the higher meet the requirements for the level of viable cells in probiotic
storage temperature of 30
C. A similar finding was reported by products.
Wang et al. (2004) where the viability of spray-dried Streptococcus
thermophilus and Bifidobacterium longum decreased as the storage Prediction of the storage stability of L. lactis 1464 in shrimp feed
temperature increased. This was also consistent with Gardiner et al. pellets
(2002), who reported that the viability of a probiotic was main-
tained at 108 CFU/g during storage at 4
C, while viable cells In this study, the accelerated storage test was used to predict the
declined to 106 CFU/g after 49 d when stored at 30
C. storage stability of the strain in dried feed pellets. The changes of
In addition, it was also observed that the reduction rate of viable L. lactis 1464 viability in the 70
C-dried pellets with the addition of
cells at 30
C was markedly influenced by the drying temperature. MSG under storage temperatures of 50
C, 60
C, 70
C and 80
C are
The viability of the strain dried at higher temperature declined shown in Fig. 3 and the slope of each line is equal to the specific
faster than at lower temperature. For example, the viability of the degradation rate per hour (k) as shown in Table 3. The correlation
strain dried at 50
C (Fig. 2A) slightly decreased approximately between the temperature and the k value can be described by the
2e4 log CFU/g (from approximately 108 CFU/g to approximately Arrhenius equation as shown in Equation (2) (Lee, 1991).
103e105 CFU/g) after 6 mth. In contrast to the strain dried at 80
C,
viable cells reduced about 1.5e3 log CFU/g in 3 mth and decreased k ¼ k0 eðEa =RTÞ (2)
rapidly to an undetectable level after 4 mth (Fig. 2D). A similar
trend was found in the pellets dried at 70
C (Fig. 2C). Furthermore, where R is the universal gas constant (8.32 J/mol.K), Ea is the apparent
the viability of the strain during storage was enhanced by the activation energy in kilojoules per mol, T is the absolute temperature
addition of protectants. All protectants showed a similar ability to in degrees kelvin and k0 is the pre-exponential constant. In Fig. 4,
protect cells in the first 3 mth. After 4 mth, acacia gum and MSG when the log k values were plotted against 1/T, the regression
exhibited significant protective ability for the strain dried at 50
C equation was obtained as log k ¼ 28.048e9.4984 [(1/T)  1000]
and 60
C whereas no significant protective effect on the strain which was used for determination of the k value at 4
C and 30
C

Table 2
Survival of L. lactis 1464 in shrimp feed pellets with various protective agents after fluidized bed drying at various temperatures, not adjusted pH in culture in shrimp feed
pellets.

Protective agent Survival* (%)

50
C, 15 min 60
C, 10 min 70
C, 5 min 80
C, 5 min

a ab ab
Control 98.42 ± 1.50 97.20 ± 0.16 96.98 ± 0.08 86.02 ± 0.23d
MSGy 97.47 ± 0.13a 98.21 ± 0.07ab 99.36 ± 0.41a 92.43 ± 0.14a
Acacia gum 97.93 ± 0.40a 98.78 ± 0.11a 98.50 ± 0.88a 85.31 ± 0.05e
Milk powder 97.38 ± 0.59a 97.37 ± 0.70b 98.49 ± 1.08a 91.25 ± 0.18b
Maltodextrin 97.76 ± 0.37a 96.09 ± 0.12c 95.83 ± 0.06b 87.50 ± 0.13c

*Values in the same column with different superscripted lowercase letters (ae) are significantly different using Duncan's multiple range test (p < 0.05).
y
MSG ¼ monosodium glutamate.
 M. Wirunpan et al. / Agriculture and Natural Resources 50 (2016) 1e7 5

Fig. 2. Viability loss of L. lactis 1464 in feed pellet dried at: (A) 50
C for 15 min; (B) 60
C for 10 min; (C) 70
C for 5 min; (D) 80
C for 5 min during storage at 4
C (solid lines) and
30
C (short dash lines) for 6 mth with various protectants. (C; Control, ;; MSG, -; acacia gum, A; milk powder, :; maltodextrin). MSG ¼ monosodium glutamate.

(k4 ¼ 6  107/h and k30 ¼ 5  104/h). Therefore, the prediction

models of stability at 4
C and 30
C were obtained by replacing the log N ¼ log N0  6  107 t (3)
value of N0 and k4 or k30 in Equation (1), as shown in Equations (3) and
(4), respectively:
log N ¼ log N0  5  104 t (4)
To validate Equations (3) and (4), the theoretical viability
calculated from these equations and the experimental viability
obtained from stability tests were compared. As shown in Fig. 5,
there was no significant difference between the predicted and
experimental viability during storage at 4
C over 12 mth, con-
firming that the theoretical viability from Equation (3) can be used
as a prediction model of stability of this strain in dried feed pellets.
On the other hand, a great difference between the predicted
experimental viability was found at 30
C storage. The experi-
mental viability followed the linear regression equation within

Table 3
Specific degradation rate at 50
C, 60
C, 70
C and 80
C of L. lactis 1464 in pellets
dried at 70
C.

Storage temperature (
C) Specific rate of degradation* (k, per h) (R2)

50 0.0383a (0.981)
60 0.3642b (0.961)
70 2.9332c (0.828)
80 11.2660d (0.855)

Fig. 3. Viability in the 70
C-dried pellets with the addition of monosodium glutamate *Values with different superscripted lowercase letters (ad) are significantly
stored at various temperatures. different using Duncan's multiple range test (p < 0.05).
6 M. Wirunpan et al. / Agriculture and Natural Resources 50 (2016) 1e7

type of protectants. However, the remaining viable cells in pellets

were 106e108 CFU/g with a moisture content below 11% after
drying at every drying temperature which met the level re-
quirements for probiotic products. The study of probiotic product
storage revealed that the drying and storage temperatures are
critical factors for microbial survival. The addition of some pro-
tective agents enhanced viability during drying and storage.
Moreover, the accelerated storage testing was a rapid and simple
technique for estimating the long-term shelf life of this probiotic
product but at a low specific temperature of 4
C only in the model.

Conflict of interest

There is no conflict of interest.

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