DOI: 10.1111/are.13701
ORIGINAL ARTICLE
Effects of synbiotic containing Lactobacillus plantarum 7–40 and galactooligosaccharide on the growth performance of white shrimp, Litopenaeus vannamei Truong-Giang Huynh1,2 | Chia-Chun Chi1 | Thanh-Phuong Nguyen2 | Tran-Thi-Thanh Hien Tran2 | Ann-Chang Cheng3 | Chun-Hung Liu1,4 1 Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan
Abstract This study aimed to develop a synbiotic combination with probiotic, Lactobacillus
2
College of Aquaculture and Fisheries, Can Tho University, Can Tho, Vietnam 3 Department and Graduate Institute of Aquaculture, National Kaohsiung University of Science and Technology, Kaohsiung, Taiwan 4
Research Center for Animal biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan
plantarum 7–40 and one of three prebiotics, fructooligosaccharide (FOS), galactooligosaccharide (GOS) and mannan oligosaccharide (MOS). The best in vitro growth was observed when probiotic was cultured in the medium containing either FOS or GOS as the sole of carbon source. The analysis of enzyme activity revealed that GOS induced the highest activities of protease and b-galactosidase of probiotic. Based on the findings, probiotic + GOS were selected as synbiotic to evaluate if it could promote the growth of white shrimp, Litopenaeus vannamei. For this, four
Correspondence Chun-Hung Liu, Department of Aquaculture, National Kaohsiung University of Science and Technology of Science and Technology, Pingtung, Taiwan. Email:
[email protected] and Ann-Chang Cheng, Department of Aquaculture, National Kaohsiung University of Science and Technology Kaohsiung, Taiwan. Email:
[email protected] Funding information Ministry of Science and Technology, ROC, Grant/Award Number: MOST 104-2313-B020-006-MY3
diets, including a basal diet with no GOS or probiotic (control), 0.4% GOS (PRE), 108 CFU probiotic kg otic kg
1
1
(PRO) and 0.4% GOS in combination with 108 CFU probi-
(SYN), were fed to shrimp for 60 days, and then the growth performance,
intestinal microbiota (including total Vibrio counts, VBCs; and lactic acid bacteria, LAB) and digestive enzyme (including protease, leu-aminopeptidase and b-galactosidase) were evaluated. The weight gain (WG) of shrimp fed the PRO did not significantly differ from those of control (p > .05). Shrimp fed the SYN had significantly higher WG compared with the other treatments (p < .05). In addition, the SYN-fed shrimp had significantly higher LAB and protease, leu-aminopeptidase and b-galactosidase activity (p < .05). The lowest presumptive Vibrio count (VBC) was also observed in intestines of SYN-fed shrimp. Therefore, we suggested that Lac. plantarum 7–40+ GOS can be used as a synergistic synbiotic for shrimp culture. KEYWORDS
growth, Lactobacillus plantarum 7–40, Litopenaeus vannamei, prebiotic, synbiotic
1 | INTRODUCTION
been widely used in intensive white shrimp culture to improve the feed value and digestion efficacy, which contribute to feed efficiency
According to recent investigations, feed costs in intensive culture of
and growth improvements (Kongnum & Hongpattarakere, 2012; Liu,
white shrimp, Litopenaeus vannamei, may reach 68% of total produc-
Chiu, Ho, & Wang, 2009; Wang, 2007; Zhang et al., 2012; Zokaeifar
tion costs (Hung & Quy, 2013). Indeed, shrimp farmers desire to
et al., 2012).
minimize the costs of production in which feed cost is of the great-
Synbiotics, a combination of prebiotics and probiotics, have been
est concern (Hoseinifar, Zare, & Merrifeld, 2010). Among practical
used since 2005 (Panigrahi et al., 2005). Synbiotics have been widely
approaches, supplements such as prebiotics and probiotics have
applied and are assumed to be growth- and immunity-promoting
Aquaculture Research. 2018;1–13.
wileyonlinelibrary.com/journal/are
© 2018 John Wiley & Sons Ltd
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factors in freshwater fish (Azimirad, Meshkini, Ahmadifard, &
diet could promote the immune responses and disease resistance of
Hoseinifar, 2016; Hoseinifar, Dadar, & Ringø, 2017; Hoseinifar, Este-
L. vannamei, but no growth-promoting effect of probiotic was found
ban, Cuesta, & Sun, 2015; Hoseinifar, Mirvaghefi, Amoozegar, Merri-
(Chiu, Guu, Liu, Pan, & Cheng, 2007). However, we believed that the
field, & Ringø, 2017) and marine fish (Ai et al., 2011; Reyes-Becerril
combination of Lac. plantarum with a selected prebiotic would
et al., 2014). It is surprising that although growth-enhancing effect
induce higher growth rates of L. vannamei compared to the single
of synbiotics on the growth of fish is well documented, little infor-
form. Therefore, the first aim of this study was to determine the
mation was reported in cultured Penaeid shrimp. In fact, several pre-
best synbiotic combination between Lac. plantarum 7–40 and three
vious studies reported that synbiotics decreased the prevalence of
different prebiotics, including MOS, FOS and GOS. The second aim
intestinal pathogenic bacteria of Vibrio spp. and increased immune
was to assess the in vivo effects of Lac. plantarum 7–40, GOS and
responses and disease resistance against Vibrio alginolyticus and
Lac. plantarum 7–40+ GOS on growth performance, production of
white spot syndrome virus (WSSV) in L. vannamei and Kuruma
digestive enzymes and intestinal microbiota of L. vannamei.
shrimp, Penaeus japonicas (Li, Tan, & Mai, 2009; Zhang et al., 2011). Meanwhile, a study by Partida-Arangure, Luna-Gonzalez, Fierro-Coronado, Flores-Miranda and Gonzalez-Ocampo (2013) reported that a synbiotic did not affect the growth of L. vannamei. Synbiotics are perceived as single products in which a probiotic and prebiotic work together and beneficially affect the host (Lauzon,
2 | MATERIALS AND METHODS 2.1 | Preparations of prebiotics, probiotics and L. vannamei
Dimitroglou, Merrifield, Ringø, & Davies, 2014). Kolida and Gibson
Commercial prebiotic products used in this study including MOS
(2011) stated that both synergistic and complementary synbiotic
from yeast (Saccharomyces cerevisiae), FOS and GOS were purchased
approaches should be considered when applying synbiotics in animal
from Carbosynth Ltd. (Old Station Business Park, Compton, UK). The
science. The synergistic effect of a probiotic is chosen based on its
probiotic was the Lac. plantarum 7–40 strain (Chiu et al., 2007). The
effects on the host, and the prebiotic must specifically stimulate the
probiotic bacterium was kept at
growth and enzyme activities of the probiotic. In addition, the probi-
growth in De Man, Rogosa and Sharpe (MRS) broth (DifcoTM Labora-
otic can directly activate digestive enzyme activities in the hep-
tories, Sparks, MD, USA) at 37°C. For experimental shrimp, 104
atopancreas of shrimp, can serve as a provider of exogenous
L. vannamei postlarvae were obtained from a private shrimp hatchery
enzymes, and assist in making food more digestible (Liu et al., 2009).
in Linyuan, Kaohsiung, Taiwan. Shrimp were acclimated and reared
Therefore, the combination of a prebiotic and probiotic may lead to
in a cement tank with a capacity of 15 m3 filled with 20& brackish
higher health benefits to shrimp.
water. Shrimp were fed a commercial shrimp diet (Lee-Hye, Ping-
Fructooligosaccharide (FOS), galactooligosaccharide (GOS) and
80°C and revitalized by overnight
tung, Taiwan) twice daily until ready for the experiment.
mannan oligosaccharide (MOS) are commonly used prebiotics and were recently applied in aquaculture as additives (reviewed by Ringø et al., 2010). These compounds have also been used as parts of syn-
2.2 | Experimental design
biotics to improve the growth of fish (Hoseinifar, Hoseini, & Bagheri,
This study encompassed in vitro and in vivo trials. In the in vitro trial,
2017; Hoseinifar, Mirvaghefi et al., 2017; Modanloo, Soltanian, Akh-
prebiotics were screened to establish a relevant synbiotic in combina-
laghi, & Hoseinifar, 2017; Rodriguez-Estrada, Satoh, Haga, Fushimi,
tion with the probiotic Lac. plantarum 7–40. Prebiotics, including FOS,
& Sweetman, 2009; Zhang et al., 2015). However, their effects of
GOS and MOS, were first screened based on the growth of the probi-
improving the growth of L. vannamei remain a subject of intense
otic Lac. plantarum 7–40 by culturing in different media containing 2%
debate. For instance Zhang et al. (2012) revealed that dietary MOS
of the prebiotic. Prebiotics which showed the highest growth-enhan-
at 2%~4% improved the growth performance of L. vannamei,
cing effect on Lac. plantarum 7–40 were examined for ability to induce
whereas Li et al. (2007) found no effect when using FOS. Therefore,
bacterial enzyme activities, including protease, leu-aminopeptidase
it is recommended that instead of probiotic alone, several prebiotics,
and b-galactosidase. In addition, the selected prebiotics were also
such as FOS, GOS and MOS, should be jointly used to improve the
investigated for supporting the growth of pathogenic bacteria, includ-
survivability and implantation of the targeted probiotic in the intes-
ing V. alginolyticus (Liu, Cheng, Hsu, & Chen, 2004) and V. para-
tine, and confer the health benefits in shrimp. However, prebiotics
haemolyticus (Yeh, Chiu, Shiu, Huang, & Liu, 2014), as references.
may be utilized via diverse mechanisms dependent on sugar linkages
GOS, which was able to induce higher production of enzyme activity
and the bacterial strain; therefore, an in vitro test for optimal combi-
of a probiotic and exhibited significantly lower support for the growth
nation between probiotic and prebiotic must be done prior to
of pathogenic bacteria, was used to formulate a synbiotic with
in vivo test in shrimp (Huynh et al., 2017).
Lac. plantarum 7–40 and was incorporated in the diets of shrimp,
Among Lactobacillus species, Lactobacillus plantarum is known to
including 0.4% GOS (PRE), Lac. plantarum 7–40 at 108 colony-forming
produce proteolytic and glycoside hydrolase enzymes that aid pro-
units (CFU)/kg (PRO), synbiotic (0.4% GOS/Lac. plantarum 7–40 at
tein and carbohydrate digestion, and is used as a probiotic (Cebeci &
108 CFU/kg, SYN) and a basal diet control (no prebiotic or probiotic)
Gurakan, 2003; Macedo, Tavares, & Malcata, 2003; Marathe &
in the second experiment. The level of probiotic used in the experi-
Ghosh, 2009), such as the Lac. plantarum 7–40 used as probiotic in
mental diets is according to Chiu et al. (2007), and Lac. plantarum
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7–40 at 108 CFU/kg + 0.4% GOS (SYN). After 60 days of feeding, the
containing 95 ml of m-MRS (Table 1), and then each culture was dis-
survival rate, growth performance, digestive enzyme activities and
tributed in several aliquots to 15-ml sterile tubes. Growth of
intestinal microbiota of shrimp were evaluated.
Lac. plantarum 7–40 in each culture was observed by examining the OD600 at 0, 2, 4, 6, 12, 24, 36 and 48 hr, using a spectrophotometer (Jasco V-630, Hachioji, Tokyo, Japan). pH values were also recorded
2.3 | In vitro studies for establishing a synergistic synbiotic
as an indirect parameter of growth and sugar metabolism using a pH metre (Suntex SP-2100, Taipei, Taiwan). Each sample was run in triplicate.
2.3.1 | Screening of the prebiotics FOS, GOS and MOS for synbiotic preparation The evaluation of prebiotic utilization by Lac. plantarum 7–40 was modified based on the method of Cebeci and Gurakan (2003); Rur-
2.3.3 | Inducing growth of pathogenic bacteria by prebiotics
angwa et al. (2009); Hoseinifar, Mirvaghefi et al. (2017). Lactobacillus
Prebiotics, including FOS and GOS, were used as the sole or carbon
plantarum 7–40 was first cultured in MRS broth for 24 hr at 37°C,
sources to assess the growth of V. alginolyticus and V. parahaemolyti-
then centrifuged at 3,000 9 g for 10 min at 4°C. Subsequently, the
cus under aerobic culture condition. The method was the same as
bacterium was washed twice and re-suspended in modified MRS
described in Section 2.3.2. Bacteria were cultured on medium (VM)
broth without sugar (m-MRS-no sugar) (Table 1). The bacterium was
containing 1.7% tryptone (a pancreatic digest of casein), 2% sodium
then streaked onto m-MRS agar that contained 2% of a prebiotic
chloride, 0.25% dipotassium hydrogen phosphate and 2% of a prebi-
(FOS, GOS or MOS) with bromcresol purple (Sigma-Aldrich, Saint
otic at 27°C (with the prebiotic as the sole carbon source). Media
Louis, MO, USA) as an indicator. Prebiotic utilization was observed as
containing 2% glucose and no sugar, respectively, served as the posi-
a change in the colour of the medium surrounding the colonies from
tive and negative controls. The growth of bacteria in each culture
purple to yellow.
was also observed as the OD600 at 0, 2, 6, 12, 24, 36 and 48 hr. Each sample was run in triplicate.
2.3.2 | Growth stimulation of Lac. plantarum 7–40 by prebiotics
2.3.4 | Assessment of prebiotic scores
A Lac. plantarum 7–40 cell suspension was prepared as described
According to a definition given by Huebner, Wehling and Hutkins
above. Five microlitres of each cell suspension was added to bottles
(2007), the prebiotic score reflects the ability of a given substrate to
T A B L E 1 Ingredients of modified De Man, Rogosa and Sharpe broth and modified De Man, Rogosa and Sharpe agar media (g 100 ml Ingredients
m-MRS-glucose††
m-MRS-no sugar†
m-MRS-FOS
m-MRS-GOS
1
m-MRS-MOS
m-MRS broth composition Proteose peptone No.3
1
1
1
1
1
Beef extract
1
1
1
1
1
Yeast extract
0.5
0.5
0.5
0.5
0.5
Polysorbate 80
0.1
0.1
0.1
0.1
0.1
Ammonium citrate
0.2
0.2
0.2
0.2
0.2
Magnesium sulphate
0.01
0.01
0.01
0.01
0.01
Manganese sulphate
0.005
0.005
0.005
0.005
0.005
Dipotassium phosphate
0.2
0.2
0.2
0.2
0.2
Glucose
2
FOS
2
GOS
2
MOS Distilled water
2 100
100
100
100
100
m-MRS agar consists of the same above ingredients with L-Cystein
0.1
Agar
1.5
1.5
1.5
1.5
1.5
Bromcresol purple
0.003
0.003
0.003
0.003
0.003
FOS, fructooligosaccharide; GOS, galactooligosaccharide; MOS, mannan oligosaccharide; m-MRS, modified De Man, Rogosa and Sharpe. The ingredients were following composition from Difco, Laboratories. Symbols represent negative control (†) and positive control (††).
)
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support the growth of a targeted bacterium relative to the indige-
bacterial suspension. The absorbance change at 410 nm was moni-
nous intestinal flora and is relative to its growth on glucose. Carbo-
tored at 37°C for 1 hr. One unit of enzyme activity was defined as
hydrates have a positive prebiotic score if they (i) are metabolized as
the amount of enzyme which produces L lmol of p-NA per hour at
well as glucose by the probiotic strain and (ii) are selectively metabo-
37°C.
lized by the probiotic but not by pathogenic bacteria. Therefore, in
The method for b-galactosidase activity was based on Vinderola
this study, the prebiotic with a higher prebiotic score supported
and Reinheimer (2003) with slight modifications. Lactobacillus plan-
good growth of Lac. plantarum 7–40. A negative prebiotic score was
tarum 7–40 cells were harvested by centrifugation at 3,000 9 g for
obtained if Lac. plantarum 7–40 grew less on the prebiotic compared
10 min at 4°C and then washed twice with phosphate-buffered saline
with that on glucose and/or had less growth on the prebiotic than
(PBS, pH 7.0). Cells were re-suspended in PBS, and then the OD560
the growth of pathogenic bacteria on the prebiotic.
was measured. One millilitre of the cell suspension was permeabilized
Assessment of the prebiotic scores for FOS and GOS was modi-
with 50 ll of a toluene: acetone (1:9 v/v) solution, vortexed for 7 min
fied based on the method of Mazzola, Aloisio, Biavati and Gioia
and immediately assayed for b-galactosidase activity. One hundred
(2015). Lactobacillus plantarum 7–40 was cultured in m-MRS with 2%
millilitres of sample was placed in a tube containing 900 ll of PBS, and
glucose or 2% of the prebiotic as the sole carbon source. Similarly, a
then 200 ll of o-nitrophenyl-b-D-galactopyranoside (ONPG) at a con-
bacterium V. alginolyticus or V. parahaemolyticus was also prepared
centration of 4 mg/ml was added. Tubes were placed in a water bath
by growing on VM with 2% glucose or 2% of the prebiotic. For the
(37°C) for 15 min, and 0.5 ml of 1 M Na2CO3 was added to stop the
enteric mixture, each V. alginolyticus or V. parahaemolyticus strain
reaction, followed by recording the OD420 and OD560. b-Galactosidase
was cultured on VM with 2% glucose and then mixed in a 1:1 ratio
activity is expressed in Miller units. The specific activity of the enzyme
and inoculated (20 ml/L) on VM containing 2% glucose or 2% of the
is presented in units/mg protein, and total soluble protein was deter-
prebiotic. Lactobacillus plantarum 7–40 culture was incubated at
mined using a Bio-Rad protein assay kit with bovine serum albumin
37°C, whereas the cultures of V. alginolyticus, V. parahaemolyticus
(BSA) as the standard (Bradford, 1976).
and the enteric mixture were incubated at 27°C. OD600 values were recorded at 0 and 24 hr. Each culture was run in triplicate. The prebiotic score was calculated as follows: Prebiotic score = ([OD600 of Lac. plantarum 7–40 on the prebiotic at 24 hr
OD600 of Lac. plantarum 7–40 on the prebiotic at
2.4 | Effects of the synbiotics on shrimp growth 2.4.1 | Preparation of diets
OD600
Lactobacillus plantarum 7–40 was cultured in a sterilized 2-L flask
of Lac. plantarum 7–40 on glucose at 0 hr]) – ([OD600 of pathogenic
and harvested by centrifugation as described above. The pelleted
bacteria on the prebiotic at 24 hr
bacterial cells were mixed with skim milk at a ratio of 1:4 and stored
0 hr]/[OD600 of Lac. plantarum 7–40 on glucose at 24 hr
OD600 of pathogenic bacteria
on the prebiotic at 0 hr]/[OD600 of pathogenic bacteria on glucose
at
at 24 hr
homogenized into a probiotic powder. The viability of the bacterium
OD600 of pathogenic bacteria on glucose at 0 hr]).
80°C. The frozen sample was dried in a freeze-dryer and then
was determined by plate counting on MRS agar. After determining the viability, the experimental diets were prepared according to Liu
2.3.5 | Evaluation of the effect of prebiotics on enzyme activity of Lac. plantarum 7–40
et al. (2009). The basal diet was formulated to be sufficient to support the optimal growth of L. vannamei (Table 2). GOS and probiotic
Lactobacillus plantarum 7–40 was cultured in m-MRS medium con-
powder containing Lac. plantarum 7–40 at 108 CFU/g were added to
taining 2% FOS or 2% GOS. The culture with glucose served as a
the test diets with respective decreases in the amounts of cellulose
control group. After 24 hr of incubation, cultures were used to mea-
and skim milk. Four dietary treatments were tested: 0.4% GOS (w/
sure the protease, leu-aminopeptidase and b-galactosidase enzyme
w) (PRE); Lac. plantarum 7–40 at a dose of 108 CFU/kg of feed
activities. Each sample was run in triplicate. The protease activity
(PRO); synbiotic (0.4% GOS + Lac. plantarum 7–40 at 108 CFU/kg of
was assayed at 40°C in 100 mmol/L Tris-HCl buffer (pH 9.0) (Liu
feed, SYN); and the control diet (no GOS or Lac. plantarum 7–40).
et al., 2009). The culture (100 ll) was incubated with 100 ll of a 1%
The ingredients were ground in a hammer mill to pass through a 60-
casein solution (prepared in Tris-HCl buffer, pH 7.0) for 10 min at
mesh screen. Experimental diets were prepared by mixing the dry
37°C. The reaction was stopped by adding 500 ll of 5% (v/v) tri-
ingredients with fish oil, and then water was added until a stiff
chloroacetic acid. After 20 min, the contents were centrifuged at
dough resulted. Each diet was then passed through a mincer with a
3000 9 g and 4°C for 20 min, and the supernatant was measured
die, and the resulting spaghetti-like strings were dried at room tem-
by a modified Lowry’s method. One unit of protease was equivalent
perature to a moisture level of
