ORIGINAL RESEARCH

published: 25 February 2022

doi: 10.3389/fmicb.2022.826151

The Combined Use of Pediococcus

pentosaceus and
Fructooligosaccharide Improves
Growth Performance, Immune
Response, and Resistance of
Whiteleg Shrimp Litopenaeus
vannamei Against Vibrio
parahaemolyticus
Nguyen Thi Xuan Hong 1 , Nguyen Thi Hue Linh 1 , Kartik Baruah 2 , Do Thi Bich Thuy 3 and
Edited by: Nguyen Ngoc Phuoc 1*
Mehdi Raissy, 1
Faculty of Fisheries, University of Agriculture and Forestry, Hue University, Hue, Vietnam, 2 Department of Animal Nutrition
Islamic Azad University, Iran
and Management, Aquaculture Nutraceuticals Research Group, Faculty of Veterinary Medicine and Animal Sciences,
Reviewed by: Swedish University of Agricultural Sciences, Uppsala, Sweden, 3 Faculty of Engineering and Food Technology, University
Peng Luo, of Agriculture and Forestry, Hue University, Hue, Vietnam
South China Sea Institute
of Oceanology (CAS), China
Ghasem Rashidian, In this study, we evaluated the effect of probiotic bacteria Pediococcus pentosaceus
Tarbiat Modares University, Iran supplemented at different inclusion levels in a control diet [basal diet containing
*Correspondence: 0.5% fructooligosaccharide (FOS)] on the growth performance, feed conversion ratio,
Nguyen Ngoc Phuoc
[email protected]; immune response, and the disease resistance of whiteleg shrimp Litopenaeus vannamei
[email protected] juveniles against Vibrio parahaemolyticus. A control diet with 0.5% FOS but without
P. pentosaceus supplementation (Control) was prepared. In addition, three other test
Specialty section:
This article was submitted to diets were also formulated: control diet supplemented with P. pentosaceus at (i)
Food Microbiology, 1 × 106 cfu g−1 diet (P1), (ii) 1 × 107 cfu g−1 diet (P2), or (iii) 1 × 108 cfu g−1
a section of the journal
Frontiers in Microbiology
diet (P3). After a 60-day feeding trial, the experimental shrimps were challenged with
Received: 30 November 2021
V. parahaemolyticus. The results showed that dietary supplementation of P. pentosaceus
Accepted: 14 January 2022 significantly improved the growth performance and immune responses of L. vannamei
Published: 25 February 2022 juveniles. The juveniles that were fed with a P2 or P3 diet recorded the maximum
Citation: increase in the final body weight, final length, weight gain, and survival rate. The total
Hong NTX, Linh NTH, Baruah K,
Thuy DTB and Phuoc NN (2022) The hemocyte counts, phenoloxidase, and lysozyme activity of shrimp fed with either of
Combined Use of Pediococcus these two diets were significantly enhanced. The results also showed that juveniles
pentosaceus
and Fructooligosaccharide Improves
fed with a P2 or P3 diet exhibited significantly lower mortality when challenged with
Growth Performance, Immune V. parahaemolyticus. Overall results suggested that a combination of P. pentosaceus
Response, and Resistance at the inclusion level of 1 × 107 cfu g−1 diet (P2) and 0.5% FOS could be considered
of Whiteleg Shrimp Litopenaeus
vannamei Against Vibrio as a potential synbiotic formulation for improving the growth, health, and robustness of
parahaemolyticus. L. vannamei.
Front. Microbiol. 13:826151.
doi: 10.3389/fmicb.2022.826151 Keywords: P. pentosaceus, fructooligosacharide, L. vannamei, growth performance, immune response

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Hong et al. Pediococcus pentosaceus and Whiteleg Shrimp

INTRODUCTION (FOS), and has been studied on fish species, such as rainbow
trout, Japanese flounder, yellow croaker, and cobia (Cerezuela
Aquaculture accounted for 46% of the total global fish production et al., 2011; Huynh et al., 2017; Villumsen et al., 2020). Synbiotic
and 52% of fish for human consumption (FAO, 2020). It plays improves the survival, disease resistance, and microbial ecology
an important role in providing the growing population with of the gastrointestinal tract of the host leading to greater
high-quality animal protein, and creating job all over the world, benefits than the application of individual probiont (Ohtani
including in Vietnam and many Southeast Asian countries. The et al., 2020. Knipe et al., 2021). Pediococcus pentosaceus, a lactic
whiteleg shrimp Litopenaeus vannamei is one the most popular acid bacteria species, has been demonstrated as a promoter
cultured penaeid species in Asian countries, reaching 5.4 million of growth, immune response, and disease resistance, while
tons in 2019 (FAO, 2021). Diseases outbreak caused by viral and replacing antibiotics in farmed shrimp (Leyva-Madrigal et al.,
bacterial pathogens has been the main obstacle to the sustainable 2011; Adel et al., 2017; Won et al., 2020). The supplementation
production of this species both in Asia and in other parts of of FOS in the feed has proven beneficial for improving growth
the world. In particular, the outbreaks of acute hepatopancreatic performance, immune response, and survival of whiteleg shrimp
necrosis disease (AHPND) in farmed marine shrimp, which (Hu et al., 2019; Mustafa et al., 2020). However, the modes
is caused by virulent strains of bacteria belonging to Vibrio of action of P. pentosaceus combined with FOS on the host’s
parahaemolyticus and related species, caused mass mortalities in immune responses are still unclear (Cerezuela et al., 2011;
cultured L. vannamei and giant tiger prawn (Penaeus monodon) Huynh et al., 2018). This study aims to examine whether the
(Shinn et al., 2018; Tang et al., 2020). The economic losses due growth performance, immune response, and resistance towards
to this disease have amounted to over USD 7 billion annually V. parahaemolyticus challenge could be improved when the
(Tang et al., 2020), and therefore AHPND was listed by the World L. vannamei were fed diets supplemented with a combination of
Organization for Animal Health (OIE) as a notifiable disease in P. pentosaceus and FOS.
2019 (OIE, 2019).
The application of antibiotic has been an effective strategy for
preventing an extensive range of Gram-negative/positive bacteria MATERIALS AND METHODS
in farmed shrimp (Baticados and Paclibare, 1992; Yano et al.,
2014). However, the indiscriminate use of antibiotics in shrimp Preparations of Probiotic
aquaculture has led to the rapid development of resistant bacterial The selected P. pentosaceus R6 evaluated in this study was isolated
strains, which eventually constitute a direct threat to human from home-made fermented anchovy sauce, a commercial fish
health and to the environment (Cabello, 2006; DeLorenzo et al., sauce product in Thua Thien Hue province, Vietnam according
2016; Knipe et al., 2021). Antimicrobial resistance (AMR) is to the modified method of Adel et al. (2017). The fish sauce was
currently one of the most important human and farmed animal rinsed, homogenized, and suspended in sterile phosphate buffer
health-threatening issues worldwide (Watts et al., 2017; Okocha saline (PBS) at a ratio of one fish sauce and 9 PBS (v/v). Then,
et al., 2018). Consequently, there is an urgent need to develop the suspension was serially diluted to 10−3 , 10−4 , 10−5 , and
a strategy to prevent pathogen propagation and enhance shrimp 10−6 . Finally, the suspension was plated on the De Man Rogosa
immunity during farming operations. Probiotics are defined as and Sharp (MRS) agar (Merck, Damstadt, Germany) and was
live microorganisms that confer a health benefit on the host incubated under the aerobic condition at 28◦ C for 24 h. Among
when administered in adequate amounts (Hill et al., 2014). 11 strains isolated from the anchovy fish sauce, six isolates were
Over the past few years, probiotics are becoming increasingly identified as P. pentosaceus according to the results of 16S rDNA
popular alternatives to antibiotics for promoting growth and sequencing using the general-purpose primers of prokaryotic 16S
health status and preventing disease in shrimp aquaculture. rDNA: 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ), and 1492R
Several studies have shown the important role of probiotics in (50 -AAGGAGGTGATCCAGCCGCA-30 ) (Lane, 1991). The six
the competitive exclusion of pathogenic bacteria, nutrient, and isolates of P. pentosaceus were tested for antagonistic effect to
enzymatic contribution to shrimp digestion, enhancement of V. parahaemolyticus by using the agar-diffusion method (Bernal
the shrimp immune response, and antiviral effects (Hoseinifar et al., 2015) and hemolysis activity in whiteleg shrimp blood
et al., 2018; Ringø, 2020). Probiotics can be administered orally according to the description of Chang et al. (2000). Briefly, the
with the feed, or directly into the shrimp rearing water as P. pentosaceus isolates were grown on MRS broth for 24 h at 28◦ C.
purified cultures or spores (Ringø, 2020), or in a fermented One hundred microliters of V. parahaemolyticus suspension at
growth media, for example, Bacillus subtilis E20-fermented a concentration of 106 cfu ml−1 , which was recovered from
soybean meal (Tsai et al., 2019; Wang et al., 2019). Similarly, AHPND outbreaks in Thua Thien Hue province (Vietnam), was
probiotics may be administered in combination with a non- plated on the TSA (Tryptone Soya Agar, Oxoid, Hamsphire,
digestible food ingredient (i.e., prebiotics) that beneficially United Kingdom) with 2% NaCl added. After 30 min, from
affects the host by causing synergistic effects, an approach the TSA plate, six 2-mm agar plugs were removed by a cork
referred to as “synbiotics” (Schrezenmeir and de Vrese, 2001; borer (2 mm diameter) and 100 µl of overnight culture of
Li et al., 2018; Knipe et al., 2021). The synbiotic formulation each P. pentosaceus isolate was placed on these wells. The
comprised of evaluated probiotics corresponds to two bacterial plates were then incubated at 28◦ C for 24 h and the diameter
genera, Enterococcus spp. and Bacillus spp., with prebiotics, such of the zone of inhibition was measured for determining the
as mannan oligosaccharide (MOS) and fructooligosaccharide antagonistic activity.

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Hong et al. Pediococcus pentosaceus and Whiteleg Shrimp

A shrimp blood agar consisting of 1 ml of whiteleg shrimp TABLE 1 | Ingredients and composition (% dry weight basis) of the control diet for
whiteleg shrimp used in this study.
hemolymph in medium containing 200 ppm Rose Bengal
was used to determine the hemolytic activity of 6 isolates of Ingredient g kg−1
P. pentosaceus (Chang et al., 2000). Hemolymph (1 ml) was
drawn between the first and second pleopods from whiteleg Fish meal1 350.0
shrimp previously surface-disinfected with 70% ethanol. The Soybean meal1 200.0

hemolymph (1 ml) was immediately transferred into a sterilized Wheat meal1 285.0

tube containing 0.2 ml of citrate-EDTA buffer (0.1 M glucose, Lecithin1 20.0

30 mM trisodium citrate, 26 mM citric acid, and 10 mM EDTA Fish oil1 30.0

dissolved in 20 ppt seawater) and stained by the addition of Vitamin mixture2 20.0

133 µl of 3% (w/v) Rose Bengal (dissolved in citrate EDTA buffer) Mineral mixture2 20.0

with gentle rotation to achieve a complete mixture. Rose Bengal- Calcium dihydrogen phosphate3 25.0

stained hemolymph (1 ml) was added to 15 ml of the Nutrient Cholesterol 25.0

Agar (Himedia, Mumbai, India) with l.5% NaCl, which cooled to Corn starch1 20.0

45–50◦ C in a water bath with gentle shaking for proper mixing Fructooligosaccharide (FOS) 5.0

and pouring into petri dishes. The six isolates of P. pentosaceus Proximate composition %
were inoculated onto the shrimp blood agar plate. The plates were
Moisture (g kg−1 ) 11.5
incubated for 48 h at 28◦ C and observed for the formation of a
Protein (g kg−1 dry matter) 40.2
transparent zone around the inoculated colonies.
Lipid (g kg−1 dry matter) 7.3
Based on the results of the antagonistic effect and non-
Ash (g kg−1 dry matter) 12.3
hemolytic (see Supplementary Data) on whiteleg shrimp blood
agar, the P. pentosaceus R6 isolate was selected for preparing 1 The GreenFeed Co., Ltd., Vietnam.
2 Vemedim 3 Sigma-
probiotic. The P. pentosaceus R6 was cultured and incubated at (Vietnam). Premix detailed by Won et al. (2020),
Aldrich, Vietnam.
28◦ C for 48 h on MRS agar. From a pure bacterial growth plate of
P. pentosaceus R6 on MRS agar, a single colony was isolated and
placed directly into 20 ml of sterile MRS broth and incubated for through a laboratory pelleting machine (3A, Vietnam) to obtain
48 h at 28◦ C in the shaking incubator (Kuhner shaker, ISF-1-W, pellets of size 0.5 to 1 mm diameter. The pellets were air-dried for
Switzerland; 140 rpm). After 48 h, the bacterial suspension was 72 h to a moisture level of <10%, and then stored at 4◦ C in the
centrifuged at 3,500 rpm (Sanyo NSE Mistral 2000R, Japan) for refrigerator until use. New batches of feed were produced every
30 min, washed twice in sterile PBS containing 0.02 M phosphate 2 weeks to keep up P. pentosaceus viability.
and 0.15 M NaCl, and the cell pellets obtained were re-suspended
in PBS to achieve an OD600nm value of 1, which was equivalent Shrimp and Experimental Design
to 1 × 109 cfu ml−1 based on standard bacterial growth curves Juveniles of L. vannamei were purchased from The National
(data not presented). The actual bacterial concentration was Breeding Centre for Central Aquaculture at Phu Hai Commune,
determined by viable colony counts according to the method of Phu Vang district, Thua Thien Hue province, Vietnam. They were
Miles et al. (1938). The pellets were collected by centrifugation transferred by air-conditioned car to the wet lab of Fish Pathology
at 3,500 rpm for 30 min, mixed with 20% skimmed milk, and Laboratory of the University of Agriculture and Forestry, Hue
stored at −70◦ C. The frozen bacterial sample was concentrated city, Vietnam. The juveniles were maintained in 1,000-L fiberglass
in a freeze-dryer and homogenized, followed by storage of the tanks using continuous flow-through water at 0.38 L min−1 at
bacterial powder at 4◦ C until used. The viability of the bacterial 28 ± 2◦ C. They were acclimatized to the experimental conditions
mixture was determined according to the method of Miles et al. for 14 days during which they were fed four times a day with
(1938). the control diet. Shrimp used in this study were quarantined
for free AHPND, white spot disease, and yellow head disease
Preparations of Experimental Diets at Veterinary Clinic, sub-department of Animal Husbandry and
In total, one control and three test diets were formulated in Veterinary Medicine in Thua Thien Hue. Before the start of the
this study. Feed formulation and proximate composition of the feeding experiment, health checks of shrimp were performed
control diet prepared with the supplementation of 0.5% FOS in by sampling the hepatopancreas of five shrimp directly onto
the basal diet are shown in Table 1. The control diet was not Thiosulfate Citrate Bile Salts (TCBS, Himedia, Mumbai, India)
supplemented with P. pentosaceus (Control), whereas the three agar and checking for bacterial growth (Phuoc et al., 2021).
other test diets were formulated prepared by supplementing the A total of 1,200 juveniles with an initial body weight of
control diet with P. pentosaceus at the inclusion level of 1 × 106 0.5( ± 0.1) g were distributed randomly into 12 fiberglass tanks
cfu g−1 diet (P1), 1 × 107 cfu g−1 diet (P2), or 1 × 108 cfu g−1 diet (120 L, 3 tanks per diet, 100 shrimp per tank) equipped with
(P3). The preparation and storage of the diets were conducted continuous aeration. Shrimp were fed four times a day (07:00 h,
following Adel et al. (2017). All the dry ingredients were weighed, 12:00 h, 16:00 h, and 20:00 h) with the control or test diets at a
ground in a hammer mill to pass through a 250-µm-mesh screen, rate of 5–7% of wet body weight. During the experimental period,
and then mixed in a mixing machine. After that, fish oil and water the water parameters were maintained at 27 ± 1◦ C temperature,
were added until a dough was formed. Each diet was then passed pH 7.7–8.3, dissolved oxygen at 5.5–7.3mgL−1 , salinity at 20–22g

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Hong et al. Pediococcus pentosaceus and Whiteleg Shrimp

L−1 , and a 12-h light:12-h dark cycle for 60 days. The condition (10 mM sodium cacodylate, 450 mM sodium chloride, 10 mM
of the tanks was maintained by siphoning off all residual, feces, calcium chloride, and 260 mM magnesium chloride; pH 7.0), and
molts, and dead shrimp in the morning. Uneaten feeds were equal aliquots (cell suspension) were placed into two tubes. One
siphoned after 2 h feeding from tanks. After the 60-day feeding tube was used for measuring the PO activity, and the other tube
regime, 30 shrimp from each experimental group were collected was measured for background PO activity. The cell suspension
for the challenge test with V. parahaemolyticus. Six shrimp from (100 µl) was incubated for 10 minat 25–26◦ C with 50 µl of
each group were collected for evaluation of immune responses, trypsin (1 mgml−1 ), which served as an elicitor. Fifty microliters
including the total hemocyte count, phenoloxidase activity, and of l-DOPA was added, followed by 800 µl of cacodylate buffer
lysozyme activity, and 6 other shrimp from each group were used 5 min later. The control solution, which consisted of 100 µl of
for the phagocytic activity assay. the cell suspension, 50 µl of cacodylate buffer (to replace the
trypsin), 50 µl of l-DOPA, and 800 µl of cacodylate buffer, was
Analysis of Growth Performance used for the background PO activity. The shrimp’s PO activity
At the end of the feeding trial, the juveniles were fasted for 24 h, was measured at an OD of 490 nm using a spectrophotometer
and then weighed and counted. Based on the weight of each (Jasco V-630, Hachioji, Tokyo, Japan).
shrimp and the number of juveniles survived, the specific growth
Lysozyme Activity
rate (SGR), weight gain (WG), feed conversion ratio (FCR), and
The lysozyme (LYS) activity was quantified following the
survival rate (SR) were calculated following the standard formula
procedures described by Chiu et al. (2007). Briefly, 500 µl of
(Adel et al., 2017):
diluted hemolymph were centrifuged, and the precipitate was
mixed with 1 ml (0.02%) of Micrococcus lysodeikticus (Sigma, St.
ln of final weight g − ln of initial weight (g)


Louis, MO, United States). The reaction was carried out at room
SGR = × 100 temperature, and the absorbance at 530 nm was measured after
number of days
0.5 and 4.5 min. A unit of LYS activity was defined as the amount
of enzyme producing a decrease in absorbance of 0.01 min−1 , and
Final weight g − initial weight (g)


the specific activity was expressed as U (g protein−1 ).
WG = × 100
initial weight (g)
Phagocytic Activity Assay
Feed intake (g) Twenty microliters of a bacterial suspension of
FCR = V. parahaemolyticus (2 × 106 cfuml−1 ) was injected into
Weight gain (g)
the ventral sinus. After being injected, the shrimp were kept
Final number of live shrimp in separate tanks containing 40L of seawater (20h) for 2 hat
SR = × 100 27 ± 1◦ C. Hemolymph (100 µl) from six shrimp was withdrawn
Number of initial shrimp
and mixed with 900 µl of an anticoagulant solution. One
Non-specific Immune Responses hundred microliters of a diluted hemolymph sample was mixed
with 100 µl of 0.1% paraformaldehyde and incubated for
Analysis 30 minat 4◦ C to fix the hemocytes. Then, 50 µl of the suspension
Total Hemocyte Count was spread on a glass slide. The slide was placed in a cytospin
The total hemocyte count (THC) was conducted as described by centrifuge and centrifuged at 113 × g for 3 min. The slide was
Chiu et al. (2007). Briefly, hemolymph (100 µl) was withdrawn then air-dried, stained with Diff-Quick stain, and observed using
from the ventral sinus of each shrimp using a 1-ml sterile syringe a light microscope. Two hundred hemocytes were counted.
(25-gauge) containing 0.9 ml anticoagulant solution (30 mM Phagocytic activity (PA) was expressed as:
trisodium citrate, 0.34 M sodium chloride, and 10 mM EDTA,
at a pH of 7.55 and with the osmolality adjusted with glucose Phagocytic hemocytes
PA(%) = × 100
to 780 Osm kg−1 ). A drop of each diluted hemolymph sample Total hemocytes
was placed in a hemocytometer and observed under a light
microscope to determine its total hemocyte count. Challenge Test
Pathogenic V. parahaemolyticus was obtained from the
Phenoloxidase Activity laboratory of Fish Pathology, Faculty of Fisheries, University
The Phenoloxidase (PO) activity was measured of Agriculture and Forestry, Hue University, Vietnam. A single
spectrophotometrically by recording the formation of colony of V. parahaemolyticus grown on TCBS agar was sub-
dopachrome produced from l-dihydroxyphenylalanine (l- cultured in 10 ml of Tryptone Soya Broth (Oxoid, Hamsphire,
DOPA). One microliter of diluted hemolymph from each tube United Kingdom) containing 2% NaCl at 28◦ C for 24 h to
was centrifuged at 800 × g and 4◦ C for 20 min. The supernatant achieve exponential growth. The broth culture was centrifuged at
was discarded, and the pellet was rinsed, re-suspended gently 3,000 rpm for 30 minat 4◦ C. The supernatant was removed, and
in 500 µl cacodylate citrate buffer (10 mM sodium cacodylate, the bacterial pellet was re-suspended in saline solution (0.85%
450 mM sodium chloride, and 100 mM trisodium citrate; pH NaCl), which corresponds to approximately 109 cfu ml−1 for the
7.0), and then centrifuged again. The supernatant was discarded, challenge test. The viable colony counts were performed using
and the pellet was resuspended in 100 µl of cacodylate buffer the method of Miles et al. (1938).

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Hong et al. Pediococcus pentosaceus and Whiteleg Shrimp

After 60 days, shrimp fed with different experimental diets A significant increase (p < 0.05) in the THC and PO activity was
(30 shrimp per tank × 3 tanks per treatment = 90 shrimp noted in the group fed with P. pentosaceus-supplemented diets
per treatment) were challenged with V. parahaemolyticus by when compared to the control group. In the case of THC, the
immersion for 1 h. Shrimps were immersed in 10 L of 20h highest value was recorded in the P3 group followed by the P2
seawater containing bacteria at 1.1 × 106 cfu ml−1 at 28 ± 1◦ C, group, whereas in the case of PO activity, the P3 group recorded
removed after 1 h, and placed into the flow-through experimental maximum PO activity, but was not significantly different from
tanks (120 L) at 0.38 L min−1 , with water temperature at 26 ± 2◦ C that of the P1 and P2 groups (p > 0.05). Feeding of shrimp
for 14 days to record any abnormal behavior, clinical signs, with a diet supplemented with P. pentosaceus in the P2 diet
and daily mortality. The bacterial concentration was determined exhibited a maximum increase in the activity of the LYS enzyme.
from previous pilot studies and was designed to give 60% total However, no significant difference was recorded with the P3
mortalities (data not shown). Aeration was supplied through an group (p > 0.05). The lysozyme activity of shrimp fed the P1
air stone to each tank and the shrimp were fed with control diet to diet and that of the control group did not differ significantly
apparent satiation twice daily. At the end of the challenge test, the (p > 0.05) (Table 3).
cumulative mortality percentage (%) was calculated as previously In the present study, the phagocytic activities of shrimp fed
described (Kongnum and Hongpattarakere, 2012). P. pentosaceus-supplemented diets were significantly higher than
the group fed a control diet. Among the treatment groups,
Statistical Analysis those fed with P. pentosaceus-supplemented P3 diet exhibited the
The data were expressed as mean ± standard deviation. They highest phagocytic activity followed by the P2 and P1 groups.
were first checked for underlying Gaussian distribution of However, there is no significant difference between the P2 and
data using a Shapiro–Wilks test. Once this distribution was P3 groups (p > 0.05) (Table 4).
confirmed, data were analyzed by one-way ANOVA, and multiple
comparisons were performed using a Tukey post hoc test. All Challenge Test
statistical analyses were performed using the SPSS 20.0 (SPSS Feeding of diet supplemented with P. pentosaceus caused a
Inc, Chicago, IL, United States). Differences were considered significant effect on the cumulative mortality percentage of the
significant at p 0.05. shrimp, regardless of the dose of P. pentosaceus in the feed that
was used, noting that isolate was used at its LD60 (Figure 1).
Mortality was recorded highest in the control group fed with
RESULTS a control diet without P. pentosaceus added. The mortality
decreased for each treatment as the dose of P. pentosaceus
Growth Performances increased (Figure 1). The mortality of shrimp fed with the P3 diet
Growth performances of L. vannamei were presented in Table 2. was lowest but was not significantly different from the treatment
The final body weight, WG, and SGR of shrimp increased in group that was fed with the P2 diet (p > 0.05). The mortality
all the groups that were fed with P. pentosaceus-supplemented of these two treatment groups was significantly lower when
diets when compared with the control diet-fed group. The compared with the P1 group (p < 0.05 for both groups). The
final body weight and WG of shrimp fed P2 or P3 diet were mortality of shrimp fed with the P3 diet was lower but was not
significantly (p < 0.05) higher when compared with the control significantly different from the control group (p > 0.05).
and P1 groups. These values of shrimp in the group fed with Mortality was first observed on day 3 post-challenge in all the
the P1 diet were higher but not significantly different from experimental groups except in the P3 group that was fed with a
that of shrimp in the control treatment (p > 0.05). The FCR diet supplemented with 1 × 108 cfu g−1 P. pentosaceus. In the
decreased and was significantly lower in all the treatment groups P3 group, mortality was noted on day 4 post-challenge. Lower
fed with P. pentosaceus-supplemented diets compared to that in percentage mortality was associated with later onset of mortality.
the control group (p < 0.05). Similarly, the survival rate was Mortality continued until day 12 post-challenge in the control
significantly higher in all the P. pentosaceus fed groups when group. Whereas in the treatment groups, mortality continued
compared with the control diet-fed group. The highest survival only till day 10 except in the P3 group, in which mortality
rate was observed in P2 treatment where shrimp were fed with continued until day 11 post-challenge (Figure 1).
P. pentosaceus-supplemented diet at the inclusion level of 1 × 107
cfu g−1 , significantly higher than that observed in P1 treatment
where shrimp were fed with P. pentosaceus at 1 × 106 cfu DISCUSSION
g−1 (p < 0.05) but was not significantly different from that
of shrimp fed with P. pentosaceus at 1 × 108 cfu g−1 (P3) Lactic acid bacteria strains have been used as probiotics in
(p > 0.05). aquaculture for a long time, in which P. pentosaceus has
received more attention in recent years for application in
Immune Response farmed shrimp. It is because of its wide range of beneficial
Feeding of diets supplemented with P. pentosaceus caused a effects, especially on the growth performance, digestive enzyme
significant improvement in the immune responses of juvenile activity (Adel et al., 2017; Won et al., 2020), immunity, and
L. vannamei especially with reference to THC, lysozyme, PO, tolerance towards pathogenic vibrios (Adel et al., 2017). In the
and phagocyte activity against V. parahaemolyticus (Tables 3, 4). present study, we combined the probiotic strain P. pentosaceus

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Hong et al. Pediococcus pentosaceus and Whiteleg Shrimp

TABLE 2 | Growth performance of shrimp fed with different levels of P. pentosaceus.

Parameters Treatments

Control P1 P2 P3

Initial weight (g shrimp−1 ) 0.53 ± 0.05a 0.55 ± 0.03a 0.57 ± 0.04a 0.58 ± 0.07a
Final weight (g shrimp−1 ) 4.1 ± 0.1a 4.3 ± 0.3a 4.9 ± 0.3b 5.0 ± 0.3b
Gain weight (%) 677.0 ± 22.1a 685.0 ± 23.2a 768.0 ± 21.0b 770.0 ± 21.0b
FCR 1.38 ± 0.16a 1.20 ± 0.05b 1.05 ± 0.04c 1.02 ± 0.03c
SGR (% day) 3.41 ± 0.09a 3.42 ± 0.07a 3.60 ± 0.06b 3.60 ± 0.10b
Survival (%) 70.0 ± 1.8a 75.0 ± 1.6b 85.3 ± 1.2c 84.7 ± 1.4c

Data are means ± SD of triplicate groups of shrimps. Values in each row with different superscripts are significantly different (p < 0.05). Diets: Control = Basal diet with
0.5% FOS, refer to Table 1; P1: P. pentosaceus at 1 × 106 cfu g−1 ; P2: P. pentosaceus at 1 × 107 cfu g−1 ; P3: P. pentosaceus at 1 × 108 cfu g−1 .

TABLE 3 | Phenoloxidase (PO) and lysozyme activities, and the total hemocyte count (THC) of shrimp in response to feeding different experimental diets.

Parameters Treatments

Control P1 P2 P3

THC ( × 106 cells ml−1 ) 12.20 ± 0.89a 13.80 ± 0.58b 14.50 ± 0.85c 17.70 ± 1.04d
PO (100 µl−1 ) 0.19 ± 0.00a 0.22 ± 0.01b 0.25 ± 0.00b 0.26 ± 0.03b
Lysozyme (U) 0.66 ± 0.01a 0.68 ± 0.04a 1.03 ± 0.15b 0.94 ± 0.08b

Values are means ± SD of triplicate replicates. Values in the row with different superscripts letters are significantly different (p < 0.05).

TABLE 4 | Phagocyte activity of shrimp in treatments with different levels of P. pentosaceus.

Parameters Treatments

Control P1 P2 P3
Phagocytic activity (no. of cells) 178.0 ± 2.0a 192.0 ± 3.0b 196.0 ± 2.0c 197.0 ± 2.0c
Non-phagocytic activity (no. of cells) 22.0 ± 2.0a 8.0 ± 3.0b 4.0 ± 2.0c 3.0 ± 2.0c

Values are means ± SD of three replicates. Values in the row with different superscripts letters are significantly different (p < 0.05).

FIGURE 1 | Cumulative mortality percentage of juvenile whiteleg shrimp fed with different doses of P. pentosaceus for 60 days and experimentally challenged with
V. parahaemolyticus for 14 days. Data with different letters are significantly different among groups (p < 0.05).

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Hong et al. Pediococcus pentosaceus and Whiteleg Shrimp

at different inclusion levels with a fixed level of a known vitamins, such as ascorbate (vitamin C), folate (vitamin B9),
prebiotic component FOS to develop a synbiotic formulation and cyanocobalamin (vitamin B12) (Rossi et al., 2005; Rodrigues
to determine its impact on the growth indices, immune status, et al., 2011). These biologically active compounds might play
and infection stress tolerance trait in commercially important an important role in the food assimilation, absorption, and
L. vannamei. Our results showed that when P. pentosaceus was growth of aquatic animals. Besides, the positive effect of the
combined with 0.5% FOS especially in the treatment groups conjoint administration of FOS and P. pentosaceus, in the
P2 and P3, a significant increment in the final weight was form of synbiotics, on the growth performances of whiteleg
achieved. The improvement in the weight gain of the juveniles shrimp could be due to an increase in the digestive enzyme
in response to feeding P2 or P3 diet resulted in an FCR activities induced by the P. pentosaceus. The presence of this
that was significantly lower than for the other diets. It is also LAB strain might stimulate the production of endogenous
noteworthy to mention that in the control group, there was enzymes in the host or contribute to the total enzyme activity
about 63% mortality at the end of the feeding trial. However, of the gut as reported previously in shrimps (Adel et al., 2017;
in the treatment groups, there was a significant reduction in Wanna et al., 2021).
the mortality, with the P2 and P3 groups having a mortality Several lines of evidence suggested that the crustacea
of 15%. This suggests that the probiotic strain at the inclusion hemocytes, PO, and lysozyme activity play an important role
levels of 1 × 107 cfu g−1 (P2) or 1 × 108 cfu g−1 (P3) in generating a protective immune response against bacterial
and in combination with 0.5% FOS had no adverse effect on diseases including those caused by vibrios (Oktaviana et al.,
the survival of the shrimp juveniles, at least in our described 2014; Zubaidah et al., 2015; Kuo et al., 2021). Shrimps
experimental condition, and hence can be considered safe for possess three hemocyte types named hyaline cells, semigranular
use in L. vannamei feed. While all experimental feeds in the cells, and granular cells (Johansson et al., 2000). Hemocyte
present study are formulated with a prebiotic supplement, count can vary greatly in response to infection, environmental
the observed improvement in the growth performance was stress, and the life cycle of the animal (Zubaidah et al.,
associated with differences in the probiotic component of 2015; Kuo et al., 2021). An increase in the THC provides
the synbiotic formulation. To our knowledge, there is no enhanced immune capability during a period of stress leading
available report on the effect of the dietary supplementation to disease resistance in crustacean (Chiu et al., 2007). The
of P. pentosaceus in combination with FOS on the growth prophenoloxidase plays an important role in eliciting protective
and survival performances in L. vannamei juveniles. However, immune responses in the crustaceans. The proposed mechanism
in earlier studies, Adel et al. (2017) and Won et al. (2020) of prophenoloxidase is that active PO induces oxidation of
reported that dietary supplementation of P. pentosaceus could phenols to quinones, and results in the production of melanin,
cause a significant improvement in the growth performance and which can hold and barricade infectious pathogens leading
nutritional utilization in L. vannamei, findings in agreement with to induced phagocytosis, and cytotoxic reactant production
that of our study. There are also studies on whiteleg shrimp (Chiu et al., 2007; Amparyup et al., 2012). In the current
in which improved growth and feed utilization in response to study, the supplemental P. pentosaceus at the inclusion level
feeding diets supplemented with probiotics, such as Bacillus 1 × 106 cfu g−1 (P1), or 1 × 107 cfu g−1 (P2), or
subtilis (Munaeni et al., 2014; Tsai et al., 2019; Won et al., 2020), 1 × 108 cfu g−1 (P3) in the diets with 0.5% FOS induced
Bacillus licheniformis (Fan et al., 2021), and Lactococcus lactis the most significant improvement in PO activity, compared
(Won et al., 2020), were reported. In another study, the use to the control diets. Phagocytosis is an important cellular
of 0.2–0.4% FOS in shrimp diets was shown to improve the defense mechanism, whereas lysozyme is an important humoral
growth performance, FCR, and the microbial diversity, and to defense mechanism in crustaceans. Results of the present
suppress several potential pathogens, such as Vibrio tubiashii, study are in line with previous studies that demonstrated
V. parahaemolyticus, and Photobacterium damselae-like strains that synbiotic increased proliferation of hemocytes and activity
in the intestine of whiteleg shrimp (Hu et al., 2019). However, of PO and lysozyme enzymes (Adel et al., 2017). Our
it is noteworthy to mention that dietary supplementation of results suggest that the improvement in the tested immune
FOS at 0, 0.15, and 0.30% did not cause any significant effector molecules in the group fed with a diet supplemented
beneficial effect on the survival, weight gain, and immune with 1 × 107 cfu g−1 (P2) or 1 × 108 cfu g−1 (P3)
responses of the shrimp juveniles (Mustafa et al., 2020). Previous was strongly associated with a significant reduction in the
studies reported that combining prebiotics, such as MOS, mortality of the V. parahemolyticus-challenged L. vannamei
FOS, or galactooligosaccharide (GOS), with probiotic bacteria, shrimp fed with P. pentosaceus-supplemented diets. This
Enterococcus spp., Bacillus spp., or LAB strains improved nutrient result suggests that besides having a positive effect on the
utilization (Zhou et al., 2009; Won et al., 2020) and growth growth, the probiont P. pentosaceus have additional health-
performance in shrimps by increasing the absorptive surface beneficial effects.
area of intestinal microvilli (Das et al., 2017; Huynh et al., In the gut, bacteria can adhere to the intestinal
2017; Chen et al., 2020; Butt et al., 2021; Yao et al., 2021). epithelial surfaces by specific attachment of bacterial
During synbiotic fermentation, many essential amino acids are surface proteins to complementary oligosaccharides on
released, such as histidine, isoleucine, leucine, lysine, tryptophan, the tissue surface. The oligosaccharides such as FOS
and nonessential amino acids such as alanine, glutamate, and can modulate the adhesive of selective bacteria by
tyrosine (Ndagijimana et al., 2009; Rodrigues et al., 2011), and acting as antiadhesion agents to opportunistic pathogens

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Hong et al. Pediococcus pentosaceus and Whiteleg Shrimp

(Van den Abbeele et al., 2009; Altamimi et al., 2016). Moreover, study was conducted. In the absence of a regulatory framework
oligosaccharides can produce short-chain fatty acids (SCFA) for formal ethical approval, the work was conducted according
that lower intestinal pH in the colon, consequently creating an to the ethical standards of the UK Home Office, based on
unsuitable condition for the growth and survival of pathogenic training received by the corresponding author at the Institute of
bacteria (Chen et al., 2020). This evidence might explain for Aquaculture, University of Stirling, Stirling, United Kingdom.
higher survival of the L. vannamei that was observed in the
treatment groups (P2 and P3) after being challenged with
V. parahaemolyticus when P. pentosaceus was supplemented in AUTHOR CONTRIBUTIONS
the basal diet with FOS.
In conclusion, a synbiotic formulation was developed NH, DT, KB, and NP conceived the study. NH and NL conducted
combining P. pentosaceus at the inclusion level of 1 × 107 cfu the challenge experiments. NH acquired the funding. NP, NH,
g−1 or 1 × 108 cfu g−1 with 0.5% FOS, exhibiting a beneficial and KB wrote the manuscript. All authors read and approved the
effect on the growth performance and immune response of final manuscript.
whiteleg shrimp. Our results also showed that an improvement
in the resistance of the juveniles towards V. parahaemolyticus
challenge was associated with a significant improvement in FUNDING
the immune response as manifested by an increase in the
activity of LYS and PO enzymes and hemocyte counts in the This study was financially supported by a grant from Hue
synbiotic fed groups. Overall results suggest that a combination of University, Vietnam (No. DHH2020-02-141).
P. pentosaceus and FOS can be considered as a potential synbiotic
for farmed shrimp species.
ACKNOWLEDGMENTS
DATA AVAILABILITY STATEMENT We would like to thank the Hue University, who provided the
funding for the study. We also would like to thank Le Bao for
The original contributions presented in the study are included
assistance with technical advices.
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author/s.
SUPPLEMENTARY MATERIAL
ETHICS STATEMENT
The Supplementary Material for this article can be found
The Animal Ethics Committee at University of Agriculture and online at: https://www.frontiersin.org/articles/10.3389/fmicb.
Forestry, Hue University was not established during the time this 2022.826151/full#supplementary-material

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Yano, Y., Hamano, K., Satomi, M., Tsutsui, I., Ban, M., and Aueumneoy, Conflict of Interest: The authors declare that the research was conducted in the
D. (2014). Prevalence and antimicrobial susceptibility of Vibrio species absence of any commercial or financial relationships that could be construed as a
related to food safety isolated from shrimp cultured at inland ponds potential conflict of interest.
in Thailand. Food Control 38, 30–36. doi: 10.1016/j.foodcont.2013.
09.019 Publisher’s Note: All claims expressed in this article are solely those of the authors
Yao, W. X., Li, X. Q., Zhang, C. Y., Wang, J., Cai, Y. W., and Leng, X. J. (2021). and do not necessarily represent those of their affiliated organizations, or those of
Effects of dietary synbiotics supplementation methods on growth, intestinal the publisher, the editors and the reviewers. Any product that may be evaluated in
health, non-specific immunity and disease resistance of Pacific white shrimp, this article, or claim that may be made by its manufacturer, is not guaranteed or
Litopenaeus vannamei. Fish Shellfish Immunol. 112, 46–55. doi: 10.1016/j.fsi. endorsed by the publisher.
2021.02.011
Zhou, X. X., Wang, Y. B., and Li, W. F. (2009). Effect of probiotic on larvae shrimp Copyright © 2022 Hong, Linh, Baruah, Thuy and Phuoc. This is an open-access
(Penaeus vannamei) based on water quality, survival rate and digestive enzyme article distributed under the terms of the Creative Commons Attribution License
activities. Aquaculture 287, 349–353. (CC BY). The use, distribution or reproduction in other forums is permitted, provided
Zubaidah, A., Yuhana, M., and Widanarni (2015). Encapsulated synbiotic dietary the original author(s) and the copyright owner(s) are credited and that the original
supplementation at different dosages to prevent Vibriosis in white shrimp, publication in this journal is cited, in accordance with accepted academic practice. No
Litopenaeus vannamei. HAYATI J. Biosci. 22, 163–168. use, distribution or reproduction is permitted which does not comply with these terms.

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