Received Date : 24-Mar-2016

Revised Date : 24-Jul-2016

Accepted Article
Accepted Date : 16-Aug-2016

Article type : Original Article

Screening of pigmented Bacillus aquimaris SH6 from the intestinal tracts of

shrimp to develop a novel feed supplement for shrimp

Huong Thi Ngo1†, Trang Thi Nhu Nguyen1†, Quang Minh Nguyen2†, Anh Van Tran2†, Huong Thi Viet Do3,

Anh Hoa Nguyen4, Tuan-Nghia Phan1, Anh Thi Van Nguyen1*

1
Key Laboratory of Enzyme and Protein Technology, VNU University of Science, 334 Nguyen

Trai, Hanoi, Vietnam

2
High School for Gifted Students, VNU University of Science, 182 Luong The Vinh, Hanoi,

Vietnam

3
Faculty of Chemistry, VNU University of Science, 19 Le Thanh Tong, Hanoi, Vietnam

4
ANABIO Research & Development JSC, No. 22, Lot 7, Van Khe urban, Hadong, Hanoi, Vietnam

†
These authors contribute to this work equally.

*Correspondence: A. T. V. Nguyen, Key Laboratory of Enzyme and Protein Technology, VNU

University of Science, 334 Nguyen Trai, Hanoi, Vietnam. Email: vananhbiolab@gmail.com

This article has been accepted for publication and undergone full peer review but has not
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/jam.13274
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 Running head: Bacillus from shrimp intestine
Accepted Article
Abstract

Aims: To develop a novel feed supplement for shrimp using pigmented spore-forming bacterial strains

isolated from their gastrointestinal tracts.

Methods and Results: Eight pigmented Bacillus strains were selected from the isolates based on high

production of heat-stable spores, typical UV-Vis spectra of produced carotenoids (400–550 nm), and free

radical scavenging activity of their extracts. Of the eight strains, the red-orange pigmented B. aquimaris

SH6 was selected because it showed the highest abundance in shrimp guts (70% population). Whiteleg

shrimp (n = 30 group-1) fed with SH6 spores, at >3 × 106 CFU g-1 pellet for four weeks had redder colour

(score of 21–23 vs. 20–22), 2.7-fold higher astaxanthin level (0.69 vs. 0.25 µg g-1 shrimp), 34% higher

weight gain (7.18 vs. 5.32 g shrimp-1), and 85% higher phenoloxidase activity (OD490 = 0.265 vs. 0.143)

than shrimp in the control group.

Conclusions: The result supports the potential use of B. aquimaris SH6 as a feed supplement for

promoting the colourisation and weight gain, and for enhancing innate immunity of whiteleg shrimp.

Significance and Impact of Study: This study demonstrates that carotenoids produced by B. aquimaris

SH6 can be successfully absorbed and converted to astaxanthin in whiteleg shrimp.

Key words: pigmented, Bacillus, carotenoid, supplement, shrimp, gastrointestinal tract.

Introduction

The need for sustainable shrimp aquaculture drives studies on the use of probiotics in growing

shrimp, such as whiteleg shrimp (Litopenaeus vannamei) or black-tiger shrimp (Litopenaeus

monodon). Probiotics can have a beneficial effect on the digestive processes in shrimp because

of their ability to synthesize extracellular enzymes such as proteases, amylases, and lipases and

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 to provide growth factors such as vitamins, carotenoids, fatty acids, and amino acids (Araya et al.

2002). Therefore, nutrients are absorbed more efficiently when feed is supplemented with
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probiotics.

The commercial value of shrimps is predominately based on the visual appeal of their body colour. The

red carotenoid, astaxanthin, has been identified as the predominant pigment in Penaeus shrimp (Yamada

et al. 1990). However, these shrimp are unable to produce astaxanthin de novo; only plants and protists

(bacteria, algae, and fungi) are capable of synthesizing carotenoids (Schmidt-Dannert 2000). Therefore,

astaxanthin must be available in either their native habitat or manufactured diet to meet metabolic

nutritional requirements. The red colour of cooked shrimp is produced by the release of the individual

carotenoid prosthetic group (astaxanthin) from the carotenoproteins when denatured by the heat of

cooking. Lack of dietary astaxanthin in cultured shrimp has been shown to cause "blue colour syndrome"

(Latscha 1989). Supplementing shrimp feed with synthesized red-carotenoid astaxanthin is common to

improve colourisation of shrimp. D’ Abramo et al. (1983) have demonstrated that pure carotenoids such

as -carotene, echinenone, and canthaxanthin are converted to astaxanthin in cultured lobsters and that

the level of pigmentation produced by these biosynthetic precursors is related to the proximity to the

astaxanthin end product. In recent years, many research groups have attempted to isolate pigmented

bacteria that can produce carotenoids to develop novel natural food and feed supplements (Pane et al.

1996; Duc et al. 2006; Kaneja et al. 2009; Sy et al. 2013). Yellow, orange, red, and pink Bacillus species

have been isolated from seawater, sand, and soil. For example, Pane and his colleagues have identified a

red-pigmented Bacillus firmus strain producing astaxanthin, which was isolated from a seawater rock

pool. Their result suggests the potential use of this bacterium in aquaculture and in the pharmaceutical

field (Pane et al. 1996). Yoon and his colleagues have isolated two yellow pigmented strains from

seawater and identified them as new species B. aquimaris and B. marisflavi (Yoon et al. 2003). In another

study by Suresh et al. an arsenic-resistant yellowish-orange pigmented bacterium was isolated from a

sand sample obtained from an arsenic-contaminated aquifer. The strain was identified as a new species

named B. indicus (Suresh et al. 2004). Khaneja et al. (2009) isolated several other carotenoid-producing

Bacillus species from seawater, soil, and fermented rice condiment. These included B. marisflavi, B. cibi,

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 and B. altitudinis strains producing yellow and yellow-orange pigments; B. aquimaris and B. pumilus

strains producing orange and orange-red pigments; and some B. firmus strains producing pink and deep
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pink pigments. Carotenoids produced by these strains were determined to have absorption maxima at

455, 467, and 492 nm, corresponding to the visible colours yellow, orange and pink, respectively (Khaneja

et al. 2009). In other studies, yellow pigmented B. indicus HU36 of human intestinal origin and red

pigmented B. firmus GB1 isolated from soil have been investigated for their ability to produce natural

antioxidant carotenoids (Duc et al. 2006; Hong et al. 2008; Khaneja et al. 2009; Cutting et al. 2011); this

was followed by in vivo studies on the bioaccessibility and bioavailability of these carotenoids, which

were found to be even better than those of commercial available synthesized carotenoids (Sy et al.

2013).

Many protocols on the isolation and characterization of pigmented Bacillus species from seawater,

shrimp ponds, and human faeces have been implemented (Yoon et al. 2003; Duc et al. 2006; Khaneja et

al. 2009); however, the isolation of pigmented Bacillus species from gastrointestinal tracts (GITs) of

shrimp, to develop probiotics for use as feed supplements for shrimp themselves, has not been reported.

This is especially important as it is generally recommended that probiotic strains should be isolated from

the GITs of their host; such strains are thought to have the best chance of surviving and colonizing the

intestine, allowing beneficial microbial flora to thrive (Dunne et al. 2001). Therefore, we investigated

whether reintroduction of pigmented Bacillus sp. strains, isolated from the shrimp gut, back into shrimp,

would create beneficial microbiota. Thus, we attempted to isolate pigmented strains of Bacillus species

directly from the GITs of shrimp to screen for the production of high levels of antioxidant carotenoids and

for abundance in the shrimp gut. Spores of the selected strain were produced for laboratory-20scale

blind trials in whiteleg shrimp to evaluate shrimp colourisation (colour score and astaxanthin level),

weight gain, and phenoloxidase activity, reflecting innate immune status.

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 Materials and methods
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Reference strains

Yellow-pigmented B. indicus HU36 and B. subtilis HU58 isolated from human faeces (Duc et al. 2006; Tam

et al. 2009) and non-pigmented laboratory B. subtilis PY79 was used as the reference strain in most

experiments described here; B. cereus ATCC 10876 was used as a control for the haemolysis test.

Preparation of intestinal samples and isolation of pigmented Bacillus colonies

Twelve natural shrimp of different species including L. vannamei and L. monodon were collected

from rivers and coastal regions (Binh Dai, Phu An, Thanh Thuy) in Ben Tre Province, Vietnam.

Each shrimp GIT was prepared to collect the mucosa in 0.9% NaCl and then vigorously

resuspended by vortexing until a homogenous suspension was obtained. To recover heat-

resistant spores, 1 mL of the suspension was heated at 65°C for 20 min and serial dilutions were

made with 0.9% NaCl before plating on tryptone soy agar (TSA) (Oxoid; Hampshire, UK) and

incubating for 1 d at 37°C to obtain individual colonies. For each sample, different pigmented

colonies were picked randomly and transferred to new Difco Sporulation Medium (DSM) agar

plates, and then checked by microscopy for morphological traits and presence of spores.

General methods

Each isolate was grown on DSM agar or in DSM broth (Oxoid; Hampshire, UK) for 48 h and assessed for

sporulation percentage by determining the titre of heat-resistant cells (from 40°C to 80°C, 20 min) versus

the total viable cell count. Bacterial growth was monitored in tryptone soy broth (TSB) (Oxoid;

Hampshire, UK) at 30°C, or at 37°C; absorbance of cell cultures was measured after 24 h at 600 nm

(OD600) using a spectrophotometer Biomate 3 (Thermo Scientific, Waltham, MA, USA). Characterization of

Bacillus strains using microbiological and biochemical methods was performed following Bergey’s Manual

of Systematic Bacteriology (The Firmicutes 1984, 2nd Ed. 2009). The shapes of bacteria and positions of

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 spores of selected strains were observed under a conventional microscope Primo Star (Carl Zeiss,

Oberkochen, Germany). To determine the ability of bacterial isolates to grow under aerobic or anaerobic
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conditions, strains were streaked into test tubes of thioglycolate semi-solid media with 0.3% agar and

then incubated at 37°C for 24 h (Evans and Kloos 1972). Metabolism of glucose and citrate was assessed

using a conventional Voges-Proskauer (VP) reaction and citrate utilization test. To test for haemolysis,

isolates were inoculated on tryptose agar containing sheep blood and incubated for 24 h at 37°C to

observe haemolysis zones. To evaluate antibiotic resistance and sensitivity, antibiograms were obtained

for each strain using the radial diffusion method, according to the recommendations of the National

Committee for Clinical Laboratory Standards (NCCLS 1997). Inhibition zones were measured for 14

common antibiotics as listed in Table 1. Amylase activity was assayed using the starch hydrolysis test on

agar plates containing 1% soluble starch. Proteolytic activity was examined with casein and gelatin

hydrolysis tests on agar plates.

16S rRNA sequence analysis

Total genomic DNA was extracted from Bacillus species overnight LB cultures. The genomic DNA was used

as template for PCR and partial 16S rRNA sequencing of a PCR-amplified 1500 bp fragment using primers

27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1527R: 5′-AAAGGAGGTGATCCAGCC-3′ (Khaneja et al. 2010;

Nguyen et al. 2015). The resultant partial 16S rRNA sequences were assembled and aligned with ApE®

software (The University of Utah, UTAH, USA). Obtained sequences were compared to sequences in the

GenBank non-redundant nucleotide database by BLAST analysis. The closest species were identified and

the per cent identity was recorded. Phylogeny was inferred from aligned nucleotide sequences of the 16S

rRNA genes using MEGA6 software (Tamura et al. 2013). Evolutionary relationships of the Bacillus strains

were estimated using the neighbour-joining method (Saitou and Nei 1987) with 1,000 bootstrap

replicates. Evolutionary distance was computed using the p-distance model and are given in units of

number of base differences per site. Branch lengths are proportional to the amount of evolutionary

change.

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 Pigment extraction and analysis

Pigment extraction and analysis from Bacillus isolates were performed following a previously reported
Accepted Article
method (Khaneja et al. 2010). Extracts (200-L) from 1.2 mg dry weight of cells or 1-mL cultures were

prepared to measure absorbance values at optimal peaks, such as OD460 for the SH8 and SH20 strains,

OD470 for the SH6 and SH14 strains, or OD490 for the SH1, SH4, and SH5 strains. Concentrations of

carotenoids in the extracts were determined from the standard curve of astaxanthin at concentrations

ranging from 1.05 to 16.75 µmol L-1. High performance liquid chromatography (HPLC) analysis of the

selected SH6 strain was performed on 20 AD-UFLC system (Shimadzu, Kyoto, Japan) consisting of a

photodiode array (PDA) detector using a reverse-phase (RP) C30, 5-µm column (250 mm × 4.6 mm)

coupled to a C30 guard column (20 mm × 4.6 mm) (Thermo Scientific, Waltham, MA, USA). The mobile

phase and elution conditions were identical to those described by Khaneja et al. (2010). The carotenoids

were identified by comparison of spectral and chromatographic characteristics with those published for

reference carotenoids (Britton et al. 2004).

Free radical scavenging activity of pigmented extractions

2,2-Diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity was measured following a standard method

(Sharma and Bhat 2009). In brief, individual 1.5-mL extracts from approximately 0.5 g (wet weight) of

vegetative cells were incubated with 500 µL of 250 µmol L-1 1,1-diphenyl-2-picryl-hydrazyl (DPPH) (Sigma,

St. Louis, MO, USA). The DPPH absorbance at 517 nm was measured before and after the reactions.

Inhibition level was calculated using following equation: Inhibition (%) = [(ODcontrol -

ODsample)/ODcontrol]*100. In parallel experiments, 1.5 mL of ascorbic acid (6.25–25 µmol L-1) was added as

a quantitative standard. The experiment was repeated three times.

Production of pigmented spores

Growth of bacterial strains and spore formation were optimized using various media (DSM, LB, TSB), pH

(6–9), and temperatures (25–45°C) in a flask. To induce spore production under the optimized conditions,

Bacillus strains were cultured in suitable medium for 48 h in a fermenter (ANABIO R&D built in-house) at

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 the optimal temperature (30–37°C) and pH (7.0–8.0) for each strain. The purification of spores has been

described previously (Nicholson & Setlow 1990). The purified spore suspensions were then spray-dried at
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160°C at an atomizer speed of 28,000 × g (ANABIO R&D built in-house) for collection of spore powder.

Preparation of feed supplemented with spores or synthesized astaxanthin

Commercial shrimp feed (Uni President, Tainan, Taiwan) was used as the basal diet, supplemented with

either spores or astaxanthin. For selection of the best surviving strain in shrimp guts, a mixture of spores

from five strains including SH1, SH5, SH6, SH8, and SH14 were mixed with feed pellets at equal

concentrations for each spore (>3 × 106 CFU g-1 feed); the feed pellets were then coated with cod liver oil

Seven Seas® (Merck Consumer Health, Darmstadt, Germany). For evaluating the probiotic effects of SH6

spores, feed pellets were supplemented with either B. aquimaris SH6 spores or B. indicus HU36 spores at

concentrations of >3 × 106 CFU g-1 feed, followed by coating the pellets with cod liver oil. As a negative

control, the feed pellets were coated with cod liver oil. As a positive control, the commercialized

Carophyll Pink® (DSM, Heerlen, the Netherlands) powder containing 10% synthesized astaxanthin was

used as supplements. Three grams of Carophyll Pink® powder was dissolved in 30 mL water (70°C) then

sprayed on 600 g commercial shrimp feed (ratio: 5 mg Carophyll powder to 1 g pellets) to have a final

astaxanthin concentration of 0.5 mg g-1 pellets; feed pellets were then coated with the liver oil. The feed

pellets with or without spores were coded and stored at room temperature for the duration of the

experimental trials in whiteleg shrimps.

Trials of probiotic treatment in whiteleg shrimp on a laboratory scale

Forty-five-day-old whiteleg shrimp (Litopenaeus vannamei), weighing approximately 3 g, were used for

probiotic treatment on a laboratory scale. Approval of animal care and use protocol form for conducting

trials in shrimps is not required as shrimp species are invertebrates. Trials in whiteleg shrimp followed

biosecurity guidelines of the Department of Aquaculture, Vietnam Ministry of Agriculture and Rural

Development

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 For the in vivo survival assays of Bacillus spores in shrimp guts, shrimp were divided into two groups (n =

10 shrimp per tank). Shrimp in each group were fed as follows: commercial feed only (control group),
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feed supplemented with a mixture of SH1, SH5, SH6, SH8, and SH14 spores at >3 × 106 CFU g-1 pellet

(Spore group). After 7 d of feeding, five shrimp guts from each group were taken at each time point: (i) 3

h after the last feeding and (ii) 8 h after the last feeding. Shrimp guts were prepared to collect the

mucosa in 0.9% NaCl and then vigorously resuspended by vortexing until a homogenous suspension was

obtained. Serial dilutions with 0.9% NaCl were made before plating on TSA and incubating for 1 d at 37°C

to obtain individual colonies. Colonies were observed based on their typical morphologies and colours,

and then counted to calculate their initial population in shrimp guts.

For evaluating the probiotic activity of SH6 spores, a blind trial was performed. Shrimps (n = 30 per tank)

in each group were fed with coded feed pellets as follows: commercial feed only (control group), feed

supplemented with SH6 spores at >3 × 106 CFU g-1 pellet (SH6 group), feed supplemented with HU36

spores at >3 × 106 CFU g-1 pellet (HU36 group), and feed supplemented with the commercialized

Carophyll® at 0.5 mg synthesized astaxanthin g-1 pellet (Carophyll group). The shrimp were maintained in

a water bath thermostatically controlled at 28°C and fed 5 g feed per day. For measuring shrimp weight

and PO activity, ten shrimp (n = 10) from each group were taken and data were recorded after 14 and 28

d. The SD was calculated based on data collected from ten shrimp. For comparing colours, five shrimp (n

= 5) in each group were taken after 28 d, and then boiled comparing colour using the Roche index,

SalmoFan™ standard colour (Brun and Frédéric 2006). For measuring astaxanthin concentration, five

shrimp (n = 5) in each group were taken and data were recorded after 28 d. SD was calculated based on

data collected from five shrimp.

For evaluating the safety of the SH6 probiotics, a similar experimental design was set up for the four

groups (n = 30 per tank; 2 tanks per group) feed was supplemented with spores at an extremely high

concentration (>1 × 109 CFU g-1 pellet). Survival of shrimp in each group was recorded after 7, 14, 21, and

28 d for calculation of survival rate at each time point. SD was calculated based on data collected from

two tanks.

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 Extraction and measurement of astaxanthin in shrimp

Shrimp (n = 5) from each experimental group were treated as follows. The chitin shell was removed, and
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the shrimp were pulped well in liquid nitrogen. Pigments, including astaxanthin, were extracted from 3-g

shrimp samples by adding 2 mL methanol, followed by 4 mL chloroform. The suspension was incubated

on ice for 20 min to minimize degradation of astaxanthin. To the suspension, 1 mL water was added and

the sample was vortexed for 15 s. To form a partition, the suspension was centrifuged for 3 min at 10,000

× g. The lower phase (organic hypophase) was collected and the upper phase (aqueous hyperphase) re-

extracted twice with chloroform until no colour was observed in the debris. Protein contamination was

removed from extractions, which were concentrated in PBS. The extracts were stored at -20°C at this

stage. Before measuring, 1-mL samples were concentrated five-fold using 0.2 mL

acetonitrile/methanol/dichloromethane (ratio 75:20:5) and injected onto the HPLC column. Astaxanthin

in extract (A) was first separated and then detected online, using the Waters Alliance (Milford, MA, USA)

HPLC system with an online photo diode array (PDA 2996 at 450 nm) detector. Injections were made, and

separations performed on a reverse phase (RP) C18 (2) 5-m column (150 mm × 4.6 mm) (Agilent, San

Francisco, CA, USA). The mobile phase, acetonitrile/methanol/dichloromethane (75:20:5), eluted at a rate

of 1.5 mL min-1, was maintained at a constant temperature of 30°C. A defined sample of astaxanthin (2.68

µmol L-1), used as control, was measured at the same time to compare retention time (Rt).

Concentrations of astaxanthin in the extract were measured based on height and area of peaks. Total

astaxanthin concentration per 1 gram of shrimp Cs (μg g-1 shrimp-1) was calculated using the equation: Cs

= k × 1.67 × Ce/W, where k was the dilution ratio of the samples (k = 6), Ce was astaxanthin concentration

in the extract (µmol L-1), and W was the weight of shrimp (g).

Phenoloxidase (PO) activity measurement

PO activities in shrimp were measured following the protocol of a previous report by Luciane et al. (1997)

and Nguyen et al. (2014).

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 Statistical analysis

Data of astaxanthin concentrations, weight gains, and PO activities among the four treatment groups
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were compared using a Student’s t-test at a significance level of 0.05, 0.01, 0.001, and 0.0001. Statistical

analyses were performed using the Analysis ToolPak in Microsoft Excel Software. An F-test for two-

sample variance was used before performing a t-test for two unpaired samples. ANOVA single factor

analysis was used to compare more than two samples. Survival rate data among the four treatment

groups were compared using a Chi-Squared X2 test and P values were considered significant at a level of

<0.05.

Results

Characterization of pigmented Bacillus strains isolated from shrimp gastrointestinal tracts

We isolated 23 pigmented, spore-forming aerobic strains of bacteria from the guts of 12 shrimp collected

from rivers and coastal regions of Ben Tre (Vietnam). To develop heat-stable probiotics as feed

supplements to colourise shrimp and improve shrimp health, we screened strains of Bacillus. We first

screened strains based on the following criteria: (i) diverse pigment found in bacterial strains ranging

from yellow, orange, red and pink, (ii) high absorbance value of methanol-chloroform extracted

carotenoids at a typical UV-Vis wavelength of 400–550 nm (equivalent to more than 250 µmol L-1), and

(iii) a high sporulation efficiency of more than 85%. Our primary investigation of the 23 pigmented

Bacillus strains indicated that they had much different characteristics. As shown in Table 2, we screened

eight representative pigmented, spore-forming strains named SH1, SH4, SH5, SH6, SH8, SH12, SH14, SH20

having different colours and peaks of absorbance wavelengths. For example, the pink-red extract of SH1

had the highest peak at 495 nm, the red-orange extract of SH6 had the highest peak at 468 nm, and the

yellow extract of SH8 had three peaks at 435, 465 and 487 nm. The data were confirmed by a negative

absorbance value obtained from the reference non-pigmented B. subtilis PY79 strain and positive VIS

absorbance peak of extract from strain HU36 at 454 nm (data not shown). Among the eight strains, SH1,

SH5, SH6, SH8, SH12, and SH20 produced high levels of carotenoids equivalent to 250 g mL-1 or higher.

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 All strains were able to sporulate quickly in DSM medium with sporulation efficiency ranging from 85–

100%. Most of the pigmented spores were not very heat-resistant, except that of SH8, which retained its
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haft-count (50% survival) after treatment at 80°C for 20 min. With lower temperature treatment (55°C for

20 min), 50% survival of SH5, SH6, and SH12 spores were retained, whereas live counts of SH1, SH14, and

SH20 spores remained at only 1–10%.

The eight-pigmented strains were further characterized based on physiological and biochemical

properties according to Bergey’s Manual of Systematic Bacteriology and 16S rRNA sequence analysis

(Table 2). In all strains, width of vegetative cells was less than 1 µm. The spore position and

characteristics of isolates were different from each other; some were sub-terminal, and others were

terminal and central. All strains were able to grow aerobically at 30°C, the temperature for culturing

shrimp. Only the SH8 strain was able to growth in both aerobic and anaerobic conditions. In the presence

of 6.5% NaCl, all strains also grew well. Most strains were able to hydrolyse starch at different levels

except SH8. Different strains exhibited different levels of caseinase, except SH5. In the lipase test, only

three strains, SH1, SH6, and SH8, could hydrolyse Tween 80, as indicated by observable opaque halos

around these colonies. Among the eight screened strains, SH6 exhibited strong amylase, caseinase and

lipase activities. For assessing the safety and toxicity of these eight pigmented strains, we used sheep

blood agar and observed haemolysis zones; all isolates were found to be negative in haemolysis. All

strains were evaluated for their antibiotic sensitivity to a panel of antibiotics, including those highlighted

by the European Food Safety Authority (EFSA 2005) and recommended by the NCCLS (1997). As shown in

Table 1, all strains were sensitive to most of the 14 tested antibiotics. Among these, 11 antibiotic groups

were included with different modes of action, including a β-lactam (ampicillin), a quinolone

(ciprofloxacin), a lincosamide (clindamycin), a macrolide (erythromycin), four aminoglycosides

(gentamicin, streptomycin, kanamycin, neomycin), a glycopeptide (vancomycin), and rifampicin,

tetracycline, chloramphenicol, and cotrimoxazol (trimethoprim and sulfamethoxazole). Unexpectedly, all

strains were either resistant or moderately sensitive to clindamycin; SH4 was moderately sensitive to

erythromycin and rifampicin, and SH12 was resistant to rifampicin. To determine species identity, strains

were assessed by 16S rRNA sequencing and BLAST analysis. Identification at the species level using 16S

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 rRNA sequence analysis indicated that strains were closely related to Bacillus aquimaris (SH1, SH4, SH6,

SH14, SH20), B. firmus (SH5, SH12), and B. marisflavi (SH8) with identity scores equal to or greater than
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0.98.

Assessing all characterization data listed in Table 1 and 2, we determined that SH6 was the most

promising strain due to the following: (i) high production level of carotenoids, (ii) sporulation efficiency of

100%, and (iii) good production of three kinds of enzymes including amylase, protease and lipase. By

analysing the phylogenetic relationship (based on rRNA sequences) between SH6 (GenBank accession No:

KF443807) and other reference strains (Fig. 1), SH6 was confirmed to belong to the B. aquimaris species.

Antioxidant activities of pigmented extracts

An important criterion for identifying carotenoids is antioxidant activity. Thus, to confirm that the

pigments found in selected strains were carotenoids, we used a standard scavenging assay of free DPPH

radicals. As shown in Fig. 2, pigmented extracts of four isolated strains including SH1, SH5, SH6, and SH8

exhibited significantly high scavenging activity 76–98%, whereas the four remaining extracts of SH4, SH12,

SH14, and SH20 had less than 70% scavenging activities (data not shown). The pigmented extracts of SH5

and SH6 had the highest DPPH radical scavenging activities (SH5: 98.5 ± 0.2%; SH6: 87.0 ± 0.9%) among all

strains, which was equal to that of ascorbic acid (96.0 ± 0.2% at a concentration of 9.38 µmol L-1) and

higher than that of the positive control HU36 (78.4 ± 0.9%). The data were confirmed using non-

pigmented B. subtilis PY79 as a negative control, wherein no detectable DPPH scavenging activity was

observed. Based on this data, we concluded that the pigments found in the eight selected strains showed

the potential to be antioxidant carotenoids, and that carotenoids of the SH5 and SH6 strains exhibited

stronger antioxidant activity than those of the other strains (SH1: 77.1 ± 1.1%; SH8: 84.8 ± 0.5%).

Survival and colonisation of strain SH6 in shrimp intestines

After the in vitro characterization of Bacillus strains, we wanted to screen strains for the in vivo

characteristics of survival and colonization in shrimp guts, which are important probiotic properties. For

convenience, we selected five strains with distinguished colony morphology, including SH1, SH5, SH6, SH8,

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 and SH14, for assessing viability in shrimp guts. We set up two time points for counting bacterial

populations in the shrimp gut, 3 h and 8 h after the last feeding. This was because transit time of feed in
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the shrimp intestine is 4–5 h. At 3 h, our results showed that colonies of the SH6 strain were the most

abundant, accounting for 70% of the total microbiota (Fig. 3A). Meanwhile, SH1 accounted for only for

30% of the population, and colonies of SH5, SH8, and SH14 were not detectable. Thus, it was concluded

that SH6 spores survive better than spores of other pigmented strains and were the most abundant

population in the shrimp gut during the passage of feed. To further access adhesion and colonisation

capability of pigmented Bacillus strains in the shrimp intestinal mucosa, we collected spores 8 h after

feeding, when feed was completely extruded from the shrimp intestine. The results (Fig. 3B) indicated

that there was an increase in non-pigmented Bacillus colonies (~79%) and a significant reduction in

pigmented Bacillus colonies (21%). However, colonies of the SH6 strain were still the most abundant

among pigmented strains (20% of the population), followed by SH1 (1% of the population). Thus, even

though it was not comparable to that of non-pigmented Bacillus strains, colonisation of the SH6 strain in

the shrimp gut was still the best among pigmented Bacillus strains. As SH6 showed promising probiotic

properties in vitro (as mentioned above) and in vivo (based on colonisation assays), we decided to

characterize the SH6 strain for the development of probiotics for use in whiteleg shrimp.

Carotenoid profiling of SH6 and characterisation of SH6 spores

To clarify the type of carotenoids produced by the SH6 strain, the carotenoid extract was analysed by

HPLC-PDA. Typical HPLC chromatographic profiles for the SH6 extract were recorded at 450 nm and are

presented in Fig. 4. The physical characteristics of the carotenoids detected were concordant with the

visible colour of the orange-red SH6 colonies, as fraction no. 3 (Rt at 29.437 min), showing absorbance

peaks at 492 and 522 nm (orange, pink, red), was the most abundant.

Bacillus strains are conventionally thought to form heat resistant spores. However, our primary data,

shown in Table 2, indicates that pigmented Bacillus strains do not possess this ability, as most were not

stable during heat-treatment at 80°C for 20 min. However, to utilize SH6 as a probiotic strain in feed

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 production, we needed to assess sporulation and its exact level of heat-stability. For this, we attempted

to culture SH6 spores in DSM broth for 48 h using a fermenter; the resulting spore suspension was spray-
Accepted Article
dried to obtain a heat-stable spore powder. As controls, we also produced spore powders of the medium-

sporulating B. indicus HU36 yellow-pigmented strain (about 50% sporulation efficiency) and the high-

sporulating non-pigmented B. subtilis HU58 strain (almost 100% sporulation efficiency), both reference

strains. As expected, from 50 L of we obtained 500 g of red-orange SH6 spore powder at the very high

concentration of 4.5 × 1011 CFU g-1 (Fig. 5A) with almost 100% spore purity (Fig. 5B). This concentration

was almost equal to that obtained for HU58 spores (5.8 × 1011 CFU g-1), and was 4-fold higher than that

obtained for HU36 spores (data not shown). To explain this difference in spore concentration, the heat-

stability of the SH6 spores was compared to that of HU36 and HU58. As shown in Fig. 5C, HU36 spores

were surprisingly heat-sensitive as the live count was reduced by 8% at only 50°C. In contrast, SH6 spores

were stable at 50°C (60% survival), and 15% survival was maintained at 70°C, equivalent to 50% survival

at 55°C. However, the heat-stability of SH6 spores was much less than that of the B. subtilis HU58 spores

(55% survival at 80°C, 23% survival at 90°C); the heat-stability of HU58 has been characterised for use as a

food ingredient (Permpoonpattana et al. 2012).

Improved pigmentation of whiteleg shrimp by supplementing with SH6 spores

We first evaluated the effects of SH6 probiotics on the pigmentation of whiteleg shrimp. Four groups

were designed for pigmentation assays, including SH6 (fed with SH6 spores at >3 x 10 6 CFU g-1 pellet),

HU36 (fed with the reference HU36 spores at >3 x 106 CFU g-1 pellet), control (without supplements), and

Carophyll (fed with commercial Carophyll®, at 0.5 mg synthesized astaxanthin g-1 pellet). After 4 weeks of

feeding, shrimp colour after boiling was compared for the four groups (Fig. 6A). The Carophyll group had

the reddest colour (score: 24–25), followed by the SH6 group (score: 21–23), the HU36 group (score: 21–

23), and the control group (score: 20–22).

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 To correlate the relationship between the shrimp colour and the presence of astaxanthin in shrimp

tissue, we determined the astaxanthin levels in shrimp of each group. As shown in Fig. 6B, we found that
Accepted Article
the level of astaxanthin in the SH6 group (0.69 ± 0.03 µg g-1 shrimp-1) was 2.7-fold higher than that of the

control (0.25 ± 0.09 µg g-1 shrimp-1) (P < 0.05) but 2.3-fold lower than that of the Carophyll group (1.59 ±

0.08 µg g-1 shrimp-1) (P < 0.01). Unexpectedly, the difference in astaxanthin levels between the HU36

group (0.47 ± 0.13 µg g-1 shrimp-1) and control group was not statistically significant (P > 0.05).

Weight gain in whiteleg shrimp by supplementing with SH6 spores

At time points of two weeks and four weeks, there was an apparent disparity between spore and non-

spore groups. Shrimp in both the SH6 and HU36 groups gained weight much faster than the control and

Carophyll groups (P < 0.0001) at both time points. For example, the SH6 group gained 5.36 ± 0.3 g and

7.18 ± 0.24 g at two weeks and four weeks, respectively; the HU36 group gained 5.22 ± 0.19 g and 6.71 ±

0.21 g at two weeks and four weeks, respectively. By contrast, the control group gained only 4.26 ± 0.35

at two weeks and 5.32 ± 0.26 g at four weeks (Fig. 7A). In the non-spore Carophyll group, shrimp were

heavier (4.79 ± 0.22 g and 6.00 ± 0.26 g at two weeks and four weeks, respectively; P < 0.0001) than

those in the control group, but not as heavy as those in the SH6 group.

Increased immune-related phenoloxidase activity by supplementing with SH6 spores

Shrimp have no adaptive memory, and therefore depend on innate defence systems to protect against

pathogens. Phenoloxidase (PO) activity is the parameter most used to represent the innate immune

response, which protests against pathogenic attacks (Sarathi et al. 2007; Nguyen et al. 2014). We found

that PO activity in the SH6 group was clearly higher than that of other groups after 2 weeks of continuous

feeding (OD490 = 0.134 for the SH6 group vs. 0.09 for the HU36 group, 0.076 for the control group, and

0.081 for the Carophyll group; P < 0.01). At week four, an 85% increase in PO activity was observed in the

SH6 group (OD490 = 0.265) compared to that of the control group (OD490 = 0.143) (P < 0.01), whereas the

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 PO activities in the HU36 (OD490 = 0.1.21) and Carophyll groups (OD490 = 0.1.21) were not significant

different (P > 0.05) from those of the control group (Fig. 7B).
Accepted Article
Safety of SH6 spores

SH6 is a new probiotic strain that has not been well documented in the Qualified Presume as Safe (QPS)

list. We therefore determined the toxicity dose of SH6 spores in whiteleg shrimp through increasing the

dose of probiotics up to >1 x 109 CFU g-1, which was approximately 300-fold higher than the probiotic

concentration (>3 x 106 CFU g-1). After 2 and 4 weeks of feeding, we found that the SH6-fed or HU36-fed

groups did not die, but experienced an even better survival rate (SH6: 84.9 ± 3.6% and HU36: 79.4 ± 0.9%),

compared to that of the control group (77.4 ± 2.4%) (Fig. 8). The Carophyll-fed group also had a good

survival rate of 83.5 ± 4.9%. Nevertheless, the increases in survival rates for SH6-fed (9.5%), HU6-fed

(2.3%), and Carophyll-fed groups (7.4%) in comparison to those of the control group were not statistically

significant (P > 0.05). The data confirms that the lethal doses of both SH6 and HU36 spores in whiteleg

shrimp were above 1 x 109 CFU g-1 for up to a 4-week treatment.

Discussion

As mentioned in the introduction to this article, a number of publications have reported the identification

and characterisation of pigmented bacilli isolated from seawater, water, and mud in shrimp ponds, soil,

and human faeces. To our knowledge, this is the first study to isolate and characterise carotenoid-

producing Bacillus strains from the shrimp GIT. To develop a novel supplement for promoting the

colourisation and health of shrimp, we hypothesized that probiotic strains for shrimp should be isolated

from the intestinal tracts of shrimp, because these strains have the best chance of surviving and

colonising in the intestine. We have tried isolating and screening spore-forming bacteria, which belong to

the genus Bacillus, because their spores are normally stable in acidic conditions of the stomach. Thus,

they can pass through the stomach unscathed and subsequently germinate in the small intestine and

grow (Tam et al. 2006). Then, we can characterize and screen the best strain of pigmented bacteria,

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 which produce carotenoids, to develop probiotics for improving the colour of whiteleg shrimp and

providing antioxidants and nutrition for shrimp. Here, whiteleg shrimp was chosen for trials because it is
Accepted Article
one of the most exported species from Vietnam (Lan 2013) and its colour change is easy to observe.

Based on this hypothesis, we set up a protocol to isolate pigmented colonies from GIT of shrimp. All

shrimp were collected from wild rivers and coastal regions in the Ben Tre province to avoid the use of any

commercial probiotics and to obtain biodiversity of the intestinal microorganisms.

Among 23 coloured isolates, eight strains were selected from in vitro conditions due to their potential

properties for development of a novel heat-stable supplement for shrimps. These include (i) high

sporulation efficiency (>85%) of heat-stable spores (>10% survival at 55°C treatment for 20 min); (ii)

typical UV-Vis spectra of produced carotenoids (400–550 nm); (iii) free radical scavenging activity to

DPPH of their extracts (>90%); (iv) high production of beneficial digestive enzymes such as amylase,

caseinase, and lipase; (v) and growth at 30°C, which is the optimal temperature of shrimp ponds. The

data of negative haemolysis and sensitivity to 14 common antibiotics of the eight strains indicate their in

vitro safety. Of the eight strains, B. aquimaris SH6 strain was selected because it was shown to possess

several interesting properties. SH6 produced antioxidant carotenoids (with 90% scavenging activity of

DPPH free radicals) at the high level of 423 µg [g DW]-1. It also had high sporulation efficiency (100%)

yielding high concentrations of spores at 4.5 × 1011 CFU g-1 in the sprayed powder. The spores were also

more heat-stable (15% survival at 70°C for 20 min) than those of the reference HU36 strain of human

intestinal origin. Finally, this strain had the ability to survive and colonise in the whiteleg shrimp GIT

(comprising 70% of the microflora population 3 h after feeding). SH6 remained only 20% viable after only

8 h when feed was completely extruded from the shrimp intestine. This evidence suggests the human

safety of SH6 when probiotics of this strain are used as a feed supplement for shrimp. Given that shrimp

are continuously fed SH6 spores, then changed to un-coated feed pellets just several days before

harvesting, SH6 spores and bacteria would be virtually absent from the flora of the shrimp gut and would

not cause any toxicity to humans.

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 We further characterised the HPLC chromatographic profile of extracts of SH6 bacteria and the heat-

stability of the SH6 spores, which are basic properties of carotenoid producing spore formers. The
Accepted Article
absorbance spectra of the collected fractions of SH6 extracts, with three peaks at approximately 400–500

nm, suggests that the SH6 strain produced at least three different carotenoid types. Comparison with

reference spectra, identified the carotenoids in the three fractions, no. 1-3, as being closely related to

keto/hydroxyl derivatives of -carotene (Britton et al. 2004; Le et al. 2006). This finding also suggests that

carotenoids are the sole pigments responsible for the colour of SH6. In terms of the heat-stability of

spores, although SH6 spores (50% survival at 55°C) were less heat-stable than those of the non-

pigmented B. subtilis HU58 spores (50% survival at 85°C), they were much better than those of yellow-

pigmented HU36 spores (50% survival at 41°C). This result demonstrates that the SH6 spores could

survive industrial feed processing to a greater extent than those of HU36, as this process normally

requires heat treatment at approximately 70–80°C for 15–20 min. For mass production of SH6 spores as a

commercial shrimp feed ingredient, one might scale-up SH6 spore production using pilot- or industrial-

scale fermenters, followed by concentration and spray-drying to obtain pigmented spore powder.

In a laboratory-scale trial in whiteleg shrimp, we used a primary dose of SH6 spores at >3 x 106

CFU g-1 pellets, in accordance with previous reports regarding the effective doses of probiotics

for general use in animals, and for shrimp specifically (Castexa et al. 2008; Tran et al. 2013,

Nguyen et al. 2015). As a result, SH6 showed greater ability to colourise shrimp guts and

increased astaxanthin production by three-fold (a score of 21–23 with SH6 vs. 20–22 with the

control group), compared to those of the control group. However, the SH6 group had a lower

colour score than the Carophyll group (score: 24–25). The positive correlation between shrimp

pigmentation and astaxanthin concentration in shrimp tissue of the four groups (in descending

order, specifically the Carophyll group, SH6 group, HU36 group, and control group, confirmed

that astaxanthin plays a key role in shrimp pigmentation (Chein and Jeng 1992; Brun and

Frédéric 2006). This result is similar to that of Yamada et al. (1990); therein the level of tissue

astaxanthin in groups fed β-carotene or cantaxanthin was lower than that of a group fed

astaxanthin, but higher than that of a group fed an exclusive carotenoid diet. Taken together, we

can conclude that in terms of shrimp colourisation and astaxanthin level, supplementation with

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 SH6 spores was less efficient than supplementation with Carophyll (astaxanthin), but was slightly

better than HU36 spores and obviously better than uncoated feed.
Accepted Article
In terms of weight gain, the SH6 group was heaviest (7.18 ± 0.24 g), followed by the HU36 group (6.71 ±

0.21 g), the Carophyll group (6.00 ± 0.26 g), and the control group (5.32 ± 0.26 g). The improvement on

weight gain in groups treated with carotenoids or carotenoid-producing bacteria over the control group

can be explained by the fact that carotenoids may perform some physiological function as an intracellular

oxygen reserve in low dissolved oxygen conditions (Chein and Jeng 1992). Thus, the dietary carotenoid

requirement in aquatic animals, which survive in low dissolved oxygen conditions, may by higher than

that for species living in normal conditions. The SH6 and HU36 groups gained more weight than the

Carophyll group because Bacillus strains not only produce carotenoids, but also produce beneficial

enzymes such as amylase and caseinase to maximise the digestion of shrimp feed (Table 2 of this work;

Duc et al. 2006). This could result in the observed 34% and 26% increased weight gain, by SH6 and HU36

strains, respectively, compared to that of the control group, after 4 weeks of feeding. The better weight

gain of the SH6 group compared to that of the HU36 group might be due to better colonisation and

growth of SH6 in shrimp guts (Fig. 3), which would result in better digestive health.

The most common parameter reflecting the innate immune response of shrimp is active phenoloxidase

(PO). PO catalyzes the oxidation of tyrosine to produce toxic quinones and other short-lived reaction

intermediates leading to the formation of melanin. Melanin then binds to the surface of bacteria and

increases the adhesion of hemocytes to bacteria, thus accelerating their removal through the formation

of small nodes (Cerenius and Soderhall 2004). In this study, the slight increase in PO activity over time for

all groups (from week two to week four) suggests that the immune system was improved during shrimp

maturation. Similar data was previously reported by Nguyen et al., in which increasing PO activities in

whiteleg shrimp groups from day 0 to day 14 was observed during feeding with pellets of either uncoated

or coated spores of B. subtilis PY79 reference strain (Nguyen et al. 2014). The effective 85% increase in

PO activity in the SH6 group can be attributed to the immunostimulatory effects of the cell wall

components of this strain, such as β-glucan and lipopolysaccharide (Rengpipat et al. 2000; Gullian et al.

2004). Alternatively, mucosal immunity in the shrimp might have been improved through the efficient

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 colonisation of B. aquimaris SH6 in the shrimp gut. This could nullify the deregulated and suppressed

immune responses induced by pathogenic bacteria, and optimize immune parameters (Gatesoupe 1999).
Accepted Article
In conclusion, the probiotic properties of the SH6 strain of shrimp origin conferred greater health benefits

to shrimp than those of the HU36 strain of human origin in terms of shrimp colourisation, weight gain,

and immune enhancement. In comparison to conventional supplementation with astaxanthin in shrimp

feed, supplementation with SH6 spores was more advantageous in terms of weight gain and immune

enhancement, although less efficient in terms of colourisation. Biosafety data indicate that SH6 spores, at

a 300-fold higher concentration than that of the conventional probiotic concentration, did not cause

toxicity to shrimps. Thus, SH6 shows potential as a carotenoid-producing probiotic strain for further

development of a novel feed supplement. Further interesting questions to be addressed are how SH6

spores germinate, proliferate, and interact with the intestine of whiteleg shrimp to produce carotenoids,

to confer these beneficial effects. In addition, dose- and time-dependent trials on the effects of SH6

probiotics in whiteleg shrimps are necessary to find an optimal feeding regime applicable for shrimp

aquaculture.

Acknowledgements

This work was supported by a grant from Ministry of Science and Technology, Key Laboratory of Enzyme

and Protein Technology (code KLEPT.12.03) to N.T.V.A. We thank Simon M. Cutting for the kind gift of the

reference strains, Vu T. M. Duc and Tran T. My for technical assistance, Le H. Dung for measuring

carotenoids and astaxanthin levels, Do M. Ha for statistical analyses, Pham T.T. Huong and Bui T. V. Ha for

fruitful discussion.

Conflict of interest

The authors report no declarations of interest.

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Accepted Article
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Table 1: Antibiotic susceptibility of pigmented Bacillus strains from shrimp GI tracts

Antibiotic discs* SH1† SH4† SH5† SH6† SH8† SH12† SH14† SH20†

43.23 ± 0.42 31.73 ± 0.00 46.74 ± 0.33 22.44 ± 1.25 38.51 ± 0.49 47.55 ± 0.16 36.98 ± 0.19 18.60 ± 0.55
Ampicillin (10)
S S S S S S S S

Chloramphenicol 29.18 ± 0.21 20.77 ± 0.35 31.02 ± 0.30 26.12 ± 0.92 28.83 ± 0.15 31.61 ± 0.14 27.01 ± 0.78 18.83 ± 0.50

(30) S S S S S S S S

38.80 ± 0.45 17.10 ± 0.10 35.62 ± 0.58 30.87 ± 0.53 27.87 ± 0.21 31.48 ± 0.31 47.08 ± 0.00 32.67 ± 0.47
Ciprofloxacin (5)
S S S S S S S S

14.89 ± 0.01 12.70 ± 0.42 14.80 ± 0.01 9.57 ± 0.09 8.11 ± 0.02 18.22 ± 0.01 10.35 ± 0.20 17.83 ± 0.05
Clindamycin (2)
R R R R I I R R

Co-trimoxazole 33.72 ± 0.07 23.97 ± 0.22 30.60 ± 0.30 24.63 ± 0.41 30.70 ± 0.22 29.02 ± 0.01 43.52 ± 0.67 26.74 ± 0.25

(25) S S S S S S S S

21.18 ± 0.12 20.56 ± 0.30 30.55 ± 0.10 22.55 ± 0.61 25.30 ± 0.04 29.28 ± 0.17 40.80 ± 1.54 28.01 ± 0.41
Erythromycin (15)
I I S S S S S S

29.02 ± 0.29 19.15 ± 1.19 24.57 ± 0.00 19.24 ± 0.46 20.80 ± 0.07 26.49 ± 0.00 31.89 ± 0.45 25.01 ± 0.14
Gentamicin (10)
S S S S S S S S

28.63 ± 0.16 17.49 ± 0.57 30.79 ± 0.06 25.33 ± 1.19 24.90 ± 0.63 28.54 ± 0.01 36.75 ± 0.87 23.94 ± 0.15
Kanamycin (30)
S S S S S S S S

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 27.27 ± 0.67 17.14 ± 0.42 24.38 ± 0.01 17.15 ± 0.05 27.79 ± 0.24 21.31 ± 0.20 25.03 ± 0.30 22.55 ± 0.18
Neomycin (30)
S S S S S S S S
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32.59 ± 0.26 16.58 ± 0.04 36.73 ± 0.32 15.51 ± 0.35 30.70 ± 0.37 33.48 ± 0.30 29.14 ± 0.86 15.81 ± 0.34
Rifampicin (30)
S I S S S R S S

27.87 ± 0.33 14.28 ± 0.00 28.19 ± 0.01 29.40 ± 1.47 16.60 ± 0.26 25.85 ± 0.16 29.65 ± 0.58 21.00 ± 0.14
Streptomycin (10)
S S S S S S S S

30.79 ± 0.18 26.74 ± 0.48 33.78 ± 0.21 28.28 ± 0.36 31.01 ± 0.05 33.41 ± 0.40 40.16 ± 0.00 23.27 ± 0.21
Tetracycline (30)
S S S S S S S S

37.48 ± 0.00 6.45 ± 0.00 34.38 ± 0.16 24.01 ± 0.73 6.76 ± 0.00 31.52 ± 0.27 50.06 ± 0.54 30.28 ± 0.07
Trimethoprim (5)
S S S S S S S S

19.57 ± 0.01 14.77 ± 0.26 19.51 ± 0.08 16.65 ± 0.44 16.70 ± 0.11 20.44 ± 0.06 24.29 ± 0.57 13.74 ± 0.09
Vancomycin (30)
S S S S S S S S

S, sensitive; I, intermediate; R, resistant.

*
Antibiotic-impregnated discs (6 mm) with amount in µg shown in brackets.

†
Average diameter of inhibition from three individual experiments.

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 Table 2: Characterisation of pigmented Bacillus strains isolated from shrimp GI tracts
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Strains SH1 SH4 SH5 SH6 SH8 SH12 SH14 SH20

Colour* PR PO PO RO Y PO YO RP

UV-Vis spectral
495 494 490 468 435, 465, 487 504 470 460
characteristics (nm)

Carotenoid production ++++ +++ ++++ ++++ ++++ ++++ ++ ++++

Sporulation (%) 85 80 95 100 100 95 80 90

Survival during heat

++ + +++ +++ ++++ +++ + ++
treatment†

Size of bacteria‡ <1 μm <1 μm <1 μm <1 μm <1 μm <1 μm <1 μm <1 μm

Spore position§ S S T C S T S T

Aerobic + + + + + + + +

Anaerobic - - - - + - - -

Growth at 30°C + + +++ +++ ++ +++ + +++

6.5% NaCl + + + + + + + +

Amylase ++ ++ +++ ++ - ++ ++ ++

Caseinase + ++ - ++ + + + +

Lipase ++ + - ++++ ++ + - -

Haemolysis γ γ γ γ γ γ γ γ

VP Test - + + - - - - -

B. aquimaris B. aquimaris B. firmus B. aquimaris B. marisflavi B. firmus B. aquimaris B. aquimaris

¶
Closest match
(0.99) (0.99) (0.98) (0.99) (0.99) (0.98) (0.99) (0.99)

*Colour of vegetative cell, P - pink, R - red, Y - yellow

†
Heat treatment at 55°C, 20 min

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 ‡
Width size of vegetative cell

§
T: Terminal, S: Subterminal, C: Central
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¶
Using 16S rDNA sequence analysis in this work. The similarity score is shown in brackets.

-, negative; +, weak or positive; ++, average; +++, good/high; ++++, very good/very high

Figure legends

Figure 1. Phylogenetic relationship between the selected pigmented Bacillus strains. Dendrograms of

strains based on 16S rRNA sequence alignment using MEGA6 software. A selected Bacillus strain is

highlighted in bold and GenBank accession numbers are shown in brackets. Statistical (bootstrap) values

and a scale bar representing evolutionary distance are shown. The 16S rRNA gene sequence of the lactic

acid bacterium Lactobacillus acidophilus, an out-group of the Bacillus genus, was used as the root of the

phylogenetic tree.

Figure 2. Inhibition of DPPH free radical formation by pigmented extracts. (■) Samples include extracts

of SH1, SH5, SH6, SH8, and reference strains (HU36, PY79). ( ) Control was ascorbic acid at

concentrations of 18.75 µM, 9.38 µM, and 4.69 µM. Inhibition(%) = [(ODcontrol -ODsample)/ODcontrol]*100.

Figure 3. Survival and colonization of pigmented Bacillus strains in the shrimp gut. Panel A. Pie chart of

the major population of SH6 & SH1 strains isolated from shrimp guts after 3-h-feeding of the

experimental group (fed with a mixture of SH1, SH5, SH6, SH8, and HU36 spores at >3 x 106 CFU g-1).

Panel B. Pie chart of population (B2) of major non-pigmented Bacillus strains and SH6 and SH1 strains

isolated from shrimp guts after 8-h-feeding in the experimental group.

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 Figure 4. High-performance liquid chromatography profiles of SH6 pigment extract. UV/VIS recorded at

450 nm indicating two carotenoid types at low absorbance levels of peak no. 1, Rt at 20.109 min, λ max
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(484; 496), peak no. 2, Rt at 22.239 min, λ max (466; 489), and one carotenoid type at high absorbance

level of peak no. 3, Rt at 29.437 min, λ max (492; 522). Peaks having higher absorbance values were

underlined.

Figure 5. SH6 spores and their heat resistance. Panel A. Spray-dried powder of orange SH6 spores (>3 ×

1011 CFU g-1). Panel B. Microscopy observation of 100% SH6 spores in the powder. Panel C. Heat stability

of SH6 in comparison to that of HU58 and HU36 spores. Heat-counts of (■) SH6, ( ) HU58, and (□) HU36

after treatment at various temperatures ranging from RT to 90°C. Data are presented as arithmetic

means and error bars are standard deviations (n = 3).

Figure 6. Colour and astaxanthin concentration in L. vannamei after 28 d feeding with SH6 spores.

Experiment groups include SH6 (fed with SH6 spores at >3 × 106 CFU g-1), HU36 (fed with HU36 spores at

>3 × 106 CFU g-1), control (without supplements), and Carophyll (fed with synthesized astaxanthin at 0.5

mg g-1). Panel A. Image of boiled shrimps and their variable colour scores indicating the levels of red

pigmentation. Panel B. Astaxanthin concentrations in shrimp. Data are presented as arithmetic means

and error bars are standard deviations (n = 5). P values were generated by ANOVA using the Student’s t-

test for multiple comparisons to the control (*P < 0.05; **P < 0.01).

Figure 7. Weight gain (A) and phenoloxidase activity (B) of L. vannamei after 14 d and 28 d feeding with

SH6 spores. Experimental groups include (■) SH6 (fed with SH6 spores at >3 × 106 CFU g-1), ( ) HU36 (fed

with HU36 spores at >3 × 106 CFU g-1), ( ) control (without supplements), and (□) Carophyll (fed with

synthesized astaxanthin at 0.5 mg g-1). Data are presented as arithmetic means and error bars are

standard deviations (n = 10). P values were generated by ANOVA using the Student’s t-test for multiple

comparisons to the control (**P < 0.01; ****P < 0.0001).

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 Figure 8. Survival rate of L. vannamei at an extremely high concentration of SH6 spores. Experimental

groups include (■) SH6 (fed with SH6 spores at >1 × 109 CFU g-1), ( ) HU36 (fed with HU36 spores at >1 ×
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109 CFU g-1), ( ) control (without supplements), and (□) Carophyll (fed with synthesized astaxanthin at 0.5

mg g-1). Survival rates were assessed after 7, 14, 21, and 28 d. Data are presented as arithmetic means

and error bars are standard deviations (n = 60).

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