Screening of pigmented Bacillus aquimaris SH6 from ... - Anabio

ORIGINAL ARTICLE

Screening of pigmented Bacillus aquimaris SH6 from theintestinal tracts of shrimp to develop a novel feedsupplement for shrimpH.T. Ngo1,*, T.T.N. Nguyen1,*, Q.M. Nguyen2,*, A.V. Tran2,*, H.T.V. Do3, A.H. Nguyen4, T.-N. Phan1

and A.T.V. Nguyen1

1 Key Laboratory of Enzyme and Protein Technology, VNU University of Science, Hanoi, Vietnam

2 High School for Gifted Students, VNU University of Science, Hanoi, Vietnam

3 Faculty of Chemistry, VNU University of Science, Hanoi, Vietnam

4 ANABIO Research & Development JSC, Hanoi, Vietnam

Keywords

Bacillus, carotenoid, gastrointestinal tract,

pigmented, shrimp, supplement.

Correspondence

Anh Thi Van Nguyen, Key Laboratory of

Enzyme and Protein Technology, VNU

University of Science, 334 Nguyen Trai,

Hanoi, Vietnam.

E-mail: [email protected]

*These authors contribute to this work

equally.

2016/0625: received 24 March 2016, revised

24 July 2016 and accepted 16 August 2016

doi:10.1111/jam.13274

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 Bacillus aquimaris

SH6 was selected because it showed the highest abundance in shrimp guts

(70% population). Whiteleg shrimp (n = 30 per group) fed with SH6 spores,

at >3 9 106 CFU g�1 pellet for 4 weeks had redder colour (score of 21–23 vs

20–22), 2�7-fold higher astaxanthin level (0�69 vs 0�25 lg 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 colourization and weight gain, and for

enhancing innate immunity of whiteleg shrimp.

Significance and Impact of the Study: This study demonstrates that

carotenoids produced by B. aquimaris SH6 can be successfully absorbed and

converted to astaxanthin in whiteleg shrimp.

Introduction

The need for sustainable shrimp aquaculture drives stud-

ies 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 to provide

growth factors such as vitamins, carotenoids, fatty acids

and amino acids (Araya et al. 2002). Therefore, nutrients

are absorbed more efficiently when the feed is supple-

mented with 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 pre-

dominant 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 synthe-

sized red carotenoid astaxanthin is common to improve

Journal of Applied Microbiology 121, 1357--1372 © 2016 The Society for Applied Microbiology 1357

Journal of Applied Microbiology ISSN 1364-5072

colourization of shrimp. D’Abramo et al. (1983) have

demonstrated that pure carotenoids such as b-carotene,echinenone and canthaxanthin are converted to astaxan-

thin in cultured lobsters and that the level of pigmenta-

tion 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; Khaneja 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 Bacillus aquimaris and Bacillus 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 Bacillus indicus (Suresh et al. 2004).

Khaneja et al. (2009) isolated several other carotenoid-

producing Bacillus species from seawater, soil and fer-

mented rice condiment. These included B. marisflavi,

Bacillus cibi and Bacillus altitudinis strains producing yel-

low and yellow-orange pigments; B. aquimaris and Bacil-

lus pumilus strains producing orange and orange-red

pigments; and some B. firmus strains producing pink and

deep pink pigments. Carotenoids produced by these

strains were determined to have absorption maxima at

455, 467 and 492 nm, corresponding to the visible col-

ours 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 carote-

noids (Duc et al. 2006; Hong et al. 2008; Khaneja et al.

2009; Cutting 2011); this was followed by in vivo studies

on the bioaccessibility and bioavailability of these carote-

noids, 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); how-

ever, the isolation of pigmented Bacillus species from gas-

trointestinal 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 sur-

viving 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 pro-

duction of high levels of antioxidant carotenoids and for

abundance in the shrimp gut. Spores of the selected

strain were produced for laboratory-20 scale blind trials

in whiteleg shrimp to evaluate shrimp colourization (col-

our score and astaxanthin level), weight gain and phe-

noloxidase activity, reflecting innate immune status.

Materials and methods

Reference strains

Yellow-pigmented B. indicus HU36 and Bacillus subtilis

HU58 isolated from human faeces (Duc et al. 2006; Tam

et al. 2006) and nonpigmented laboratory B. subtilis

PY79 was used as the reference strain in most experi-

ments described here; Bacillis 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 pre-

pared to collect the mucosa in 0�9% NaCl and then vig-

orously 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, Hamp-

shire, UK) and incubating for 1 day 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 pres-

ence of spores.

General methods

Each isolate was grown on DSM agar or in DSM broth

(Oxoid) for 48 h and assessed for sporulation percentage

Journal of Applied Microbiology 121, 1357--1372 © 2016 The Society for Applied Microbiology1358

Bacillus from shrimp intestine H.T. Ngo et al.

by determining the titre of heat-resistant cells (from 40°Cto 80°C, 20 min) vs the total viable cell count. Bacterial

growth was monitored in tryptone soy broth (TSB;

Oxoid) 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, Wal-

tham, MA). Characterization of Bacillus strains using

microbiological and biochemical methods was performed

following Bergey’s Manual of Systematic Bacteriology

(Vos et al. 2009). The shapes of bacteria and positions of

spores of selected strains were observed under a conven-

tional microscope Primo Star (Carl Zeiss, Oberkochen,

Germany). To determine the ability of bacterial isolates

to grow under aerobic or anaerobic 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 con-

taining sheep blood and incubated for 24 h at 37°C to

observe haemolysis zones. To evaluate antibiotic resis-

tance 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: 50-AGAGTTTGATCMTGGCTCAG-30 and 1527R: 50-AAAGGAGGTGATCCAGCC-30 (Khaneja et al. 2009; Nguyen

et al. 2015). The resultant partial 16S rRNA sequences

were assembled and aligned with APE� software (The

University of Utah, Salt Lake, UT). Obtained sequences

were compared to sequences in the GenBank nonredun-

dant 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 MEGA ver. 6 soft-

ware (Tamura et al. 2013). Evolutionary relationships of

the Bacillus strains were estimated using the neighbour-

joining method (Saitou and Nei 1987) with 1000 boot-

strap 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.

Pigment extraction and analysis

Pigment extraction and analysis from Bacillus isolates

were performed following a previously reported method

(Khaneja et al. 2009). Extracts (200-ll) 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 deter-

mined from the standard curve of astaxanthin at concen-

trations ranging from 1�05 to 16�75 lmol 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 photodi-

ode array (PDA) detector using a reverse-phase (RP) C30,

5-lm column (250 mm 9 4�6 mm) coupled to a

C30 guard column (20 mm 9 4�6 mm; Thermo Scien-

tific). The mobile phase and elution conditions were

identical to those described by Khaneja et al. (2009). 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 activ-

ity was measured following a standard method (Sharma

and Bhat 2009). In brief, individual 1�5-ml extracts from

approx. 0�5 g (wet weight) of vegetative cells were incu-

bated with 500 ll of 250 lmol l�1 1,1-diphenyl-2-picryl-

hydrazyl (DPPH; Sigma, St. Louis, MO). The DPPH

absorbance at 517 nm was measured before and after the

reactions. Inhibition level was calculated using following

equation: Inhibition (%) = [(ODcontrol � ODsample)/

ODcontrol] 9 100. In parallel experiments, 1�5 ml of

ascorbic acid (6�25–25 lmol l�1) was added as a quanti-

tative 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, Bacil-

lus strains were cultured in suitable medium for 48 h in

a fermenter (ANABIO R&D built in-house, Hanoi, Viet-

nam) at the optimal temperature (30–37°C) and pH

(7�0–8�0) for each strain. The purification of spores has been

described previously (Nicholson and Setlow 1990). The


H.T. Ngo et al. Bacillus from shrimp intestine

Table

1Antibioticsuscep

tibility

ofpigmen

tedBacillusstrainsfrom

shrimpgastrointestinal

tracts

Antibioticdiscs*

SH1†

SH4†

SH5†

SH6†

SH8†

SH12†

SH14†

SH20†

Ampicillin

(10)

43�23

�0�42

S

31�73

�0�00

S

46�74

�0�33

S

22�44

�1�25

S

38�51

�0�49

S

47�55

�0�16

S

36�98

�0�19

S

18�60

�0�55

S

Chloramphen

icol(30)

29�18

�0�21

S

20�77

�0�35

S

31�02

�0�30

S

26�12

�0�92

S

28�83

�0�15

S

31�61

�0�14

S

27�01

�0�78

S

18�83

�0�50

S

Ciprofloxacin(5)

38�80

�0�45

S

17�10

�0�10

S

35�62

�0�58

S

30�87

�0�53

S

27�87

�0�21

S

31�48

�0�31

S

47�08

�0� 00

S

32�67

�0�47

S

Clindam

ycin

(2)

14�89

�0�01

R

12�70

�0�42

R

14�80

�0�01

R

9�57

�0�09

R

8�11

�0�02

I

18�22

�0�01

I

10�35

�0�20

R

17�83

�0�05

R

Co-trimoxazole

(25)

33�72

�0�07

S

23�97

�0�22

S

30�60

�0�30

S

24�63

�0�41

S

30�70

�0�22

S

29�02

�0�01

S

43�52

�0�67

S

26�74

�0�25

S

Erythromycin

(15)

21�18

�0�12

I

20�56

�0�30

I

30�55

�0�10

S

22�55

�0�61

S

25�30

�0�04

S

29�28

�0�17

S

40�80

�1�54

S

28�01

�0�41

S

Gen

tamicin

(10)

29�02

�0�29

S

19�15

�1�19

S

24�57

�0�00

S

19�24

�0�46

S

20�80

�0�07

S

26�49

�0�00

S

31�89

�0�45

S

25�01

�0�14

S

Kan

amycin

(30)

28�63

�0�16

S

17�49

�0�57

S

30�79

�0�06

S

25�33

�1�19

S

24�90

�0�63

S

28�54

�0�01

S

36�75

�0�87

S

23�94

�0�15

S

Neo

mycin

(30)

27�27

�0�67

S

17�14

�0�42

S

24�38

�0�01

S

17�15

�0�05

S

27�79

�0�24

S

21�31

�0�20

S

25�03

�0�30

S

22�55

�0�18

S

Rifam

picin

(30)

32�59

�0�26

S

16� 58

�0�04

I

36�73

�0�32

S

15�51

�0�35

S

30�70

�0�37

S

33�48

�0�30

R

29�14

�0�86

S

15�81

�0�34

S

Streptomycin

(10)

27�87

�0�33

S

14�28

�0�00

S

28�19

�0�01

S

29�40

�1�47

S

16�60

�0�26

S

25�85

�0�16

S

29�65

�0�58

S

21�00

�0�14

S

Tetracycline(30)

30�79

�0�18

S

26�74

�0�48

S

33�78

�0�21

S

28�28

�0�36

S

31�01

�0�05

S

33�41

�0�40

S

40�16

�0�00

S

23�27

�0�21

S

Trim

ethoprim

(5)

37�48

�0�00

S

6�45

�0�00

S

34�38

�0�16

S

24�01

�0�73

S

6�76

�0�00

S

31�52

�0�27

S

50�06

�0�54

S

30�28

�0�07

S

Van

comycin

(30)

19�57

�0�01

S

14�77

�0�26

S

19�51

�0�08

S

16�65

�0�44

S

16�70

�0�11

S

20�44

�0�06

S

24�29

�0�57

S

13�74

�0�09

S

S,sensitive;I,interm

ediate;R,resistan

t.

*Antibiotic-im

pregnated

discs

(6mm)witham

ountin

lgshownin

brackets.

†Averagediameter

ofinhibitionfrom

threeindividual

experim

ents.



purified spore suspensions were then spray-dried at 160°Cat an atomizer speed of 28 000 g (ANABIO R&D built

in-house, Hanoi, Vietnam) 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 9 106 CFU g�1 feed); the feed pellets were

then coated with cod liver oil Seven Seas� (Merck Con-

sumer Health, Darmstadt, Germany). For evaluating the

probiotic effects of SH6 spores, feed pellets were supple-

mented with either B. aquimaris SH6 spores or B. indicus

HU36 spores at concentrations of >3 9 106 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 Caro-

phyll Pink� (DSM, Heerlen, the Netherlands) powder

containing 10% synthesized astaxanthin was used as sup-

plements. 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 (L. vannamei), weigh-

ing approx. 3 g, were used for probiotic treatment on a

laboratory scale. Approval of animal care and use proto-

col 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.

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), feed

supplemented with a mixture of SH1, SH5, SH6, SH8,

and SH14 spores at >3 9 106 CFU g�1 pellet (Spore

group). After 7 days 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 day at 37°C to obtain individual colo-

nies. Colonies were observed based on their typical mor-

phologies 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 9 106 CFU g�1 pellet (SH6

group), feed supplemented with HU36 spores at

>3 9 106 CFU g�1 pellet (HU36 group), and feed sup-

plemented 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 measur-

ing shrimp weight and phenoloxidase (PO) activity, ten

shrimp (n = 10) from each group were taken and data

were recorded after 14 and 28 days. The SD was calcu-

lated based on data collected from ten shrimp. For com-

paring colours, five shrimp (n = 5) in each group were

taken after 28 days, and then boiled comparing colour

using the Roche index, SalmoFanTM standard colour

(Brun and Fr�ed�eric 2006). For measuring astaxanthin

concentration, five shrimp (n = 5) in each group were

taken and data were recorded after 28 days. SD was cal-

culated based on data collected from five shrimp.

For evaluating the safety of the SH6 probiotics, a simi-

lar experimental design was set up for the four groups

(n = 30 per tank; 2 tanks per group) feed was supple-

mented with spores at an extremely high concentration

(>1 9 109 CFU g�1 pellet). Survival of shrimp in each

group was recorded after 7, 14, 21 and 28 days for calcu-

lation of survival rate at each time point. SD was calcu-

lated based on data collected from two tanks.

Extraction and measurement of astaxanthin in shrimp

Shrimp (n = 5) from each experimental group were treated

as follows. The chitin shell was removed, and 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 col-

lected 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 fivefold using

0�2 ml acetonitrile/methanol/dichloromethane (ratio

75 : 20 : 5) and injected onto the HPLC column. Astaxan-

thin in extract (A) was first separated and then detected

online, using the Waters Alliance (Milford, MA) HPLC sys-

tem 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-lm column

(150 mm 9 4�6 mm; Agilent, San Francisco, CA). The

mobile phase, acetonitrile/methanol/dichloromethane

(75 : 20 : 5), eluted at a rate of 1�5 ml min�1, was main-

tained at a constant temperature of 30°C. A defined sample

of astaxanthin (2�68 lmol l�1), used as control, was mea-

sured at the same time to compare retention time (Rt).

Concentrations of astaxanthin in the extract were mea-

sured based on height and area of peaks. Total astaxanthin

concentration per 1 g of shrimp Cs (lg g�1 shrimp�1) was

calculated using the equation: Cs = k 9 1�67 9 Ce/W,

where k was the dilution ratio of the samples (k = 6), Ce

was astaxanthin concentration in the extract (lmol l�1),

and W was the weight of shrimp (g).

Phenoloxidase activity measurement

PO activities in shrimp were measured following the pro-

tocol of a previous report by Luciane and Margherita

(1997) and Nguyen et al. (2014).

Statistical analysis

Data of astaxanthin concentrations, weight gains and PO

activities among the four treatment groups 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 (Micro-

soft, Redmond, WA). An F-test for two-sample variance

was used before performing a t-test for two unpaired sam-

ples. 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

colourize 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 lmol 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 pig-

mented, 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 neg-

ative 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 caro-

tenoids equivalent to 250 lg ml�1 or higher. All strains

were able to sporulate quickly in DSM medium with

sporulation efficiency ranging from 85 to 100%. Most of

the pigmented spores were not very heat resistant, except

that of SH8, which retained its haft-count (50% survival)

after treatment at 80°C for 20 min. With lower tempera-

ture 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 lm. The spore

position and characteristics of isolates were different from

each other; some were subterminal, and others were ter-

minal and central. All strains were able to grow aerobi-

cally 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 antibi-

otic groups were included with different modes of action,

including a b-lactam (ampicillin), a quinolone (ciproflox-

acin), a lincosamide (clindamycin), a macrolide (ery-

thromycin), four aminoglycosides (gentamicin,

streptomycin, kanamycin, neomycin), a glycopeptide

(vancomycin) and rifampicin, tetracycline, chlorampheni-

col and cotrimoxazol (trimethoprim and sulfamethoxa-

zole). 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

rRNA sequence analysis indicated that strains were closely

related to B. aquimaris (SH1, SH4, SH6, SH14, SH20),

B. firmus (SH5, SH12) and B. marisflavi (SH8) with iden-

tity scores equal to or greater than 0�98.Assessing all characterization data listed in Tables 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 phyloge-

netic relationship (based on rRNA sequences) between

SH6 (GenBank accession No: KF443807) and other refer-

ence 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

Table 2 Characterization of pigmented Bacillus strains isolated from shrimp gastrointestinal tracts

Strains SH1 SH4 SH5 SH6 SH8 SH12 SH14 SH20

Colour* PR PO PO RO Y PO YO RP

UV-Vis spectral

characteristics (nm)

495 494 490 468 435, 465, 487 504 470 460

Carotenoid

production

++++ +++ ++++ ++++ ++++ ++++ ++ ++++

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

Survival during

heat treatment†

++ + +++ +++ ++++ +++ + ++

Size of bacteria

(lm)‡

<1 <1 <1 <1 <1 <1 <1 <1

Spore position S S T C S T S T

Aerobic + + + + + + + +

Anaerobic � � � � + � � �Growth at 30°C + + +++ +++ ++ +++ + +++

6�5% NaCl + + + + + + + +

Amylase ++ ++ +++ ++ � ++ ++ ++

Caseinase + ++ � ++ + + + +

Lipase ++ + � ++++ ++ + � �Haemolysis c c c c c c c c

VP Test � + + � � � � �Closest match§ Bacillus aquimaris

(0�99)B. aquimaris

(0�99)Bacillus

firmus

(0�98)

B. aquimaris

(0�99)Bacillus

marisflavi

(0�99)

B. firmus



(0�99)

T, Terminal; S, subterminal; C, central.

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

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

†Heat treatment at 55°C, 20 min.

‡Width size of vegetative cell.

§Using 16S rDNA sequence analysis in this work. The similarity score is shown in brackets.

Peaks having higher absorbance values were underlined



http://www.ncbi.nlm.nih.gov/nuccore/KF443807

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 lmol l�1) and higher than that of the positive con-

trol HU36 (78�4 � 0�9%). The data were confirmed

using nonpigmented B. subtilis PY79 as a negative con-

trol, wherein no detectable DPPH scavenging activity was

observed. Based on this data, we concluded that the pig-

ments found in the eight selected strains showed the

potential to be antioxidant carotenoids, and that carote-

noids 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 colonization 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 morphol-

ogy, including SH1, SH5, SH6, SH8, and SH14, for

assessing viability in shrimp guts. We set up two time

points for counting bacterial populations in the shrimp

gut, 3 and 8 h after the last feeding. This was because

transit time of feed in 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

Bacillus marisflavi strain TF-11 (NR025240)

Bacillus marisflavi strain SNS4 (LC009453)

Bacillus aquimaris strain CH9 (KM516787.1)

Bacillus aquimaris strain SH6 (KF443807)

Bacillus aquimaris strain TF-12 (NR025241.1)

Bacillus lichenifomis strain AIX64 (GU967449)

Bacillus pumilis strain GC43 (KF158227)

Bacillus megaterium isolate OS-223 (AM237398)

Bacillus cereus strain LKT 1/2 (AJ577284)

Lactobacillus acidiphillus strain ATCC 4356 (AB008203)

0·01

93

97

87

98

Bacillus lichenifomis strain LZBL-13 (JX847119)

76

Bacillus pumilis strain ESR21 (KC915229)

Bacillus megaterium strain WN606 (DQ275183)

Bacillus cereus strain G56 (KM019745)

100

100

100

100

Figure 1 Phylogenetic relationship between the selected pigmented Bacillus strains. Dendrograms of strains based on 16S rRNA sequence align-

ment using MEGA ver. 6 software. A selected Bacillus strain is highlighted in bold and GenBank accession numbers are shown in brackets. Statisti-

cal (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.



and SH14 were not detectable. Thus, it was concluded

that SH6 spores survive better than spores of other pig-

mented strains and were the most abundant population

in the shrimp gut during the passage of feed. To further

access adhesion and colonization capability of pigmented

Bacillus strains in the shrimp intestinal mucosa, we col-

lected 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 nonpigmented

Bacillus colonies (~79%) and a significant reduction in

pigmented Bacillus colonies (21%). However, colonies of

the SH6 strain were still the most abundant among pig-

mented strains (20% of the population), followed by SH1

(1% of the population). Thus, even though it was not

comparable to that of nonpigmented Bacillus strains, col-

onization 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 men-

tioned above) and in vivo (based on colonization assays),

we decided to characterize the SH6 strain for the devel-

opment of probiotics for use in whiteleg shrimp.

Carotenoid profiling of SH6 and characterization 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, indicate 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 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 sus-

pension was spray-dried to obtain a heat-stable spore

powder. As controls, we also produced spore powders of

the medium-sporulating B. indicus HU36 yellow-pigmen-

ted strain (about 50% sporulation efficiency) and the

high-sporulating nonpigmented 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 concentra-

tion of 4�5 9 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 9 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 surpris-

ingly 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

characterized for use as a food ingredient (Permpoonpat-

tana et al. 2012).

0

10

20

30

40

50

60

70

80

90

100

SH1SH5

SH6SH8

HU36PY79

Sca

veng

ing

perc

enta

ge o

f DP

PH

(%

)

Group

37·5

µm

ol l–1

18·7

5 µm

ol l–1

9·38

µm

ol l–1

4·69

µm

ol l–1

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�75,9�38 and 4�69 lmol l�1. Inhibition (%) =

[(ODcontrol � ODsample)/ODcontrol] 9 100.



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 9 106 CFU g�1 pellet), HU36

(fed with the reference HU36 spores at

>3 9 106 CFU g�1 pellet), control (without supple-

ments), 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 com-

pared 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).

To correlate the relationship between the shrimp colour

and the presence of astaxanthin in shrimp tissue, we deter-

mined the astaxanthin levels in shrimp of each group. As

shown in Fig. 6b, we found that the level of astaxanthin in

the SH6 group (0�69 � 0�03 lg g�1 shrimp�1) was 2�7-fold higher than that of the control (0�25 � 0�09 lg g�1

shrimp�1; P < 0�05) but 2�3-fold lower than that of

the Carophyll group (1�59 � 0�08 lg g�1 shrimp�1;

P < 0�01). Unexpectedly, the difference in astaxanthin

levels between the HU36 group (0�47 � 0�13lg 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 2 and 4 weeks, there was an apparent

disparity between spore and nonspore groups. Shrimp in

both the SH6 and HU36 groups gained weight much fas-

ter 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 2 and 4 weeks

SH1: 30%

SH6: 70%

SH1: 1%

SH6: 20%

Nonpigmented Bacillus: 79%

(b)(a)

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 9 106 CFU g�1).

Panel (b) Pie chart of population (B2) of major

nonpigmented Bacillus strains and SH6 and

SH1 strains isolated from shrimp guts after 8-

h-feeding in the experimental group.

5·0 10·0 15·0 20·0 25·0 30·0 min

0·0

2·5

5·0

7·5

10·0

mAU450 nm,4 nm (1·00)

20·1

09

22·2

39

29·4

37

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, k max

(484; 496), peak no. 2, Rt at 22�239 min, k

max (466; 489) and one carotenoid type at

high absorbance level of peak no. 3, Rt at

29�437 min, k max (492; 522). Peaks having

higher absorbance values were underlined.



respectively; the HU36 group gained 5�22 � 0�19 g and

6�71 � 0�21 g at 2 and 4 weeks respectively. By contrast,

the control group gained only 4�26 � 0�35 at 2 weeks

and 5�32 � 0�26 g at 4 weeks (Fig. 7a). In the nonspore

Carophyll group, shrimp were heavier (4�79 � 0�22 g

and 6�00 � 0�26 g at 2 and 4 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.

PO activity is the parameter most used to represent the

innate immune response, which protests against patho-

genic 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 contin-

uous feeding (OD490 = 0�134 for the SH6 group vs 0�09for 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 PO

activities in the HU36 (OD490 = 0�121) and Carophyll

groups (OD490 = 0�121) were not significant different

(P > 0�05) from those of the control group (Fig. 7b).

Safety of SH6 spores

SH6 is a new probiotic strain that has not been well doc-

umented 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 9 109 CFU g�1, which was approx. 300-fold

higher than the probiotic concentration

(>3 9 106 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 Caro-

phyll-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 confirm that the lethal doses of both SH6 and HU36

spores in whiteleg shrimp were above 1 9 109 CFU g�1

for up to a 4-week treatment.

(a) (b)

0

0.5CF

U (

× 1

09 m

l–1 )

1

1.5

2

2.5

3

Temperature (°C)

25 40 50 60 70 80 90

2 µm

(c)Figure 5 SH6 spores and their heat

resistance. Panel (a) Spray-dried powder of

orange SH6 spores (>3 9 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). Colour figure can be viewed at

wileyonlinelibrary.com.



wileyonlinelibrary.com

Discussion

As mentioned in the introduction to this article, a num-

ber of publications have reported the identification and

characterization of pigmented bacilli isolated from seawa-

ter, water and mud in shrimp ponds, soil, and human

faeces. To our knowledge, this is the first study to isolate

and characterize carotenoid-producing Bacillus strains

from the shrimp GIT. To develop a novel supplement for

promoting the colourization 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 colo-

nizing 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 germi-

nate in the small intestine and grow (Tam et al. 2006).

Then, we can characterize and screen the best strain of

pigmented bacteria, 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 one of the most exported species from Viet-

nam (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 probi-

otics and to obtain biodiversity of the intestinal micro-

organisms.

Among 23 coloured isolates, eight strains were selected

from in vitro conditions due to their potential properties

0

0·2

0·4

0·6

0·8

1

1·2

1·4

1·6

1·8

SH6 HU36 Control Carophyll

µg a

stax

anth

in g

–1 s

hrim

p–1

Group

*

**

Score: 21–23 21–23 20–22 24–25

Group: SH6 HU36 Control CarophyII(a)

(b)

Figure 6 Colour and astaxanthin concentration in Litopenaeus van-

namei after 28 days feeding with SH6 spores. Experiment groups

include SH6 (fed with SH6 spores at >3 9 106 CFU g�1), HU36 (fed

with HU36 spores at >3 9 106 CFU g�1), control (without supple-

ments), and Carophyll (fed with synthesized astaxanthin at

0�5 mg g�1). Panel (a). Image of boiled shrimps and their variable col-

our scores indicating the levels of red pigmentation. Panel (b) Astax-

anthin 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 compar-

isons to the control (*P < 0�05; **P < 0�01). Colour figure can be

viewed at wileyonlinelibrary.com.

0·0

1·0

2·0

3·0

4·0

5·0

6·0

7·0

8·0

14 days 28 days

Wei

ght (

g sh

rimp–

1 )

Time

Time

**** ********

********

****

0

0·05

0·1

0·15

0·2

0·25

0·3

0·35

14 days 28 daysPhe

nolo

xida

se a

ctiv

ity (

O.D

. 490

nm

)

**

**

****

(a)

(b)

Figure 7 Weight gain (a) and phenoloxidase activity (b) of Litope-

naeus vannamei after 14 and 28 days feeding with SH6 spores.

Experimental groups include (■) SH6 (fed with SH6 spores at

>3 9 106 CFU g�1), ( ) HU36 (fed with HU36 spores at >3 9 106

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).



wileyonlinelibrary.com

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°Ctreatment for 20 min); (ii) typical UV-Vis spectra of pro-

duced carotenoids (400–550 nm); (iii) free radical scav-

enging 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 interest-

ing properties. SH6 produced antioxidant carotenoids

(with 90% scavenging activity of DPPH free radicals) at

the high level of 423 lg (g DW)�1. It also had high

sporulation efficiency (100%) yielding high concentra-

tions of spores at 4�5 9 1011 CFU g�1 in the sprayed

powder. The spores were also more heat-stable (15% sur-

vival 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 colonize 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 toxi-

city to humans.

We further characterized the HPLC chromatographic

profile of extracts of SH6 bacteria and the heat stability

of the SH6 spores, which are basic properties of carote-

noid-producing spore formers. The absorbance spectra of

the collected fractions of SH6 extracts, with three peaks

at approx. 400–500 nm, suggests that the SH6 strain pro-

duced at least three different carotenoid types. Compar-

ison with reference spectra, identified the carotenoids in

the three fractions, no. 1–3, as being closely related to

keto/hydroxyl derivatives of c-carotene (Britton et al.

2004; Duc et al. 2006). This finding also suggests that

carotenoids are the sole pigments responsible for the col-

our 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 nonpigmented 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 nor-

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

mercial shrimp feed ingredient, one might scale-up SH6

spore production using pilot- or industrial-scale fer-

menters, 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 9 106 CFU g�1 pel-

lets, 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 colourize shrimp guts and increased

astaxanthin production by threefold (a score of 21–23with 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 pigmen-

tation 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

Fr�ed�eric 2006). This result is similar to that of Yamada

et al. (1990); therein the level of tissue astaxanthin in

groups fed b-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 colourization

and astaxanthin level, supplementation with SH6 spores

50

55

60

65

70

75

80

85

90

95

100

7 days 14 days 21 days 28 days

Sur

viva

l rat

e (%

)

Time

Figure 8 Survival rate of Litopenaeus vannamei at an extremely high

concentration of SH6 spores. Experimental groups include (■) SH6

(fed with SH6 spores at >1 9 109 CFU g�1), () HU36 (fed with HU36

spores at >1 9 109 CFU g�1), () control (without supplements), and

(□) Carophyll (fed with synthesized astaxanthin at 0�5 mg g�1). Sur-

vival rates were assessed after 7, 14, 21 and 28 days. Data are pre-

sented as arithmetic means and error bars are standard deviations

(n = 60).



was less efficient than supplementation with Carophyll

(astaxanthin), but was slightly better than HU36 spores

and obviously better than uncoated feed.

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 car-

otenoid-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 aqua-

tic animals, which survive in low dissolved oxygen condi-

tions, 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 maxi-

mize 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 colonization 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 PO. PO catalyses

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 haemocytes to

bacteria, thus accelerating their removal through the for-

mation 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 matu-

ration. Similar data were previously reported by Nguyen

et al. (2014), 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. The effective 85%

increase in PO activity in the SH6 group can be attribu-

ted to the immunostimulatory effects of the cell wall

components of this strain, such as b-glucan and

lipopolysaccharide (Rengpipat et al. 2000; Gullian et al.

2004). Alternatively, mucosal immunity in the shrimp

might have been improved through the efficient coloniza-

tion 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).

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 colourization, weight gain and

immune enhancement. In comparison to conventional

supplementation with astaxanthin in shrimp feed, supple-

mentation with SH6 spores was more advantageous in

terms of weight gain and immune enhancement, although

less efficient in terms of colourization. Biosafety data

indicate that SH6 spores, at a 300-fold higher concentra-

tion than that of the conventional probiotic concentra-

tion, 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 research is funded by Vietnam Ministry of Science

and Technology, Viet Nam National University, Hanoi

(VNU) under project number 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 carote-

noids 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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