Screening and Identification of Bacillus amyloliquefaciens from the

Gut of Aquatic Animals

Hedong Lu

1, 2 a

, Chengyuan Gu

, Tao Yan

, Qihan Zhang

, Chengxin Geng

1,* e

and Can Lv

School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian, China

School of Food Science and Technology, Jiangnan University, Wuxi, China

19851781556@163.com

Keywords: Bacillus amyloliquefaciens, Gut, Appraisal, Probiotics.

Abstract: Probiotics have the dual effects of inhibiting pathogenic bacteria and promoting growth, while being green

and environmentally friendly, and will not cause problems such as increased drug resistance, veterinary

drug residue, and meat quality deterioration, making them to be good substitute for antibiotic. In order to

realize healthy and ecological culture and provide good microbial resources for probiotics, this experiment

isolated probiotics from the intestinal tracts of cultured Procambarus clarkii and crab. One strain LPB-5 was

identified according to traditional colony morphological characteristics observation, physiological and

biochemical tests and 16S rDNA sequence analysis. The results showed that the strain had the closest

relationship with Bacillus amyloliquefaciens MPA 1034, the 16S rDNA sequence similarity was 99%, and

the physiological and biochemical reaction spectrum was consistent with the 16S rDNA sequence analysis,

which identified the strain as Bacillus amyloliquefaciens. Procambarus clarkii and crab are important

aquaculture animals. The probiotics isolated from the intestinal tract of procambarus clarkii have their own

biosafety and high efficiency, which lays a foundation for the later development of probiotics application in

aquatic feed.

1 INTRODUCTION

There are a very large number of microorganisms in

the intestinal tract, which are generally divided into

neutral bacteria, probiotics and harmful bacteria. In

the long-term evolution of intestinal flora, as the

host adapts to the environment and natural selection,

the microbial community, host and environment

depend on each other to form a system of mutual

restriction, which is always in a state of relative

dynamic balance (Yan, 2017). The relationship

between the host and its intestinal microbes also has

a very important impact on the health of aquaculture

animals (Chaiyapechara, 2012. Liu, 2011). A large

number of studies have found that intestinal flora

https://orcid.org/0000-0002-1440-9902

https://orcid.org/0000-0002-6723-9572

https://orcid.org/0000-0001-8430-018X

https://orcid.org/0000-0001-6518-3432

https://orcid.org/0000-0002-5830-3106

https://orcid.org/0000-0002-0610-8464

can help and promote the body's antagonism to

pathogenic bacteria, enhance the body's immunity

and absorb nutrients (Ravi, 2007. Olmos, 2011).

Using probiotics to replace antibiotics and other

drugs with great side effects to achieve true

ecological aquaculture has become the trend and

research hotspot of the entire aquaculture (Chauhan,

2019). So far, some probiotics have been used as

alternative antibiotics or disease control agents for

the prevention and control of diseases in aquaculture

animals (Das, 2008. Kuebutornye, 2019. Waldroup,

2003). Probiotics inhibit the growth of harmful

bacteria in the gut (Hazel, 2020). The experiment of

Li et al. verified the inhibitory effect of competition

between two kinds of pathogenic bacteria and five

kinds of probiotics on the gastrointestinal mucosa of

fish (Li, 2012). Probiotics can produce non-specific

immune regulatory factors to stimulate the host's

immune function, effectively increase the

concentration of immunoglobulin in the host,

significantly enhance the activity of macrophages

and interferon, and strengthen the host's immunity to

Lu, H., Gu, C., Yan, T., Zhang, Q., Geng, C. and Lv, C.

Screening and Identiﬁcation of Bacillus amyloliquefaciens from the Gut of Aquatic Animals.

DOI: 10.5220/0011297900003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 855-861

ISBN: 978-989-758-595-1

855

viruses, bacteria, fungi and parasites

(Elsabagh,2018. Douglas, 2008. Opiyo, 2019).

Abarike ED et al. showed that Nile tilapia can

increase the activity of relevant enzymes in their

bodies and increase their own immunity by

consuming a commercial probiotic called BS (a

mixture of Bacillus subtilis and Bacillus

licheniensis) (Abarike, 2018). Probiotics can secrete

various digestive enzymes (amylase, protease,

lipase, etc.) in the body of aquaculture animals,

promote the absorption and transformation of

nutrients in the feed of aquaculture animals, and

improve feed digestibility, which may play a role in

animal nutrition and feed utilization rate (Das, 2019.

Hai, 2009. Balcáza, 2006). Probiotics can also

improve the constitution and meat quality of aquatic

animals, and improve their tolerance to aquatic

environment (Valente,2016. Wang, 2020). Studies

have shown that after feeding Western King prawns

with probiotics, the water content of the shrimp body

decreases, while the protein content increases, and

their constitution and meat quality are significantly

improved (Hai, 2009).

In this study, procambarus clarkii and crab were

used as experiment subjects and their intestinal

microorganisms were selected, separated and

identified. The isolated probiotics have great

potential in developing probiotics for aquaculture

feed due to their biosafety and high efficiency.

2 MATERIALS AND METHODS

2.1 Test Raw Materials and Media

Five alive procambarus clarkii and five crabs, which

were purchased from dajiang Aquatic Market in

Huai 'an, were used as raw materials.

Nutrient broth medium (per liter): 10 g peptone,

3 g beef extract, 5 g sodium chloride, pH was

adjusted to 7.2-7.4. Nutrient broth agar medium: 17

g agar was added on the basis of the above. It was

sterilized at 121℃ for 20 min in a high pressure

steam sterilizer.

Soluble starch medium (per liter): 10 g peptone,

5 g sodium chloride, 5 g beef extract, 2 g soluble

starch. It was sterilized at 115℃ for 30 min in a high

pressure steam sterilizer.

Gelatin liquefication medium (per liter): 10 g

peptone, 3 g beef extract, 5g sodium chloride, 12g

gelatin; the pH value was adjusted to 7.0 and

sterilize at 115℃ for 30 min in a high-pressure

steam sterilization pot.

Sugar fermentation medium (per liter): 5 g

peptone, 5 g tryptone, 5 g sodium chloride, 5 mL

Tween-80, 1.4 mL 1.6% Bromocresol violet

solution, 10 g sugar, 6 g agar. It was sterilized at

115℃ for 30 min in a high pressure steam sterilizer.

2.2 Incubation and Isolation of the

Strain

Procambarus clarkii and crabs were washed with

sterile water and the body surfaces were disinfected

with 75% ethanol. The intestinal tracts were

dissected in a super clean table under sterile

conditions. They were placed in test tubes with 10

mL sterilized normal saline, shaken and mixed, and

left for 10 min. The supernatant was inoculated in

the nutrient broth medium and placed in a constant

temperature and oscillation incubator for 24 h at

37℃ and 200 rpm. The cultured bacteria solution

was placed in a water bath at 80℃ for 15 min to kill

the non-spore cells, and then inoculated again into

the nutrient broth medium for enrichment and

cultured for 24 h. Appropriate gradient dilution was

performed on the bacterial solution, which was

coated in nutrient broth agar medium, and the

colonies with growth advantage were selected for

repeated lineation separation until pure strains were

obtained.

2.3 Physiological and Biochemical

Identification of Strain

The isolated strains were selected and inoculated on

the nutrient broth agar medium and placed in a

constant temperature incubator at 37℃. Colony

morphological characteristics were observed after 12

h culture, and the bacteria were selected for gram

staining test, contact enzyme test, starch hydrolysis

test and other physiological and biochemical tests.

2.3.1 Gram Stain Test

Gram dyeing test generally includes four steps:

initial dyeing, medium dyeing, decolorization and

redyeing. The specific operation methods are as

follows: 1) Take strains, smear them and fix them on

the slide. 2) Stain with crystal violet of ammonium

oxalate for 1.5min and rinse with fine water. (3)

Cover the coated surface with iodine solution and

dye it for about 1min. Rinse with fine water and

absorb the water with absorbent paper. 4) Add a few

drops of 95% alcohol, and gently shake for

decolorization, after 20 seconds, wash with water to

absorb the water. (5) After dyeing with sand yellow

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

856

solution for 1min, rinse with distilled water. Dry and

examine under microscope.

2.3.2 Contact Enzyme Test

The single colonies were inoculated on the medium

for 24 h, and then inoculated on the glass with 3%

hydrogen peroxide by inoculation ring. If there were

bubbles, it was positive; otherwise, it was negative.

2.3.3 Starch Hydrolysis Test

Single colonies were inoculated on soluble starch

medium and incubated at 37℃ for 48 h. Then the

iodine solution was dropped on the petri dish. If the

colonies formed a transparent circle around them,

they were positive; if they remained blue and black,

they were negative.

2.3.4 Gelatin Liquefaction Test

The strains were punctured and inoculated into the

test tubes filled with gelatin liquefactional medium,

and the uninoculated test tubes were used as blank

control, and cultured in 37℃ incubator for 24h.

Then the test tube was placed in cold water, and the

phenomenon was observed after the medium in the

blank control group solidified. If the experimental

test tube liquefaction, it was positive, otherwise it

was negative.

2.3.5 Sugar Fermentation Experiment

The strains cultured for 24 h were punctured and

inoculated into a test tube containing sugar

fermentation medium, and then inverted small glass

tubes were added into the test tube. Each sugar was

divided into three parallel groups. The uninoculated

group was cultured in 30℃ incubator for one week.

The phenomenon was observed and recorded, if acid

and ga were produced (from purple to yellow), it

was positive and no change was negative.

2.4 Identification of Strains by

Molecular Biology

Using the kit method, the bacterial DNA was

extracted according to the operation steps of the

bacterial genome extraction kit of Sangon

Biotechnology Company, and then the bacterial 16S

rDNA universal primers 27F/1492R (Table 1) were

used for amplification. Each sample consisted of 50

μL solution containing DNA template, forward

primers, reverse primers, sterile water (Table 2). The

5 μL PCR product was tested by 1% agarose gel

electrophoresis, and the purified PCR sample was

sent to Shanghai Biotechnology Co., LTD for

sequencing. According to sequences returned by

sequencing companies, phylogenetic trees were

constructed using MEGA 5.0 software.

Amplification procedure: 1) pre-denaturation at

94℃ for 3 min; Denaturation at 94℃ for 1 min; 2)

Annealing at 55℃ for 1 min; 3) Extended at 72℃

for 2 min for 25 cycles; 4) Extend at 72℃ for 6 min;

5) Store at 4 ℃.

Table 1: Universal primers for bacterial 16S rDNA.

Prime Genetic sequence

27F 5’-AGA GTT TGA TCC TGG CTC AG-3’

1492R 5’-GGT TAC CTT GTT ACG ACT T-3’

Table 2: PCR amplification system.

Com

ositio Volume /

Master Mix 25

Primer 1 1

Primer 2 1

DNA template 2

Sterile wate

3 RESULTS AND ANALYSIS

3.1 Screening Results

After being treated at 80℃ for 15 min, a total of 5

strains were inoculated on the medium, and the

dominant strain LPB-5 was selected as the

experimental strain. The strain grew round colonies

with creamy white opacity, neat edges, smooth and

moist surface on nutrient broth agar medium (FIG.

1). The microscopic results of gram staining are

shown in Figure 2. The sand yellow staining solution

cannot stain the bacteria red, so the bacteria is gram-

positive.

Figure 1: Colony morphology of the strain.

Screening and Identiﬁcation of Bacillus amyloliquefaciens from the Gut of Aquatic Animals

857

Figure 2: Microscopic diagram of gram stain of strain

(Dye according to the four steps of initial dyeing, medium

dyeing, decolorization and redyeing, and then observe

under the 1000x oil microscope).

3.2 Physiological and Biochemical

Identification

The physiological and biochemical identification of

strain LPB-5 (contact enzyme test, starch hydrolysis

test, gelatin liquefication test, Glucose fermentation

test, sucrose fermentation test, maltose fermentation

test) were carried out, and the results were shown in

Table 3. According to Berger's Manual of

Systematic Bacteriology, the strain could be

preliminarily identified as Bacillus.

Table 3: Physiological and biochemical characteristics of

LPB-5("+" means a positive result, "-" means a negative

result.).

Project Result

Gram stain test +

Contact enzyme test

－

Starch hydrolysis test

－

Gelatin liquefaction

test

Sterile water

(Glucose)+

(Sucrose)+

(Maltose)＋

3.3 Molecular Identification

The PCR purified product of this strain was sent to

Shanghai Shenggong Bioengineering Technology

Service Co., LTD for sequencing. The agarose gel

electrophoresis of this strain was shown in Figure 3.

According to the 16S rDNA gene sequence returned

by the company, Blast comparison was performed

with the nucleic acid sequence database in GenBank,

and the phylogenetic tree was constructed by

neighbor-joining method with MEGA 5.0 software

(FIG. 4). Strain LPB-5 was in the same branch with

Bacillus amyloliquefaciens MPA 1034, and had the

highest homology with the closest evolutionary

distance and the similarity was 99%. Therefore, the

strain was identified as Bacillus amyloliquefaciens.

Figure 3:16S rDNA electrophoresis results (Condition:

Voltage =120V, time =30min).

Figure 4: Phylogenetic tree of strain LPB-5(LPB-5 had the

closest relationship with Bacillus amyloliticus MPA 1034,

and the similarity was 99%.).

4 DISCUSSIONS

In this study, nutrient broth, a common medium, was

used to isolate strains from the intestines of healthy

cultured procambarus clarkii and crabs. It was

identified as Bacillus amyloliticus by morphological

observation, physiological and biochemical tests and

16S rDNA sequence analysis. In strain

identification, if a single application of molecular

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

858

biology of the strains identified is likely to affect the

accuracy of the appraisal result, eventually lead to

the wrong conclusion, so the paper also combines

the bacteria morphology observation and

physiological and biochemical test evaluation, to

ensure the accuracy of the appraisal results and

academic.

In order to avoid the problems caused by the use

of antibiotics such as the enhancement of drug

resistance, veterinary drug residues, and the

deterioration of meat quality, and to realize healthy

ecological aquaculture, the aquaculture industry has

an increasingly strong demand for good alternatives

to antibiotic feed (Watts, 2017.Didier, 2020).

Probiotics can improve feed digestibility, promote

growth, inhibit pathogens, improve immunity, and

improve water quality, etc., and are considered as

the most potential alternatives (Wang, 2020.El

Saadony Mohamed T, 2021). In a report in 1893,

Kozasa used spores of Bacillus Toyota as feed

additives for yellowtail fish and found that the

growth rate of yellowtail fish was improved, which

was the first application of probiotics in practice and

proved the beneficial effect of probiotics in

aquaculture (Cruz, 2012).

In recent years, the aquaculture industry has

started to add some probiotics to the feed, and

practice has proved that it is feasible to use

probiotics to prevent and control the diseases of

cultured fish and shrimp, and the use of probiotics in

aquaculture can significantly reduce the need for

antibiotics (Opiyo, 2019. Abarike, 2018). Sun et al.

exposed the fertilized zebrafish larvae to

perfluorinated butanesulfonic acid (PFBS) and

applied the probiotic lactobacillus rhamnosus during

this period (Sun, 2021). The results showed that

probiotics supplement effectively inhibited the

growth retardment caused by PFBS, Probiotics can

secrete various digestive enzymes (amylase,

protease, lipase, etc.) in the body of aquaculture

animals, promoting the absorption and

transformation of nutrients in the feed of aquaculture

animals, improving feed digestibility, and promoting

individual growth (Das, 2019. Hai, 2009). After

entering the digestive tract of the host, probiotics

mainly prevent the growth of pathogens on the

intestinal surface by producing antibacterial

substances, regulating intestinal pH and competing

with harmful bacteria for adsorption sites

(Kuebutornye, 2019. Kuebutornye, 2020.

Chabrillon, 2005). Studies have shown that

maintaining a high level of probiotics in fish ponds

can not only reduce the accumulation of dissolved

organic carbon and granular organic carbon during

the growing season, but also balance the production

of phytoplankton (Cruz, 2012. Kazun, 2019).

Most of the probiotics that make up the

probiotics used in aquaculture come from the

digestive tracts of mammals, or soil and seawater,

etc. The probiotics used in aquaculture were

originally commercial probiotics designed for land

animals. In the development of probiotics for

aquaculture, the probiotics in the intestinal tract of

aquaculture animals are good raw materials, because

they are not only biosafe, but also can effectively

improve the survival rate and colonization rate of

probiotics in the intestinal tract (Ramlucken, 2020).

In this study, only one strain with this characteristic

was screened, but the development of relevant

probiotic preparation products still needs to consider

various factors such as hemolysis, antibiotic

resistance, compliance analysis, pH and bile salt

tolerance. (Balcázar, 2008.). In addition, in some

studies, to test the safety of probiotics, probiotics

were injected directly into fish intraperitoneally to

study mortality without inducing severe pathogenic

symptoms (El-Rhman, 2009.Aly, 2008). In any case,

safety is always the most important, so further

experiments are needed to verify the addition of this

strain as a probiotic preparation to aquatic feed.

5 CONCLUSIONS

In this study, a probiotic strain with quantity

advantage was screened and isolated from the

intestinal tract of procamblus clarkii and crab which

were purchased from dajiang Aquatic Market of

Huai 'an city. The strain was identified by

observation of colony morphology, physiological

and biochemical tests and 16S rDNA sequence

analysis. The results showed that the strain had the

closest relationship with Bacillus amyloliquefaciens

MPA 1034. The 16S rDNA sequence similarity was

99%, and the physiological and biochemical reaction

spectrum was consistent with the 16S rDNA

sequence analysis. The biosafety and high efficiency

of the strain in aquaculture feed can improve the

excellent microbial resources for further

development of probiotics and other products.

ACKNOWLEDGEMENTS

This work was supported by the National Natural

Science Foundation of China (31801524), and

Screening and Identiﬁcation of Bacillus amyloliquefaciens from the Gut of Aquatic Animals

859

Natural Science Foundation of Jiangsu Province

(BK20170461, BK20181063).

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