7.2
CiteScore
3.7
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
ABUNDANCE ESTIMATION IN AN ARID ENVIRONMENT
Case Study
Correspondence
Corrigendum
Editorial
Full Length Article
Invited review
Letter to the Editor
Original Article
Retraction notice
REVIEW
Review Article
SHORT COMMUNICATION
Short review
7.2
CiteScore
3.7
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
ABUNDANCE ESTIMATION IN AN ARID ENVIRONMENT
Case Study
Correspondence
Corrigendum
Editorial
Full Length Article
Invited review
Letter to the Editor
Original Article
Retraction notice
REVIEW
Review Article
SHORT COMMUNICATION
Short review
View/Download PDF

Translate this page into:

Original article
32 (
2
); 1641-1646
doi:
10.1016/j.jksus.2019.12.023

Growth promoting activity of Penaeus indicus by secondary metabolite producing probiotic bacterium Bacillus subtilis isolated from the shrimp gut

, , , , , , , , ,
a Department of Bio-Environmental Chemistry, College of Agriculture and Life Sciences, Chungnam National University, 99 Daehak-Ro, Yuseung-Gu, Daejeon 34134, Republic of Korea
b Department of Zoology, College of Science King Saud University, Riyadh 11451, Saudi Arabia
c Bioprocess Engineering Division, Smykon Biotech Pvt Ltd, Nagercoil, Kanyakumari District, Tamil Nadu, India
d Department of Zoology, Nesamony Memorial Christian College, Marthandam, Kanyakumari, Tamil Nadu 629 165, India
e Department of Clinical Pharmacology, College of Medicine, Soonchunhyang University, Cheonan, Republic of Korea
f The Comfort Animal Hospital, Sungbuk-gu, Soonginro-50, Seoul, Republic of Korea

⁎Corresponding author. hak3962@sch.ac.kr (Hak-Jae Kim)

Received: , Accepted: ,
© The Author(s) 2020
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

In this study, 104 bacteria were isolated from the gut of Penaeus indicus. The morphologically different bacterial isolates were screened for probiotic properties. The strain D5 showed potent activity against various bacteria. The isolated strain Bacillus subtilis showed 17 mm zone of inhibition against Bacillus sp., 19 mm against Pseudomonas sp., 22 mm against Vibrio sp., and 22 mm against Micrococcus sp. The bacterial growth and bacterocin production in relation with incubation time was evaluated. Bacteriocin production was found to be high at the stationary phase and reduced considerably after fifth day. Bacterial growth and bacterocin production was maximum at pH 7.0 and optimum between pH 6.0 and 8.0. Incubation temperature is one of the important factors which significantly influences on the growth and production of bacteriocin. Bacterial growth and bacterocin production (17 ± 2 mm) were maximum at 30 °C. Glucose stimulated growth and bacterocin production (21 ± 3 mm). Among the supplemented nitrogen sources, glycine positively influenced on growth and metabolite production. In this study, bacteriocin supplemented diet enhanced the growth than control shrimp Bacteriocin was administered at various doses ranged between 10 and 50 mg/100 gm level. Shrimp length was 3.3 ± 0.13 cm and shrimp weight was 9.7 ± 0.12 g in 100 mg. The present finding revealed enhanced growth at high bacteriocin concentration. At 2 × 102 CFU/100 g probiotics in feed, the juvenile shrimp attained 16.8 ± 0.11 g after 40 days. The weight gain was 16.8 ± 0.11 CFU/100 g at 10 × 102 CFU/100 g probiotic concentration.

Keywords

Shrimp gut
Bacillus subtilis
Probiotics
Penaeus indicus
Feed conversion ratio
1

1 Introduction

Aquaculture is a significant economic activity in many countries and is an important source of protein for human consumption (Sathyamoorthi et al., 2019; Kumaresan et al., 2019; Ravichandran et al., 2018;Sathyamoorthi et al., 2017). Based on Food and Agriculture Organization (FAO) report, the production of molluscs, fish and crustaceans in aquaculture increased rapidly from 4% to 27% in 2000. Also, this sector growing very rapidly than that of animal-food sector. In Asia, China plays a significant role than Asia Pacific region and the production was reported about 89% (FAO, 2005). In aquaculture, antibiotics have been frequently used; however, drug resistance is an important problem in aquaculture sector (Ravichandran et al., 2017;Arasu et al., 2017a, Arasu et al., 2017b;Ravichandran et al., 2016; Arasu et al., 2016a,b). The antibiotics administered in aquaculture easily transmitted to human beings through food chain. Also, antibiotics will inhibit or kill useful microorganisms from the gut of fishes and affect digestibility, immunity and fish nutrition (Maynard et al., 2012). There are many findings showing the transferral of highly antibiotic resistant genes among bacteria (Schwarz et al., 2001). Further, the antimicrobial resistance bacteria from the fish could slowly transfer plasmids to other non-resistant bacterium and this may affect human health (Phillips et al., 2004). To avoid the risk of antibiotics resistance, Government institutions implemented strict regulations for the usage of antibiotics in aquaculture practice. European Union (EU) earlier ban on the application of avoparcin in aquaculture practice, later ban on bacitracin, virginiamycin, tylosin and spiramcin, these were included with animal feed as growth promoters to improve the growth of fishes (Delsol et al., 2005).

In shrimp culture, antibiotics is widely used used to improve the production (Chaurasia et al., 2016a; Kumaresan et al., 2016; Chaurasia et al., 2016b; Kumaresan et al., 2015a; Arockiaraj et al., 2015a). The continuous application of these antibiotics in the aquaculture ponds enhane drug resistance among bacterial pathogens in shrimps. The usage of antibiotics significantly affected shrimps and also allows transmit of resistant genes in food chain (Grave et al., 1999). In general, Asian countries have limited guidelines and regulations to control the use of antibiotics in aquaculture sector. In Thailand, antibiotics such as, chloramphenicol has been widely used to control various bacterial diseases in 1990s, however very small quantity of this antibiotic is frequently detected from the cultured shrimp of Thailand. This leads to complete ban of shrimp export in Thailand (Heckman, 2004). Also, chloramphenicol has detected in shrimp farm in Myanmar, Vietnam and India.

To avoid the use of antimicrobial agents, probiotic bacteria were used in aquaculture farms to improve the fish health (Cruz et al., 2012; Palanisamy et al., 2015; Arockiaraj et al., 2015b; Chaurasia et al., 2015; Kumaresan et al., 2015b; Rao et al., 2015). These probiotic organisms significantly improved the growth of fishes and also involved in the production of various extracellulare enzymes. Recently, organisms suh as, bacteria and yeast were screened for the production of various antibiotics for aquaculture industry (Banerjee et al., 2016). Probiotic organisms showed antibacterial potentials and has the ability to alter the microbial flora in the intestine. Probiotic bacteria produce various natural antibacterial substances such as, organic acids and bacterocin (Aarti et al., 2018; Arasu and Al-Dhabi, 2017; Aarti et al., 2017; Arasu et al., 2014a; Arasu et al., 2014b). These agents readily stimulate the production of toxic substances and thus improve the growth (Ringo and Vadstein, 1998). Probiotics has also been used to enhance reproductive performance and stress tolerance in fishs (Mohapatra et al., 2014). The introduction of these probiotics in aquaculture sector significantly replaced the use of commercial synthetic antibiotics. Probiotics release various antibacterial agents with either bactericidal or bacteriostatic properties that inhibit colonization of pathogenic organism in the intestine of shrimps(Aarti et al., 2016; Arasu et al., 2016a,b; Arasu et al., 2014c; Arasu et al., 2014d). Probiotics have been applied along with feed in commercial aquaculture sector to control or prevent bacterial or fungal infections. The applied probiotics effectively control diseases in both fin fishes and shell fishes (Castex et al., 2014). Also, alter the microbial consortium in the gut, reduce microbial diversity and increase richness of beneficial microbes in the gut. Probiotic bacteria have the potential to produce various extracellular enzymes namely proteases, amylases and lipases and also produce the growth promoting substances such as, amino acids, vitamins and fatty acids (Dimitroglou et al., 2011).

2

2 Materials and methods

2.1

2.1 Isolation of probiotic bacteria from the gut of Penaeus indicus

In this study 20P. indicus was collected from the coastal region under disease free conditions. It was maintained in glass tank at 30 ± 2 ppt salinity, pH 7.5 and at 30 ± 2 °C temperature. From the experimental tank, healthy animals were sorted out and subjected for the analysis of intestinal microbial flora. Intestine was carefully removed using a sterile needle and washed with sterile seawater to eliminate non-adherant bacteria. The sample was homogenized using a glass homogenizer. Homogenized sample was serially diluted with sterile seawater and spreaded on Zobell Marine Agar (ZMA) plates by pour plate method. After 48 h incubation, the colonies appeared were purified by repeat straking on ZMA plates (Sivakumar et al., 2012).

2.2

2.2 Screneing of probiotic bacteria

Antibacterial potential of the morphologically distinct bacterial isolates were initially screened by cross streak method using indicator bacterium (Pseudomonas sp). Morphologically distinct bacterial isolates were streaked on ZMA on a straight line and incubated for 24 h at 37 °C. After 24 h incubation, indicator strain (Pseudomonas sp.) was streaked across the probiotic bacterium and further incubated for 24 h at 37 °C. Antibiotic producing ability of the isolate showed zone of inhibiton around the test organism (Karthik et al., 2013). Isolates showed inhibitory activity against indicator organism was further used for secondary screening.

2.3

2.3 Screnning of bacteriocin production by bacteria

Acid production test has been used to identify bacteriocin production by the selected bacteria. The bacterial isolates were cultured in submerged fermentation at 37 °C for 48 h. Then the culture was centrifuged (10,000×g) for 10 min and the cell free supernatant was obtained. It was subjected for acid production test using bromothymol blue indicator. Developement of blue colour into yellow colour indicated the production of acid. Bacterocin positive bacteria did not show any colour change.

2.4

2.4 Screening of antibiotic potentials by well diffusion method

The bacterial strains with acid production negative were confirmed bacteriocin production. Then quantitative assays of the selected culture were performed using agar well diffusion method. The selected bacterial isolate was inoculated in 250 ml Erlenmeyer flask containing nutrient broth (100 ml) and incubated at 37 °C for 48 h. The culture was centrifuged (10,000×g, 10 min) and loaded (10 µl) on Meuller Hinton Agar (MHA) plates. Four pathogenic bacterial isolates, namely, Vibrio sp., Pseudomonas sp., Micrococcus sp. and Bacillus sp. were selected and the zone of inhibition against these bacteria was registered.

2.5

2.5 Characterization of the probiotic bacterium

A probiotic bacterium was selected based on the above screening protocol and sub cultured periodically for every two months and stored at 4 °C. Morphological and biochemical identification of bacterium was performed by standard methods. 16S rDNA analysis was performed to identify the probiotic bacterium in molecular level.

2.6

2.6 Optimized production of bacteriocin by a traditional method

To find the effect of incubation period on the growth and the production of bacteriocin, the selected bacteria strain Bacillus subtilis was cultured in submerged fermentation for 7 days. To study the influence of pH on bacteriocin production, the culture medium pH was adjusted at various pH values (pH 4.0 – 9.0). After 72 h incubation, bacterial growth and bacteriocin production were determined from the cell free supernatant. To evaluate the impact of temperature on the growth and the production of bacteriocin, the strain was incubated in culture medium for 20–45 °C. To analyze the impact of carbon source, glucose, starch, glycerol, maltose, lactose and sucrose were supplemented with the culture medium at 1% (w/v) level. To the control, carbon source was not incorporated. The nitrogen sources such as, sodium nitrate, ammonium chloride, peptone, yeast extract, beef extract and glycine were incorporated with the culture medium at a level of 1% (w/v) to explore best nitrogen sources. These experiments were performed in triplicates and Mean ± SD was calculated. One-way analysis of variance (ANOVA) was used to analyze the significance level.

2.7

2.7 Shrimp feed formulation

The experimental diet consists of protein (47%), lipid (8.2%), ash (13.2%), moisture content (8.1%) and fibre level was 2.1%. These ingredients were used for the formulation of artificial shrimp diet as suggested by Boonyaratpalin and New (1982). These feed ingredients were mixed with gelatine 5% (w/v) and required water was added. The pH of the feed was adjusted as 7.0 ± 0.20. The formulated diet consists of (g) fish (2 8 0), shrimp head waste (1 0 0), squid meal (20), squid liver (30), wheat (60), wheat flour (2 0 0), soy bean (1 0 0), broken rice (1 0 0), fish oil (25), vitamin (10), minerals (40) and gelatine (15). To the feed bacteriocin was added (10 mg–100 mg) and used for experiment (Swapna et al., 2015).

2.8

2.8 Effect of bacteriocin on the growth performance of P. Indicus

P. indicus (8 ± 2 g) was purchased from the shrimp farm and stock was maintained in culture tank (25 L). Continuous air flow was attached with the tank and the flow rate was maintained as 2.0 L/min. The experimental animal was fed with bacteriocin containing diet at various concentrations (10 mg–100 mg) for 40 days at the rate of 10% total body weight. The control and experimental group animals were fed twice in a day (morning and evening). Debris and uneaten pellet diet was removed every day. The other factors such as, salinity, temperature and pH was also monitiored continuously.

2.9

2.9 Effect of probiotic on the growth and survival of juvenile P. Indicus

The screened probiotic strain was used for the production of secondary metabolites. The probiotic bacterium was grown in nutrient broth medium for 48 h. It was centrifuged at 10,000 rpm and the cells were collected. The obtained pellet was washed with physiological saline (0.9% NaCl, w/v) and resuspended with the same. To the artificial diet, probiotic organism was sprayed and the feed was dried at room temperature. Probioic organism was sprayed at five different concentrations viz., 2 × 102, 4 × 104, 6 × 106, 8 × 108 and 10 × 1010 CFU/100 g of feed. Inoculum was not included with the control shrimp. 20 experimental animals were introduced into the experimental tank (12 ± 0.5 g each) and growth performance was analyzed (Ajitha et al., 2004).

3

3 Results and discussion

3.1

3.1 Gut probiotics

A total of 104 bacterial isolates were isolated from the gut of P. indicus. The morphologically different bacterial isolate were further screened for probiotic properties. Based on inital screening only seven bacterial isolates were tested against indicator bacterium. The zone of inhibition against the tested bacterial pathogens was tabulated (Table 1). The present finding revealed that B. subtilis showed potent ablity to control the growth of marine pathogens. There are various bacterial strains from the genus Bacillus produce various bioactive molecules such as, polymyxin, bacitracin and colistin. Bacillus species are aerobic bacteria, Gram’s-positive rod which are widely distributed. These organisms were found in gastrointestinal tracts, rocks and aquatic environment (Nicholson, 2002). Previously, many bacteriocin producing bacterial isolates have been screened from various sources. In a study, Schallmey et al. (2004) reported the ability of Bacillus sp. to produce bacitracin A.

Table 1 Antibacterial activity of probiotic bacteria isolated from the shrimp gut of P. indicus.
Gut bacteria Zone of inhibition (mm)
Bacillus sp. Pseudomonas sp. Vibrio sp. Micrococcus sp.
D1 12 ± 2 12 ± 2 21 ± 3 12 ± 2
D2 10 ± 1 18 ± 3 – 18 ± 1
D3 11 ± 2 10 ± 2 15 ± 2 16 ± 3
D4 – 12 ± 1 10 ± 1 –
D5 17 ± 1 19 ± 1 22 ± 2 21 ± 1
D6 11 ± 2 – – 20 ± 2
D7 – 11 ± 2 – –

The isolated probiotic bacteria showed activity against these bacteria. 16S rDNA sequencing was performed and identified as strain D5. The strain D5 showed potent activity against all tested organisms. The isolated strain D5 showed 17 mm zone of inhibition against Bacillus sp., 19 mm against Pseudomonas sp., 22 mm against Vibrio sp., and 22 mm against Micrococcus sp. Based on these experiments antibacterial potential of B. subtilis was studied and it was selected for further studies. The identified bacterial isolate was Gram –positive, motile, rod shaped, hydrolyzed casein and starch. It was not able to hydrolyze urea, nitrate reduction-negative, indole-negative and citrate-negative. Bacteriocins have potent activity against various bacterial pathogens. In a study, Bacillus licheniformis for bacillocin production and effectively inhibited the growth of Micrococcus flavus. Likewise, Mendo et al. (2004) isolated a probiotic Bacillus licheniformis strain from the extreme environment which inhibited the growth of various Gram-positive bacteria. Kugler et al. (1990) isolated the bacteria such as, Bacillus subtilis, B. cereus, and B. licheniformis for the production of seconday metabolites and the produced metabolites were found to be effective against both Gram-positive and Gram-negative bacteria.

3.2

3.2 Optimization of growth of bacteria and bacteriocin production in submerged fermentation

The influence of bacterial growth and bacterocin production in relation with incubation time was evaluated. Bacteriocin production was found to be high at the stationary phase and was detected only after 48 h and reduced considerably after fifth day (Table 2). Likewise, Eppelmann et al. (2001) found that Bacillus sp. produce secondary metabolites after 48 h incubation and reached maximum level after 96 h. In L. brevis FPTLB3 and W. paramesenteroides DFR-8 bacteriocin synthesis were found to be maximum after 96 h of incubation (Banerjee et al., 2013). However, in B. cereus Bc7, production of bacteriocin was maximum before stationary phase (Oscáriz and Pisabarro, 2000).

Table 2 Effect of incubation time on the growth and bacteriocin production from B. subtilis. Data represent mean ± SD (n = 3).
Incubation time (days) Optical Density (600 nm) Zone of inhibition (mm)
1 0.329 ± 0.021 0 ± 0
2 1.572 ± 0.057 7 ± 2
3 2.195 ± 0.081 15 ± 1
4 1.986 ± 0.038 21 ± 3
5 1.822 ± 0.042 18 ± 5
6 1.712 ± 0.061 12 ± 2
7 0.713 ± 0.049 10 ± 1

Bacterial growth and bacterocin production was maximum at pH 7.0, however secondary metabolite production was found to be optimum between pH 6.0 and 8.0 (Table 3). Hydrogen ion concentration (pH) of the culture medium is one of the significant factors for the production of bacteriocin because it significantly affects the adsorption of secondary metabolites to their cell surface and attachment or aggregation of the newly produced cells. Also, the pH of the culture medium critically plays significant role in the degradation of bacteriocins by proteases (Cheigh et al., 2002). The influence of pH on bacteriocins production was analyzed from L. mesenteroides FR52, Bacillus spp. P11 and L. plantarum 17.2b and reported earlier (Delgado et al., 2007). The present findings are in accordance with observation made previously with other Bacillus sp. In Bacillus subtilis KIBGE IB-17, bacteriocin snynthesis was optimum at pH 7.0 (Ansari et al., 2012). Also, in B. licheniformis, bacteriocins production was found to be high from 7.0 to 8.0 (Martirani et al., 2002).

Table 3 Effect of pH on the growth and bacteriocin production from B. subtilis. Data represent mean ± SD (n = 3).
pH Optical Density (at 600 nm) Zone of inhibition (mm)
4 0.126 ± 0.033 0 ± 0
5 0.572 ± 0.039 7 ± 1
6 1.893 ± 0.057 12 ± 0
7 1.972 ± 0.064 27 ± 5
8 1.426 ± 0.092 18 ± 3
9 1.172 ± 0.074 11 ± 2
10 0.724 ± 0.059 ± 0

Incubation temperature is one of the important factors which significantly influences on growth and production of bacteriocin. Bacterial growth and bacterocin production (17 ± 2 mm) were maximum at 30 °C (Table 4). Fermentation temperature is a critical factor which significantly influenced on bacterocin production. In our study, bacteriocin production was maximum at 30 °C. However, various temperatures have been reported for the production of secondary metabolites by bacteria. Kim et al. reported that secondary metabolite production was maximum at 37 °C in the case of Micrococcus sp. GO5.

Table 4 Effect of temperature on the growth and bacteriocin production from B. subtilis. Data represent mean ± SD (n = 3).
Temperature (°C) Optical Density (600 nm) Zone of inhibition (mm)
20 1.127 ± 0.057 14 ± 1
25 1.565 ± 0.033 19 ± 2
30 1.946 ± 0.029 22 ± 3
35 1.707 ± 0.032 17 ± 2
40 1.632 ± 0.044 13 ± 2
45 0.501 ± 0.024 10 ± 1
50 0.305 ± 0.019 10 ± 0

The influence of various carbon sources on the growth and bacteriocin production were analyzed. Glucose stimulated growth and bacteriocin production (21 ± 3 mm) (Table 5). In the case of bacteria, glucose is one of the important nutrients and it significantly influenced on bacteriocin production. In a study, Todorov and Dicks (2005) reported maximum production of bacteriocin in the medium containing glucose. Likewise, in E. faecium ST311LD glucose stimulated the production of bacteriocin and has been reported by Todorov (2008). The present results suggest that the glucose moiety of sucrose was very essential for the production of bacteriocin. Todorov and Dicks also found the positive regulation of glucose on bacteriocin production by Lactobacillus pentosus ST151. Among the supplemented nitrogen sources, glycine positively influenced on growth and metabolite production than other sources (Table 6). Our findings are in accordance the results reported previoiusly with many bacteria. In lactic acid probiotic bacteria and B. cereus XH25, glycine significantly stimulated bacteriocin production (Meera and Devi, 2012). Nitrogen sources enhance bacterial biomass and bacteriocin production in Lactococcus lactis (Kim et al., 2007) (Tables 7 and 8).

Table 5 Effect of carbon sources on the growth and bacteriocin production from B. subtilis. Data represent mean ± SD (n = 3).
Carbon source (1%) Optical Density (600 nm) Zone of inhibition (mm)
Glucose 2.208 ± 0.101 21 ± 3
Glycerol 1.529 ± 0.068 12 ± 2
Starch 1.796 ± 0.031 20 ± 2
Lactose 1.097 ± 0.042 14 ± 1
Maltose 1.927 ± 0.062 18 ± 3
Sucrose 1.508 ± 0.079 12 ± 3
Control 2.042 ± 0.051 17 ± 4
Table 6 Effect of nitrogen sources on the growth and bacteriocin production from B. subtilis. Data represent mean ± SD (n = 3).
Nitrogen source (1%) Optical Density (at 600 nm) Zone of inhibition (mm)
Ammonium chloride 1.629 ± 0.072 13 ± 2
Sodium nitrate 1.803 ± 0.052 17 ± 1
Peptone 1.554 ± 0.042 13 ± 2
Beef extract 1.992 ± 0.039 18 ± 2
Yeast extract 1.982 ± 0.041 16 ± 4
Glycine 2.109 ± 0.038 19 ± 3
Control 1.434 ± 0.061 16 ± 2
Table 7 Administration of bacteriocin with formulated diet and its effect on the growth and survival of P. indicus. Data represent mean ± SD (n = 10).
Bacteriocin (mg) Shrimp length (cm) Shrimp weight (g) Survival (%)
10 3.3 ± 0.13 9.7 ± 0.12 98
20 4.9 ± 0.15 11.3 ± 0.15 98
30 5.7 ± 0.2 12.2 ± 0.19 100
40 5.3 ± 0.2 13.2 ± 0.13 100
50 4.8 ± 0.15 12.8 ± 0.11 100
Table 8 Effect of probiotics on the growth of P. indicus. Data represent mean ± SD (n = 10).
Probiotics (CFU/100 g of feed) Shrimp length (cm) Shrimp weight (g)
2 × 102 4.4 ± 0.13 13.7 ± 0.12
4 × 104 5.9 ± 0.12 14.3 ± 0.15
6 × 106 6.1 ± 0.21 15.2 ± 0.19
8 × 108 6.2 ± 0.23 16.2 ± 0.13
10 × 1010 6.2 ± 0.16 16.8 ± 0.11

3.3

3.3 Influence of bacteriocin and probiotics on the growth and survival of P. Indicus

In the present study, bacteriocin supplemented diet enhanced the growth than control shrimp. Bacteriocin was administered at various doses ranged between 10 and 50 mg/100 gm level. Shrimp length was 3.3 ± 0.13 cm and shrimp weight was 9.7 ± 0.12 g in 100 mg bacteriocin group. The present finding revealed enhanced growth at high bacteriocin concentration. The administered bacteriocin enhanced the immunity and induced immune modulation. Previously, Chiu et al. (2007) reported increased superoxide dismutase and phenoloxidase activity in probiotic administered diet. In a study Itami et al. (1998) reported immuno stimulatory effect of peptidoglycans of L. plantarum in Penaeus japonicas and were effective against Vibrio penaeicida. Probioic organism was administered at five different concentrations (2 × 102, 4 × 104, 6 × 106, 8 × 108 and 10 × 1010 CFU/100 g) with feed and growth performance was analyzed. Probiotic feed stimulated the growth of P. indicus. At 2 × 102 CFU/100 g feed, the juvenile shrimp attained 16.8 ± 0.11 g weight after 40 days. The weight gain was 16.8 ± 0.11 g at 10 × 102 CFU/100 g probiotic concentration. In a study, Rengpipat et al., 1998 used Bacillus S11 as a probiotic isolate in shrimp culture and reported better survival rate in probiotic treated animals than control. The application of probiotics with feed enhanced digestibility. The administered probiotics significantly improved the growth, survival and Feed Conversion Ratio (FCR) in shrimps (Hai et al., 2009).

4

4 Conclusions

Intestinal microbial flora may significantly influence the diversity of the gut microbiota in shrimps. The present finding shows bacteria isolated from Penaeus indicus have the ability to produce secondary metabolites and these metabolites showed activity against various bacteria. P. indicus fed with probiotic diet enhanced growth rate in juvenile shrimps. The applied proiotic organism could colonize and synthezise various secondary metabolites in the gut. Also, probiotic may involve in the synthesis of various enzymes to enhance the digestibility of shrimp feed.

Acknowledgements

The authors (KAG, SM and FAM) express their sincere appreciation to the Researcher Supporting Project No. RSP-2019/48, King Saud University, Riyadh, Saudi Arabia. The authors Hak-Jae Kim thank the support received from Soochunhyang University research fund for this research work.

References

  1. , , , , , , , . In vitro investigation on probiotic, anti-Candida, and antibiofilm properties of Lactobacillus pentosus strain LAP1. Arch. Oral Biol.. 2018;89:99-106.
    [Google Scholar]
  2. , , , , , , , , . In vitro studies on probiotic and antioxidant properties of Lactobacillus brevis strain LAP2 isolated from Hentak, a fermented fish product of North-East India. LWT – Food Sci. Technol.. 2017;86(2017):438-446.
    [Google Scholar]
  3. Aarti, C., Khusro, A., Arasu, M.V., Agastian, P., & Al–Dhabi, N.A. (2016). Biological potency and characterization of antibacterial substances produced by Lactobacillus pentosus isolated from Hentak, a fermented fish product of North–East India. SpringerPlus, 5, Article Number: 1743.
  4. , , , , , . Probiotic effects of lactic acid bacteria against Vibrio alginolyticus in Penaeus (Fenneropenaeus) indicus (H. Milne Edwards) Asian Fish. Sci.. 2004;17:71-80.
    [Google Scholar]
  5. , , , , , . Bacteriocin (BAC IB17): screening, isolation and production from Bacillus subtilis KIBGE IB 17. Pak. J. Pharm. Sci.. 2012;25(1):195-201.
    [Google Scholar]
  6. , , , , , , , . Bactericidal activity of fish galectin 4 derived membrane-binding peptide tagged with oligotryptophan. Dev. Comp. Immunol.. 2017;71:37-48.
    [Google Scholar]
  7. , , , , , , , . Bacterial membrane binding and pore formation abilities of carbohydrate recognition domain of fish lectin. Dev. Comp. Immunol.. 2017;67:202-212.
    [Google Scholar]
  8. , , , , , , . Coagulation profile, gene expression and bioinformatics characterization of coagulation factor X of striped murrel Channa striatus. Fish Shellfish Immunol.. 2016;55:149-158.
    [Google Scholar]
  9. , , , , , , , , . Characterization, phylogenetic affiliation and probiotic properties of high cell density Lactobacillus strains recovered from silage. J. Sci. Food Agric.. 2014;94:2429-2440.
    [Google Scholar]
  10. , , . In vitro antifungal, probiotic, and antioxidant functional properties of a novel Lactobacillus paraplantarum isolated from fermented dates in Saudi Arabia. J. Sci. Food Agric. 2017
    [CrossRef] [Google Scholar]
  11. , , , , , . In vitro importance of probiotic Lactobacillusplantarum related to medical field. Saudi J. Biol. Sci. 2016
    [CrossRef] [Google Scholar]
  12. , , , , , , , , , . Identification and characterization of Lactobacillus brevis P68 with antifungal, antioxidant and probiotic functional properties. Indian J. Microbiol.. 2014;55:19-28.
    [Google Scholar]
  13. , , , , , , , , , . Enhancing nutritional quality of silage by fermentation with Lactobacillus plantarum. Indian J. Microbiol.. 2014;54:396-402.
    [Google Scholar]
  14. , , , , , , , , , . In vitro antifungal, probiotic and antioxidant properties of novel Lactobacillusplantarum K46 isolated from fermented sesame leaf. Ann. Microbiol.. 2014;64:1333-1346.
    [Google Scholar]
  15. , , , , , . Molecular and functional roles of 6C CC chemokine 19 in defense system of striped murrel Channa striatus. Fish Shellfish Immunol.. 2015;45(2):817-827.
    [Google Scholar]
  16. , , , , , , , , . Fish chemokines 14, 20 and 25: A comparative statement on computational analysis and mRNA regulation upon pathogenic infection. Fish Shellfish Immunol.. 2015;47(1):221-230.
    [Google Scholar]
  17. , , , , , . Mode of association, enzyme producing ability and identification of autochthonous bacteria in the gastrointestinaltract of two Indian air-breathing fish, murrel (Channa punctatus) and stinging catfish (Heteropneustes fossilis) Proc. Zool. Soc.. 2016;1(1):20-25.
    [Google Scholar]
  18. , , , , . Characterization and identification of enzyme-producing autochthonous bacteria from the gastrointestinal tract of two Indian air-breathing fish. Benef. Microbes.. 2013;4:277-284.
    [Google Scholar]
  19. , , , . Probiotic applications in crustaceans, in aquaculture nutrition: gut health, probiotics and prebiotics. Chichester, UK: John Wiley & Sons Ltd; .
  20. , , , , , , , , . Molecular importance of prawn large heat shock proteins 60, 70 and 90. Fish Shellfish Immunol.. 2016;48:228-238.
    [Google Scholar]
  21. , , , , , , , , . In-silico analysis and mRNA modulation of detoxification enzymes GST delta and kappa against various biotic and abiotic oxidative stressors. Fish Shellfish Immunol.. 2016;54:353-363.
    [Google Scholar]
  22. , , , , , , . Molecular profiles and pathogen-induced transcriptional responses of prawn B cell lymphoma-2 related ovarian killer protein (BOK) Fish Shellfish Immunol.. 2015;45(2):598-607.
    [Google Scholar]
  23. , , , . Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi. J. Biotechnol.. 2002;95:225-235.
    [Google Scholar]
  24. , , , , , . Immune response and gene expression in white shrimp, Litopenaeus vannamei, induced by Lactobacillus plantarum. Fish Shellfish Immun.. 2007;23:364e77.
    [Google Scholar]
  25. , , , , . Use of probiotics in aquaculture. ISRN Microbiol. 2012:96845.
    [Google Scholar]
  26. , , , . Optimum bacteriocin production by Lactobacillus plantarum 17.2b requires absence of NaCl and apparently follows a mixed metabolite kinetics. J. Biotechnol.. 2007;130:193-201.
    [Google Scholar]
  27. , , , , , , . Effect of the growth promoter avilamycin on emergence and persistence of antimicrobial resistance in enteric bacteria in the pig. J. Appl. Microbiol.. 2005;98:564-571.
    [Google Scholar]
  28. , , , , , , , , . Microbial manipulations to improve fish health and production a Mediterranean perspective. Fish Shellfish Immunol.. 2011;30(1):1-16.
    [Google Scholar]
  29. , , , . Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis. J. Biol. Chem.. 2001;276:34824-34831.
    [Google Scholar]
  30. FAO. In: Serrano PH, ed. 2005. Responsible Use of Antibiotics in Aquaculture. Rome: FAO; FAO Fisheries Technical Paper. 98, 469.
  31. , , , , . Surveillance of the overall consumption of antibacterial drugs in humans, domestic animals and farmed fish in Norway in 1992 and 1996. J. Antimicrob. Chemother.. 1999;43:243-252.
    [Google Scholar]
  32. , , , . The use of customised probiotics in the cultivation of western king shrimps (Penaeus latisulcatus Kishinouye, 1896) Fish Shellfish Immunol.. 2009;27:100-104.
    [Google Scholar]
  33. , . What else can happen? Other problems for fish production. Aquacult. Magaz.. 2004;30(3):27-40.
    [Google Scholar]
  34. , , , , , , . Enhancement of disease resistance of kuruma shrimp, Penaeus japonicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilum. Aquaculture. 1998;164:277-288.
    [Google Scholar]
  35. , , , , . Efficacy of bacteriocin from Lactobacillus sp. (AMET 1506) as a biopreservative for seafood’s under different storage temperature conditions. J. Modern Biotechnol.. 2013;2(3):59-65.
    [Google Scholar]
  36. , , , . Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss) J. Appl. Microbiol.. 2007;102:1654-1664.
    [Google Scholar]
  37. , , , , , . Rhizocticin an antifungal phosphono-oligopeptide of Bacillus subtilis ATCC 6633: biological properties. Arch. Microbiol.. 1990;153:276-281.
    [Google Scholar]
  38. , , , , , , , , . Arockiaraj J.A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica. Mol. Immunol. 2015,68,:421-433.
    [Google Scholar]
  39. , , , , , , , . Comparative analysis of CsCu/ZnSOD defense role by molecular characterization: gene expression-enzyme activity-protein level. Gene. 2015;564:53-62.
    [Google Scholar]
  40. , , , , , , , , . A comparative transcriptome approach for identification of molecular changes in Aphanomycesinvadans infected Channastriatus. Mol. Biol. Rep.. 2019;45:2511-2523.
    [Google Scholar]
  41. , , , , , , , , . Multifunctional murrel caspase 1, 2, 3, 8 and 9: Conservation, uniqueness and their pathogen-induced expression pattern. Fish Shellfish Immunol.. 2016;49:493-504.
    [Google Scholar]
  42. , , , , . Purification and partial characterization of bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis. Microb. Cell Factories.. 2002;1:1-5.
    [Google Scholar]
  43. , , , , . Reciprocal interactions of the intestinal microbiota and immune system. Nature. 2012;489:231-241.
    [Google Scholar]
  44. , , . Partial characterization and optimization of parameters for Bacteriocin production by Probiotic Lactic acid bacteria. J. Microbiol. Biotechnol. Res.. 2012;2(2):357-365.
    [Google Scholar]
  45. , , , , , . Purification and characterization of a new peptide antibiotic produced by a thermotolerant Bacillus licheniformis strain. Biotechnol. Lett.. 2004;26:115-119.
    [Google Scholar]
  46. , , , , , . Beneficial effects of dietary probiotics mixture on hemato-immunology and cell apoptosis of Labeo rohita fingerlings reared at higher water temperatures. PLoSONE. 2014;9(6) 100929
    [Google Scholar]
  47. , . Roles of Bacillus endospores in the environment. Cell Mol. Life Sci.. 2002;59:410-416.
    [Google Scholar]
  48. , , . Characterization and mechanism of action of cerein 7, a bacteriocin produced by Bacillus cereus Bc7. J. Appl. Microbiol.. 2000;89(2):361-369.
    [Google Scholar]
  49. , , , , , , . Functional roles and gene regulation of tumor necrosis factor receptor 1 in freshwater striped murrel. Mol. Immunol.. 2015;66:240-252.
    [Google Scholar]
  50. , , , , , , , , , . Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J. Antimicrob. Chemother.. 2004;53:28-52.
    [Google Scholar]
  51. , , , , , , , . Defense properties in the epidermal mucus of different freshwater fish species. Aquacult., Aquar., Conservat. Legisl.. 2015;8:184-190.
    [Google Scholar]
  52. , , , , , , , , , . Pellino-1 derived cationic antimicrobial prawn peptide: bactericidal activity, toxicity and mode of action. Mol. Immunol.. 2016;78:171-182.
    [Google Scholar]
  53. , , , , , , . A cumulative strategy to predict and characterize antimicrobial peptides (AMPs) from protein database. Int. J. Pept. Res. Ther.. 2017;23:281-290.
    [Google Scholar]
  54. , , , , , , , , , . Bactericidal and fungistatic activity of peptide derived from GH18 domain of prawn chitinase 3 and its immunological functions during biological stress. Int. J. Biol. Macromol.. 2018;106:1014-1022.
    [Google Scholar]
  55. , , , , . Effects of a probiotic bacterium on black tiger shrimp Penaeus monodon survival and growth. Aquaculture. 1998;167:301-313.
    [Google Scholar]
  56. , , . Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae. J. Appl. Microbiol.. 1998;84(2):227-233.
    [Google Scholar]
  57. , , , , , , , . Gene expression and in silico analysis of snakehead murrel interleukin 8 and antimicrobial activity of C-terminal derived peptide WS12. Vet. Immunol. Immunopathol.. 2017;190:1-9.
    [Google Scholar]
  58. , , , , , , , , , , , . Therapeutic cationic antimicrobial peptide (CAP) derived from fish aspartic proteinase cathepsin D and its antimicrobial mechanism. Int. J. Pept. Res. Therapeut.. 2019;25:93-105.
    [Google Scholar]
  59. , , , . Developments in the use of Bacillus species for industrial production. Can. J. Microbiol.. 2004;50:1-17.
    [Google Scholar]
  60. , , , . Use of antimicrobial agents in veterinary medicine and food animal production. Int. J. Antimicrob. Agents.. 2001;17:431-437.
    [Google Scholar]
  61. , , , . Probiotic effect of Lactobacillus acidophilus against vibriosis in juvenile shrimp (Penaeus monodon) Afr. J. Biotechnol.. 2012;11(91):15811-15818.
    [Google Scholar]
  62. , , , . Effect of probiotic bacteria Bacillus licheniformis and Lactobacillus rhamnosus on growth of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) Eur. J. Exp. Biol.. 2015;5(11):31-36.
    [Google Scholar]
  63. , , . Optimization of bacteriocin ST311LD production by Enterococcus faecium ST311LD, isolated from spoiled black olives. J. Microbiol.. 2005;43:370-374.
    [Google Scholar]

Fulltext Views
21

PDF downloads
7
View/Download PDF
Download Citations
BibTeX
RIS
Show Sections

Suggested read for related articles:

© Copyright 2025 – Journal of King Saud University – Science.

Published by Scientific Scholar on behalf of King Saud University.

ISSN (Print): 1018-3647
ISSN (Online): 2213-686X

Scientific Scholar CrossRef Creative Commons Open Acess Portico