Fate of carotenoid-producing Bacillus aquimaris SH6 colour spores in shrimp gut and their dose-dependent probiotic activities

PLoS One. 2018 Dec 21;13(12):e0209341. doi: 10.1371/journal.pone.0209341. eCollection 2018.


Bacillus aquimaris SH6 spores produce carotenoids that are beneficial to white-leg shrimp (Litopenaeus vannamei) health. However, the optimal dose and mechanisms behind these effects are not well understood. We investigated the fate of SH6 spores in the gut of L. vannamei. Shrimp were divided into six groups administrated with either feed only (negative control) or SH6 spores at 5 × 106 CFU/g pellet (high dose, SH6 spore-H group), 1 × 106 CFU/g pellet (medium dose, SH6 spore-M group), 2 × 105 CFU/g pellet (low dose, SH6 spore-L group), astaxanthin at 0.5 mg/g pellet (Carophyll group), or carotenoids from SH6 vegetative cells at 5 μg/g pellet (SH6 carotenoid group). The growth rate was highest in SH6 spore-H (3.38%/day), followed by SH6 spore-M (2.84%/day) and SH6 spore-L (2.25%/day), which was significantly higher than the control (1.45%/day), Carophyll (1.53%/day) or SH6 carotenoid (1.57%/day) groups. The astaxanthin levels (1.9-2.0 μg/g shrimp) and red-colour scores (21-22) in SH6 spore-H/M were higher than the control (astaxanthin: 1.2 μg/g shrimp; red score: 20) or SH6 spore-L, but lower than the Carophyll and SH6 carotenoids. Feeding with medium and high doses of SH6 spores after 28 days resulted in respective 1.3-2-fold increases in phenol oxidase activity and 8-9 fold increases in Rho mRNA expression compared to the control and low dose group. The live-counts of SH6 in the gut gradually increased during the 28-day feeding period with SH6 spores at different concentrations, starting from 4.1, 8.2, and 5.4 × 104 CFU/g gut at day 1 and reaching 5.3, 5.1, and 4.4 × 105 CFU/g gut in the SH6-H/M/L groups, respectively, at day 28. Gut microbiota became more diversified, resulting in a 2-8-fold increase in total bacterial live-counts compared to the controls. SH6 spore germination was detected by measuring the mRNA expression of a specific sequence coding for SH6 amylase at 4 h, reaching saturation at 24 h. Our results confirm that SH6 spores colonize and germinate in the gut to improve the microbial diversity and boost the immune system of shrimp, exhibiting beneficial effects at >1 × 106 CFU/g pellet.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animal Feed / microbiology
  • Animals
  • Aquaculture / methods
  • Bacillus / physiology
  • Carotenoids / administration & dosage*
  • Carotenoids / immunology
  • Carotenoids / metabolism
  • Color
  • Epithelium / microbiology
  • Epithelium / physiology
  • Gastrointestinal Microbiome / physiology*
  • Penaeidae / microbiology*
  • Penaeidae / physiology
  • Probiotics / administration & dosage*
  • Shellfish
  • Spores, Bacterial / immunology
  • Spores, Bacterial / metabolism*
  • White spot syndrome virus 1


  • Carotenoids

Grant support

This study was funded by a grant from Third World Academy of Science (TWAS) to A.T.V. Nguyen under project number 16-549 RG/BIO/AS_G - FR3240293311, and a grant from Vietnam National University_Hanoi, Key Laboratory of Enzyme and Protein Technology (KLEPT) to Dr. Thang Dinh Nguyen and A.T.V. Nguyen under project number KLEPT.18.01. While Dr. Thang Dinh Nguyen is the PI for KLEPT.18.01 and A.T.V. Nguyen is a key member, Dr. Thang Dinh Nguyen is not an author of the present study. The authors would like to express sincere thanks to TWAS and KLEPT for funding the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.