Assessment of Encapsulated Probiotic Lactococcus lactis A12 Viability Using an In Vitro Digestion Model for Tilapia.

Probiotics face harsh conditions during their transit through the gastrointestinal tract (GIT) of fish because of low-pH environments and intestine fluid. Therefore, the evaluation of probiotic viability under simulated gastrointestinal conditions is an important step to consider for probiotic supplementation in fish feed prior to in vivo trials. Therefore, this study aimed to evaluate the effect of stomach and intestinal simulated conditions on the viability of encapsulated Lactococcus lactis A12 using an in vitro digestion model for tilapia. A Box Behnken design was used to evaluate the potential effect of three factors, namely stomach pH, residence time in the stomach, and enzyme quantity, on the viability of encapsulated Lactococcus lactis A12. As the main results, low pH (4.00), long residence time (4 h), and enzyme quantity (2.68 U of total protease activity) led to lower final cell counts after the phases of the stomach and intestine. Encapsulated probiotic bacteria showed higher viability (p < 0.05) and antibacterial activity (p < 0.05) against the pathogen Streptococcus agalactiae than non-encapsulated bacteria. The results suggest that L. lactis A12 survives in GIT conditions and that the proposed in vitro model could be used to explore the viability of probiotic bacteria intended for fish feed supplementation.

Production of a potential multistrain probiotic in co-culture conditions using agro-industrial by-products-based medium for fish nutrition.

BACKGROUND: Probiotics are viable microorganisms that when administered in adequate amounts confer health benefits to the host. In fish, probiotic administration has improved growth, and immunological parameters. For this reason, it is necessary production of probiotic bacteria, however, commercial culture mediums used for probiotic growth are expensive, so the design of a "low" cost culture medium is necessary. Therefore, this research aimed to produce a potential multistrain probiotic preparation composed of L. lactis A12 and Priestia species isolated from Nile tilapia (Oreochromis niloticus) gut using an agro-industrial by-products-based culture medium. RESULTS: A Box-Behnken design with three factors (whey, molasses, and yeast extract concentration) was used. As the main results, a high concentration of three components enhanced the viability of L. lactis A12, however, viable cell counts of Priestia species were achieved at low molasses concentrations. The Optimal conditions were 1.00% w/v whey, 0.50% w/v molasses, and 1.50% w/v yeast extract. L. lactis A12 and Priestia species viable counts were 9.43 and 6.89 Log10 CFU/mL, respectively. L. lactis A12 concentration was higher (p < 0.05) in the proposed medium compared to commercial broth. CONCLUSIONS: It was possible to produce L. lactis A12 and Priestia species in co-culture conditions. Whey and molasses were suitable components to produce the multistrain preparation. The cost of the proposed culture medium was 77.54% cheaper than the commercial medium. The proposed culture medium could be an alternative to commercial mediums for the production of this multistrain probiotic.

Evaluation of dietary single probiotic isolates and probiotic multistrain consortia in growth performance, gut histology, gut microbiota, immune regulation, and infection resistance of Nile tilapia, Oreochromis niloticus, shows superior monostrain performance.

The probiotic potential of a designed bacterial consortia isolated from a competitive exclusion culture originally obtained from the intestinal contents of tilapia juveniles were evaluated on Nile tilapia alevins. The growth performance, intestinal histology, microbiota effects, resistance to Streptococcus agalactiae challenge, and immune response were assessed. In addition, the following treatments were included in a commercial feed: A12+M4+M10 (Lactococcus lactis A12, Priestia megaterium M4, and Priestia sp. M10), M4+M10 (P. megaterium M4, and Priestia sp. M10) and the single bacteria as controls; A12 (L. lactis A12), M4 (P. megaterium M4), M10 (Priestia sp. M10), also a commercial feed without any probiotic addition was included as a control. The results showed that all probiotic treatments improved the growth performance, intestinal histology, and resistance during experimental infection with S. agalactiae in comparison to the control fish. Also, the administration of probiotics resulted in the modulation of genes associated with the innate and adaptive immune systems that were non-dependent on microbial colonization. Surprisingly, L. lactis A12 alone induced benefits in fish compared to the microbial consortia, showing the highest increase in growth rate, survival during experimental infection with S. agalactiae, increased intestinal fold length, and the number of differentially expressed genes. Lastly, we conclude that a competitive exclusion culture is a reliable source of probiotics, and monostrain L. lactis A12 has comparable or even greater probiotic potential than the bacterial consortia.

Competitive Exclusion Bacterial Culture Derived from the Gut Microbiome of Nile Tilapia (Oreochromis niloticus) as a Resource to Efficiently Recover Probiotic Strains: Taxonomic, Genomic, and Functional Proof of Concept.

This study aims to mine a previously developed continuous-flow competitive exclusion culture (CFCEC) originating from the Tilapia gut microbiome as a rational and efficient autochthonous probiotic strain recovery source. Three isolated strains were tested on their adaptability to host gastrointestinal conditions, their antibacterial activities against aquaculture bacterial pathogens, and their antibiotic susceptibility patterns. Their genomes were fully sequenced, assembled, annotated, and relevant functions inferred, such as those related to pinpointed probiotic activities and phylogenomic comparative analyses to the closer reported strains/species relatives. The strains are possible candidates of novel genus/species taxa inside Lactococcus spp. and Priestia spp. (previously known as Bacillus spp.) These results were consistent with reports on strains inside these phyla exhibiting probiotic features, and the strains we found are expanding their known diversity. Furthermore, their pangenomes showed that these bacteria have indeed a set of so far uncharacterized genes that may play a role in the antagonism to competing strains or specific symbiotic adaptations to the fish host. In conclusion, CFCEC proved to effectively allow the enrichment and further pure culture isolation of strains with probiotic potential.

Probiotics in tilapia (Oreochromis niloticus) culture: Potential probiotic Lactococcus lactis culture conditions.

Tilapia is one of the most extensively farmed fish on a global scale. Lately, many studies have been carried out to select and produce probiotics for cultured fish. Bacteria from the genera Bacillus, Lactiplantibacillus (synonym: Lactobacillus), and Lactococcus are the most widely studied with respect to their probiotic potential. Among these microorganisms, Lactococcus lactis has outstanding prospects as a probiotic because it is generally recognized as safe (GRAS) and has previously been shown to exert its probiotic potential in aquaculture through different mechanisms, such as competitively excluding pathogenic bacteria, increasing food nutritional value, and enhancing the host immune response against pathogenic microorganisms. However, it is not sufficient to simply select a microorganism with significant probiotic potential for commercial probiotic development. There are additional challenges related to strategies involving the mass production of bacterial cultures, including the selection of production variables that positively influence microorganism metabolism. Over the last ten years, L. lactis production in batch and fed-batch processes has been studied to evaluate the effects of culture temperature and pH on bacterial growth. However, to gain a deeper understanding of the production processes, the effect of hydrodynamic stress on cells in bioreactor production and its influence on the probiotic potential post-manufacturing also need to be determined. This review explores the trends in tilapia culture, the probiotic mechanisms employed by L. lactis in aquaculture, and the essential parameters for the optimal scale-up of this probiotic.

Establishment and characterization of a competitive exclusion bacterial culture derived from Nile tilapia (Oreochromis niloticus) gut microbiomes showing antibacterial activity against pathogenic Streptococcus agalactiae.

This study reports the characterization of the microbial community composition, and the establishment and dynamics of a continuous-flow competitive exclusion culture (CFCEC) derived from gut microbiomes of Nile tilapia (Oreochromis niloticus) specimens reared on aquaculture farms in Colombia. 16S rRNA gene amplicon Illumina sequencing was used to identify taxonomical changes in the CFCEC microbial community over time. The CFCEC was developed from adult tilapia from two farms in Colombia, and CFCEC samples were collected over two months. The pH varied from 6.25 to 6.35 throughout culturing, while anaerobic and aerobic cell counts stabilized at day 9, at 109 CFU mL-1 and were maintained to day 68. A variation in the CFCEC bacterial composition was observed over time. Cetobacterium was the most abundant in the first two days and coincided with a higher CFCEC supernatant antimicrobial effect against the fish pathogen Streptococcus agalactiae. Antimicrobial activity against S. agalactiae disappeared by day 3. Changes in bacterial composition continued to day 33 with Lactococcus spp. becoming the most abundant member of the community. In conclusion, the study of the CFCEC from intestinal tract of Nile tilapia (Oreochromis niloticus) by 16S rRNA gene sequencing allowed identification of predominant bacterial genera in the continuous-flow competitive exclusion culture exhibiting antibacterial activity against the fish pathogen Streptococcus agalactiae.

Enterococcus, Myroides Y Exiguobacterium: GÉNEROS BACTERIANOS CON POTENCIAL PROBIÓTICO PARA EL CULTIVO DE TILAPIA NILÓTICA (Oreochromis niloticus) / Enterococcus, Myroides, and Exiguobacterium: Bacterial Genus with Probiotic Potential for Nile Tilapia (Oreochromis niloticus) Culture

RESUMEN Se aislaron 120 morfotipos bacterianos de intestino de tilapia y se seleccionaron según su actividad antibacteriana contra patógenos como Aeromonas hydrophila, Streptococcus agalactiae y Edwardsiella tarda, su capacidad de adherencia a mucus intestinal y cinética de crecimiento. Las bacterias seleccionadas se identificaron mediante secuenciación de 16S rRNA y se identificaron como Exigobacterium sp. l9, Enterococcus faecalis I15 y Myroides odoratimimus l19. Además, se evaluó su efecto in vivo sobre el crecimiento de los peces, mediante su adición al alimento de juveniles de Oreochromis niloticus (106 UFC / g, por 15 días). Se determinó la supervivencia luego de un desafío experimental con Edwardsiella tarda por inyección intraperitoneal (100 µL 105 UFC / mL). Las tres bacterias seleccionadas incrementaron la tasa de crecimiento específico, redujeron la mortalidad de los peces durante el desafío experimental con E. tarda y no causaron mortalidad durante la adición en el alimento. Los efectos positivos in vivo se relacionan posiblemente con actividad in vitro; sin embargo, por motivos de bioseguridad se recomienda efectuar estudios posteriores a Exigobacterium sp. l9y E. faecalis I15 dado que se han reportado miembros de este género como causantes de mortalidad en peces, mientras que en el caso de M. odoratimimus l19, es necesario efectuar futuros estudios para verificar su actividad positiva a mayor escala productiva.

ABSTRACT 120 bacteria were isolated from tilapia intestine and screened according to the antibacterial activity against pathogens such as Aeromonas hydrophila, Edwardsiella tarda and Streptococcus agalactiae, the ability to adhere to intestinal mucus and growth kinetics. The selected bacteria were identified by 16S rRNA sequencing of and were identified as Exigobacterium sp. I9, Enterococcus faecalis l15 and Myroides odoratimimus l19. Furthermore, the in vivo effect on fish growth was assessed by the addition of selected bacteria to juvenile Oreochromis niloticus feed (106 CFU / g, for 15 days). Survival was also determined after a challenge by intraperitoneal injection of E. tarda (100 µL of 105 UFC / mL). The three selected bacteria increased the specific growth rate, reduced mortality of fish during the experimental challenge with E. tarda and did not cause mortality during the addition in the feed. The positive effects observed in vivo are possibly associated with in vitro activity; however, for biosafety reasons, it is recommended that further studies of Exigobacterium sp. l9 and E. faecalis l15 may be carried out since this genus have been reported of as causative agents of fish mortality, whereas in the case of M. odoratimimus l19, verification of positive effects at a higher production scales is desirable.