Enrichment Mechanisms Of Brine Shrimp With Probiotic Bacteria Attachament To Body Surface Or Bioencapsulationdoc

=== Enrichment mechanisms of brine shrimp with probiotic bacteria – attachament to body surface or bioencapsulation ===

Enrichment mechanisms of brine shrimp (Artemia franciscana Kellog 1906) with probiotic bacteria – attachment to body surface or bioencapsulation?

Saeed Ziaei-Nejad1

1-Department of Fisheries, Natural Resources Faculty, Behbahan Khatam alanbia University of Technology, Behbahan, Iran.

Abstract

This study examined the ability of two enrichment mechanisms, bioencapsulation or/and attachment to the body surface, of brine shrimp (Artemia franciscana Kellog 1906) nauplii with two isolated probiotics. The probiotics tested were Bacillus subtilis BP6 and Lactobacillus plantarium LP3 delivered at two concentrations (103 and 106 cells.ml-1) and at two growth phases (start of the exponential phase and during the stationary phase). Results show that both species of bacteria could be bioencapsulated into and attached to brine shrimp successfully. For all treatments, the attachment of probiotic to brine shrimp increased, while bioencapsulated probiotic into brine shrimp decreased over time. The maximum attachment level occurred when nauplii were exposed to probiotic bacteria in the stationary growth phase of bacteria. With bacteria at 106 cells.ml-1 attachment levels reached 2.6 ± 0.2 × 103 CFU.nauplius-1 for B. subtilis BP6 and 1.7 ± 0.1 × 103 CFU.nauplius-1 for L. plantarum LP3 occurred. In comparison, levels of bioencapsulation were similar in the exponential and stationary growth phases. When nauplii were exposed to the probiotic bacteria in the exponential growth phase at 106 cells.ml-1, maximum bioencapsulation levels were 4.3 ± 0.2 × 103 CFU.nauplius-1 with B. subtilis BP6 and 2.4 ± 0.1 × 103 CFU.nauplius-1 with L. plantarum LP3.

Key words: Probiotic; brine shrimp (Artemia franciscana Kellog 1906); enrichment; bioencapsulation; Bacillus subtilis; Lactobacillus plantarium

1. Introduction

Probiotic bacteria are defined as a live microbial feed supplement, which beneficially affects the host animal by improving the intestinal microbial balance (Fuller 1989). In aquaculture, probiotics may be applied in several delivery methods, the success of which is directly related to the selection of the best method according to the nature of the species, its stage of life, the goal of probiotic application and the type of probiotic used (Ziaei-nejad 2010). Colonising the intestine with useful bacteria ingested orally by the host while it is a larva can provide great benefit because this pioneering community of gut-colonising bacteria may acquire a competitive advantage over possible opportunistic bacteria that may be introduced at a later developmental stage (Hansen and Olafsen 1999). One of the main aims in using probiotics in the larviculture of aquatic organisms is to enhance growth and disease resistance, and currently the best way of introducing the probiotic is via the ingestion of live food inoculated with bacteria (Ziaei-nejad 2010).

Nauplii of the Artemia spp. have good potential as probiotic vectors as they are able to filter feed on bacteria (Makridis and Vadstein 1999). Members of the genus have been used as a vector for delivering probiotic bacteria to the larval digestive tract (Planas et al. 2004), not only in fish (turbot (Scophthalmus maximus Linnaeus 1758) (Garcia de la Bande et al. 1992); Atlantic halibut (Hippoglossus hippoglossus Linnaeus 1758) (Makridis et al. 2001)), but also in shrimp (Black tiger shrimp (Penaeus monodon Fabricius 1798 (Rengpipat et al. 1998), giant freshwater prawn (Macrobrachium rosenbegii de Man 1879) (Venkat et al. 2004), and Indian white shrimp (Fenneropenaeus indicus H. Milne Edwards 1837) (Ziaei-nejad et al. 2006). The successful introduction of probiotic bacteria via brine shrimp in larviculture has been show to result in significant improvements in growth and survival of the host animal (Rengpipat et al. 1998; Ziaei-nejad et al. 2006; Immanuel et al. 2007). An alternative use of bioencapsulating bacteria into brine shrimp was used (Chair et al. 1994) whereby they introduced pathogenic Vibrio anguillarum strain into turbot larvae via brine shrimp nauplii as part of a pathogen challenge experiment.

Colonisation of brine shrimp nauplii with bacteria can occur either via attachment to the external body surface, via ingestion by the nauplii, or by a combination of the two (Grisez et al. 1996). In one study, two different bacteria were bioencapsulated into brine shrimp, however the proportion of the bacteria colonised by either of these pathways was not determined (Gomez-Gil et al. 1998). In another study, only the attachment ability of candidate probiotics to the external surfaces of brine shrimp was assessed (Vine 2004). In this study also mentioned that bacterial attachment to livefood organisms is influenced by various factors such as probiotic preparation method and metabolic stage of the probionts.

In order to select the optimal protocol for probiotic delivery for future studies, the present study was conducted to determine the optimal concentration and growth phase of two species of probiotic bacteria for attachment to the external surfaces and bioencapsulation using brine shrimp as a vector.

2. Materials and methods

2.1. Probiotic preparation

The probiotic cultures, Bacillus subtilis BP6 and Lactobacillus plantarum LP3, were originally isolated from the intestinal tract of cultured yellow tail sea bream, Acantopagrus latus, and were selected because of their in vitro inhibitory activities against various pathogenic bacteria (Vibrio harveyi, Aeromonas hydrophila and Elizabethkingia meningoseptica), their production of beneficial compounds and their contribution to improvement of survival and growth of sea bream larvae (Ziaei-nejad 2010). The strains were cultured in tryptic soy broth (Difco) supplemented with 2% (wt.vol-1) NaCl and stored frozen (-80°C) in 10% (wt.vol-1) glycerol (Difco). The probiotic strains subsequently were reconstituted in marine broth (Difco) at 30 °C for 24 h prior to use. The broth was then centrifuged (10 min, 2000 x g), and the cell pellet was washed in normal saline solution (NSS; NaCl 8.5 g.l-1) three times. Fresh bacterial cells were harvested and maintained at 4 °C for a maximum of one week or at -20 °C for 2 weeks prior to being used. Routine checks on the purity of the cultures were conducted during the course of this investigation.

2.2. Brine shrimp hatching:

Artemia franciscana Kellog 1906 was used in all of the experiments (Arjan Zist yar Co., Iran). All cysts were decapsulated according to the method of Sorgeloos et al (1977). Decapsulated cysts were incubated and allowed to hatch under optimum hatching conditions (3.5% salinity; 27 ± 1°C temperature and 1000 lux light) under sterile conditions (using sterile seawater and oxygenated through mechanical agitation) (Gomez-Gil et al. 1998). For bacterial disinfection of the nauplii, the antibiotics chloramphenicol (30 mg.l-1) and trimethoprim (40 mg.l-1) were added to the hatching water of the nauplii (Gomez-Gil et al. 1998). The newly hatched nauplii were collected by exploiting the positive phototactic behavior of the nauplii and washed thoroughly with sterile distilled water. A disk diffusion test on Muller-Hinton agar plates (supplemented with 2% NaCl) was used (with Escherichia coli, Vibrio harveyi, B. subtilis BP6 and L. plantarum LP3 as reference bacteria) to check whether any antibiotic remained in the nauplii after rinsing (Gomez-Gil et al. 1998).

2.3. Enrichment process

Brine shrimp nauplii (instar 2) were exposed to the bacterial suspension (sterile seawater with the desired bacterium) in 1-liter flasks at a density of approximately 50 nauplii.ml-1. Concentrations of 103 and 106 bacterial cells.ml-1 were used for each species of bacteria. Treatments B3 and B6 consisted of B.subtilis BP6 at 103 and 106 bacterial cells.ml-1, respectively, and treatments L3 and L6 consisted of L. plantarum LP3 at 103 and 106 bacterial cells.ml-1, respectively. The control was nauplii only (brine shrimp to which no bacteria had been added). All treatments were performed in triplicate. To determine the effect of probiont metabolic stage on brine shrimp attachment efficiency, each treatment was repeated for each isolate at two different growth phases, namely the exponential phase (phase 1) and the stationary phase (phase 2). In determining the growth phase of the probiont, the optical density (OD) was measured in triplicate samples for each species of probiont. For each probiont, a 2 ml volume of 24-hour old bacterial culture was added to 30 ml of marine broth at 30°C with continuous agitation. The OD was recorded at 640 nm every 15 minutes for 24 hours using a spectrophotometer (DR- 2800; Hach.) (Vine et al. 2004). Readings of the profiles for each triplicate were averaged. Over this 24h period, cell counts were determined by collecting 100 µl samples from each bacterial culture every 2 hours and plating them onto either Bacillus cerius agar or MRS agar by the spread plate technique. For rapid calculation of the probiont cell concentration, (cells.ml-1) was related to OD.

The aliquot volume of probiont culture to be added to each enrichment container was calculated on the basis of the above data. The aliquot was centrifuged (10 min, 2000 g), and the resulting bacterial pellet was resuspended and washed in normal saline solution (NSS; NaCl 8.5 g.l-1) three times before being added to an enrichment container.

2.4. Sampling procedures

Samples were collected from each enrichment container at 2, 6, 10 and 24 hour after the start of incubation. Samples of brine shrimp nauplii (5000 nauplii, approximately) from each treatment were drained through a sterile 100 µm screen and washed with 500 ml Sterile Seawater (SSW) to remove any non-adhering probiotic bacteria and then resuspended in 50 ml SSW. To separate the attached probiotics from the brine shrimp, the nauplii were sonicated at 40 ± 5 KHz for ten minutes in a bath sonicator (Parsonic , ParsNahand Co.) (15; with some modification, as he used 50 KHz). This method removed almost all of the attached bacteria while not killing the brine shrimp. Any damage to the brine shrimp could result in the release of ingested bacteria, thereby elevating the estimate of the number of attached bacteria (Vine 2004). After collection of the nauplii, serial dilutions were made of the remaining sonicated water with 100 µl aliquots being plated onto Bacillus cereius agar and MRS agar for isolates of B. subtilis BP6 and L. plantarum LP3, respectively. The plates were incubated at 30°C for 48 hours to determine the number of probiotic bacteria attached to the external surface of nauplii (CFU.nauplius-1).

After the sonication process, brine shrimp nauplii were killed by dipping in cold sterile distilled water. To remove any bacteria that may have remained attached to the external surface, nauplii were rinsed with sterilized distilled water, followed by benzalkonium chloride (0.1%), and then rinsed again thoroughly with sterilized distilled water (Gatesoupe 1999; Ziaei-nejad et al. 2006). The nauplii were then homogenized to determine the quantity of ingested probiotics bacteria (CFU.nauplius-1) within their gut. Serial dilutions of homogenized samples were used to determine probiotic counts as described above.

2.5. Statistical analysis

A completely randomized design was used in each experiment. Normality of data was tested using the Anderson–Darling test (MINITAB 13.31 software). Means were compared using One-Way ANOVA analysis. Differences between groups were determined using Duncan’s multiple range test (SPSS software). A significance level of P <0.05 was used for all tests. Data are reported as means ± standard errors.

3. Results

There were no probiotic bacteria present in any control treatments (nauplii without bacteria). Both probionts were capable of attachment onto and bioencapsulation within brine shrimp nauplii (fig.1 – fig.4). We observed that, levels of attachment of probiotic to the brine shrimp increased, while levels of bioencapsulation of probiotic within brine shrimp decreased with incubation time in all treatments. However, both the rate of increase of attachment and the rate of decrease for bioencapsulation were greater with L. plantarum LP3 (Fig. 3 and Fig. 4) than with B. subtilis BP6 (Fig. 1 and Fig. 2). For example, in treatment B3, where 103 cells.ml-1 of B. subtilis BP6 isolates were added at the start of the exponential growth phase (phase 1), 2.5 ± 0.1 × 102 CFU.nauplius-1 were observed 2h after the start of the enrichment process, but at 24h the concentration had decreased to 1.9 ± 0.1 × 102 CFU.nauplius-1.

In treatment B3, phase 1, attached probiotic increased from 1.3 ± 0.1 × 102 CFU.nauplius-1 at 2 h to 2.0 ± 0.2 × 102 CFU.nauplius-1 at 24 h and in phase 2 of the treatment, the isolate (B. subtilis BP6) attached to brine shrimp at higher level (2.3 ± 0.1 × 102 CFU.nauplius-1 at 2 h to 3.5 ± 0.1 × 102 CFU.nauplius-1 at 24 h) but had no significant difference compare to phase 1 (P > 0.05).

Enrichment studies using LP3 (Fig.3 and Fig.4) showed similar patterns to that of isolate BP6, however there were differences in the probiotic counts (CFUs) which attached to or bioencapsulated into brine shrimp and their increasing or decreasing procedures.

4. Discussion

In aquaculture, live foods such as brine shrimp are important carriers of bacteria to the larval digestive tract (Blanch et al. 1997). With the concept of probiotics in aquaculture fast gaining interest, this mode of probiotic delivery warrants investigation as a means of intentionally colonising the gut of aquatic larvae with beneficial microorganisms (probiotics) thereby improving the intestinal microbial balance hopefully resulting in better growth and survival. In Several investigations (Garcia de la Bande et al. 1992; Rengpipat et al. 1998; Makridis et al. 2001; Gatesoupe 2002; Venkat et al. 2004; Ziaei-nejad et al. 2006) successful application of enriched brine shrimp with probiotic bacteria have been reported.

Rengpipat et al. (1998) showed that by feeding black tiger shrimp (P. monodon Fabricius 1798) larvae with probiotic (Bacillus S11) enriched brine shrimp development time was shorter and survival higher. Similarly, Ziaei-nejad et al. (2006) observed higher survival and digestive enzyme activity in indian white shrimp (F. indicus H. Milne Edwards 1837) larvae, after feeding brine shrimp enriched with Bacillus sp. bacteria. In addition, Venkat et al. (2004) reported better growth and food conversion ratios in giant freshwater prawn (M. rosenbegii de Man 1879) fed Lactobacillus sporogenes enriched brine shrimp with. Gatesoupe (2002) fed two commercial probiotics, Bactocell (Pediococcus acidilactici) and Levucell (Saccharomyces cerevisiae) to pollack larvae (Pollachius pollachius Linnaeus 1758) via brine shrimp and reported improved larval growth.

The enrichment process of brine shrimp with probiotic bacteria has two pathways – attachment to the external surfaces and bioencapsulation (or ingestion by the brine shrimp). In the present study we separated each pathway when brine shrimp was enriched with two probiotic isolates (B. subtilis BP6 and L. plantarum LP3) added at two concentrations (103 and 106 cells.ml-1) which had been harvested at one of two growth phases (start of exponential and stationary phase).

The results indicate that both isolates can attach to and bioencapsulate into brine shrimp successfully, however, the degree of attachment and bioencapsulation is influenced by certain factors. These factors include – time of enrichment process, type of bacteria, and the concentration of the bacterial suspension. Probiotic cells harvested at the two different growth phases did not show any differences in brine shrimp bioencapsulation. It is possibly due to the role that brine shrimp plays in the bioencapsulation process as non-selective filtration of ambient environment and irrelevant for brine shrimp that the probiotic is in exponential or stationary phase. Gomez-Gil et al. (1998) confirmed that accumulation of bacteria in brine shrimp depends on the type of bacteria used, time of exposure and status (live or dead) of the bacteria.

Another pathway for enrichment of brine shrimp with probiotics is attachment to the external surfaces of the brine shrimp. Vine (2004) mentioned that the attachment ability of suitable candidate probionts for larviculture should be investigated as a selective criterion, although it seems that the stability of attached probiotics to brine shrimp is less that of bioencapsulated one, because the attached bacteria may detach by some factors such as washing (Ziaei-nejad 2010).

However, both isolate (B. subtilis BP6 and L. plantarum LP3) show good ability to attach to brine shrimp nauplii. We found a maximum attachment of 2.6 ± 0.2 × 103 CFU.nauplius-1 for isolate B. subtilis BP6 and 1.7 ± 0.1 × 103 CFU.nauplius-1 for isolate L. plantarum LP3 when nauplii exposed to the probiotic bacteria at 106 cells.ml-1 in the stationary phase. The results were similar to those of Gomez-Gil et al. (1998) and Vine (2004) who respectively showed a maximum attachment of 4.55 × 103 CFU.nauplius-1 for isolate HL57 and 7.2 ± 0.1 × 103 CFU.nauplius-1 for isolate Kocuria AP4, although the authors used higher bacterial concentrations (108 cells.ml-1).

Results indicate that the number of attached probiotic to the brine shrimp affected by type of bacteria, time of enrichment period, bacterial concentration and bacterial growth phase (see Fig.1, Fig.2, Fig.3 and Fig.4). Type of bacteria has a main effect on attachment property and the property is relate to the surface structure of the bacteria (Santos et al. 1991). Although the structure and condition of surfaces which bacteria attach to them may have an effect on the attachment efficiency.

Furthermore, bacterial attachment to livefood organisms is influenced by 1) the already present microflora the livefood (Munro et al., 1993), 2) preparation method and metabolic stage of the probionts (Fletcher 1990; Blum et al. 1999) and 3) the probiotic.livefood-1 incubation environment (Gomez-Gil et al. 1998; Gatesoupe 2002).

We found that with the change of bacterial growth phase from start of exponential phase (phase 1) to stationary phase (phase 2) that maybe affected the metabolic stage of the probiotics directly, the number of attached probiotic increased (compare Fig.1 with Fig.2 and Fig.3 with fig.4). Similarly, Blum et al. (1999) stated that the attachment ability of probiotics is greater during the stationary phase compared to the logarithmic growth phase. It is determined that progression from lag to log (exponential) to stationary growth phases of L. salivarius in vitro correlated with increasing prominence of an 84 kD protein associated with in vitro adherence ability (Kelly et al. 2005).

5. Conclusion

In conclusion, the results from this study indicate that both isolates can attach to and bioencapsulate into brine shrimp successfully, however, the degree of attachment and bioencapsulation is influenced by different factors. According to this study, Nauplii of the brine shrimp have good potential as a probiotic vectors for delivering the bacteria to the aquatic larval digestive tract.

Acknowledgments

We would like to thank the Microbial Laboratory of Iran Veterinary Organization for their technical assistance during the experiments. A special thanks to Dr. B. Gomez-Gil and Dr. N. Vine that provided invaluable assistance in the design of the study and development of methodology. We also give special acknowledgment to Behbahan khatam alanbia University of Technology for grant support.

References

Blanch AR., Alsina M, Simn M. & Jofre J., 1997. Determination of bacteria associated with reared turbot (Scophthalmus maximus) larvae. Journal of Applied Microbiology 82: 729–734.

Blum, S., Reniero, R., Schiffrin, E.J., Crittenden, R., Mattila-Sandholm, T., Ouwehand, A.C., Salminen, S., von Wright, A., Saarela, M., Saxelin M., Collins, K. and Morelli, L., 1999. Adhesion studies for probiotics: need for validation and refinement. Trends in Food Science and Technology 10: 405-410.

Chair, M., Dehasque, M., Van Poucke, S., Nelis, H., Sorgeloos, P., De Leenheer, A. P., 1994. An oral challenge for turbot larvae with Vibrio anguillarum. Aquaculture International 2:270–272.

Fletcher, M., 1990. Methods for studying adhesion and attachment to surfaces. Methods of Microbiology 22:251-283.

Fuller, R., 1989. Probiotics in man and animal. Journal of Applied Bacteriology 66:365–378.

Garcia de la Bande, I., Chereguini, O., Rasines, I., 1992. Influence of lactic bacterial additives on turbot (Scophthalmus maximus L.) larvae culture. Boletin Instituto Espanol de Oceanografia 8:247-254.

Gatesoupe, F.J., 1999. The use of probiotics in aquaculture. Aquaculture 180:147– 165.

Gatesoupe, F.J. 2002. Probiotic and formaldehyde treatments of Artemia nauplii as food for larval pollack, Pollachius pollachius. Aquaculture 212:347-360.

Gomez-Gil, B., Herrera-Vega, M.A., Abreu-Grobois, F.A., Roque, A., 1998. Bioencapsulation of two different Vibrio species in nauplii of the brine shrimp (Artemia franciscana). Applied Environmental Microbiology 64:2318– 2322.

Grisez, L., M. Chair, P. Sorgeloos, and F. Ollevier. 1996. Mode of infection and spread of Vibrio anguillarum in turbot Scophthalmus maximus larvae after oral challenge through live feed. Disease of Aquatic Organisms 26:181–187.

Hansen, G.H., Olafsen, J.A. 1999. Bacterial interactions in early life stages of marine cold water fish. Microbial Ecology 38:1-26.

Immanuel, G., Citarasu, T., Sivaram, V., Michael Babu, M., Palavesam, A., 2007 . Delivery of HUFA, probionts and biomedicine through bioencapsulated Artemia as a means to enhance the growth and survival and reduce the pathogenesity in shrimp Penaeus monodon postlarvae. Aquaculture International 15:137–152.

Kelly, P., Maguire, P. B., Bennett, M., Fitzgerald, D. J., Edwards, R. J., Thiede, B., Treumann, A., Collins, J. K., O,Sullivan, G.C., Shanahan F., Dunne C. 2005. Correlation of probiotic Lactobacillus salivarius growth phase with its cell wall-associated proteome. FEMS Microbiology Letters 252:153–159.

Makridis P., and Vadstein O., 1999. Food size selectivity of Artemia franciscana at three developmental stages. Journal of Plankton Research 21:2191–2201.

Makridis, P., Bergh, Ø., Skjermo, J., Vadstein, O., 2001. Addition of bacteria bioencapsulated in Artemia metanauplii to a rearing system for halibut larvae. Aquaculture International 9:225–235.

Munro, P.D., Birkbeck, T.H., Barbour, A., 1993. Influence of rate of bacterial colonisation of the gut of turbot larvae on larval survival. In Fish Farming Technology – Proceedings of the First International Conference of on Fish Farming Technology. Reinerstein,H., Dahle,L.A., Jogensen,L., and Tvinnereim,K. (eds.). A.A. Balkema, Rotterdam p. 85-92.

Planas, M., Vazquez, J.A., Marques, J., Perez-Lomba, R., Gonzalez, M.P. and Murado, M. (2004) Enhancement of rotifer (Brachionus plicatilis) growth by using terrestrial lactic acid bacteria. Aquaculture 240:313-329.

Rengpipat, S., Phianphak, W., Piyatiratitivorakul, S., Menasveta, P., Sirirat, R., Wannipa, P., Somkiat, P. and Piamsak, M., 1998. Effects of a probiotic bacterium on black tiger shrimp Penaeus monodon survival and growth. Aquaculture 167:301-313.

Santos, Y., Bandin, I., Nieto, T.P., Barja, J.L. and Toranzo, A.E., 1991, Cell surface-associated properties of fish pathogenic bacteria. Aquatic Animal Health 3:297-301.

Sorgeloos, P., E. Bossuyt, E. Lavin˜a, M. Baeza-Mesa, and G. Persoone. 1977. Decapsulation of Artemia cysts: a simple technique for the improvement of the use of brine shrimp in aquaculture. Aquaculture 12:311-315.

Venkat, H.K., Sahu, N.P. and Jain, K.K., 2004. Effect of feeding Lactobacillus-based probiotics on the gut microflora, growth and survival of postlarvae of Macrobrachium rosenbergii (de Man). Aquaculture Research 35:501-507.

Vine, N.G., 2004. Towards the development of a protocol for the selection of probiotics for marine fish larviculture. PhD Thesis. Rhodes University, Grahamstown, South Africa. 206pp.

Vine N.G., Leukes W.D., and Kaiser H. 2004. In vitro growth characteristics of five candidate aquaculture probiotics and two fish pathogens grown in fish intestinal mucus. FEMS Microbiology Letters 231:145–152.

Ziaei-Nejad, S., Habibi-Rezaei, M., Azari-Takami G., Lovett, DL., Mirvaghefi, A. & Shakouri, M., 2006. The effect of Bacillus spp. bacteria used as probiotics on digestive enzyme activity, survival and growth in the Indian white shrimp Fenneropenaeus indicus. Aquaculture 252:516-524.

Ziaei-Nejad, S., Rafiee, Gh., Marammazi, J., Mirvaghefi, A., Farahmand, H., 2009. Screening of probiotic bacteria from marine fish, yellowfin seabream (Acanthopagrus latus) gastrointestinal tract. Final report of Research Project .Shahid Chamran University, Iran. 200pp.

Fig. 1. Amount of isolate B. subtilis BP6 attached to or bioencapsulated into brine shrimp nauplii after probiotic enrichment in either treatment B3 (103 cells.ml-1) or treatment B6 (106 cells.ml-1) when the probiotic was administered in the exponential growth phase (phase 1). Mean ± S.E. indicated (n = 3). Means within the same enrichment time with the same letter are not significantly different (P>0.05). art.—brine shrimp nauplii; treat—treatment; treatment B3— 103 cells.ml-1 of isolate B. subtilis BP6 provided for enrichment process; treatment B6— 106 cells.ml-1 of isolate B. subtilis BP6 provided for enrichment process. Note the change of scale.

Fig. 2. Amount of isolate B. subtilis BP6 attached to or bioencapsulated into brine shrimp nauplii after probiotic enrichment in either treatment B3 (103 cells.ml-1) or treatment B6 (106 cells.ml-1) when the probiotic was administered in the stationary phase (phase 2). Mean ± S.E. indicated (n = 3). Means within the same enrichment time with the same letter are not significantly different (P>0.05). art.—brine shrimp nauplii; treat—treatment; treatment B3— 103 cells.ml-1 of isolate B. subtilis BP6 provided for enrichment process; treatment B6— 106 cells.ml-1 of isolate B. subtilis BP6 provided for enrichment process. Note the change of scale.

Fig. 3. Amount of isolate L. plantarum LP3 attached to or bioencapsulated into brine shrimp nauplii after probiotic enrichment in either treatment B3 (103 cells.ml-1) or treatment B6 (106 cells.ml-1) when the probiotic was administered in the exponential growth phase (phase 1). Mean ± S.E. indicated (n = 3). Means within the same enrichment time with the same letter are not significantly different (P>0.05). art.—brine shrimp nauplii; treat—treatment; treatment L3— 103 cells.ml-1 of isolate L. plantarum LP3 provided for enrichment process; treatment L6— 106 cells.ml-1 of isolate L. plantarum LP3 provided for enrichment process. Note the change of scale.

Fig. 4. Amount of isolate L. plantarum LP3 attached to or bioencapsulated into brine shrimp nauplii after probiotic enrichment in either treatment B3 (103 cells.ml-1) or treatment B6 (106 cells.ml-1) when the probiotic was administered in the stationary phase (phase 2). Mean ± S.E. indicated (n = 3). Means within the same enrichment time with the same letter are not significantly different (P>0.05). art.—brine shrimp nauplii; treat—treatment; treatment L3— 103 cells.ml-1 of isolate L. plantarum LP3 provided for enrichment process; treatment L6— 106 cells.ml-1 of isolate L. plantarum LP3 provided for enrichment process. Note the change of scale.