Characterization of Lactic Acid Bacteria from the Gut of Penaeus vannamei as Potential Probiotic

Penaeus vannamei gut associated beneficial bacteria were isolated from brackishwater shrimp ponds with salinity ranging from 5 to 25 ppt in Kanchipuram, Chengalpattu, Tiruvallur and Villupuram districts of Tamil Nadu, during 2021-22. The pond and farming details such as location, pond size, stocking density and shrimp size at the time of sampling are given in Table 1. The ponds were provided with commercial branded feeds such as Avanti, CP and Growel with the feed management practices suggested by the respective company. Farmers used commercial environmental probiotics as and when required during the culture period for the improvement of water and soil quality.  The gut of 25 shrimp from each pond (n=10) were pooled, homogenized and serially diluted and inoculated in Lactobacillus MRS Agar (Himedia-M641I) supplemented with 1% CaCO₃ and incubated anaerobically 30°C for 48 hrs. The bacterial isolates were stored in glycerol suspensions at -80°C for further screening and characterisation (Wang et al., 2020).


 
Genomic DNA was extracted from bacterial isolates by cetyltrimethylammonium bromide (CTAB) method (Minas et al., 2011) and stored -80°C. Bacterial 16S rRNA genes was amplified using universal primers 27F (5'-AGAGTTT GATCCTGGCTCAG-3') and 1492R (5'-TACGGYTACC TTGTTACGACTT-3') (Lane, 1991) sequenced and blasted against nucleotide database using NCBI-BLASTn program and the bacterial strains were identified. Construction of phylogenetic tree of the lactic acid bacterial isolates from shrimp gut was done based on 16S rRNA gene sequences. The Maximum Likelihood approach and the Tamura-Nei model were used to infer the evolutionary history (Tamura et al., 1993). MEGA11 was used to perform evolutionary analysis (Tamura et al., 2021).
 
The gut bacterial isolates were evaluated for digestive enzyme activity viz., the protease (Bhowmik et al., 2015), amylase, lipase and cellulase (Das et al., 2014) using plate screening method. Antibacterial activity of the isolates was studied against the Vibrio campbelli, V. harveyi and V. parahaemolyticus obtained from Aquatic Animal Health and Environment Division, ICAR-CIBA, Chennai, by agar well diffusion assay (Wanna et al., 2021; Kaewchomphunuch et al., 2022; Rajyalakshmi et al., 2021). The plates were examined for clearing zones around the wells after 24 hours of incubation at 30°C, with sterile MRS broth as negative control and tetracycline disc (TE: SD037- Hi media) as positive control. Six isolates of lactic acid bacteria (LAB) spread across four genera, which exhibited highest antibacterial and enzymatic activity were evaluated in vitro against varied NaCl concentrations (1,2,3,4,5 and 6%); pH (3.0,7.0 and 10.0); bile salt concentrations (0.4, 0.6, 0.8 and 1%) (Li et al., 2020) and kinetics were measured using a Multimode reader (Spark 10M, TECAN) at 600 nm.
 
The auto aggregation test was used to detect specific cell-cell interactions. The isolates were grown in MRS broth at 30°C for 20 hours and the bacterial pellet obtained by centrifugation at 5000 G for 10 minutes, washed twice and resuspended to a final count of 10⁸ CFU/ml with PBS. The auto-aggregation test was done and auto-aggregation percentage calculated (Wanna et al., 2021). Hydrophobicity of the isolates was performed by growing the isolates in MRS broth at 30°C for 24 hours, centrifuged for 15 minutes at 3000 G. The resultant pellet was used for measurement cell-surface hydrophobicity (Liu et al., 2020). Cross streaking assay was done by streaking single colony of the isolates ON495586, ON491817, ON479264, ON478992, ON478991 and ON564885, incubating them anaerobically at 30°C for 24 h on the MRS agar plates and examining for inhibition zone near the contact point of the streaking lines (Kaewchomphunuch et al., 2022). The statistical analysis was performed with SPSS Version 17.0 using one way ANOVA and mean comparison employing Duncan’s multiple range test.

Molecular characterization of the LAB from shrimp gut
 
Thirty LAB strains were isolated, identified, their sequences submitted to the NCBI and accession numbers were obtained. A phylogenetic tree (Fig 1) was constructed using the partial 16S rRNA sequences. In the present study, thirty isolates of LAB spread across four genera were identified from the shrimp gut. Research has shown that in shrimp, P. pentosaceus can improve shrimp innate immunity, physiological stability and pathogen resistance (Truc et al., 2021); L. plantarum was used effectively as a potential probiotic in shrimp farming to improve P. vannamei production efficiency, immunity strength and disease resistance (Wei et al., 2022). Supplementation of the bacteria viz., Lactococcus lactis, Pediococcus pentosaceus and Bacillus subtilis in white leg shrimp augmented growth, enhanced digestive enzyme function, resistance to disease, enhanced immunity and gene expression (Won et al., 2020). Enterococcus faecium and Enterococcus durans involved the production of various antimicrobial compounds against both gram positive and negative bacteria (Hanchi et al., 2018). Enterococcus hirae isolated from the seabass intestine has the antibacterial activity against Vibrio harveyi (Masduki et al., 2020).
 

 
Enzyme activity
 
Thirty isolates of LAB were screened for the enzyme activity viz., amylase, lipase, cellulase and protease. The growth of isolates on tributyrin agar plates revealed clearing around colony depicting better lipase activity (Fig 2-Panel A); growth of isolates on starch agar plates presented transparent halo zone surrounding the colony when flooded with Gram’s iodine solution after 24 h, showing better amylase activity (Fig 2-Panel B); growth of isolates on carboxy methyl cellulose agar exhibited halo surrounding a colony and after addition of 1% Congo red, representing better cellulase activity (Fig 2-Panel C) and growth of isolates on gelatin peptone agar showed halo after flooding with 15% Mercuric chloride followed by washing with 1M NaCl, indicating better protease activity (Fig 2-Panel D). Feed supplemented with Lactococcus  lactis Sub sp. Lactis isolated from shrimp gut showed enhanced cellulose, lipase, amylase and protease levels substantially (Adel et al., 2017) contributed by Lactobacillus enzyme secretions as well as secretion from probiotic stimulated cells (Zuo et al., 2019). The isolates which had the better enzyme activity from each genus were further subjected to antibacterial activity, pH resistance, bile salt tolerance, NaCl tolerance, aggregation and hydrophobicity studies for further selection as candidate probiotic.
 

 
Antibacterial activity
 
The isolates from the gut of the Penaeus vannamei ON491817, ON495586, ON478992, ON479264, ON478991 and ON564885 exhibited antibacterial activity against shrimp bacterial pathogens V. campbelli, V. harveyi and V. parahaemolyticus with zone of inhibition ranging between 12.33 to 21.00 mm. Among the isolates, ON479264 had no effect on growth of V. campbelli and V. harveyi. However, the isolate, ON491817 showed excellent antibacterial activity against V. campbelli and V. harveyi, whereas the isolates ON478991 and ON564885 exhibited better antibacterial activity versus V. parahaemolyticus (Fig 3). Prior investigations on antibacterial activities expressed results with bacterial strains Lactobacillus paracasei, Pediococcus acidilactici and Lactobacillus rhamnosus inhibited Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus and Vibrio cholera most effectively with exhibition of zone of inhibition of 23-24 mm (Rajyalakshmi et al., 2021), whereas P. pentosaceus against shrimp pathogens V. harveyi and V. parahaemolyticus (Wanna et al., 2021). Enterococcus hirae isolated from intestine of Seabass found to inhibit the growth of V. harveyi with a zone of inhibition of 11±6 mm (Masduki et al., 2020). Organic acids, bacteriocins and hydrogen peroxide are the antimicrobial metabolites produced by probiotics (Ispirli et al., 2015). The cell free culture supernatant (CFCS) of L. acidophilus and P. pentosaceus found to inhibit the growth of E. coli strains, that may be due to the fact that several active constituents function together in CFCS (Kaewchomphunuch et al., 2022). Furthermore, the CFCS in our investigation requires additional research to identify active compounds in order to substantiate the inhibitory activity shown against the Vibrio spp.
 

 
NaCl, pH and bile salt tolerance
 
The strains ON478991, ON564885 showed better growth at 1-2% of NaCl concentration compared to higher concentrations at 12 hours of incubation (Fig 4a). All the strains of LAB showed better growth at pH 7.0 at 12 hours of incubation. The viability was not there for all the strains at pH 3. However, except the strain ON495586, all other strains showed better tolerance to show viability even at pH of 10, showing that these bacterial strains can be used as probiotic even when there is a change in the pH to 10 (Fig 4b). It was found that the all six strains (ON478992, ON79264, ON491817, ON495586, ON478991 and ON564885) tolerated the bile salts up to 1% concentration (Fig 4c). Probiotics are exposed to a variety of environmental variables upon intake by the host and throughout passage through the gastrointestinal tract.
 

 
P. pentosaceus grow in a broad range of salt between 1 to 6% as well as bile salt concentrations 0.6 to 1%, further can be acclimated to acidic conditions of pH 3 (Wanna et al., 2021). LAB strains were particularly resistant to acid and bile salt (Li et al., 2020).  Enterococcus faecium was shown to grow at pH levels ranging from 2.0 to 4.0 for 8 hours, bile contents ranging from 0.2 to 1.2% (Mao et al., 2020). Enterococcus hirae grow at pH levels between 2 to 10, with the optimum growth occurring at pH 7, propagated up to 4% NaCl with excellent growth at 1.5% NaCl (Masduki et al., 2020). Exposure of L. plantarum tolerated NaCl concentrations up to 6% (Wang et al., 2018). In our study, the LAB strains grow showed better growth at pH 7.0; tolerated the bile salts up to 1% concentration and salt concentrations ranging up to 6.0% and hence its selection as candidate probiotic.
 
Aggregation, hydrophobicity and cross streaking assay
 
The probiotic bacteria must have the auto-aggregation property to build a barrier and inhibit unwanted microorganisms from attaching (Saito et al., 2019). All of the isolates (ON491817, ON495586, ON478992, ON479264, ON478991 and ON564885) examined in this investigation showed auto-aggregation percentage of 74.45 to 91.14% at 24 hrs. Thus, the bacterial isolates could serve as the promising gut probiotic for usage in shellfish culture (Fig 5). Similar auto aggregation activity of bacterial strains was seen in; Pediococcus pentosaceus, 40.40 to 75.00% (Wanna et al., 2021); Lactobacillus, 39.58 to 56.37% (Liu et al., 2020).
 

 
In the current investigation, LAB isolates from the gut of shrimp demonstrated excellent hydrophobicity ranging between 77 to 99.93%, showing better ability to adhere onto intestinal epithelial cells (Ortiz et al., 2015) and hence as candidate gut probiotic (Fig 5). The isolates ON495586, ON491817, ON479264, ON478992, ON478991 and ON564885 showed no inhibition zone near the contact point of the streaking lines, showing no antagonist activities among all LAB strains, similar to observations of Kaewchomphunuch et al., 2022. Hence, these strains could be used in the multiple strain probiotic preparation, (Puvaneswari et al., 2021).

The screening, characterization and probiotic evaluation of host endogenous probiotic strains from P. vannamei’s gut provided six candidate probiotic strains showing better enzyme activities, exhibited anti-bacterial activity, withstanding pH change, better growth in presence of bile salt and tolerant to varying NaCl concentrations. Further, these isolates possessed aggregation and hydrophobicity properties with no antagonism between isolates. Thus, these isolates have the required probiotic properties for use as single as well as multiple strain probiotic for brackishwater shrimp farming. Further in vivo studies on evaluation of these candidate probiotic strains in P. vannamei is being carried out for its development as the probiotic with functional feed in shrimp aquaculture.

Authors thank the Tamil Nadu Veterinary and Animal Sciences University, Chennai and The Director, ICAR-Central Institute of Brackishwater Aquaculture, Chennai for the facilities provided.

None.