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Paraprobiotics (dead/inactivated probiotics) are promising candidates in functional feeds to promote growth performance, and modulate intestinal microbiota and enhance the immune response of fish. These benefits were studied at in vivo and in vitro levels in Salmonids, here we present the results.
The rapid development of aquaculture in recent decades can significantly contribute to meeting global demand for animal-derived protein.
However, the aquaculture industry faces considerable economic losses during he grow-out period due to smolts of low quality combined with exposure to multi-stressor conditions such as stress due to handling, sea water transportation, delousing, poor water quality and exposure to pathogens, leading to diseases and high mortality.
“A cost-effective way to prevent such losses is the use of functional feeds with immunomodulatory properties during the freshwater phase to promote a more resilient fish.”
The intestine is an interesting target for this type of approach, since the mucosa associated lymphoid tissue (MALT) in this organ (GALT) can coordinate both local and systemic immune responses (e.g., inflammation, antigen presentation process, T cell polarization, production of effector molecules and cytokines), which modulate the fish’s response to a challenge.
In addition, intestinal microbiota, due to its modulatory capacity, could be the link between diet, immune system and host health.
HK L-137 (Lactiplantibacillus plantarum), has a large potential for functional feeds. Its use has been associated with increased growth performance or immunomodulation in aquaculture species like giant freshwater prawns (Macrobrachium rosenbergii), sea cucumber (Apostichopus japonicus), Red Sea bream (Pagrus major) and Nile tilapia (Oreochromis niloticus).
“The research presented in this article evaluates the effect of the paraprobiotic HK L-137 as a bioactive component in functional feed for Atlantic salmon.”
To study the possible immunomodulating effects of HK L-137, an in vitro trial was performed using a rainbow troutderived intestinal epithelium cell line (RTgutGC) exposed to different concentrations of pre-digested HK L-137 to mimic in vivo gastrointestinal digestion.
Following the in vitro trial, an in vivo salmon feeding trial was performed with increasing doses of HK L137.
Materials and methods
The heat-killed strain of L. plantarum (HK L-137) was produced and provided by House Wellness Foods Corporation (Itami, Japan) in the commercial name Feed LP20™. L. plantarum was heat-killed at 70° C for 10 minutes, with a final concentration in the dry product of 2×1011 CFU g-1.
As an in vitro model, RTgutGC cells line derived from rainbow trout (Oncorhynchus mykiss) intestinal cells were used.
To mimic in vivo gastro-intestinal digestion, Feed LP20™ was pre-digested before exposing to cells in a two-step in vitro system. To 2 g of Feed LP20™, 9.6 mL pepsin-solution (pepsin 416.7 U mL-1 in 0.084 mM HCl, 35 mM NaCl, pH 2.0) and 0.4 mL chloramphenicol solution (0.1% w v-1) were added in a 50 mL screw cap tube.
The solution was incubated in a shaking water bath at 37°C for 2 hours. After incubation, 675 µL 1 M NaOH was added to inactivate pepsin activity and pH were adjusted to 7.8 by adding 10 mM phosphate buffered saline (PBS). The solution went through another incubation in a shaking water bath at 37°C for 1 hour to stabilize the temperature again.
Later, 0.6 mL of an intestinal enzyme cocktail (trypsin 2100 U mL-1, chymotrypsin 100 U mL-1 and elastase 0.2 U mL-1 in 10 mM PBS pH 7.8), was added and incubated in the shaking water bath at 37°C for 6 hours. After digestion, the solution was immediately kept in boiling water for 5 minutes to deactivate the enzymes and the total solution was freeze-dried.
A stock solution was made with a resuspension in PBS at 20 mg mL-1 for later in vitro assays. All compounds and enzymes were supplied by SigmaAldrich, Germany. The fish trial was conducted at the Center of Sustainable Aquaculture at the Norwegian University of Life Science (NMBU), Ås, Norway.
“A total of 825 Atlantic salmon (AquaGen Atlantic QLT-innOva SHIELD) were randomly distributed into 15 fiberglass tanks (300 L) with an average biomass per tank of 1,500 ± 2.7 g (55 fish per tank with an average individual weight of 27.3 g).”
Fish were kept under continuous light in recirculated fresh water with a water supply of 8 L min-1 and average water temperature of 14.9°C.
Dissolved oxygen levels were kept above 7.0 mg L-1 in the water outlet. Each experimental diet was supplied in triplicate tanks over a perio of 61 days. Fish were fed ad libitum with 10% excess by electrically driven belt feeders once a day for 6h.
The amount of feed in the feeders was adjusted daily based on the expected fish biomass in the tanks and the uneaten feed in each tank.
Uneaten pellets were collected within one hour after feeding from the outlet water settling on a screen for each tank, according to a method described previously. Feed intake, biomass gain and feed conversion ratio and specific growth rate were calculated.
Results
In vitro trial
After exposing RTgutGC cells to different doses of Feed LP20™ for 24 hours, no difference in cell viability was observed even, at the higher exposure levels. RTgutGC monolayer showed no changes in the transepithelial electrical resistance (TEER) after 6- and 24-hours exposure to HK L-137.
Nevertheless, a significantly decreased (p-value = 0.002) paracellular permeability was detected after 6-hours exposure to HK L-137. The detection of immune biomarkers showed that IL-1b production by RTgutGC was higher in cells exposed to HK L-137 after 6 (p-value = 0.011) and 24 hours (p-value = 0.021).
At the same time, Anxa1 production was significantly lower, but only at the 24-hour time point (p-value = 0.031). Production of E-cad did not change significantly after HK L-137 exposure at any time point.
In vivo experiment
No significant difference was found between dietary groups regarding feed intake, body weight gain, and specific growth rate and feed conversion ratio, as presented in Table 1.
Regarding digesta samples, more than 95% of all bacteria in digesta belong to the phyla Proteobacteria, Firmicutes and Actinobacteriota (Figure 1A).
Proteobacteria and Firmicutes relative abundances did not differ from the control group, while Actinobacteriota abundance was significantly increased by the highest inclusion levels of HK L-137 (p-value < 0.0001 and p-value = 0.021, respectively).
At the lower taxonomic level, Arenimonas, Limosilactobacillus, Psychrobacter and Mycobacterium were the most abundant genera (Figures 1B). The relative abundance of Mycobacterium was significantly different from the CD group (except in the LP20 group), while the higher inclusion of HK L-137 led to a lower relative abundance of the genera Stenotrophomonas and Ligilactobacillus.
“With a prevalence threshold of 80%, 53 ASVs were identified as core microbiota, with 28 of those shared by all experimental groups. Three ASVs, two classified as Limosilactobacillus and one as HT002, were identified as core ASVs in all the digesta samples.”
When studying microbial diversity, alpha-diversity analysis showed that the control diet group (CD) was only significantly different from the positive control (MG) at the observed number of AVSs (p-value = 0.015), indicating increased richness.
Histological analyses showed no significant difference in single fold height between groups or any signs of inflammation in the distal intestinal samples. The transcriptomic analysis showed that increasing the inclusion level of HK L-137 in the diets induced an increase in differentially expressed genes in the distal intestine.
Moreover, DEGs from each diet comparison were used for enriched pathway analysis. LP20 diet induced a down-regulation of 2 terms related to oocyte meiosis and tight junction, but also up-regulated other 5 terms related to ECM-receptor interaction, focal adhesion, vascular smooth muscle contraction, regulation of the actin cytoskeleton and the TGF-beta signaling pathway.
Regarding protein detection, the results showed modulation of biomarkers related to the immune response. The levels of Anxa1 were decreased in the distal intestine of fish fed LP500 (p-value = 0.0335). The same group also had a significant increase in total IgM production in plasma (p-value = 0.0378) compared to the control diet.
Protein levels of IL-1β and Ecad in distal intestine did not differ between experimental diet groups or in positive control MG. Moreover, when measuring the production of specific IgM against HK L-137, there was no significant difference between groups.
Discussion
Immunonutrition is a promising cost-effective strategy to prevent considerable economic losses during fish production. The effects of fish diseases, and subsequent mortality, are aggravated by several unavoidable stress factors such as handling, seawater transfer and pathogens.
By providing functional feeds, the immune response of fish can be enhanced, leading to a more robust and healthy fish, able to cope better with such challenges. With this, stress, susceptibility to diseases and mortality can be mitigated leading to improved animal welfare and more sustainable aquaculture production.
At the in vitro level, after exposure to HK L-137, the lack of differences in TEER analysis confirmed the similar integrity of the monolayer in both the control and experimental groups due to the proper development of tight-junctions between adjacent cells.
“When intestinal integrity is impaired, there is a reduction of digestive function and fish growth and fish become more vulnerable to bacterial infection. Moreover, when Lucifer yellow was added apically, cells treated with HK L-137 reduced the passage of it, indicating a strengthened barrier function of the monolayer.”
Interestingly, after 24 hours’ treatment, there was a significant lower production of Anxa1, which supports the previous idea of activation of pro-inflammatory pathways, since Anxa1 has anti-inflammatory properties.
To characterize potential benefits for Atlantic salmon, an in vivo experiment was designed. After 61 days of feeding, no mortality was observed as were no differences in fish performance between groups.
Also, there were no signs of inflammation, and the villi height did not change among the groups. Together, these results suggest that HK L-137, at the current inclusion levels, is safe to feed to Atlantic salmon without any deleterious effects.
“When studying the intestinal microbiota of Atlantic salmon in this experiment, at the phylum level, there was an increase on the relative abundance of Actinobacteriota in the LP100 and LP500 groups due to the presence of Mycobacterium genus.”
Members of the genus Mycobacterium are found in wild and captive fish, and several species are considered pathogens. The route of infection is the oral-intestinal pathway and when infected, fish develop skin ulcers which lead to infection and increased mortality.
However, none of these symptoms were observed in this experiment. Moreover, not all the fish reared in the same tank harbored this genus nor was it identified as top abundant genera in feed and water samples. Interestingly, fish fed MG also presented a relative higher abundance of the genus Mycobacterium compared to the CD group.
There was no indication of any consequences of the presence of this genus in the current study and no reports of Atlantic salmon were found. Overall, the modulation of the microbiota in the digesta of the distal intestine was minor, which was demonstrated by the lack of significant differences in diversity between the CD and the remaining groups.
“At the same time, such an observation is not so surprising because we used paraprobiotic HK L-137 in small inclusion levels, ranging from 20, 100 and 500 mg of Feed LP20™ kg-1 feed, (which means 5, 20 and 100 mg HK L-137 kg-1 feed). Because the bacterium was heat-killed, there was no obvious colonization, as was also observed in previous studies.”
Therefore, there was no competition for space and nutrients with commensal bacteria. Instead, the observed effects of the paraprobiotic on the modulation of the microbiota would need to act through other pathways, such as epithelial cell metabolism and the immune response, which in turn could modulate the microbiota.
The intestinal epithelium regulates nutrient uptake and represents a crucial barrier function to the extrinsic environment, which when disrupted, is commonly associated with an inflammatory response. In fish fed LP100 and LP500 diets, other terms related to immune response and barrier function were also upregulated, such as phagosome term, MAPK signaling pathways, tight junctions, gap junctions and cell adhesion molecules.
Finally, the current study was performed on healthy fish, while future studies should combine HK L-137 with multi-stressor conditions or pathogen challenges to create an environment closer to commercial fish production.
Conclusion
In the current study, we investigated the potential of HK L-137 at the in vitro level, by exposing a rainbow trout-derived intestinal epithelial cell line (RTgutGC) to HK L-137, and at in vivo level, with different inclusion levels of HK L-137 in pre-smolt feed. This approach was used to improve the experimental design when evaluating novel additives using an immunonutrition approach to promote animal health and welfare.
In both levels, evidence of immunostimulant properties was observed in HK L-137 by modulating cytokines and effector molecules, suggesting its potential in functional feeds for salmonids without compromising fish performance and gut microbiota.
This approach creates a baseline for future work involving HK L-137 and other pro and paraprobiotics, both in vitro and in vivo.
This is a summarized version developed by the editorial team of Aquaculture Magazine based on the review article titled “FROM A CELL MODEL TO A FISH TRIAL: IMMUNO-MODULATORY EFFECTS OF HEAT-KILLED LACTIPLANTI-BACILLUS PLANTARUM AS A FUNCTIONAL INGREDIENT IN AQUAFEEDS FOR SALMONIDS” developed by: Sérgio Domingos Cardoso Rocha, Peng Lei,Byron Morales-Lange, Liv Torunn Mydland and Margareth Øverland – Norwegian University of Life Sciences, Ås, Norway.
The original article, including tables and figures, was published on MARCH, 2023 through FRONTIERS.
The full version can be accessed online through this link: DOI 10.3389/fimmu.2023.1125702.
