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The Effects of Nisin-Producing Lactococcus Lactis Strain Used As Probiotic on Gilthead Sea Bream (Sparus Aurata) Growth, Gut Microbiota, and Transcriptional Response

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The Effects of Nisin-Producing Lactococcus Lactis Strain Used As Probiotic on Gilthead Sea Bream (Sparus Aurata) Growth, Gut Microbiota, and Transcriptional Response fmars-08-659519 April 9, 2021 Time: 13:57 # 1 ORIGINAL RESEARCH published: 12 April 2021 doi: 10.3389/fmars.2021.659519 The Effects of Nisin-Producing Lactococcus lactis Strain Used as Probiotic on Gilthead Sea Bream (Sparus aurata) Growth, Gut Microbiota, and Transcriptional Response Federico Moroni1†, Fernando Naya-Català2†, M. Carla Piazzon3, Simona Rimoldi1, Edited by: Josep Calduch-Giner2, Alberto Giardini4, Inés Martínez5, Fabio Brambilla6, Fotini Kokou, 2 1 Wageningen University and Research, Jaume Pérez-Sánchez and Genciana Terova * Netherlands 1 Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy, 2 Nutrigenomics and Fish Growth Reviewed by: Endocrinology, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellón, Spain, 3 Fish Pathology, Institute Carlo C. Lazado, of Aquaculture Torre de la Sal (IATS-CSIC), Castellón, Spain, 4 Centro Sperimentale del Latte S.r.l., Zelo Buon Persico, Italy, Norwegian Institute of Food, Fisheries 5 Sacco S.r.l., Cadorago, Italy, 6 VRM S.r.l. Naturalleva, Cologna Veneta, Italy and Aquaculture Research (Nofima), Norway Yun-Zhang Sun, The present research tested the effects of dietary nisin-producing Lactococcus lactis Jimei University, China on growth performance, feed utilization, intestinal morphology, transcriptional response, *Correspondence: and microbiota in gilthead sea bream (Sparus aurata). A feeding trial was conducted Genciana Terova [email protected] with fish weighting 70–90 g. Fish were tagged with passive, integrated transponders †These authors have contributed and distributed in nine 500 L tanks with 40 fish each. Fish were fed for 12 weeks with equally to this work either a control (diet A) or experimental diets (diets B and C) in triplicate (3 tanks/diet). Extruded pellets of diets B and C were supplemented with a low (2 × 109 CFU/kg) and Specialty section: 9 This article was submitted to a high (5 × 10 CFU/kg) dose of probiotic, respectively. No significant differences were Marine Fisheries, Aquaculture found between groups for the feed conversion ratio or specific growth rates. However, and Living Resources, the final body weight of fish fed diet C was significantly higher than the control group a section of the journal Frontiers in Marine Science with intermediate values for fish fed diet B. Histological analysis conducted using a Received: 27 January 2021 semi-quantitative scoring system showed that probiotic did not alter the morphology Accepted: 22 March 2021 of the intestine and did not trigger inflammation. With regard to the transcriptomic Published: 12 April 2021 response, a customized PCR array layout was designed to simultaneously profile a panel Citation: Moroni F, Naya-Català F, of 44 selected genes. Significant differences in the expression of key genes involved in Piazzon MC, Rimoldi S, innate and acquired immunity were detected between fish fed probiotic and control Calduch-Giner J, Giardini A, diets. To analyze the microbiota associated to the feeds and the gut autochthonous Martínez I, Brambilla F, Pérez-Sánchez J and Terova G (2021) microbial communities, we used the Illumina MiSeq platform for sequencing the 16S The Effects of Nisin-Producing rRNA gene and a metagenomics pipeline based on VSEARCH and RDP databases. The Lactococcus lactis Strain Used as Probiotic on Gilthead Sea Bream analysis of gut microbiota revealed a lack of colonization of the probiotic in the host’s (Sparus aurata) Growth, Gut intestinal mucosa. However, probiotic did modulate the fish gut microbiota, confirming Microbiota, and Transcriptional that colonization is not always necessary to induce host modification. In fact, diets B and Response. Front. Mar. Sci. 8:659519. doi: 10.3389/fmars.2021.659519 C were enriched with Actinomycetales, as compared to diet A, which instead showed a Frontiers in Marine Science| www.frontiersin.org 1 April 2021| Volume 8| Article 659519 fmars-08-659519 April 9, 2021 Time: 13:57 # 2 Moroni et al. LAB-Fortified Feed for Fish higher percentage of Pseudomonas, Sphyngomonas, and Lactobacillus genera. These results were confirmed by the clear separation of gut bacterial community of fish fed with the probiotic from the bacterial community of control fish group in the beta-diversity and PLS-DA (supervised partial least-squares discriminant analysis) analyses. Keywords: aquaculture, gilthead sea bream, probiotic, Lactococcus lactis, gut microbiota, transcriptomic INTRODUCTION human and veterinary medicine (EFSA Panel on Biological Hazards (BIOHAZ) et al., 2020). The definition of “Probiotics” has changed many times during The increase in the use of probiotics in aquaculture is mostly this century. However, according to (Food and Agriculture related to the need to decrease or even avoid the use of antibiotics, Organization of the United [FAO] and World Health increasing at the same time the sustainability of the aquaculture Organisation [WHO], 2001) probiotics are “live microorganisms industry. The negative effects of antibiotics overuse include the that confer a health benefit on the host when administered in accumulation of residue in the aquatic environment, particularly adequate amounts.” The most commonly used probiotics are in the marine sediments where antibiotics can persist for months, bacteria belonging to Lactobacillus, Bifidobacterium, Bacillus, favoring the selection of multi-antibiotic-resistant bacterial and Enterococcus genera (European Medicines Agency [EMA], strains. Indeed, there is an increasing risk that antibiotic-resistant and European Food Safety Authority [EFSA], 2017; EFSA bacteria, initially derived from food-producing animals, could FEEDAP [EFSA Panel on Additives and Products or Substances render the latest generation of antibiotics virtually ineffective for used in Animal Feed] et al., 2018), but some fungal genera have humans (Cabello, 2006; World Health Organisation [WHO] et also been reported as novel probiotics. al., 2006). Another negative outcome of antibiotics being used as In the last 25–30 years, the use of probiotics in animal growth promoters in cultured fish is the reduction of biodiversity production has increased (Chaucheyras-Durand and Durand, and quantity of indigenous gut microbiota, which can impair fish 2010; Ezema, 2013). Indeed, several publications have reported immune responses (Borch et al., 2015). numerous beneficial effects associated with the supplementation For these reasons, the use of antibiotics as growth promoters of live yeast or bacteria (mostly Lactobacillus) in the diet in animal production has been fully banned in the EU since 2006 of terrestrial animals, including amelioration of resistance to (Casewell et al., 2003; European Parliament and the Council of pathogens, improvement in growth parameters (in swine and the European Union, 2003, 2019; European Medicines Agency poultry), increase in productivity and quality of eggs in laying [EMA], and European Food Safety Authority [EFSA], 2017) and hens, and enhancement of milk production in cattle (Gallazzi many research efforts have been undertaken to replace them with et al., 2008; Shabani et al., 2012; Puphan et al., 2015; Uyeno et al., probiotics for animal health management (Ezema, 2013). 2015; De Cesare et al., 2017; Wang et al., 2017; Dowarah et al., Several studies have demonstrated that probiotics can 2018; Forte et al., 2018). reduce pathogenic bacteria due to direct competition-colonizing In aquaculture, a great number of bacterial species are dynamics, through which microorganisms can partition spatial currently used as probiotics (for a review, please see Newaj- niche habitats in the intestinal mucosa (Balcázar et al., 2007b; Fyzul et al., 2014). These microorganisms can be administered Sugimura et al., 2011). Probiotics can also produce inhibitory as multi-species (multi-strain) or single-species (single-strain) molecules, such as bacteriocins, siderophores, enzymes, and (Food and Agriculture Organization of the United [FAO], 2016) hydrogen peroxide, or inhibit pathogenic bacteria by decreasing and provided either as a suspension in water, or added to the intestinal pH through the release of organic acids (Ringø, the feed. However, use in feed is considered the best option; 2008; Zhou X. et al., 2010; Ustyugova et al., 2012; Perez et al., 2014; therefore, this approach is employed most frequently (Nayak, Dahiya et al., 2020). 2010; Jahangiri and Esteban, 2018). In the European Union In addition, probiotics enhance the host immune system (EU), probiotic strains, must obtain a market authorization by generating systemic and/or local responses (Balcázar et al., 1 by the EFSA (European Safety Food Authority) , which grants 2006b; Salinas et al., 2008) that include activation of various a QPS (Qualified Presumption of Safety) status. The QPS is antioxidant pathways and an increase in several innate immune based on reasonable evidence. No microorganism belonging to parameters, such as phagocytosis, lysozyme levels, respiratory a QPS status group needs to undergo a full safety assessment, burst peroxidase and antiprotease activity, cytokine production, but microorganisms that pose a safety concern to humans, and white blood cell count (Nayak, 2010; Lazado and Caipang, animals, or environment are not considered suitable for 2014; Newaj-Fyzul et al., 2014; Simó-Mirabet et al., 2017). QPS status and must undergo a full safety assessment. The In cultured fish, probiotics improve fish growth and feed QPS assessment requires: (1) the identity of the strain to conversion rates, too, due to an increase in feed digestibility
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