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Probiotics are a viable alternative to traditional chemotherapy agents to control infectious diseases in aquaculture. In this regard

Weissella cibaria P71 is a lactic acid bacterium that was isolated from common octopus ( Octopus vulgaris ) and previously showed interesting probiotic properties for turbot ( Scophthalmus maximus L.) farming. The draft genome sequence of this strain provides further data to support its potential as a probiotic for aquaculture.

Adaptation to life in the deep-sea can be dramatic, with fish displaying behaviors and appearances unlike those seen in any other aquatic habitat. However, the extent of which adaptations may have developed at a microbial scale is not as clear. Shotgun metagenomic sequencing of the intestinal microbiome of 32 species of deep-sea fish from across the Atlantic Ocean revealed that many of the associated microbes differ extensively from those previously identified in reference databases. 111 individual metagenome-assembled genomes (MAGs) were constructed representing individual microbial species from the microbiomes of these fish, many of which are potentially novel bacterial taxa and provide a window into the microbial diversity in this underexplored environment. These MAGs also demonstrate how these microbes have adapted to deep-sea life by encoding a greater capacity for several cellular processes such as protein folding and DNA replication that can be inhibited by high pressure. Another intriguing feature was the almost complete lack of genes responsible for acquired resistance to known antibiotics in many of the samples. This highlights that deep-sea fish microbiomes may represent one of few animal-associated microbiomes with little influence from human activity. The ability of the microbes in these samples to bioluminesce is lower than expected given predictions that this trait has an important role in their life cycle at these depths. The study highlights the uniqueness, complexity and adaptation of microbial communities living in one of the largest and harshest environments on Earth., Sí

peer-reviewed Nisin, an antimicrobial peptide showing activity against a broad range of Gram-positive bacteria, is widely used as a food preservative and has potential as a therapeutic for a range of infectious diseases. Here, we present a simple purification method, based on a salting-out approach, which can produce a powder containing ∼33% nisin, from a nisin-producing culture in a whey permeate-based medium. This process removes over 99% of the lactic acid, NaCl, lactose and non-nisin proteins from the cell-free culture supernatant. The approach can also enrich a commonly used commercial nisin preparation over 30-fold to a purity of ∼58%. These are higher purities than comparable published methods. The simplicity of this approach facilitates its use in research and also its scale-up.

Analysis of 1245 lactic acid bacteria (LAB) isolates obtained from fish, seafood and fish products showed that 197 exerted direct antimicrobial activity against 20 spoilage and food-borne pathogenic microorganisms. Further evaluation of 64 LAB isolates selected on the basis of their direct antimicrobial activity revealed 25 secreted bacteriocins. Biochemical characterization, PCR analysis and/or DNA sequencing of the superoxide dismutase gene (sodA) and/or 16S rRNA gene (16S rDNA), and/or SDS-PAGE analyses of the 64 selected isolates allowed the identification of 24 of them as Enterococcus faecium, 22 as Enterococcus faecalis, seven as Pediococcus pentosaceus, five as Weissella cibaria, three as Lactobacillus sakei subsp. carnosus, one as L. sakei subsp. sakei, one as Lactobacillus curvatus subsp. curvatus and one as Leuconostoc mesenteroides subsp. cremoris. PCR analyses for the detection of genes encoding previously described bacteriocins performed on the 25 bacteriocinogenic strains showed that 19 strains (18 enterococci and 1 lactobacilli) amplified, at least, one of the tested genes, and up to four and two target genes were simultaneously detected in a single enterococcal and lactobacilli strain, respectively. Moreover, in vitro safety evaluation of E. faecium strains was carried out by detection of potential virulence factors, analysis of hemolysin, gelatinase and protease production, and susceptibility testing to antibiotics of relevance for human and veterinary industry. The results reported herein demonstrate the suitability of fish, seafood and fish products for the isolation of LAB (mainly enterococci), including (multi)bacteriocinogenic strains, encoding bacteriocins active against Listeria monocytogenes and other food-borne pathogens of interest for the food industry.

Lactobacilli constitute a large genus of Gram-positive lactic acid bacteria which have widespread roles ranging from gut commensals to starters in fermented foods. A combination of in silico and laboratory-based screening allowed us to determine the overall bacteriocin producing potential of representative strains of each species of the genus. The genomes of 175 lactobacilli and 38 associated species were screened for the presence of antimicrobial producing genes and combined with screening for antimicrobial activity against a range of indicators. There also appears to be a link between the strains’ environment and bacteriocin production, with those from the animal and human microbiota encoding over twice as many bacteriocins as those from other sources. Five novel bacteriocins were identified belonging to differing bacteriocin classes, including two-peptide bacteriocins (muricidin and acidocin X) and circular bacteriocins (paracyclicin). In addition, there was a clear clustering of helveticin type bacteriolysins in the Lactobacillus acidophilus group of species. This combined in silico and in vitro approach to screening has demonstrated the true diversity and complexity of bacteriocins across the genus. It also highlights their biological importance in terms of communication and competition between closely related strains in diverse complex microbial environments.

The antimicrobial activity of Lactobacillus curvatus BCS35 is due to the production of, at least, two class IIa bacteriocins: sakacin P-H12Y (SakP-H12Y), which is a sakacin P (SakP) variant with tyrosine (Tyr) instead of histidine (His) at position 12, and sakacin X (SakX). Moreover, DNA sequence analysis revealed the presence of the genes encoding the two-peptide bacteriocin sakacin T. In order to compare the specific antimicrobial activities of SakP-H12Y and SakX with other class IIa bacteriocins (SakP from Lactobacillus sakei LTH673 and pediocin PA-1 (PedPA-1) from Pediococcus acidilactici 347), and to determine if the substitution His12/Tyr12 in SakP-H12Y/SakP exerts any effect on the bacteriocin potency and/or specificity, these four bacteriocins were purified and challenged against different indicator strains. Our results showed that (i) PedPA-1 was, in general, the most active peptide, and (ii) SakP-H12Y, which contains one less positively charged residue in the N-terminal half (His12/Tyr12), has a reduced antimicrobial potency against most of the tested indicators compared to SaKP, confirming the importance of the N-terminal cationic patch of SakP-H12Y/SakP in target-cell binding, potency and specificity. The in vitro safety of Lb. curvatus BCS35 was established by antibiotic susceptibility testing concluding that it may be considered as a safe strain.

et al., Cloning of the enterolysin A (EnlA) gene (enlA) from Enterococcus faecalis DAC9 into the pMSP3545-derived pMLG2-protein expression vector encoding EnlA under control of the inducible PnisA promoter permitted the controlled release and heterologous expression of mature EnlA by Lactococcus lactis subsp. cremoris NZ9000 and Lactococcus lactis subsp. lactis IL1403. The nisin-induced expression of enlA by L.lactis NZ9000 (pMLG2) and L.lactis IL1403 (pMLG2), grown in GM17 or bovine skim milk (BSM), caused a noticeable reduction of the optical density (OD600) of the cultures and death of the growing cells. However, a high angiotensin converting enzyme (ACE)-inhibitory activity (ACE-IA) was only observed in the BSM-derived hydrolysates of L.lactis IL1403 (pMLG2) after 48h-induction with nisin. Analysis of these hydrolysates by reversed phase high performance liquid chromatography-tandem mass spectrometry permitted the identification of major peptide fragments with known ACE-IA or sharing at least three C-terminal residues with those displaying ACE-IA. © 2013 Elsevier Ltd., This work was partially supported by project AGL2012-34829 from the Ministerio de Economía y Competitividad (MINECO), by projects AGL2009-08348 and AGL2011-24643 from the Ministerio de Ciencia e Innovación (MICINN), and by the CENIT project (2006–2009) from the MITC-CDTI. Funding by grants GR35-10A from the BSCH-UCM and S2009/AGR-1489 from the Comunidad de Madrid (CAM) are also acknowledged. L. Gútiez holds a fellowship (FPU) from the Ministerio de Educación y Ciencia (MEC), Spain.

Enterococcus faecium L50 produces enterocin L50 (L50A and L50B) (EntL50, EntL50A and EntL50B), enterocin P (EntP) and enterocin Q (EntQ) and displays a broad antimicrobial spectrum against the most relevant beer-spoilage lactic acid bacteria (LAB) (i.e., Lactobacillus brevis and Pediococcus damnosus), which is mainly due to the production of EntL50 (EntL50A and EntL50B). Bacteriocin assays using in vitro-synthesized EntL50 (EntL50A and EntL50B) showed that both individual peptides possess antimicrobial activity on their own, EntL50A being the most active, but when the two peptides were combined a synergistic effect was observed. The only virulence genes detected in E. faecium L50 were efaAfm (cell wall adhesin) and ccf (sex pheromone), and this strain was susceptible to most clinically relevant antibiotics. E. faecium L50 survived but did not grow nor showed antimicrobial activity in hopped and unhopped wort, and alcoholic (1 and 5% ethanol, v/v) and non-alcoholic (0% ethanol, v/v) commercial lager beers. However, when unhopped wort was supplemented with 50% (v/v) MRS broth, E. faecium L50 grew and exerted antimicrobial activity similarly as in MRS broth. The enterocins produced by this strain were bactericidal (5 log decrease) against P. damnosus and Lb. brevis in a dose- and substrate-dependent manner when challenged in MRS broth, wort (hopped and unhopped), and alcoholic (1 and 5% ethanol, v/v) and non-alcoholic (0% ethanol, v/v) lager beers at 32 degrees C, and no bacterial resistances were detected even after incubation for 6-15 days. The enterocins in wort and lager beer (5% ethanol, v/v) withstood the heat treatments commonly employed in the brewing industry during mashing, wort boiling, fermentation, and pasteurization, and retained most of their antimicrobial activity in lager beer (5% ethanol, v/v) after long-term storage at 8 and 25 degrees C.

Hiracin JM79 (HirJM79), a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, was cloned and produced in Lactococcus lactis , Lactobacillus sakei , Enterococcus faecium , Enterococcus faecalis , and Pichia pastoris . For heterologous production of HirJM79 in lactic acid bacteria (LAB), the HirJM79 structural gene ( hirJM79 ), with or without the HirJM79 immunity gene ( hiriJM79 ), was cloned into the plasmid pMG36c under the control of the constitutive promoter P 32 and into the plasmid pNZ8048 under the control of the inducible P NisA promoter. For the production of HirJM79 in P. pastoris , the gene encoding the mature HirJM79 protein was cloned into the pPICZαA expression vector. The recombinant plasmids permitted the production of biologically active HirJM79 in the supernatants of L. lactis IL1403, L. lactis NZ9000, L. sakei Lb790, E. faecalis JH2-2, and P. pastoris X-33, the coproduction of HirJM79 and nisin A in L. lactis DPC5598, and the coproduction of HirJM79 and enterocin P in E. faecium L50/14-2. All recombinant LAB produced larger quantities of HirJM79 than E. hirae DCH5, although the antimicrobial activities of most transformants were lower than that predicted from their production of HirJM79. The synthesis, processing, and secretion of HirJM79 proceed efficiently in recombinant LAB strains and P. pastoris .

In this work we describe the development of a biopreservation strategy for fresh fish based on the use of bacteriocinogenic LAB of marine origin. For this purpose, two multibacteriocinogenic LAB strains, Lactobacillus curvatus BCS35 and Enterococcus faecium BNM58, previously isolated from fish and fish products were selected owing to their capability to inhibit the growth of several fish-spoilage and food-borne pathogenic bacteria. Two commercially important fish species were chosen, young hake (Merluccius merluccius) and megrim (Lepidorhombus boscii), and the specimens were acquired at the Marin (Pontevedra, Spain) retail fish market, after one night in the chilled hold of a near-shore fishing vessel. The biopreservation potential and the application strategies of these two LAB strains were first tested at a laboratory scale, where several batches of fresh fish were inoculated with: (i) the multibacteriocinogenic LAB culture(s) as protective culture(s); and/or (ii) their cell-free culture supernatant(s) as food ingredient(s), and (iii) the lyophilized bacteriocin preparation(s) as lyophilized food ingredient(s). All batches were stored in polystyrene boxes, permanently filled with ice at 0-2 °C, for 14 days. Microbiological analyses, as well as sensorial analyses, were carried out during the biopreservation trials. Subsequently, Lb. curvatus BCS35 was selected to up-scale the trials, and combinations of the three application methods were assayed. For this purpose, this strain was grown in a semi-industrial scale fermentor (150l) in modified MRS broth, and three batches of fresh fish were inoculated with the protective culture and/or food ingredient, and stored on ice in a chilled chamber at 0-2 °C at the Marin retail fish market for 14 days. Microbiological analyses were carried out during the storage period, showing that when Lb. curvatus BCS35 culture or the corresponding cell-free culture supernatant was used as protective culture or food ingredient, respectively, bacterial counts were significantly lower than those of the untreated control batches, both for young hake and megrim. In addition, the presence of Listeria spp. in megrim was inhibited in both analyses. The effect of protective culture or food ingredient on the sensory characteristics of fish was evaluated by an official fish appraiser from the Marin retail fish market, who concluded that all the biopreserved batches were worth a higher price in the fish market than the respective control batches, demonstrating that the multibacteriocinogenic strain of marine origin Lb. curvatus BCS35 may be considered as a suitable candidate for its application as fresh fish biopreservative.

et al., In this work, genes encoding gelatinase (gelE) and serine proteinase (sprE), two extracellular proteases produced by Enterococcus faecalis DBH18, were cloned in the protein expression vector pMG36c, containing the constitutive P32 promoter, generating the recombinant plasmids pCG, pCSP, and pCGSP encoding gelE, sprE, and gelE-sprE, respectively. Transformation of noncaseinolytic E. faecalis P36, E. faecalis JH2-2, E. faecium AR24, and E. hirae AR14 strains with these plasmids permitted detection of caseinolytic activity only in the strains transformed with pCG or pCGSP. Complementation of a deletion (knockout) mutant of E. faecalis V583 for production of gelatinase (GelE) with pCG unequivocally supported that gelE is responsible for the caseinolytic activity of the transformed strain grown in bovine skim milk (BSM). RP-HPLC-MS/MS analysis of hydrolysates of transformed Enterococcus spp. strains grown in BSM permitted the identification of 38 major peptide fragments including peptides with previously reported angiotensin converting enzyme-inhibitory activity (ACE-IA), antihypertensive activity, and antioxidant activity. © 2014 American Chemical Society., This work was supported by Project AGL2012-34829 from the Ministerio de Economía y Competitividad (MINECO), by Projects AGL2009-08348 and AGL2011-24643 from the Ministerio de Ciencia e Innovación (MICINN), by a CENIT project (2006−2009) from the MITC-CDTI, by Grant GR35-10A from the BSCH-UCM, and by Grants S2009/AGR-1489 and P2013/ABI-2747 from the Comunidad de Madrid (CAM). L.G. holds a fellowship (FPU) from the Ministerio de Educación y Ciencia (MEC), J.B. held a research contract from the CAM, and J.J.J. was the recipient of a fellowship from the MICINN, Spain.

Enterococcus faecalis isolates from food and environmental origin were evaluated for their angiotensin-converting enzyme (ACE)-inhibitory activity (ACE-IA) after growth in bovine skim milk (BSM). Most (90% active) but not all (10% inactive) E. faecalis strains produced BSM-derived hydrolysates with high ACE-IA. Known ACE-inhibitory peptides (ACE-IP) and an antioxidant peptide were identified in the E. faecalis hydrolysates by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS). Antimicrobial activity against Pediococcus damnosus CECT4797 and Listeria ivanovii CECT913 was also observed in the E. faecalis hydrolysates. The incidence of virulence factors in the E. faecalis strains with ACE-IA and producers of ACE-IP was variable but less virulence factors were observed in the food and environmental strains than in the clinical reference strains. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) based analysis demonstrated that food and environmental E. faecalis strains were genetically different from those of clinical origin. When evaluated, most E. faecalis strains of clinical origin also originated BSM-derived hydrolysates with high ACE-IA due to the production of ACE-IP. Accordingly, the results of this work suggest that most E. faecalis strains of food, environmental and clinical origin produce BSM-derived bioactive peptides with human health connotations and potential biotechnological applications. © 2013 Elsevier B.V., This work was partially supported by project AGL2012-34829 from the Ministerio de Economía y Competitividad (MINECO), by projects AGL2009-08348 and AGL2011-24643 from the Ministerio de Ciencia e Innovación (MICINN), and by the CENIT project (2006–2009) from the MITC-CDTI. Funding by grants GR35-10A from the BSCH-UCM and S2009/AGR-1489 from the Comunidad de Madrid (CAM) are also acknowledged. L. Gútiez holds a fellowship (FPU) from the Ministerio de Educación y Ciencia (MEC), Spain.

In this work, biogenic amine production (histamine, tyramine and putrescine) by a collection of 74 lactic acid bacteria of aquatic origin has been investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thin-layer chromatography (TLC) and decarboxylase gene detection by PCR. None of the evaluated strains showed neither production of histamine and putrescine, nor presence of the genetic determinants encoding the corresponding decarboxylase activities. However, the tyrosine decarboxylase gene (tdc) was present in all the enterococcal strains, and tyramine production was detected by TLC in all of them but Enterococcus faecium BCS59 and MV5. Analysis of the tyrosine decarboxylase operon of these strains revealed the presence of an insertion sequence upstream tdc that could be responsible for their lack of tyrosine decarboxylase activity. © 2011 Elsevier B.V., This work was partially supported by projects AGL2009-08348ALI and Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD from the Ministerio de Ciencia y Tecnología (MCYT), Spain; Spanish–Portuguese Integrated Action HP2008-0070 from the Ministerio de Ciencia e Innovación (MICINN), Spain; S2009/AGR-1469 and S2009/AGR-1489 from the Comunidad de Madrid (CAM), Spain; and RM2008-00002 from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain. G. Landeta is a recipient of a predoctoral fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico. P. Natale is acknowledged for artwork.

In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal ( MF α 1 s ) and cloned, separately and together ( entL50AB ), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (P AOX1 ) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1 s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast.

This work reports the quantitative analysis of two novel antihypertensive peptides αs1-CN f(90-94), with sequence RYLGY, and αs1-CN f(143-149), with sequence AYFYPEL, by high-performance liquid chromatography– mass spectrometry in food-grade hydrolysates of milk proteins. The method was validated and showed sufficient specificity, reproducibility, linearity and recovery. Linear calibrations of the molecular ions m/z 671.2 and 902.3 were selected for the determination of the peptides RYLGY and AYFYPEL, respectively, and showed good statistical results (R2≥0.995 and with no significant lack-of-fit). The simplicity of RP-HPLC-MS method allowed the automated quantification of both antihypertensive peptides without any sample pretreatment. The application of this method permitted the evaluation of some hydrolysis variables, i.e., substrate, temperature, hydrolysis time or enzyme/substrate ratio, on the formation of antihypertensive peptides. The quantitative analysis of RYLGY and AYFYPEL showed that ultrafiltration was not effective to improve the content in active peptides, containing the hydrolysates and their respective permeates similar peptide amounts.

Samples from the intestinal content and carcasses of mallard ducks (Anas platyrhynchos) were evaluated for enterococci with antimicrobial activity, presence of genes coding bacteriocins and their expression, and potential virulence factors. Enterococcus faecalis comprised the largest enterococcal species with antagonistic activity followed by E. faecium, E. hirae, Enterococcus spp., and the non-enterococci. Although all E. faecalis isolates manifested a potent direct antimicrobial activity, no activity was detected in supernatants of most producer cultures. However, all E. faecium isolates showed antimicrobial activity in their supernatants and encoded bacteriocins, although the occurrence in the isolates of several enterocin genes did not always correlate with a higher antagonistic activity in supernatants. The efaAfm determinant was the only virulence gene detected in E. faecium, while E. faecalis showed a larger number of virulence determinants, and E. hirae did not carry any of the virulence genes examined. The rapid identification of genes coding described bacteriocins permits recognition of isolates that are potentially producers of novel bacteriocins. Purification of the antimicrobial activity of E. hirae DCH5 and Lactococcus garvieae DCC43 revealed unique chromatographic fragments after MALDI-TOF mass spectrometry analysis, suggesting the antagonistic peptides were purified to homogeneity. Bacteriocinogenic E. faecium and E. hirae isolates may be considered hygienic for production of bacteriocins, and potentially safe due to their low incidence of potential virulence genes and susceptibility to most clinically relevant antibiotics. However, the presence among the enterococci of E. faecalis strains with a potent antagonistic activity and multiple virulence factors, raises concerns regarding their potential pathogenicity to consumers.

A casein hydrolysis process was scaled up to produce the antihypertensive peptides αs1-CN f(90-94), with sequence RYLGY, and αs1-CN f(143-149), with sequence AYFYPEL. Previously, the hydrolysis process was optimized under food-grade conditions to obtain a maximum amount of active peptides, quantified by high-performance liquid chromatography-mass spectrometry. Ultrafiltration as a strategy for enrichment of active peptides was also evaluated. The casein-derived ingredients prepared at large scale showed potent angiotensin converting enzyme-inhibitory activity in vitro. Furthermore, these products produced a significant decrease in the systolic blood pressure of spontaneously hypertensive rats after oral administration (200-800 mg kg-1 of body weight). The active peptides RYLGY and AYFYPEL were stable during the processes of atomization, homogenization and pasteurization. This hydrolysate was incorporated into liquid yoghurt and no significant reduction of either peptide was detected during the shelf-life of the product. © 2011 Elsevier Ltd.

Background The microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for the aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate the antimicrobial/bacteriocin activity against fish pathogens, the antibiotic susceptibility, and the prevalence of virulence factors and detrimental enzymatic activities in 99 Lactic Acid Bacteria (LAB) (59 enterococci and 40 non-enterococci) isolated from aquatic animals regarded as human food. Results These LAB displayed a broad antimicrobial/bacteriocin activity against the main Gram-positive and Gram-negative fish pathogens. However, particular safety concerns based on antibiotic resistance and virulence factors were identified in the genus Enterococcus (86%) (Enterococcus faecalis, 100%; E. faecium, 79%). Antibiotic resistance was also found in the genera Weissella (60%), Pediococcus (44%), Lactobacillus (33%), but not in leuconostocs and lactococci. Antibiotic resistance genes were found in 7.5% of the non-enterococci, including the genera Pediococcus (12.5%) and Weissella (6.7%). One strain of both Pediococcus pentosaceus and Weissella cibaria carried the erythromycin resistance gene mef(A/E), and another two P. pentosaceus strains harboured lnu(A) conferring resistance to lincosamides. Gelatinase activity was found in E. faecalis and E. faecium (71 and 11%, respectively), while a low number of E. faecalis (5%) and none E. faecium exerted hemolytic activity. None enterococci and non-enterococci showed bile deconjugation and mucin degradation abilities, or other detrimental enzymatic activities. Conclusions To our knowledge, this is the first description of mef(A/E) in the genera Pediococcus and Weissella, and lnu(A) in the genus Pediococcus. The in vitro subtractive screening presented in this work constitutes a valuable strategy for the large-scale preliminary selection of putatively safe LAB intended for use as probiotics in aquaculture.