• Antimicrobial antagonists against food pathogens; a bacteriocin perspective

      O'Connor, Paula M.; Ross, R Paul; Hill, Colin; Cotter, Paul D.; Science Foundation Ireland; 12/RC/2273 (Elsevier, 03/02/2015)
      Efforts are continuing to find novel bacteriocins with enhanced specificity and potency. Traditional plating techniques are still being used for bacteriocin screening studies, however, the availability of ever more bacterial genome sequences and the use of in silico gene mining tools have revealed novel bacteriocin gene clusters that would otherwise have been overlooked. Furthermore, synthetic biology and bioengineering-based approaches are allowing scientists to harness existing and novel bacteriocin gene clusters through expression in different hosts and by enhancing functionalities. The same principles apply to bacteriocin producing probiotic cultures and their application to control pathogens in the gut. We can expect that the recent developments on bacteriocins from Lactic Acid Bacteria (LAB) described here will contribute greatly to increased commercialisation of bacteriocins in food systems.
    • Antimicrobials for food and feed; a bacteriocin perspective

      O'Connor, Paula M.; Kuniyoshi, Tais M.; Oliveira, Ricardo PS; Hill, Colin; Ross, R Paul; Cotter, Paul D.; Science Foundation Ireland; São Paulo Research Foundation; 12/RC/2273; 2015/24777-0; et al. (Elsevier, 2020-01-20)
      Bacteriocins are natural antimicrobials that have been consumed via fermented foods for millennia and have been the focus of renewed efforts to identify novel bacteriocins, and their producing microorganisms, for use as food biopreservatives and other applications. Bioengineering bacteriocins or combining bacteriocins with multiple modes of action (hurdle approach) can enhance their preservative effect and reduces the incidence of antimicrobial resistance. In addition to their role as food biopreservatives, bacteriocins are gaining credibility as health modulators, due to their ability to regulate the gut microbiota, which is strongly associated with human wellbeing. Indeed the strengthening link between the gut microbiota and obesity make bacteriocins ideal alternatives to Animal Growth Promoters (AGP) in animal feed also. Here we review recent advances in bacteriocin research that will contribute to the development of functional foods and feeds as a consequence of roles in food biopreservation and human/animal health.
    • Assessing the ability of Nisin A and derivatives thereof to inhibit Gram-negative bacteria from the genus Thermus

      Jonnala, Bhagya R. Yeluri; Feehily, Conor; O'Connor, Paula M.; Field, Des; Hill, Colin; Ross, R. Paul; McSweeney, P. L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D. (2020-12-09)
    • Bacteriocin Gene-Trait matching across the complete Lactobacillus Pan-genome.

      Collins, Fergus W. J.; O'Connor, Paula M.; O'Sullivan, Orla; Gómez-Sala, Beatriz; Rea, Mary; Hill, Colin; Ross, R Paul; Science Foundation Ireland; SFI/12/RC/227; 13/SIRG/2160 (Nature, 2017-06-14)
      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.
    • Bactofencin A, a New Type of Cationic Bacteriocin with Unusual Immunity

      O'Shea, Eileen F.; O'Connor, Paula M.; O'Sullivan, Orla; Cotter, Paul D.; Ross, R Paul; Hill, Colin; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; 04R; 07/CE/B1368 (American Society for Microbiology, 29/10/2013)
      Bacteriocin production is an important probiotic trait of intestinal bacteria. In this study, we identify a new type of bacteriocin, bactofencin A, produced by a porcine intestinal isolate Lactobacillus salivarius DPC6502, and assess its potency against pathogenic species including Staphylococcus aureus and Listeria monocytogenes. Genome sequencing of the bacteriocin producer revealed bfnA, which encodes the mature and highly basic (pI 10.59), 22-amino-acid defensin-like peptide. Matrixassisted laser desorption ionization–time of flight (MALDI-TOF) mass spectral analysis determined that bactofencin A has a molecular mass of 2,782 Da and contains two cysteine residues that form an intramolecular disulfide bond. Although an ABC transporter and transport accessory protein were also present within the bacteriocin gene cluster, a classical bacteriocin immunity gene was not detected. Interestingly, a dltB homologue was identified downstream of bfnA. DltB is usually encoded within the dlt operon of many Gram-positive bacteria. It is responsible for D-alanylation of teichoic acids in the cell wall and has previously been associated with bacterial resistance to cationic antimicrobial peptides. Heterologous expression of this gene conferred bactofencin A-specific immunity on sensitive strains of L. salivarius and S. aureus (although not L. monocytogenes), establishing its role in bacteriocin immunity. An analysis of the distribution of bfnA revealed that it was present in four additional isolates derived from porcine origin and absent from five human isolates, suggesting that its distribution is host specific. Given its novelty, we anticipate that bactofencin A represents the prototype of a new class of bacteriocins characterized as being cationic, with a DltB homologue providing a cognate immunity function.
    • Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

      Field, Des; Begley, Maire; O'Connor, Paula M.; Daly, Karen M.; Hugenholtz, Floor; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Science Foundation Ireland; 10/IN.1/B3027; et al. (PLOS, 08/10/2012)
      Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.
    • A Bioengineered Nisin Derivative to Control Biofilms of Staphylococcus pseudintermedius

      Field, Des; Gaudin, Noemie; Lyons, Francy; O'Connor, Paula M.; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Science Foundation Ireland; 10/IN.1/B3027 (PLoS, 19/03/2015)
      Antibiotic resistance and the shortage of novel antimicrobials are among the biggest challenges facing society. One of the major factors contributing to resistance is the use of frontline clinical antibiotics in veterinary practice. In order to properly manage dwindling antibiotic resources, we must identify antimicrobials that are specifically targeted to veterinary applications. Nisin is a member of the lantibiotic family of antimicrobial peptides that exhibit potent antibacterial activity against many gram-positive bacteria, including human and animal pathogens such as Staphylococcus, Bacillus, Listeria, and Clostridium. Although not currently used in human medicine, nisin is already employed commercially as an anti-mastitis product in the veterinary field. Recently we have used bioengineering strategies to enhance the activity of nisin against several high profile targets, including multi-drug resistant clinical pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) and also against staphylococci and streptococci associated with bovine mastitis. However, newly emerging pathogens such as methicillin resistant Staphylococcus pseudintermedius (MRSP) pose a significant threat in terms of veterinary health and as a reservoir for antibiotic resistance determinants. In this study we created a nisin derivative with enhanced antimicrobial activity against S. pseudintermedius. In addition, the novel nisin derivative exhibits an enhanced ability to impair biofilm formation and to reduce the density of established biofilms. The activities of this peptide represent a significant improvement over that of the wild-type nisin peptide and merit further investigation with a view to their use to treat S. pseudintermedius infections.
    • Bovine whey peptides transit the intestinal barrier to reduce oxidative stress in muscle cells

      Corrochano, Alberto R.; Ferraretto, Anita; Arranz, Elena; Stuknytė, Milda; Bottani, Michela; O'Connor, Paula M.; Kelly, Philip; De Noni, Ivano; Buckin, Vitaly; Giblin, Linda; et al. (Elsevier, 2019-03-06)
      Health benefits are routinely attributed to whey proteins, their hydrolysates and peptides based on in vitro chemical and cellular assays. The objective of this study was to track the fate of whey proteins through the upper gastrointestinal tract, their uptake across the intestinal barrier and then assess the physiological impact to downstream target cells. Simulated gastrointestinal digestion (SGID) released a selection of whey peptides some of which were transported across a Caco-2/HT-29 intestinal barrier, inhibited free radical formation in muscle and liver cells. In addition, SGID of β-lactoglobulin resulted in the highest concentration of free amino acids (176 nM) arriving on the basolateral side of the co-culture with notable levels of branched chain and sulphur-containing amino acids. In vitro results indicate that consumption of whey proteins will deliver bioactive peptides to target cells.
    • Compared to casein, bovine lactoferrin reduces plasma leptin and corticosterone and affects hypothalamic gene expression without altering weight gain or fat mass in high fat diet fed C57/BL6J mice

      McManus, Bettina; Korpela, Riitta; O'Connor, Paula M.; Schellekens, Harriet; Cryan, John F.; Cotter, Paul D.; Nilaweera, Kanishka (Biomed Central, 08/12/2015)
      Background Several studies in both humans and rodents have examined the use of lactoferrin as a dietary solution to weight gain and visceral fat accretion and have shown promising results in the short term (up to 7 weeks). This study examined the effects of giving lactoferrin over a longer period of time. Methods For 13 weeks, male C57/BL6J mice were given a diet containing 10 % kJ fat and 20 % kJ casein (LFD) or a diet with 45 % kJ fat and either 20 % kJ casein (HFD) or 20 % kJ lactoferrin (HFD + Lac). Physiological, metabolic, and biochemical parameters were investigated. Gene expression was investigated by Real-Time PCR and microarray. All data was assessed using t-test, ANOVA or ANCOVA. Gene Set Enrichment Analysis was used to interpret microarray data and assess the impact on gene sets with common biological roles. Results By the end of the trial, HFD + Lac fed mice did not alter energy balance, body composition, bodyweight, or weight gain when compared to the HFD group. Notably, there were no changes in subcutaneous or epididymal adipose leptin mRNA levels between high fat diet groups, however plasma leptin was significantly reduced in the HFD + Lac compared to HFD group (P < 0.05) suggesting reduced leptin secretion. Global microarray analysis of the hypothalamus indicate an overall reduction in gene sets associated with feeding behaviour (P < 0.01) and an up-regulation of gene sets associated with retinol metabolism in the HFD + Lac group compared to the HFD group (P < 0.01). Genes in the latter catergory have been shown to impact on the hypothalamic-pituitary-adrenal axis. Notably, plasma corticosterone levels in the HFD + Lac group were reduced compared to the HFD fed mice (P < 0.05). Conclusions The data suggests that prolonged feeding of full-length dietary lactoferrin, as part of a high fat diet, does not have a beneficial impact on weight gain when compared to casein. However, its impact on leptin secretion and accompanying changes in hypothalamic gene expression may underlie how this dietary protein alters plasma corticosterone. The lactoferrin fed mouse model could be used to identify leptin and corticosterone regulated genes in the hypothalamus without the confounding effects of body weight change.
    • Effect of Bioengineering Lacticin 3147 Lanthionine Bridges on Specific Activity and Resistance to Heat and Proteases

      Suda, Srinivas; Westerbeek, Alja; O'Connor, Paula M.; Hill, Colin; Cotter, Paul D.; Ross, R Paul; Science Foundation Ireland; 06/IN.1/B98 (Elsevier BV, 2010-10-28)
      Lacticin 3147 is a lantibiotic with seven lanthionine bridges across its two component peptides, Ltnα and Ltnβ. Although it has been proposed that the eponymous lanthionine and (β-methyl)lanthionine (Lan and meLan) bridges present in lantibiotics make an important contribution to protecting the peptides from thermal or proteolytic degradation, few studies have investigated this link. We have generated a bank of bioengineered derivatives of lacticin 3147, in which selected bridges were removed or converted between Lan and meLan, which were exposed to high temperature or proteolytic enzymes. Although switching Lan and meLan bridges has variable consequences, it was consistently observed that an intact N-terminal lanthionine bridge (Ring A) confers Ltnα with enhanced resistance to thermal and proteolytic degradation.
    • Efficacy of nisin A and nisin V semi-purified preparations alone and in combination with plant essential oils to control Listeria monocytogenes

      Field, Des; Daly, Karen M.; O'Connor, Paula M.; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Science Foundation Ireland; 10/IN.1/B3027; 06/IN.1/B98 (American Society for Microbiology, 06/02/2015)
      The foodborne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates reaching up to 25-30%. Listeria is a hardy organism and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semi-purified preparations (spp) containing either nisin A or an enhanced bioengineered derivative nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol and trans-cinnamaldehyde, to control L. monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing spp (25 μg/ml) with thymol (0.02%), carvacrol (0.02%) or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil/nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V + carvacrol or nisin V + cinnamaldehyde combinations was twice that of the equivalent nisin A + essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural and effective means to enhance food safety by inhibiting foodborne pathogens such as L. monocytogenes.
    • Generation of Bioactive Hydrolysates and Peptides from Bovine Hemoglobin with In Vitro Renin, Angiotensin-I-Converting Enzyme and Dipeptidyl Peptidase-IV Inhibitory Activities

      Lafarga, Tomas; Rai, Dilip K.; O'Connor, Paula M.; Hayes, Maria; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; 11/F/043 (Wiley, 02/03/2016)
      Bovine hemoglobin was selected for use in the generation of bioactive hydrolysates with potential for use as functional food ingredients for prevention of disorders such as hypertension, obesity and diabetes. Bovine hemoglobin was isolated and hydrolyzed with papain, which was selected using in silico analysis. The generated hydrolysate was enriched by ultrafiltration and further purified by high performance liquid chromatography. A number of peptides were identified using de novo peptide sequencing and these peptides were chemically synthesized to confirm their bioactivity in vitro. Three multifunctional peptides with both, ACE-I and renin-inhibitory properties and one peptide with ACE-I-inhibiting properties were identified. These included the di-peptide HR with ACE-I and renin IC50 values of 0.19 and 7.09 mM, respectively. The generated papain hydrolysate of bovine hemoglobin not only inhibited the enzymes ACE-I and renin but also the enzyme DPP-IV, which has been linked to type-2 diabetes.
    • Heat-induced Maillard reaction of the tripeptide IPP and ribose: Structural characterization and implication on bioactivity

      Jiang, Zhanmei; Rai, Dilip K.; O'Connor, Paula M.; Brodkorb, Andre; National Natural Science Foundation of China; Innovative Research Team of Higher Education of Heilongjiang Province (Elsevier, 28/09/2012)
      Maillard reaction products (MRPs) were prepared from aqueous model mixtures containing 60 g L− 1 ribose and 30 g L− 1 of the bioactive tripeptide IPP (Ile-Pro-Pro), heated at 98 °C. MRP and associated reactions with changes in IPP were observed within one hour of heat-treatment. The pH of MRPs decreased significantly during the heat treatment of IPP–ribose mixtures from 9.0 to 7.6 after one hour. The amino group content, IPP and ribose concentration decreased significantly during heat treatment. The fluorescence intensity of the IPP–ribose MRPs reached the maximum within 2 h. Modification of the UV/vis spectra for IPP–ribose MRPs was mainly due to a condensation reaction of IPP with ribose. Compounds with molecular weight between 300 and 650 Da were dominant while compounds smaller than 250 Da were also produced during the reactions, as characterized by size exclusion chromatography. Mass spectrometry revealed that IPP was conjugated to ribose at the N-terminal (m/z of 458.3) upon heat-treatment. The presence of ribose also promoted peptide degradation to dehydrated IP (m/z of 211.1). IPP–ribose MRPs lost the known angiotensin-I-converting enzyme (ACE) inhibitory activity of IPP; however, strong antioxidant properties were detected.
    • Impact of Environmental Factors on Bacteriocin Promoter Activity in Gut-Derived Lactobacillus salivarius

      Guinane, Caitriona M.; Piper, Clare; Draper, Lorraine A.; O'Connor, Paula M.; Hill, Colin; Ross, R Paul; Cotter, Paul D.; Science Foundation Ireland; SFI/11/PI/1137 (American Society for Microbiology, 04/09/2015)
      Bacteriocin production is regarded as a desirable probiotic trait that aids in colonization and persistence in the gastrointestinal tract (GIT). Strains of Lactobacillus salivarius, a species associated with the GIT, are regarded as promising probiotic candidates and have a number of associated bacteriocins documented to date. These include multiple class IIb bacteriocins (salivaricin T, salivaricin P, and ABP-118) and the class IId bacteriocin bactofencin A, which show activity against medically important pathogens. However, the production of a bacteriocin in laboratory media does not ensure production under stressful environmental conditions, such as those encountered within the GIT. To allow this issue to be addressed, the promoter regions located upstream of the structural genes encoding the L. salivarius bacteriocins mentioned above were fused to a number of reporter proteins (green fluorescent protein [GFP], red fluorescent protein [RFP], and luciferase [Lux]). Of these, only transcriptional fusions to GFP generated signals of sufficient strength to enable the study of promoter activity in L. salivarius. While analysis of the class IIb bacteriocin promoter regions indicated relatively weak GFP expression, assessment of the promoter of the antistaphylococcal bacteriocin bactofencin A revealed a strong promoter that is most active in the absence of the antimicrobial peptide and is positively induced in the presence of mild environmental stresses, including simulated gastric fluid. Taken together, these data provide information on factors that influence bacteriocin production, which will assist in the development of strategies to optimize in vivo and in vitro production of these antimicrobials.
    • Inhibitory activity of Lactobacillus plantarum LMG P-26358 against Listeria innocua when used as an adjunct starter in the manufacture of cheese

      Mills, Susan; Serrano, L Mariela; Griffin, Carmel; O'Connor, Paula M.; Schaad, Gwenda; Bruining, Chris; Hill, Colin; Ross, R Paul; Meijer, Wilco C (Biomed Central, 30/08/2011)
      Lactobacillus plantarum LMG P-26358 isolated from a soft French artisanal cheese produces a potent class IIa bacteriocin with 100% homology to plantaricin 423 and bacteriocidal activity against Listeria innocua and Listeria monocytogenes. The bacteriocin was found to be highly stable at temperatures as high as 100°C and pH ranges from 1-10. While this relatively narrow spectrum bacteriocin also exhibited antimicrobial activity against species of enterococci, it did not inhibit dairy starters including lactococci and lactobacilli when tested by well diffusion assay (WDA). In order to test the suitability of Lb. plantarum LMG P-26358 as an anti-listerial adjunct with nisin-producing lactococci, laboratory-scale cheeses were manufactured. Results indicated that combining Lb. plantarum LMG P-26358 (at 108 colony forming units (cfu)/ml) with a nisin producer is an effective strategy to eliminate the biological indicator strain, L. innocua. Moreover, industrial-scale cheeses also demonstrated that Lb. plantarum LMG P-26358 was much more effective than the nisin producer alone for protection against the indicator. MALDI-TOF mass spectrometry confirmed the presence of plantaricin 423 and nisin in the appropriate cheeses over an 18 week ripening period. A spray-dried fermentate of Lb. plantarum LMG P-26358 also demonstrated potent anti-listerial activity in vitro using L. innocua. Overall, the results suggest that Lb. plantarum LMG P-26358 is a suitable adjunct for use with nisin-producing cultures to improve the safety and quality of dairy products.
    • Insights into the Mode of Action of the Sactibiotic Thuricin CD

      Mathur, Harsh; Fallico, Vincenzo; O'Connor, Paula M.; Rea, Mary; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Science Foundation Ireland; SFI/12/RC/2273 (Frontiers, 20/04/2017)
      Thuricin CD is a two-component bacteriocin, consisting of the peptides Trnα and Trnβ, and belongs to the newly designated sactibiotic subclass of bacteriocins. While it is clear from studies conducted thus far that it is a narrow-spectrum bacteriocin, requiring the synergistic activity of the two peptides, the precise mechanism of action of thuricin CD has not been elucidated. This study used a combination of flow cytometry and traditional culture-dependent assays to ascertain the effects of the thuricin CD peptides on the morphology, physiology and viability of sensitive Bacillus firmus DPC6349 cells. We show that both Trnα and Trnβ are membrane-acting and cause a collapse of the membrane potential, which could not be reversed even under membrane-repolarizing conditions. Furthermore, the depolarizing action of thuricin CD is accompanied by reductions in cell size and granularity, producing a pattern of physiological alterations in DPC6349 cells similar to those triggered by the pore-forming single-component bacteriocin Nisin A, and two-component lacticin 3147. Taken together, these results lead us to postulate that the lytic activity of thuricin CD involves the insertion of thuricin CD peptides into the membrane of target cells leading to permeabilization due to pore formation and consequent flux of ions across the membrane, resulting in membrane depolarization and eventual cell death.
    • Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives

      Healy, Brian; Field, Des; O'Connor, Paula M.; Hill, Colin; Cotter, Paul D.; Ross, R Paul; Department of Agriculture, Food and the Marine; Science Foundation Ireland; 08/RD/C/691; 10/IN.1/B3027 (PLoS, 11/11/2013)
      Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.
    • Lactolisterin BU, a Novel Class II Broad-Spectrum Bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4

      Lozo, Jelena; Mirkovic, Nemanja; O'Connor, Paula M.; Malesevic, Milka; Miljkovic, Marija; Polovic, Natalija; Jovcic, Branko; Cotter, Paul D.; Kojic, Milan; Ministry of Education, Science and Technological Development of the Republic of Serbia; et al. (American Society for Microbiology, 2017-10-17)
      Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti, a bacteriocin with analogy to aureocin A.
    • Oral Delivery of Nisin in Resistant Starch Based Matrices Alters the Gut Microbiota in Mice

      Gough, Ronan; Cabrera-Rubio, Raul; O'Connor, Paula M.; Crispie, Fiona; Miao, Song; Hill, Colin; Ross, R Paul; Cotter, Paul D.; Nilaweera, Kanishka; Rea, Mary; et al. (Frontiers, 15/06/2018)
      There is a growing recognition of the role the gastrointestinal microbiota plays in health and disease. Ingested antimicrobial proteins and peptides have the potential to alter the gastrointestinal microbiota; particularly if protected from digestion. Nisin is an antimicrobial peptide that is used as a food preservative. This study examined the ability of nisin to affect the murine microbiota when fed to mice in two different starch based matrices; a starch dough comprising raw starch granules and a starch gel comprising starch that was gelatinized and retrograded. The effects of the two starch matrices by themselves on the microbiota were also examined. Following 16S rRNA compositional sequencing, beta diversity analysis highlighted a significant difference (p = 0.001, n = 10) in the murine microbiota between the four diet groups. The differences between the two nisin containing diets were mainly attributable to differences in the nisin release from the starch matrices while the differences between the carriers were mainly attributable to the type of resistant starch they possessed. Indeed, the differences in the relative abundance of several genera in the mice consuming the starch dough and starch gel diets, in particular Akkermansia, the relative abundance of which was 0.5 and 11.9%, respectively (p = 0.0002, n = 10), points to the potential value of resistance starch as a modulator of beneficial gut microbes. Intact nisin and nisin digestion products (in particular nisin fragment 22–31) were detected in the feces and the nisin was biologically active. However, despite a three-fold greater consumption of nisin in the group fed the nisin in starch dough diet, twice as much nisin was detected in the feces of the group which consumed the nisin in starch gel diet. In addition, the relative abundance of three times as many genera from the lower gastrointestinal tract (GIT) were significantly different (p < 0.001, n = 10) to the control for the group fed the nisin in starch gel diet, implying that the starch gel afforded a degree of protection from digestion to the nisin entrapped within it.
    • Oral Delivery of Nisin in Resistant Starch Based Matrices Alters the Gut Microbiota in Mice

      Gough, Ronan; Cabrera-Rubio, Raul; O'Connor, Paula M.; Crispie, Fiona; Brodkorb, Andre; Miao, Song; Hill, Colin; Ross, R Paul; Cotter, Paul D.; Nilaweera, Kanishka; et al. (Frontiers, 2018-06-15)
      There is a growing recognition of the role the gastrointestinal microbiota plays in health and disease. Ingested antimicrobial proteins and peptides have the potential to alter the gastrointestinal microbiota; particularly if protected from digestion. Nisin is an antimicrobial peptide that is used as a food preservative. This study examined the ability of nisin to affect the murine microbiota when fed to mice in two different starch based matrices; a starch dough comprising raw starch granules and a starch gel comprising starch that was gelatinized and retrograded. The effects of the two starch matrices by themselves on the microbiota were also examined. Following 16S rRNA compositional sequencing, beta diversity analysis highlighted a significant difference (p = 0.001, n = 10) in the murine microbiota between the four diet groups. The differences between the two nisin containing diets were mainly attributable to differences in the nisin release from the starch matrices while the differences between the carriers were mainly attributable to the type of resistant starch they possessed. Indeed, the differences in the relative abundance of several genera in the mice consuming the starch dough and starch gel diets, in particular Akkermansia, the relative abundance of which was 0.5 and 11.9%, respectively (p = 0.0002, n = 10), points to the potential value of resistance starch as a modulator of beneficial gut microbes. Intact nisin and nisin digestion products (in particular nisin fragment 22–31) were detected in the feces and the nisin was biologically active. However, despite a three-fold greater consumption of nisin in the group fed the nisin in starch dough diet, twice as much nisin was detected in the feces of the group which consumed the nisin in starch gel diet. In addition, the relative abundance of three times as many genera from the lower gastrointestinal tract (GIT) were significantly different (p < 0.001, n = 10) to the control for the group fed the nisin in starch gel diet, implying that the starch gel afforded a degree of protection from digestion to the nisin entrapped within it.