Synthesis and structure-activity study of the antimicrobial lipopeptide brevibacillin.

The antimicrobial lipopeptide brevibacillin is a non-ribosomally synthesized peptide produced by Brevibacillus laterosporus with inhibitory activity against several clinically relevant Gram-positive pathogenic bacteria such as Staphylococcus aureus, Listeria monocytogenes, and Clostridium difficile. In this study, we report the total synthesis of brevibacillin and analogues thereof as well as structure-activity relationship and cytotoxicity studies. Several novel synthetic analogues exhibited high inhibitory activities with minimal inhibitory concentration values in the low micromolar range against several bacteria including Gram-positive L. monocytogenes, S. aureus, Enterococcus faecalis, and Clostridium perfringens as well as Gram-negative Campylobacter coli and Pseudomonas aeruginosa. Of particular interest, four analogues showed a broad spectrum of action and greater antimicrobial activity versus cytotoxicity ratios than native brevibacillin. With a more accessible and efficient production process and improved pharmacological properties, these synthetic analogues are promising candidates to prevent and control the proliferation of various pathogens in the food industry as well as veterinary and human medicine.

Reuterin Enhances the Efficacy of Peracetic Acid Against Multi-species Dairy Biofilm.

Biofilms may contain pathogenic and spoilage bacteria and can become a recurring problem in the dairy sector, with a negative impact on product quality and consumer health. Peracetic acid (PAA) is one of the disinfectants most frequently used to control biofilm formation and persistence. Though effective, it cannot be used at high concentrations due to its corrosive effect on certain materials and because of toxicity concerns. The aim of this study was to test the possibility of PAA remaining bactericidal at lower concentrations by using it in conjunction with reuterin (3-hydroxypropionaldehyde). We evaluated the efficacy of PAA in pure form or as BioDestroy®, a PAA-based commercial disinfectant, on three-species biofilms formed by dairy-derived bacteria, namely Pseudomonas azotoformans PFlA1, Serratia liquefaciens Sl-LJJ01, and Bacillus licheniformis Bl-LJJ01. Minimum inhibitory concentrations of the three agents were determined for each bacterial species and the fractional inhibitory concentrations were then calculated using the checkerboard assay. The minimal biofilm eradication concentration (MBEC) of each antibacterial combination was then calculated against mixed-species biofilm. PAA, BioDestroy®, and reuterin showed antibiofilm activity against all bacteria within the mixed biofilm at respectively 760 ppm, 450 ppm, and 95.6 mM. The MBEC was lowered significantly to 456 ppm, 337.5 ppm, and 71.7 mM, when exposed to reuterin for 16 h followed by contact with disinfectant. Combining reuterin with chemical disinfection shows promise in controlling biofilm on food contact surfaces, especially for harsh or extended treatments. Furthermore, systems with reuterin encapsulation and nanotechnologies could be developed for sustainable antimicrobial efficacy without manufacturing disruptions.

Evaluation of inhibitory activity of essential oils and natural extracts on foodborne viruses.

AIMS: Enteric viruses are recognized as a major concern in health care and in the food sector in Canada. Novel clean-label strategies for controlling enteric viruses are sought in the food industry. In this study, we examined the antiviral potential of plant extracts and essential oils on murine norovirus 1 (MNV-1), hepatitis A virus (HAV), and herpes simplex virus 1 (HSV-1). METHODS AND RESULTS: Inactivation of the viruses by grape seed, blueberry, green tea, and cranberry extracts and by rosemary and thyme essential oils was measured using plaque formation assay. Concentrations ranging from 50 to 200 000 ppm with a contact time of 90 min were tested. Grape seed extract at 10 000 ppm was the most effective (P < 0.05) at reducing MNV-1 and HAV infectious titers, respectively, by 2.85 ± 0.44 log10 and 1.94 ± 0.17 log10. HSV-1 titer was reduced by 3.81 ± 0.40 log10 at 1000 ppm grape seed extract. CONCLUSIONS: Among the plant products tested, grape seed extract was found the most effective at reducing the infectious titers of MNV-1, HAV, and HSV.

Biocontrol Strategy of Listeria monocytogenes in Ready-to-Eat Pork Cooked Ham Using Peptic Hydrolysates of Porcine Hemoglobin.

Listeria monocytogenes is a foodborne pathogen that represents a serious concern for ready-to-eat (RTE) meat products due to its persistence in production facilities. Among the different strategies for the control of this pathogen, the use of antimicrobial peptides derived from food by-products, such as slaughterhouse blood proteins, has emerged as a promising biocontrol strategy. This study evaluated for the first time the use of peptic hydrolysates of porcine hemoglobin as a biocontrol strategy of L. monocytogenes in RTE pork cooked ham. Pure porcine hemoglobin (Hb-P) and porcine cruor (P-Cru) were hydrolyzed using pepsin at different temperatures (37 °C for Hb-P and 23 °C for P-Cru) for 3 h. Then, the hydrolysates were characterized in terms of their degree of hydrolysis (DH), peptide population, color, and antimicrobial activity (in vitro and in situ) against three different serotypes of L. monocytogenes. Reducing the hydrolysis temperature of P-Cru by 14 °C resulted in a 2 percentage unit decrease in DH and some differences in the peptide composition. Nevertheless, the antimicrobial activity (in situ) was not significantly impacted, decreasing the viable count of L. monocytogenes by ~1-log and retarding their growth for 21 days at 4 °C. Although the color of the product was visibly altered, leading to more saturated reddish and yellowish tones and reduced brightness, the discoloration of the hydrolysates can be addressed. This biopreservation approach holds promise for other meat products and contributes to the circular economy concept of the meat industry by valorizing slaughterhouse blood and producing new antilisterial compounds.

Screening of Lactic Acid Bacteria Strains for Potential Sourdough and Bread Applications: Enzyme Expression and Exopolysaccharide Production.

Twenty-eight strains of lactic acid bacteria (LAB) were characterized for the ability to express enzymes of interest (including protease, xylanase, α-amylase, laccase, and glucose oxidase) as well as the ability to produce exopolysaccharide (EPS). The screening of enzyme capability for all LAB strains proceeded in a progressive 3-stage manner that helps to profile the efficiency of LAB strains in expressing chosen enzymes (Stage 1), highlights the strains with affinity for flour as the substrate (Stage 2), and discerns strains that can adapt well in a simulated starter environment (Stage 3). The theoretical ability of LAB to express these enzymes was also assessed using Basic Local Alignment Search Tool (BLAST) analysis to identify the underlying genes in the whole genome sequence. By consolidating both experimental data and information obtained from BLAST, three LAB strains were deemed optimal in expressing enzymes, namely, Lb. delbrueckii subsp. bulgaricus (RBL 52), Lb. rhamnosus (RBL 102), and Lb. plantarum (ATCC 10241). Meanwhile, EPS-producing capabilities were observed for 10 out of 28 LAB strains, among which, Lactococcus lactis subsp. diacetylactis (RBL 37) had the highest total EPS yield (274.15 mg polysaccharide/L culture) and produced 46.2% polysaccharide with a molecular mass of more than 100 kDa.

Insights into Molecular Interactions between a GAPDH-Related Fish Antimicrobial Peptide, Analogs Thereof, and Bacterial Membranes.

Interactions between SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four analogs thereof with model bacterial membranes were studied using Fourier-transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. MD trajectory analyses showed that the N-terminal segment of the peptide analogs has many contacts with the polar heads of membrane phospholipids, while the central α helix interacts strongly with the hydrophobic core of the membranes. The peptides also had a marked influence on the wave numbers associated with the phase transition of phospholipids organized as liposomes in both the interface and aliphatic chain regions of the infrared spectra, supporting the interactions observed in the MD trajectories. In addition, interesting links were found between peptide interactions with the aliphatic chains of membrane phospholipids, as determined by FTIR and from the MD trajectories, and the membrane permeabilization capacity of these peptide analogs, as previously demonstrated. To summarize, the combined experimental and computational efforts have provided insights into crucial aspects of the interactions between the investigated peptides and bacterial membranes. This work thus makes an original contribution to our understanding of the molecular interactions underlying the antimicrobial activity of these GAPDH-related antimicrobial peptides from Scombridae.

High-throughput characterization of the effect of sodium chloride and potassium chloride on 31 lactic acid bacteria and their co-cultures.

Salt (NaCl) is associated with a risk of hypertension and the development of coronary heart disease, so its consumption should be limited. However, salt plays a key role in the quality and safety of food by controlling undesirable microorganisms. Since studies have focused primarily on the effect of salts on the overall counts of the lactic acid bacteria (LAB) group, we have not yet understood how salt stress individually affects the strains and the interactions between them. In this study, we characterized the effect of sodium chloride (NaCl) and potassium chloride (KCl) on the growth and acidification of 31 LAB strains. In addition, we evaluated the effect of salts on a total of 93 random pairwise strain combinations. Strains and co-cultures were tested at 3% NaCl, 5% NaCl, and 3% KCl on solid medium using an automated approach and image analysis. The results showed that the growth of LAB was significantly reduced by up to 68% at 5% NaCl (p < 0.0001). For the co-cultures, a reduction of up to 57% was observed at 5% NaCl (p < 0.0001). However, acidification was less affected by salt stress, whether for monocultures or co-cultures. Furthermore, KCl had a lesser impact on both growth and acidification compared to NaCl. Indeed, some strains showed a significant increase in growth at 3% KCl, such as Lactococcus lactis subsp. lactis 74310 (23%, p = 0.01). More importantly, co-cultures appeared to be more resilient and had more varied responses to salt stress than the monocultures, as several cases of suppression of the significant effect of salts on acidification and growth were detected. Our results highlight that while salts can modulate microbial interactions, these latter can also attenuate the effect of salts on LAB.

Antimicrobial Activity of Synthetic Enterocins A, B, P, SEK4, and L50, Alone and in Combinations, against Clostridium perfringens.

Multidrug-resistant Clostridium perfringens infections are a major threat to the poultry industry. Effective alternatives to antibiotics are urgently needed to prevent these infections and limit the spread of multidrug-resistant bacteria. The aim of the study was to produce by chemical synthesis a set of enterocins of different subgroups of class II bacteriocins and to compare their spectrum of inhibitory activity, either alone or in combination, against a panel of twenty C. perfringens isolates. Enterocins A, P, SEK4 (class IIa bacteriocins), B (unsubgrouped class II bacteriocin), and L50 (class IId leaderless bacteriocin) were produced by microwave-assisted solid-phase peptide synthesis. Their antimicrobial activity was determined by agar well diffusion and microtitration methods against twenty C. perfringens isolates and against other pathogens. The FICINDEX of different combinations of the selected enterocins was calculated in order to identify combinations with synergistic effects. The results showed that synthetic analogs of L50A and L50B were the most active against C. perfringens. These peptides also showed the broadest spectrum of activity when tested against other non-clostridial indicator strains, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Streptococcus suis, Streptococcus pyogenes, Enterococcus cecorum, Enterococcus faecalis, as well as Gram-negative bacteria (Campylobacter coli and Pseudomonas aeruginosa), among others. The selected synthetic enterocins were combined on the basis of their different mechanisms of action, and all combinations tested showed synergy or partial synergy against C. perfringens. In conclusion, because of their high activity against C. perfringens and other pathogens, the use of synthetic enterocins alone or as a consortium can be a good alternative to the use of antibiotics in the poultry sector.

Exploring the genetic basis of natural resistance to microcins.

Enterobacteriaceae produce an arsenal of antimicrobial compounds including microcins, ribosomally produced antimicrobial peptides showing diverse structures and mechanisms of action. Microcins target close relatives of the producing strain to promote its survival. Their narrow spectrum of antibacterial activity makes them a promising alternative to conventional antibiotics, as it should decrease the probability of resistance dissemination and collateral damage to the host's microbiota. To assess the therapeutic potential of microcins, there is a need to understand the mechanisms of resistance to these molecules. In this study, we performed genomic analyses of the resistance to four microcins [microcin C, a nucleotide peptide; microcin J25, a lasso peptide; microcin B17, a linear azol(in)e-containing peptide; and microcin E492, a siderophore peptide] on a collection of 54 Enterobacteriaceae from three species: Escherichia coli, Salmonella enterica and Klebsiella pneumoniae. A gene-targeted analysis revealed that about half of the microcin-resistant strains presented mutations of genes involved in the microcin mechanism of action, especially those involved in their uptake (fhuA, fepA, cirA and ompF). A genome-wide association study did not reveal any significant correlations, yet relevant genetic elements were associated with microcin resistance. These were involved in stress responses, biofilm formation, transport systems and acquisition of immunity genes. Additionally, microcin-resistant strains exhibited several mutations within genes involved in specific metabolic pathways, especially for S. enterica and K. pneumoniae.

The Physicochemical Characterization and In Vitro Digestibility of Maple Sugar Sand and Downgraded Maple Syrups.

The maple syrup industry generates substandard syrups and sugar sand as by-products, which are underused. In this study, we conducted a comprehensive analysis of the physicochemical composition of these products to assess their potential for valorization. Using HPLC analysis, we measured sugar and organic acid content as well as total polyphenol content using the Folin-Ciocalteu method. Additionally, we evaluated the in vitro digestibility using the TIM-1 model. We showed that the composition of ropy and buddy downgraded syrups is comparable to that of standard maple syrup, whereas sugar sand's composition is highly variable, with carbohydrate content ranging from 5.01 mg/g to 652.89 mg/g and polyphenol content ranging from 11.30 µg/g to 120.95 µg/g. In vitro bioaccessibility reached 70% of total sugars for all by-products. Organic acid bioaccessibility from sugar sand and syrup reached 76% and 109% relative to standard maple syrup, respectively. Polyphenol bioaccessibility exceeded 100% during digestion. This can be attributed to favorable extraction conditions, the breakdown of complex polyphenol forms and the food matrix. In conclusion, our study demonstrates that sugar sand and downgraded maple syrups exhibit digestibility comparable to that of standard maple syrup. Consequently, they hold potential as a source of polyphenols, sugar or organic acids for applications such as industrial fermentation or livestock feeds.

Assessing the Activity under Different Physico-Chemical Conditions, Digestibility, and Innocuity of a GAPDH-Related Fish Antimicrobial Peptide and Analogs Thereof.

The antimicrobial activity of SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four chemical analogs thereof was determined under different physicochemical conditions, including different pH values, the presence of monovalent and divalent cations, and after a heating treatment. The toxicity of these five peptides was also studied with hemolytic activity assays, while their stability under human gastrointestinal conditions was evaluated using a dynamic in vitro digestion model and chromatographic and mass spectrometric analyses. The antibacterial activity of all analogs was found to be inhibited by the presence of divalent cations, while monovalent cations had a much less pronounced impact, even promoting the activity of the native SJGAP. The peptides were also more active at acidic pH values, but they did not all show the same stability following a heat treatment. SJGAP and its analogs did not show significant hemolytic activity (except for one of the analogs at a concentration equivalent to 64 times that of its minimum inhibitory concentration), and the two analogs whose digestibility was studied degraded very rapidly once they entered the stomach compartment of the digestion model. This study highlights for the first time the characteristics of antimicrobial peptides from Scombridae or homologous to GAPDH that are directly related to their potential clinical or food applications.

Pathogenicity and Antibiotic Resistance Diversity in Clostridium perfringens Isolates from Poultry Affected by Necrotic Enteritis in Canada.

Necrotic enteritis (NE) caused by C. perfringens is one of the most common diseases of poultry and results in a huge economic loss to the poultry industry, with resistant clostridial strains being a serious concern and making the treatment difficult. Whole-genome sequencing approaches represent a good tool to determine resistance profiles and also shed light for a better understanding of the pathogen. The aim of this study was to characterize, at the genomic level, a collection of 20 C. perfringens isolates from poultry affected by NE, giving special emphasis to resistance mechanisms and production of bacteriocins. Antimicrobial resistance genes were found, with the tet genes (associated with tetracycline resistance) being the most prevalent. Interestingly, two isolates carried the erm(T) gene associated with erythromycin resistance, which has only been reported in other Gram-positive bacteria. Twelve of the isolates were toxinotyped as type A and seven as type G. Other virulence factors encoding hyaluronases and sialidases were frequently detected, as well as different plasmids. Sequence types (ST) revealed a high variability of the isolates, finding new allelic combinations. Among the isolates, C. perfringens MLG7307 showed unique characteristics; it presented a toxin combination that made it impossible to toxinotype, and, despite being identified as C. perfringens, it lacked the housekeeping gene colA. Genes encoding bacteriocin BCN5 were found in five isolates even though no antimicrobial activity could be detected in those isolates. The bcn5 gene of three of our isolates was similar to one previously reported, showing two polymorphisms. Concluding, this study provides insights into the genomic characteristics of C. perfringens and a better understanding of this avian pathogen.

Targeting Enterococci with Antimicrobial Activity against Clostridium perfringens from Poultry.

Necrotic enteritis (NE), caused by Clostridium perfringens, is an emerging issue in poultry farming. New approaches, other than antibiotics, are necessary to prevent NE development and the emergence of multidrug-resistant bacteria. Enterococci are commensal microorganisms that can produce enterocins, antimicrobial peptides with activities against pathogens, and could be excellent candidates for protective cultures. This study aimed to screen and characterize Enterococcus strains of poultry origin for their inhibitory activity against C. perfringens. In total, 251 Enterococcus strains of poultry origin plus five bacteriocin-producing (BP+) E. durans strains of other origins were screened for antimicrobial activity against the indicator C. perfringens X2967 strain using the "spot on the lawn" method. We detected thirty-two BP+ strains (eleven Enterococcus faecium, nine E. gallinarum, eight E. faecalis, three E. durans, and one E. casseliflavus). We further studied the antimicrobial activity of the supernatants of these 32 BP+ strains using agar well diffusion and microtitration against a collection of 20 C. perfringens strains. Twelve BP+ enterococci that were found to exhibit antimicrobial activity against C. perfringens were characterized using whole genome sequencing. Among these, E. faecium X2893 and X2906 were the most promising candidates for further studies as protective cultures for poultry farming. Both strains belong to the sequence type ST722, harbor the genes encoding for enterocin A and enterocin B, do not possess acquired resistance genes, do not carry plasmids, and present the acm gene, which is implicated in host colonization. Further research is needed to determine the utility of these strains as protective cultures.

Nisin Purification from a Cell-Free Supernatant by Electrodialysis in a Circular Economy Framework.

Nisin, an antimicrobial peptide produced by Lactococcus lactis strains, is a promising natural preservative for the food industry and an alternative to antibiotics for the pharmaceutical industry against Gram-positive bacteria. Nisin purification is commonly performed using salting out and chromatographic techniques, which are characterized by their low yields, the use of solvents and the production of large volumes of effluents. In the present work, the purification of nisin from a cell-free supernatant (CFS), after the production of nisin by fermentation on a whey permeate medium, was studied using ammonium sulfate precipitation and electrodialysis (ED) as a promising eco-friendly process for nisin purification. Results showed an increase in nisin precipitation using a 40% ammonium sulfate saturation (ASS) level with a purification fold of 73.8 compared with 34.5 and no purification fold for a 60% and 20% ASS level, respectively. The results regarding nisin purification using ED showed an increase in nisin purification and concentration fold, respectively, of 21.8 and 156 when comparing the final product to the initial CFS. Nisin-specific activity increased from 75.9 ± 4.4 to 1652.7 ± 236.8 AU/mg of protein. These results demonstrated the effectiveness of ED coupled with salting out for nisin purification compared with common techniques. Furthermore, the process was noteworthy for its relevance in a circular economy scheme, as it does not require any solvents and avoids generating polluting effluents. It can be employed for the purification of nisin and the recovery of salts from salting out, facilitating their reuse in a circular economy.

How Discoloration of Porcine Cruor Hydrolysate Allowed the Identification of New Antifungal Peptides.

Porcine blood is an important by-product from slaughterhouses and an abundant source of proteins. Indeed, cruor, the solid part of blood, is mainly composed of hemoglobin. Its enzymatic hydrolysis with pepsin generates a diversity of peptides, particularly antimicrobials. One of the downsides of using these hydrolysates as food bio-preservatives is the color brought by the heme, which can be removed by discoloration. Nonetheless, the effects of this procedure on the antimicrobial peptide population have not been completely investigated. In this study, its impacts were evaluated on the final antibacterial and antifungal activities of a cruor hydrolysate. The results demonstrated that 38 identified and characterized peptides showed a partial or total decrease in the hydrolysate, after discoloration. Antifungal activities were observed for the raw and discolored hydrolysates: MICs vary between 0.1 and 30.0 mg/mL of proteins, and significant differences were detected between both hydrolysates for the strains S. boulardii, C. guilliermondii, K. marxianus, M. racemosus and P. chrysogenum. The raw hydrolysate showed up to 12 times higher antifungal activities. Hence, peptides with the highest relative abundance decrease after discoloration were synthesized and tested individually. In total, eight new antifungal peptides were characterized as active and promising. To our knowledge, this is the first time that effective antifungal peptide sequences have been reported from porcine cruor hydrolysates.

Impact of Pulsed Electric Fields and pH on Enzyme Inactivation and Bioactivities of Peptic Hydrolysates Produced from Bovine and Porcine Hemoglobin.

The production of bioactive peptides from hemoglobin via peptic hydrolysis is a promising alternative to valorizing slaughterhouse blood proteins. Nevertheless, it has some limitations such as low yield, high cost of enzymes, and the use of chemical reagents. The latter is aggravated by the pH increase to inactivate the enzyme, which can affect the bioactivity of the peptides. Thus, this study aimed to evaluate the effect of pulsed electric fields (PEF) on the pepsin inactivation and biological activities (antimicrobial and antioxidant) of hemoglobin hydrolysates. Bovine (Hb-B) and porcine (Hb-P) hemoglobin were hydrolyzed with pepsin for 3 h and treated with PEFs to inactivate the enzyme. The degree of hydrolysis (DH) did not show significant changes after PEF inactivation, whereas peptide population analysis showed some changes in PEF-treated hydrolysates over time, suggesting residual pepsin activity. PEF treatments showed no significant positive or negative impact on antimicrobial and antioxidant activities. Additionally, the impact of pH (3, 7, and 10) on bioactivity was studied. Higher pH fostered stronger anti-yeast activity and DPPH-scavenging capacity, whereas pH 7 fostered antifungal activity. Thus, the use of hemoglobin from the meat industry combined with PEF treatments could fit the circular economy concept since bioactive peptides can be produced more eco-efficiently and recycled to reduce the spoilage of meat products. Nevertheless, further studies on PEF conditions must be carried out to achieve complete inactivation of pepsin and the potential enhancement of peptides' bioactivity.

Effects of bacterial-derived antimicrobial solutions on shelf-life, microbiota and sensory attributes of raw chicken legs under refrigerated storage condition.

In the present study, bacterial-derived antimicrobial agents included 5 mM reuterin combined with either 103.91 mM lactic acid (RL) or 0.08 µM microcin J25 (RJ) were evaluated for their effects on the microbiota and sensory attributes of raw chicken legs. Peracetic acid (13.67 mM), a conventional chemical commonly used in the poultry industry, was used as a positive control to compare efficacy. The chicken legs were sprayed with antimicrobial solutions and aerobically stored at 4 °C for 10 days. The RL treatment maintained the total viable count below the limit of 7 log CFU/g until the 8th day. Therefore, compared to the nontreated group, shelf-life was extended by 3 days in the RL treated group. The RJ treatment extended the shelf-life to 7 days, which is similar to what was achieved with the use of peracetic acid. Based on culture-independent amplicon sequencing, the RL and RJ treatments affected the microbial community on the chicken legs, inducing a delay in the increase of Pseudomonas, Psychrobacter and Carnobacterium while decreasing of Shigella. Significant decreases in sensory scores occurred in the nontreated group, while slight changes occurred in the combinations treated groups over the same period. Overall, sensory property scores for chicken legs treated with RL and RJ remained higher (P < 0.05) than those treated with peracetic acid or without antimicrobial agents. The antimicrobial combinations delayed the deterioration of sensory attributes throughout the storage period. These results suggest that RL and RJ provide a promising natural-sourced antimicrobial approach to control the growth of spoilage microorganisms on chicken legs.

Identification of Gastrointestinal Digestion Stable Antihypertensive Fish Peptides from Atlantic Mackerel (Scomber scombrus).

High blood pressure has been recognized as one of the main risk factors of cardiometabolic syndrome by health organizations. Hypertension is medicated by various classes of synthetic drugs; however, adverse effects have repeatedly been reported. Moreover, natural alternatives such as fish peptides have been effective in the treatment and prevention of hypertension. The aim of our study was to fractionate and identify antihypertensive peptides. Fractions were produced using different techniques of solid-phase extraction (SPE), pressure-driven ultrafiltration (UF), and electro04dialysis with UF membrane. According to our results, the hydrophobic fraction of SPE (IC50 5 µg) was the most potent anti-angiotensin converting enzyme (ACE) product. Findings of the study suggest that the separation technique plays an important role in the isolation efficiency of antihypertensive biopeptides. Importantly, the hydrophobic fraction's activity was retained through a static model of an in vitro gastrointestinal digestion system. In conclusion, polarity regardless of charge and size was the most important factor for anti-ACE activity of an Atlantic mackerel biopeptide. In addition, the presence of leucine at either of the extremities (C- or N-terminal) and/or leucine-rich motifs could well explain the hypotensive effect of our active fraction.

Impact of microcin J25 on the porcine microbiome in a continuous culture model.

The increased prevalence of Salmonella spp. resistance in swine spurs the search for alternatives to antibiotics. Microcin J25 (MccJ25), a bacteriocin produced by Escherichia coli, is a potent inhibitor of several pathogenic bacteria including Salmonella enterica. In this study, we aimed to evaluate in vitro the impact of MccJ25 on the composition and the metabolic activity of the swine colonic microbiota. The PolyFermS in vitro continuous fermentation model was used here with modified Macfarlane medium to simulate the porcine proximal colon. During 35 days of fermentation, a first-stage reactor containing immobilized swine fecal microbiota fed two second-stage control and test reactors operated in parallel and used to test the effects of MccJ25 on the composition and the metabolic activity of the microbiota. Reuterin, a broad-spectrum antimicrobial compound produced by Limosilactobacillus reuteri, a lactic acid bacterium naturally present in the gastro-intestinal tract of human and animals, and the antibiotic rifampicin were tested for comparison. Sequencing of 16S rRNA was performed using the Illumina MiSeq technology to evaluate microbial diversity, and liquid chromatography coupled to mass spectrometry (LC-MS) followed by multivariate analysis was used to assess the bacteriocin/antibiotic degradation products and to monitor changes in the swine colonic microbiota metabolome. The results show that MccJ25 or reuterin treatments only induce subtle changes of both the microbial diversity and the metabolome of the swine colon microbiota, while rifampicin induces significant modification in amino acid levels. Although these findings need being validated in vivo, this study affords a first proof of concept for considering MccJ25 as a possible alternative to antibiotics for veterinary and farming applications, taking into account its pathogen-selective and potent inhibitory activity.

Insights in the Development and Uses of Alternatives to Antibiotic Growth Promoters in Poultry and Swine Production.

The overuse and misuse of antibiotics has contributed to the rise and spread of multidrug-resistant bacteria. To address this global public health threat, many countries have restricted the use of antibiotics as growth promoters and promoted the development of alternatives to antibiotics in human and veterinary medicine and animal farming. In food-animal production, acidifiers, bacteriophages, enzymes, phytochemicals, probiotics, prebiotics, and antimicrobial peptides have shown hallmarks as alternatives to antibiotics. This review reports the current state of these alternatives as growth-promoting factors for poultry and swine production and describes their mode of action. Recent findings on their usefulness and the factors that presently hinder their broader use in animal food production are identified by SWOT (strength, weakness, opportunity, and threat) analysis. The potential for resistance development as well as co- and cross-resistance with currently used antibiotics is also discussed. Using predetermined keywords, we searched specialized databases including Scopus, Web of Science, and Google Scholar. Antibiotic resistance cannot be stopped, but its spreading can certainly be hindered or delayed with the development of more alternatives with innovative modes of action and a wise and careful use of antimicrobials in a One Health approach.