Susana Rubiño

The objective of this work was to assess the antimicrobial effect of crude extracts and non-polar and mid-polar subfractions of the brown macroalga Ericaria selaginoides, using a mid-polarity extraction medium. The activity was evaluated through challenge testing against Listeria monocytogenes in Catalan “mató” (fresh cheese) selected as a food matrix. Three concentrations were tested, representing 2 ×, 4 × and 10 × Minimum Bactericidal Concentration values obtained for crude extract in previous in vitro assays. Ericaria selaginoides extracts inhibited L. monocytogenes growth in a dose-dependent manner. Intermediate concentrations of the three extracts were able to notably reduce the growth rate of the pathogen. Highest concentration of crude extract and non-polar subfraction were able to completely inhibit the growth of L. monocytogenes during the 10 days of storage at 8 °C. Moreover, a slight listericidal effect was observed in presence of the non-polar subfraction, reducing the L. monocytogenes level by ca. 3 log after 10 days of refrigerated storage. These results prove the potential of E. selaginoides extracts as a source of valuable antimicrobial compounds that can be used as natural food ingredients to develop safer fresh dairy food.

Growing consumer demand for high-quality products coupled with minimally processed products and a minor use of synthetic food additives have increased the need to search for new sources of natural antimicrobials to ensure product safety. This study aimed to evaluate the antimicrobial activity of extracts from the brown algae Ericaria selaginoides against Bacillus cereus in typical Catalan fresh cheese (“mató”) by means of challenge testing. Three concentrations of a crude extract and its corresponding two subfractions (non-polar and mid-polar) obtained after purification showed an antimicrobial dose-dependent effect on B. cereus, from inhibition to inactivation. The best results were obtained with higher concentrations of the non-polar subfraction that caused a total inactivation of the inoculated pathogen after 2 or 4 days, followed by the mid-polar that inactivated B. cereus after 2 or 6 days. The results showed an improvement in the antimicrobial effect after purification compared with the effect observed when the crude extract was tested. Moreover, compounds of different chemical natures may be involved in this antimicrobial activity since it remained in both subfractions after purification. The results obtained in this work show the great potential of macroalgae extracts as natural food preservatives against B. cereus in fresh cheese.

The search for new sources of antimicrobial compounds has become an urgent need, due to the threat that the spread of bacterial resistance represents for global health and food safety. Brown macroalgae have been proposed as a great reservoir in the search for novel antimicrobial compounds. In this study, mid-polarity extracts were performed with a selection of 20 brown macroalgae species from northern Spain. The total polyphenol, carbohydrate and protein contents were quantified by spectrophotometry. The volatile organic compounds (VOCs) of whole macroalgae were also studied as a biomarker of their metabolic state in the representative species of the tested families by gas chromatography-mass spectrometry (GC-MS). The antimicrobial potential of the extracts was assessed by a disk diffusion assay against 20 target bacteria and further determinations of the minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) were performed by a microdilution assay for the active extracts. Ericaria selaginoides, Bifurcaria bifurcata and Dictyota dichotoma showed an antimicrobial effect against six Gram-positive strains: Bacillus cereus, Bacillus subtilis, Geobacillus stearothermophilus, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus haemolyticus. The phenolic content was generally higher in the extracts that showed antimicrobial activity, followed by carbohydrates and low contents of proteins. The results obtained in this study reveal the potential of brown macroalgae as a promising alternative source of antimicrobial compounds as functional ingredients for the application in industrial fields.

We report the high susceptibility of several clinical isolates of Propionibacterium acnes from different sources (skin, bone, wound exudates, abscess or blood contamination) to the head-to-tail cyclized bacteriocin AS-48. This peptide is a feasible candidate for further pharmacological development against this bacterium, due to its physicochemical and biological characteristics, even when it is growing in a biofilm. Thus, the treatment of pre-formed biofilms with AS-48 resulted in a dose- and time-dependent disruption of the biofilm architecture beside the decrease of bacterial viability. Furthermore, we demonstrated the potential of lysozyme to bolster the inhibitory activity of AS-48 against P. acnes, rendering high reductions in the MIC values, even in matrix-growing cultures, according to the results obtained using a range of microscopy and bioassay techniques. The improvement of the activity of AS-48 through its co-formulation with lysozyme may be considered an alternative in the control of P. acnes, especially after proving the absence of cytotoxicity demonstrated by these natural compounds on relevant human skin cell lines. In summary, this study supports that compositions comprising the bacteriocin AS-48 plus lysozyme must be considered as promising candidates for topical applications with medical and pharmaceutical purposes against dermatological diseases such as acne vulgaris.

Staphylococci are a group of microorganisms that can be often found in processed food and they might pose a risk for human health. In this study we have determined the content of staphylococci in 7 different fresh goat-milk cheeses. These bacteria were present in all of them, ranging from 10³ to 10⁶ CFU/g based on growth on selective media. Thus, a set of 97 colonies was randomly picked for phenotypic and genotypic identification. They could be clustered by RAPD-PCR in 10 genotypes, which were assigned by 16S rDNA sequencing to four Staphylococcus species: Staphylococcus aureus, Staphylococcus chromogenes, S. simulans, and S. xylosus. Representative strains of these species (n = 25) were tested for antibiotic sensitivity, and 11 of them were resistant to at least one of the antibiotics tested, including erythromycin, amoxicillin-clavulanic acid and oxacillin. We also tested two bacteriocins produced by lactic acid bacteria (LAB), namely the circular bacteriocin AS-48 and the lantibiotic nisin. These peptides have different mechanism of action at the membrane level. Nevertheless, both were able to inhibit staphylococci growth at low concentrations ranging between 0.16–0.73 μM for AS-48 and 0.02–0.23 μM for nisin, including the strains that displayed antibiotic resistance. The combined effect of these bacteriocins were tested and the fractional inhibitory concentration index (FICI) was calculated. Remarkably, upon combination, they were active at the low micromolar range with a significant reduction of the minimal inhibitory concentration. Our data confirms synergistic effect, either total or partial, between AS-48 and nisin for the control of staphylococci and including antibiotic resistant strains. Collectively, these results indicate that the combined use of AS-48 and nisin could help controlling (pathogenic) staphylococci in food processing and preventing antibiotic-resistant strains reaching the consumer in the final products.

A purified preparation of the nontoxic antimicrobial peptide, nisin (AMBICIN N), was used in the formulation of a germicidal sanitizer suitable for use on cow teats. The germicidal activity of the formulation against mastitis pathogens was measured on teat skin of live cows. The nisin-based formulation gave a mean log reduction of 3.90 against Staphylococcus aureus and 4.22 log reduction against Escherichia coli after exposure for 1 min to the germicide. This activity was comparable with that exhibited by a 1% iodophor teat dip but was significantly greater than that exhibited by the .1 and .5% iodophors and by the .5% chlorhexidine digluconate teat dips. The nisin-based formulation showed little or no potential for skin irritation after multiple application to skin, but iodophor and chlorhexidine digluconate teat dips showed significant potential for skin irritation in comparable studies.