Antimicrobial Activity of Crude Extracts from Ascophyllum nodosum Obtained by Microwave-Assisted Extraction †

: Ascophyllum nodosum (Linnaeus) Le Jolis is a brown alga from the Fucaceae family and a unique species from the Ascophyllum genus. This brown alga is an edible macroalga from the North Atlantic Ocean, commonly found on the European north-western coast. High-value bioactive molecules such as pigments, polyphenols, and phlorotannin were found in the macroalgae composition, which makes this alga particularly interesting for exploring potential biological activities. Among sustainable extraction technologies, microwave-assisted extraction (MAE) has many advantages such as a short extraction time and fewer solvent requirements. On the other hand, ethanol and water are eco-friendly solvents that have already been proven to be effective for obtaining bioactive compounds with antimicrobial capacity. Therefore, in this work, analytical conditions of MAE: t = 5 min; pressure = 10.5 bar; ethanol concentration (37%) as solvent were applied to obtain a polyphenol-rich extract from A. nodosum . The antimicrobial effect of the resulting extract against ﬁve food-borne microorganisms ( Bacillus cereus , Escherichia coli , Salmonella enteritidis , Pseudomonas aeruginosa , Staphylococcus aureus ), and the opportunistic bacteria Staphylococcus epidermidis was assessed. The antimicrobial activity was performed through the Kirby–Bauer disk diffusion susceptibility test protocol and the microdilution method. The analytical results indicated that the A. nodosum extract was effective against all tested bacteria except for Escherichia coli . The highest antimicrobial activity was found against Staphylococcus aureus , presenting inhibitory capacity with a concentration of 400 µ g/mL and an inhibition halo of 11.79 ± 1.92 mm.


Introduction
Nowadays, consumer demand is focused on natural, safe, and diverse food sources.Moreover, the growing interest in functional foods is leading the scientific community to search for nutraceutical characteristics in natural products as a novel approach to a healthier lifestyle.Macroalgae are traditionally part of the human diet, especially in oriental countries, but they have emerged as an important nutrition alternative because, apart from their nutritional properties, they have shown biologically active properties such as antioxidant, antimicrobial, antitumor, and anti-inflammatory properties [1][2][3].A. nodosum is a perennial edible alga belonging to the Ochrophyta phylum that can be found in cold waters, with special abundance on sheltered rocky shores of the north Atlantic ocean.This alga is a rich source of underexploited bioactive compounds.Microwave-assisted extraction (MAE) is a non-thermal emerging technique that is applied to extract bioactive compounds from macroalgae.Some works reported higher antioxidant capacity in A. nodosum extracts obtained with MAE when compared to extracts obtained by traditional extraction techniques (maceration) [4].Regarding solvents, MAE requires the use of polar solvents.In that sense, ethanol emerges as a non-toxic solvent, adequate to achieve high extraction yields [5] and has already proven to be capable of extracting bioactive molecules from alga matrix with proven antioxidant and antimicrobial capacity [6].
Food safety is a crucial topic for human health.Food-borne pathogens are responsible, worldwide, for an enormous number of diseases.For instance, it is estimated that pathogens caused 37.2 million ailments annually in the United States of America (USA), of which, 9.4 million were caused by food-borne pathogens [7].This problem led to the research of technologies and alternative antimicrobial compounds that minimize the triggering of microorganisms, increase food safety and prolong shelf life.In this work, the antimicrobial activity of a polyphenolic-rich extract from A. nodosum obtained through microwaveassisted extraction was analyzed.

Sample Preparation
Brown algae were hand-picked from the coasts of Galicia, by Algas Atlánticas Algamar SL company located in Pontevedra, Spain.Algae were carefully cleaned and washed with demineralized water, before freeze-drying.Then, algae were triturated and stored at −20 • C until use.

Microwave-Assisted Extraction
MAE was carried out using a multiwave-3000 microwave extraction system (Anton-Paar, Germany).The biomass to solvent ratio was 30 g L −1 and the extraction was performed at 10.5 bar for 5 min using aqueous ethanol 37% v/v) as solvent.These conditions were selected based on the literature and previous works [8].

Extract Preparation
A. nodosum extract obtained by MAE was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich Steinheim, Steinheim am Albuch, Germany) and brought up to 20 mg/mL concentration.Then, the solubilized extract was sterilized by passing through a 0.20 µm syringe filter.

Kirby-Bauer Plate Diffusion Test
Petri dishes containing Mueller-Hinton agar were divided into four quadrants and seeded with 50 µL of the microorganism culture and spread with sterile swabs.The test was made by putting 15 µL of DMSO (negative control) in the center of the plate and placing 15 µL of each test extract in three quadrants, and 15 µL of lactic acid 40% (v/v) (Sigma-Aldrich Steinheim, Germany) (positive control).Petri dishes were incubated at 37 • C for 24 h and the inhibition zone diameter was measured with a digital caliper rule [9,10].Determinations were conducted in triplicate.

Microdilution Assay
Minimal inhibitory concentration (MIC) determination was performed by the microdilution method [9], using multiplate reader equipment (Biotek Synergy HT).The test was performed using an extract concentration ranging from 1.2 to 8.0 mg/mL.For that, a 96-well round-bottom sterile plate was filled with a total volume of 250 µL, containing 10 6 Colony Forming Units (CFU), algae extract (100 µL), and fresh MHB media.Negative controls contained medium with extract samples and no bacteria and positive control wells were prepared with inoculated medium without extract samples.Microplates were incubated for 24 h at 37 • C at 630 nm.Determinations were conducted in triplicate.

Results and Discussion
The potential antimicrobial activity of A. nodosun extracts was tested against five common food-borne contaminants [11] E. coli, B. cereus, S. aureus, S. enteritidis, P. aeruginosa, and an opportunistic bacteria S. epidermidis.The antimicrobial capacity was firstly evaluated by the plate diffusion assay.These preliminary studies were carried out with an extract concentration of 20 mg/mL (Table 1).The results obtained showed that A. nodosun extract has an antimicrobial capacity, with greater inhibition against the Gram-positive strains, B. cereus and S. aureus, but was still active against one of the Gram-negative strains S. enteretidis.Previous studies have stated this differential interaction of algae polyphenols with Gram-positive and Gram-negative bacteria [12] and in general, Gram-positive bacteria are more sensitive to phlorotannins than Gram-negative bacteria [13].The evolution of six microorganisms' growth curves in the presence of A. nodosum extract is presented in Figure 1.As can be seen from Figure 1, it is possible to confirm that there is an effective inhibition due to a decrease in the bacterial growth rates of all the tested microorganism species, except for E. coli.[14].
By observing the growth curves, it is possible to deduce that bacterial inhibition is mostly achieved by lag time extension.Through the lag phase, the microorganisms adjusted to the new environment and cells start to adapt to the metabolic functions and synthesize the components necessary for growth [15,16].The bioactive molecules present in the extract are postponing this phase.

Conclusions
The results from the present work indicated that A. nodusum was effective against all tested bacteria except for E. coli, disclosing the potential of A. nodusum as an antimicrobial agent.Furthermore, the growth curves obtained allow for inferring that the antimicrobial action is attained due to the lag time extension.Thus, it is possible to expect that the incorporation of this extract into food products could be useful in the control of food-borne infections.For the Gram-positive bacteria, the MIC values were higher than 8.0, 4.0, and 8.0 mg/mL, respectively.It is worth mentioning that 0.4 mg/mL of alga extract produced a 20% inhibition effect in S. aureus.Concerning the Gram-negative strains, the MIC values obtained were higher than 8.0 mg/L for P. aeruginosa and 8.0 mg/mL for S. enteritidis.No inhibition effect was observed against E. coli.The MICs obtained from the Gram-negative bacteria were higher than the ones determined for Gram-positive strains.This can be attributed to the external membrane of Gram-negative microorganisms as an effective barrier to many active molecules, including antibiotics [13].A few inoculation loops were taken from the wells without measurable bacterial growth (4 mg/mL B. cereus; 8 mg/mL S. enteritidis) and spread on a Muller-Hinton agar medium.After incubation at 37 • C overnight, the existence of viable colonies was observed, therefore, it was concluded that the inhibitory effect achieved was due to the bacteriostatic effect, given that no cellular death was achieved.
It is also important to highlight that despite no inhibition activity being detected against S. epidermidis and P. aeruginosa in the plate diffusion method, different growth rates were observed in the microdilution tests (Figure 1).These results can be explained by the fact that the microdilution method allows for following the microorganism growth over time, whereas in the plate diffusion method, only the endpoint is observed.Furthermore, it was previously stated that the microdilution method is the most sensitive one [14].
By observing the growth curves, it is possible to deduce that bacterial inhibition is mostly achieved by lag time extension.Through the lag phase, the microorganisms adjusted to the new environment and cells start to adapt to the metabolic functions and synthesize the components necessary for growth [15,16].The bioactive molecules present in the extract are postponing this phase.

Conclusions
The results from the present work indicated that A. nodusum was effective against all tested bacteria except for E. coli, disclosing the potential of A. nodusum as an antimicrobial agent.Furthermore, the growth curves obtained allow for inferring that the antimicrobial action is attained due to the lag time extension.Thus, it is possible to expect that the incorporation of this extract into food products could be useful in the control of food-borne infections.

Table 1 .
Inhibition halos obtained by plate diffusion method.