Antimicrobial Activity of Spanish Propolis against Listeria monocytogenes and Other Listeria Strains

The outbreaks of Listeria associated with food consumption are increasing worldwide concurrently with public concern about the need for natural growth inhibitors. In this context, propolis seems to be a promising bioactive product collected by honeybees, due to its antimicrobial activity against different food pathogens. This study aims to evaluate the efficacy of hydroalcoholic propolis extracts for controlling Listeria under several pH conditions. The physicochemical properties (wax, resins, ashes, impurities), the bioactive compounds (phenolic and flavonoid content), and the antimicrobial activity of 31 propolis samples collected from the half North of Spain were determined. Results showed similar trends in the physicochemical composition and bioactive properties, regardless of the harvesting area. Non-limiting pH conditions (7.04, 6.01, 5.01) in 11 Listeria strains (5 from collection and 6 wild strains from meat products) exhibited MICs (Minimum inhibition concentration) and MBCs (Minimum bactericidal concentration) ranging from 39.09 to 625 μg/mL. The antibacterial activity increased under acidic pH conditions, showing a synergistic effect at pH = 5.01 (p < 0.05). These findings suggest the potential of Spanish propolis as a natural antibacterial inhibitor to control Listeria growth in food products.


Introduction
Despite advances in the food industry in technology, safety, and hygiene throughout the food chain, Listeria monocytogenes remains one of the most relevant food pathogens. The latest report issued by EFSA in December 2022 [1], which includes data on both food outbreaks and the incidence of infections caused by this microorganism during the previous year, refers to the foods involved and the countries of the European Union in which these cases have occurred. According to this report, EFSA has declared more than 2000 cases of listeriosis associated with the human population, involving 23 outbreaks and almost 10% of the samples registered for this gram-positive pathogen. The number of confirmed cases over the last five years remains more or less stable, although there has been an increase in the number of outbreaks reported in the twenty-seven member countries.
A significant increase is in the number of samples that member countries have reported in the different ready-to-eat (RTE) products responsible for most of the outbreaks of L. monocytogenes associated with the consumption of these products. In the last year, the number of sample units in meat, dairy, and fish products increased by almost 50%, highlighting the effort and importance of L. monocytogenes control.
Together with the application of the current legislation in the inspection and control of the incidence of L. monocytogenes in RTE, the agri-food industry has implemented research Mueller Hinton Agar (MHA), both from Bioser (Barcelona, Spain). Antimicrobial Susceptibility Test Discs and Resazurin (7-hydroxy-3H-phenoxazine-3-one-10-oxide) from Oxoid (Basingstoke, UK) were also employed. Petri Dishes and V-bottom microtiter plates were from Deltalab (Barcelona, Spain). The adhesive film for Culture Plates Porous was from VWR (Radnor, PA, USA).

Groups of Propolis and Sample Collection
A total of 31 samples of propolis were collected from several geographical areas in Spain throughout 2019 and 2020. Beekeepers from different regions of Spain voluntarily participated in the study, collecting propolis samples according to their usual management practices. Macroscopically, differences were observed among raw samples, including texture, color, and compactness degree. Figure 1 shows the geographical origins of the collected propolis, grouping some of them in the N-W region of the Iberian Peninsula, most of them in the province of León, Zamora, Palencia, Asturias, and Madrid (18) Zone I, Galicia (5) Zone IV, Catalonia (2) Zone II, and Navarra (2) Zone III. The areas of origin of the propolis samples were classified according to the administrative limits corresponding to the Autonomous Communities of the Kingdom of Spain (Table S1). Propolis was stored in freezing conditions (−20 °C) from collection to analysis.

Preparation of Propolis Extracts
Propolis extraction of pulverized raw propolis (10 g) comprised the addition of 600 mL of 70% hydroalcoholic solution. The extraction was carried out in two stages of 24 h, each of which involved shaking, at a controlled temperature of 20 °C, the 10 g of propolis sample, first in 300 mL of 70% ethanol, and after filtering a new extraction under the same conditions of shaking, time, and temperature [4,17]. Ethanol extracts of propolis (EEP) were kept at a refrigerated temperature (4 °C) from that moment and throughout the procedure to keep their properties intact [17].

Preparation of Propolis Extracts
Propolis extraction of pulverized raw propolis (10 g) comprised the addition of 600 mL of 70% hydroalcoholic solution. The extraction was carried out in two stages of 24 h, each of which involved shaking, at a controlled temperature of 20 • C, the 10 g of propolis sample, first in 300 mL of 70% ethanol, and after filtering a new extraction under the same conditions of shaking, time, and temperature [4,17]. Ethanol extracts of propolis (EEP) were kept at a refrigerated temperature (4 • C) from that moment and throughout the procedure to keep their properties intact [17].

Characterization of Propolis Samples
EEPs were analyzed to determine the main components responsible for their quality, bioactivity, and chemical properties. The characterization of the selected propolis samples considered their proximal composition regardless of their botanical origin, including resin, water, wax, impurity, and ash content [4,17].

Determination of Total Polyphenols Content
Total polyphenol content (PTC) was analyzed according to Folin-Ciocalteu's method (as modified by Bankova et al. [4]). The calibration graph was prepared with standard Microorganisms 2023, 11, 1429 4 of 12 methanolic solutions of a mixture of pinocembrin-galangin at a ratio of 2:1 (w/w) in a 25-300 µg/mL range. Absorbance measurements were performed in triplicate at 760 nm using a UV-vis spectrophotometer, and the results obtained were expressed as grams of pinocembrin-galangin equivalents per 100 g of raw propolis (%PGE) [4,18,19].

Determination of Total Flavonoids, Flavone, and Flavolnol Content
Total Flavonoid Content (TFC) was estimated according to Woisky and Salatino, using quercetin as a standard and expressing the results obtained as grams of quercetin equivalents per 100 g of raw propolis (%QE). Absorbance measurements at 415 nm after 40 min of incubation in the dark at room temperature against a blank were performed using a spectrophotometer [4,18]. Flavone and flavonol content was determined using the spectrophotometric method proposed by Bankova et al. [17] based on the reaction to form aluminum chloride complexes. The calibration line preparation involved a methanolic solution of galangin. Absorbance measurements were performed in triplicate at 425 nm using a UV-vis spectrophotometer. The results obtained were expressed as grams of galangin equivalents per 100 g of raw propolis (%GE) [4,18].  (Table S2).

Antimicrobial Disc Susceptibility Test
The disc diffusion method was used to evaluate the antibacterial activity of EEP. Petri dishes were prepared with MHA and inoculated with Listeria culture in MHB before overnight incubation at 37 • C (2 × 10 8 cfu/mL). Afterwards, eight peripheral sterile Whatman filter paper discs with a symmetrical arrangement template and a central one were placed in each plate. Twenty µL of each of the thirty-one EEPs were placed in the peripheral discs, using the same volume of 70% ethanol as a negative control. The growth of the inoculated strain was evaluated after incubation for 48 h at 37 • C, and the diameter of the inhibition generated around the discs was measured while using the diameter of the inhibition of the central disc of each plate as a control. The diameter zones, including the diameter of the disc, were recorded [4,20].

Minimum Inhibitory Concentration (MICs) and Minimum Bactericidal Concentration (MBCs) Determination Assay
From the EEP and based on the results of the composition analysis and determination of active compounds, further dilutions ensured a range concentration of 2500 µg of raw propolis/mL in the most concentrated dilution. MIC determinations involved three pH values, non-limiting for Listeria growth. The pH of the MHB was adjusted post-sterilization. The selected pH values were 7.04, 6.01, and 5.01 to determine if the pH conditions modified the antimicrobial activity of the EEPs. V-bottom microtiter plates contained 50 µL of MHB in each cell. The MIC values determination, namely the minimum concentration that allows for visible microbial growth, was achieved through known serial dilutions, using the last row of each plate as a growth control without EEP. The propolis' inoculated concentrations ranged from 2500 µg/mL to 39.0625 µg/mL in the most diluted one. Finally, 50 µL of the bacterial inoculums in MHB were included in each column and incubated at 37 • C for 48 h (all covered with breathable rayon film). The dilution tested was the one that achieved 1.2 × 10 5 cfu/mL. Seeding was performed in a Petri dish containing MHA, with a multi-inoculator, which was incubated at 37 • C for 24 h, thus determining the MBC values [4]. In addition, complementary and parallel staining with Resazurin at 0.01% showed a redox reaction, indicating the presence of viable cells in the dilution by a color change (it is naturally blue and turns purple) [4,21]. Results were obtained after 4 h of incubation at 37 • C using the 3-pH values selected in the test.

Data Analysis
Continuous variables were expressed as mean values ± standard deviation (SD). The Kolmogorov-Smirnov test was applied to determine data normality. Differences between zone regions were analyzed using One-way ANOVA and Kruskal-Wallis tests for normal and non-normal distribution variables, respectively. The influence of pH on the MICs values was assessed with the Kruskal-Wallis test. The software package SPSS for Windows version v.26, (IBM SPSS, Inc., Chicago, IL, USA) was used for data analysis. A p-value of <0.05 was set as representing statistical significance for all analyses.

Physicochemical Characterization
The analysis of propolis samples allowed for their classification according to the established standards. The content of soluble matter in 70% ethanol (resin fraction) was 3-0.9%. Impurities and water content showed values between 0.01 and 0.11% and 3 and 6% for impurities and moisture content, respectively. Higher wax content was found in zone III (39.04%), while the lowest wax content was found in zone IV (7.74%). Non-statistically significant differences were found between areas.

Bioactive Compounds
The analysis of the total resin content did not reveal statistically significant differences between different areas. Considering the two bioactive fractions of the soft extract of the study samples, no significant differences in TPC were observed between samples when the whole regions were considered. Neither TFC nor FFCT were found. Table 1 shows the total phenolic compounds, and the concentrations ranged between 26.65 ± 0.72% PGE in sample 16 and 78.54 ± 0.80% PGE in sample 4. The same observation was made for flavones and flavonol content, where the highest concentration was found in sample 4 (5.71 ± 1.07% GE), immediately followed by sample 3 (5.53 ± 0.28% GE) and sample 10 (5.45 ± 0.61% GE). In addition, the total flavonoid content has a concentration between 1.64 ± 0.04% PGE and 4.94 ± 0.05% PGE.

Antibacterial Activity
The tests carried out to perform a first screening of the antimicrobial activity of each EEP provided homogeneous information in terms of the results shown in Table 2. Both wild Listeria strains and CECT showed inhibition halos diameters ranging from 38 mm for L10 (L. monocytogenes isolated from a cured chorizo), which exhibited greater sensitivity to the action of the EEPs, to L. innocua CECT 910, whose lowest inhibition diameter was 5 mm. All the propolis samples showed antibacterial activity against all the Listeria strains. MIC mean values were between 39.025 and 625.00 µg/mL (Table 3).  (Table S3).
Moreover, differences in MIC mean values between pH 7.04 and 6.01 were not statistically significant for all Listeria strains, except for L10, which did not exhibit statistically significant differences between pH 6.01 and 5.01.
Although different strains of Listeria (L. monocytogenes, L. grayii, and L. innocua) were tested, the growth behavior was similar. It was only at pH 5.01 that the EEP samples from two geographical areas (Zone III and Zone IV) displayed statistically significant differences among Listeria species.
Similarly, MBC values displayed the lowest result from 115.69 µg/mL at pH 5.01, which was exhibited by Lm 4032, and the higher MBC value was 253.28 µg/mL at pH 7.04, which was shared by Lm 4032 and Lm 52.

Discussion
This study investigated the anti-listerial activity of Spanish propolis against different strains of Listeria. As far as we know, this is the first work that explores the effect of pH on different Listeria strains isolated from meat products, using simulated acidic pH conditions present in several RTE products. The propolis samples selected in this study come from a wide geographic area in the northern half of Spain, within the European or poplar-type, although showing climatic diversity depending on the geographical origin. The samples' appearance was heterogeneous in terms of the compactness degree and color, owing to the different ways of collection. Most beekeepers placed a grid or mesh for collecting, while only one sample was obtained by scraping (Propolis 18). Additionally, harvesting occurred for two consecutive years (2019-2020).
Regarding the physicochemical composition, the heterogeneity of the chemical composition of propolis rendered standardization challenging [22], making a primordial determination of sample quality, including parameters such as wax, resins, ashes, and humidity. All 31 propolis samples collected from different harvesting areas showed values that comply with the standards established for European propolis, as seen in some published works on Spanish and Portuguese propolis [23,24]. The limits of acceptable values following the protocols and specifications [4] listed in the IHC (International Honey Commission) are as follows: a minimum of 45% resins; a maximum of 6% impurities, a maximum of 8% water content, and a maximum ash content of 5%.
The total of the 31 propolis studied presented in their resinous fraction contents in polyphenols, flavonoids, and flavones that correspond to Popova et al.'s [18] previous studies for poplar-type, with a minimum of 21% in total polyphenols content (CPT) and a minimum of 4% in flavones and flavonol content (CFFT). Despite the geographical origin [25], climate, botany around the hive, year of collection [26,27], and management, among other factors, the wide range of the results obtained in the characterization analysis of the samples did not show statistically significant differences between areas. Concerning the bioactive contents, previous works have characterized European propolis (poplar-type) as rich in flavones, flavonones, and flavones [28], while the Russian and Brazilian types present other bioactive substances (diterpenoids, isoflavones, and other flavonols) [29,30]. Nevertheless, all the samples presented homogeneous values that correspond to those already shown in previous works [3,31]. TPC content (some higher than 70% PGE) and TFC (most of them higher than 4%) are higher than that reported for Portuguese propolis [31,32]. The flavonoid content did not change between regions, although there were different vegetation sources around the hive. Regarding the flavanones and flavonols subclass, both agree with the previous characterization of the poplar-type of propolis [18]. Numerous studies attribute the propolis' bioactive properties (antibacterial, anti-inflammatory, antioxidant [33], biomarker [34], and immunomodulator) to the compounds present in the resinous fraction. Therefore, polyphenols and flavonoids have been described and used in the pharmaceutical industry and the medical field for their bactericidal and bacteriostatic effects [35].
Despite the broad knowledge in this respect, few previous works have reported the control effect exerted by propolis on Listeria [36], particularly on gram-positive microorganisms and some molds [31,[37][38][39]. In the present work, involving 31 EEP against 11 strains of Listeria with different origins, the inhibition screening carried out with the disc diffusion method [40] showed that both wild and collected Listeria strains were sensitive to all the EEP studied.
The antibacterial effect of propolis on strains of Listeria demonstrated that all EEP inhibit this foodborne pathogen. Since extracted propolis samples contained ethanol, 70% ethanol was used as a control to evaluate its potential inhibition effect. Growth inhibition against the tested Listeria to the ethanol was not observed, thus suggesting that the antimicrobial effect was due only to propolis.
The ethanol extracts propolis showed an inhibition zone diameter higher than 12 mm for all the Listeria strains evaluated [40]. The results of minimum inhibitory concentration (MIC) found by the 31 propolis ethanol extracts showed high inhibitory effects against L. monocytogenes, L. grayii, and L. innocua, both the collection strains and wild ones isolated from meat products. As expected, the propolis samples showed bactericidal and bacteriostatic activity against the different strains of Listeria spp. and L. monocytogenes, independently of the EEP tested. The MIC values found when confronted with the 31 propolis against Listeria showed lower values-higher inhibition power-than previously published work with propolis of very different geographical origin than those presented in this study, demonstrating that the bactericidal effect is superior (MIC 39-625 µg/mL).
Only a few works have reported the propolis bioeffect against Listeria. Almost all of them compared results between gram-positive and gram-negative bacteria [25,[41][42][43][44] or studied Listeria in Ready-to-Eat (RTE) products, in addition to different conditions and components [11,16,31,[45][46][47][48]. Previous studies against gram-positive microorganisms [49] with European propolis revealed MIC values ranging from 80 to 5000 µg/mL, while Polish propolis (400-8000 µg/mL) [41,42] revealed higher MIC values, referred to as L. monocytogenes (1560-6250 µg/mL). In our study, all the propolis assayed exerted a high inhibitory effect against the whole Listeria strains tested. The strain origin (wild/collection) did not show significant differences in sensibility to EEP. The mean MIC value for pH 7.04 was 265 µg/mL. Although some of the independent MIC values obtained when we tested pH 6.01 were very low (199 µg/mL), the acidic assay conditions, pH 5.01, revealed a mean MIC value of 134 µg/mL, thus suggesting a potential synergistic effect between pH activity and the action EEP for the total Listeria strains tested, including both wild and collection types.
The antimicrobial effects of propolis may be influenced by many factors, such as the propolis origin, extract preparation, and chemical composition, also exhibiting considerable geographic differences. Similarly, the antimicrobial effect (expressed as MIC) of tested EEP was lower than 625 µg/mL, making this concentration compatible with their use in the food industry, as they do not modify the organoleptic characteristics of the food matrix, as reported by other natural substances in previous works [13,50]. For instance, flavor is one of the key components of propolis characterization and it should be considered when it is applied to RTE products. Nevertheless, the imitation of the pH conditions present in many RTE foods and the study of the propolis effect against 11 Listeria strains has not been previously described. The simulation of the pH values in some of the conditions for meat products and the bacterial damage observed in the most acidic pH could be extremely valuable for the food industry as an alternative way to control this food pathogen.
The low MIC found in our propolis samples evaluated against L. monocytogenes and other Listeria are compatible with its potential use in the industry for food safety control.
Despite these inherent advantages, this study has some limitations. For instance, the sample size could be homogenized within zones, considering the geobotanical characteristics. Although we provide specific results on total flavonoids, several subclasses could be further analyzed.

Conclusions
The control of Listeria in RTE products through natural bioactive compounds is of utmost importance for ensuring food safety. This study demonstrates the utility of the propolis extract from the northern half of Spain as a potent inhibitor of Listeria growth. Particularly, the studies of different pH conditions revealed that the synergistic effect of acidic values increased the antibacterial properties of propolis samples. These findings could contribute to defining future strategies for the bio-control of Listeria growth in RTE and meat products using specific pH conditions. Therefore, this investigation supports the promising integration of natural and sustainable products as efficient controllers of pathogens in the food industry. Further research is necessary to enforce the inclusion of propolis as a Clean Label product to preserve the shelf life while protecting consumers' health.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms11061429/s1, Table S1. Geographical location of the propolis samples and identification code, Table S2. Physicochemical characterization of propolis samples, and Table S3  Data Availability Statement: Data will be available upon reasonable request to the corresponding author.