Purified avocado seed acetogenins: Antimicrobial spectrum and complete inhibition of Listeria monocytogenes in a refrigerated food matrix

Figures & data

Table 1. General characteristics, oxygen requirement and growth conditions of studied microorganisms at 15 µg disc⁻¹ of acetogenins present in Avosafe®.

Tabla 1. Características generales, requerimiento de oxígeno y condiciones de crecimiento de los microorganismos estudiados a 15 µg de disc⁻¹ de acetogeninas presentes en Avosafe®.

Microorganism	ATCC	General characteristics	Oxygen requirement	Culture media	Avosafe® antimicrobial effect
Clostridium sporogenes^1	7955	Bacteria, Gram +, SFB^2	anaerobic	TPYG^3 at 37°C	Yes
Clostridium perfringens	14810	Bacteria, Gram +, SFB	anaerobic	TPYG at 37°C	Yes
Alicyclobacillus acidocaldarius	27009	Bacteria, Gram +, SFB	aerobic	MRS at 55°C	Yes
Bacillus subtilis	11774	Bacteria, Gram +, SFB	aerobic	NB^4 at 30°C	Yes
Listeria monocytogenes	35152	Bacteria, Gram +	aerobic	BHI^5 at 37°C	Yes
Staphylococcus aureus	6538	Bacteria, Gram +	aerobic	TSA^6 at 37°C	Yes
Lactobacillus plantarum	8014	Bacteria, Gram +	anaerobic	MRS^7 at 37°C	No
Leuconostoc mesenteroides	10830a	Bacteria, Gram +	anaerobic	MRS at 37°C	No
Escherichia coli	35218	Bacteria, Gram –	aerobic	NB at 37°C	No
Pseudomonas aeuroginosa	29260	Bacteria, Gram –	aerobic	TSA at 37°C	No
Saccharomyces cerevisiae	24858	Yeast	anaerobic facultative	YM^8 at 37°C	No
Candida parapsilosis	22019	Yeast	aerobic	YM,PDA^9at 37°C	No

¹C. sporogenes was tested with both vegetative cells and endospore germination. ²SFB: Spore-forming bacteria. ³Tryptone-peptone-yeast extract-glucose [17]. ⁴Nutrient broth. ⁵Brain heart infusion. ⁶Trypticase soy agar. ⁷De Man, Rogosa and Sharpe. ⁸Yeast extract-malt extract. ⁹Potato dextrose agar.

¹C. sporogenes se evaluó crecimiento de células vegetativas y germinación endosporas. ² SFB: bacterias formadoras de esporas. ³ Extracto de levadura peptona dextrosa [17]. ⁴ Caldo de nutrientes. ⁵ Infusión cerebro-corazón. ⁶ Agar de soya tríptico. ⁷ De Man, Rogosa y Sharpe. ⁸ Extracto de levadura – extracto de malta. ⁹ Agar papa dextrosa.

Figure 1. Relative concentration of individual acetogenins present in Avosafe®, a food-grade avocado seed extract enriched in acetogenins. Bars represent the mean ± standard deviation of technical replicates (n = 3). *Numbers in parenthesis for each acetogenin refer to their chromatographic elution order, additional structural information can be found in Table S1. ** Different letters indicate significant differences between each individual acetogenin concentration (Tukey HSD test, α = 0.05). ^Θ Quantified in persenone C (5) equivalents (Salinas-Salazar et al., 2016). ⁺ UPA: Unknown putative acetogenin.

Figura 1. Concentración relativa de acetogeninas individuales presentes en Avosafe®, un extracto grado alimenticio de semilla de aguacate, enriquecido en acetogeninas. Las barras representan la media ± desviación estándar de las repeticiones técnicas (n = 3). * Los números entre paréntesis para cada acetogenina se refieren a su orden de elución cromatográfica, la información adicional sobre su estructura se puede encontrar en la Tabla complementaria S1. **Letras diferentes indican diferencias significativas entre la concentración individual de cada acetogenina (Tukey HSD, α = 0.05). ^Θ Cuantificado en equivalentes de persenona C (5) (Salinas-Salazar et al., 2016). ⁺ UPA: acetogenina putativa desconocida.

Figure 2. Diameter of inhibition zones of Avosafe®, Mirenat® and Nisaplin® against the growth of different Gram-positive microorganisms. All antibacterial agents were tested at 15 µg disc⁻¹ of their active ingredients (total acetogenins, ethyl lauroyl arginate (LAE) and nisin for Avosafe®, Mirenat® and Nisaplin®, respectively). Values represent mean ± standard deviation of technical replicates (n = 2–3). *Different letters indicate significant differences between antibacterial agents for the same microorganism (Tukey HSD test, α = 0.05, n = 6–9). **Cs veg: C. sporogenes vegetative cells. Cs end: C. sporogenes endospore germination. Cp: C. perfringens. Aa: A. acidocaldarius. Bs: B. subtilis. Lm: L. monocytogenes. Sa: S. aureus. ⁺ ND: Not detected.

Figura 2. Diámetro de zonas de inhibición de Avosafe®, Mirenat® y Nisaplin® contra el crecimiento de diferentes microorganismos Gram positivos. Todos los agentes antibacterianos se probaron a 15 µg de disco⁻¹ de sus ingredientes activos (acetogeninas totales, etil lauroil arginato (LAE) y nisina para Avosafe®, Mirenat® y Nisaplin® respectivamente). Los valores representan la media ± desviación estándar de las repeticiones técnicas (n = 2–3). *Letras diferentes indican diferencias significativas entre los agentes antibacterianos para el mismo microorganismo (Tukey HSD, α = 0.05, n = 6–9). **Cs veg: Células vegetativas de C. sporogenes. Cs end: germinación de endosporas de C. sporogenes. Cp: C. perfringens. Aa: A. acidocaldarius. Bs: B. subtilis. Lm: L. monocytogenes. Sa: S. aureus. ⁺ ND: No detectado.

Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of acetogenins present in Avosafe® against vegetative cell growth (A. acidocaldarius, L. monocytogenes) and endospore germination (C. sporogenes).¹

Tabla 2. Concentración inhibitoria mínima (MIC) y concentración bactericida mínima (MBC) de acetogeninas presentes en Avosafe® contra el crecimiento de células vegetativas (A. acidocaldarius, L. monocytogenes) y la germinación de endosporas (C. sporogenes).¹

	MIC	MBC	Ratio^2
Microorganism	(ppm)	(ppm)	MBC/MIC	Reference
Alicyclobacillus acidocaldarius^3	15.63	>125	>8	In this study
Listeria monocytogenes(4°C)	7.8 to 15.63	15.6 to 31.25	2	[17]
Listeria monocytogenes(37°C)	15.63	31.25	2	[17]
Clostridium sporogenes^3	3.91	>125	>32	[16]

¹Tests were performed in triplicate. ²MBC/MIC ratio ≤ 4 indicates bactericidal activity and ˃4 sporostatic or bacteriostatic activity [37]. ³Incubation conditions are presented in Table 1.

¹Las pruebas se realizaron por triplicado. ²La relación MBC/MIC ≤ 4 indica actividad bactericida y ˃ 4 actividad esporostática o bacteriostática [37]. ³Las condiciones de incubación se presentan en la Tabla 1.

Figure 3. Listeria monocytogenes counts (log₁₀ CFU g⁻¹) in food system containing meat with 7,500 ppm Avosafe®, stored at 20 ± 2°C for 96 h (4 days). Values represent mean ± standard deviation of technical replicates (n = 3).

Figura 3. Conteo de Listeria monocytogenes (log₁₀ CFU g⁻¹) en un sistema alimenticio que contiene carne y 7,500 ppm de Avosafe®, almacenado a 20 ± 2°C durante 96 h (4 días). Los valores representan la media ± desviación estándar de las repeticiones técnicas (n = 3).

Figure 4. Listeria monocytogenes counts (log₁₀ CFU g⁻¹) in meat food system with 10,000 ppm Avosafe®, stored at 4 ± 1°C for 72 days. Values represent mean ± standard deviation of technical replicates (n = 3).

Figura 4. Conteo de Listeria monocytogenes (log₁₀ CFU g⁻¹) en un sistema alimenticio que contiene carne y 10,000 ppm de Avosafe®, almacenado a 4 ± 1°C durante 72 días. Los valores representan la media ± desviación estándar de las repeticiones técnicas (n = 3).

Salinas-Salazar, C. L., Hernández-Brenes, C., Rodríguez-Sánchez, D., Castillo, E., Navarro-Silva, J., & Pacheco, A. (2016). Inhibitory activity of Avocado seed fatty acid derivatives (Acetogenins) against Listeria monocytogenes. Journal of Food Science, 82(1), 134–144.

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