Antimicrobial activity of açaí against Listeria innocua

Accepted Manuscript Antimicrobial activity of açaí against Listeria innocua Clara Miracle Belda-Galbis, Antonio Jiménez, María Consuelo Pina-Pérez, Antonio Martínez, Dolores Rodrigo PII:

S0956-7135(15)00033-X

DOI:

10.1016/j.foodcont.2015.01.018

Reference:

JFCO 4251

To appear in:

Food Control

Received Date: 18 September 2014 Revised Date:

19 December 2014

Accepted Date: 3 January 2015

Please cite this article as: Belda-Galbis C.M., Jiménez A., Pina-Pérez M.C., Martínez A. & Rodrigo D., Antimicrobial activity of açaí against Listeria innocua, Food Control (2015), doi: 10.1016/ j.foodcont.2015.01.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Antimicrobial activity of açaí against Listeria innocua

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Clara Miracle Belda-Galbis, Antonio Jiménez, María Consuelo Pina-Pérez, Antonio Martínez,

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Dolores Rodrigo*

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Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Carrer del Catedràtic

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Agustín Escardino Benlloch 7, 46980, Paterna, València, Spain.

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Corresponding author. Tel.: +34 963 900 022 (ext. 2218). Fax: +34 963 636 301. E-mail

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address: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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A study was carried out to evaluate antimicrobial activity of açaí against Listeria innocua, as a

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non-pathogenic surrogate for Listeria monocytogenes, at different temperatures (37, 22, and 10

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°C), from a kinetic point of view. With this aim, first the Minimum Inhibitory Concentration

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(MIC) under optimal growth conditions was established (37 °C), and then the effect of 3 non-

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inhibitory doses (3, 5, and 7 g/L) at 37, 22, and 10 °C was evaluated on the basis of the kinetic

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parameters lag time (λ) and maximum specific growth rate (µ max). The Total Phenolic Content

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(TPC) of the samples tested as a function of açaí concentration was also determined. Results

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obtained showed that the MIC was 10 g/L, containing 2154.91 ± 126.10 mg of Gallic Acid

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Equivalents (GAE)/L. At non-inhibitory doses, regardless of temperature, the higher the açaí

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concentration, the higher the value of λ, and the effect of a variation in açaí concentration on λ

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was greater at high temperatures. Therefore, the addition of non-inhibitory doses of açaí could

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provide a means of controlling the growth of L. monocytogenes if a cold chain failure occurs, or

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if any other reason compromises the microbiological safety of minimally processed foods.

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Consequently, the present study is important both for the science community and for the food

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industry because it provides the first mathematical characterization of açaí antimicrobial activity

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and reveals its potential use to control the concentration and growth of pathogenic bacteria in

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foods free of non-synthetic additives.

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Keywords: Food preservatives; microbial safety; açaí; phenolic compounds; Listeria innocua;

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mathematical modelling

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ACCEPTED MANUSCRIPT 1.

Introduction

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Açaí palm (Euterpe oleracea Martius), also known as cabbage palm or palisade pine, is a

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monoecious plant native of tropical regions of northern South America, where açaí has been

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consumed since pre-Columbian times (Alonso 2012). From then until now, the distribution and

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sale of açaí and açaí-based products has increased in North America, Europe, China, and Japan,

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Brazil being the main producer of açaí pulp-based drinks (Alonso 2012). The increasing interest

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in this kind of product is partly due to studies which show that açaí consumption could have

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beneficial effects on consumer health (del Pozo-Insfran et al. 2006; Schauss et al. 2006;

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Mertens-Talcott et al. 2008; Sun et al. 2010; Horiguchi et al. 2011; Udani et al. 2011; de Moura

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et al. 2012; Feio et al. 2012; Bonomo et al. 2014).

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Nowadays, it is well known that açaí contains as much fiber as whole wheat flour or rice bran,

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and a fair amount of heart-healthy fatty acids (Sanabria and Sangronis 2007), in addition to

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flavonoids, lignans, and other polyphenols with an extremely high antioxidant potential.

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According to Schauss et al. (2006), the antioxidant capacity of freeze-dried açaí pulp (1027

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µmol of Trolox Equivalents (TE)/g) is higher than the antioxidant capacity of any other fruit or

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vegetable that has been analyzed, including blueberries (62.20 µmol of TE/g), strawberries

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(35.77 µmol of TE/g), and raspberries (49.25 µmol of TE/g) (Wu et al. 2004). For this reason,

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açaí is considered a “superfruit.”

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Although açaí health benefits and its nutritional composition have been studied in depth, its

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antimicrobial activity has not yet been exploited because, to date, the antimicrobial activity of

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açaí has scarcely been studied (Lian et al. 2012; Melhorança and Pereira 2012; Bonomo et al.

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2014). According to Lian et al. (2012), açaí antimicrobial activity depends on its phenolic

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content. Some exotic tropical fruit extracts have been reported to exert a wide spectrum of

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antimicrobial activity due to phenolic compounds with bacteriostatic and bactericidal properties

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(Kabuki et al. 2000; Esquenazi et al. 2002). These compounds are able to modify membrane

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permeability and to complex with macro- and micronutrients. Consequently, the affect the

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ACCEPTED MANUSCRIPT retention of macromolecules within the cell, the electron transport and the nutrient uptake

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(Cowan 1999; Daglia 2012).

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Until today no standardized methods for determining the antimicrobial activity of any

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compound or mix are described in the literature, but so far no standardized test has been

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developed to evaluate the antibacterial activity of possible preservatives against food-related

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microorganisms, although the need for such a test has been indicated (Burt 2004). Normally,

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researchers adapt experimental methods to provide a better representation of possible future

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applications of antimicrobials in their particular field. In this connection, a count of viable cells

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after incubation of a microorganism in the presence of a supposedly antimicrobial compound or

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mixture makes it possible to characterize the bacteriostatic or bactericidal effect of the

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compound or mixture, and also to quantify its effect in terms of kinetic parameters (maximum

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specific growth rate, µ max, and lag phase duration, λ) that define the behavior of the

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microorganism in the presence of the antimicrobial substance or mixture, and that also permit an

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exposure assessment in a risk assessment system.

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The main goal of this work was to assess the antimicrobial capability of açaí against Listeria

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innocua at different temperatures by means of mathematical modeling of microbial behavior in

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reference media, given that L. innocua is a non-pathogenic surrogate for Listeria

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monocytogenes (Char et al. 2010; Belda-Galbis et al. 2013; Jadhav et al. 2013; Belda-Galbis et

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al. 2014), a psychrotrophic facultative anaerobic pathogen, frequently found in minimally

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processed ready-to-eat foods, that is able to grow in very hostile environments (CCFH 2002;

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FSAI 2005), and that is able to form biofilms on all the materials commonly used in the industry

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(Møretrø and Langsrud 2004; Zhu et al. 2005).

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Material and methods 2.1.

Bacterial culture

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ACCEPTED MANUSCRIPT Vials containing around 9.51 × 109 (± 1.52 × 109) cfu/mL of L. innocua (CECT 910) were

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obtained from a lyophilized pure culture provided by the Spanish Type Culture Collection,

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using the method described by Saucedo-Reyes et al. (2009).

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The average cell concentration of the stock was determined by viable plate count from 4

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samples after incubation at 37 °C for 48 h. Aliquots of samples were serially diluted in 1 g/L of

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buffered peptone water (Scharlau Chemie, SA, Barcelona, Spain), and 100 µL of each dilution

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was spread on Tryptic Soy Agar (TSA; Scharlau Chemie, SA, Barcelona, Spain). 2.2.

Experimental procedure

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A commercial açaí berry extract (Nature’s Way® Products, Inc., Utah, USA) was selected. Its

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antimicrobial activity was assessed at different temperatures (37, 22, and 10 °C) by viable plate

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count. In brief, the bacteria were inoculated in Tryptic Soy Broth (TSB; Scharlau Chemie, SA,

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Barcelona, Spain) at a concentration of ca. 1 × 105 cfu/mL, in the absence and presence of açaí

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at different concentrations. Samples of the culture were taken every 60 min, up to stationary

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phase achievement, diluted in 1 g/L buffered peptone water, and plate counted in TSA after 48 h

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of incubation at 37 °C.

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As a first step, a range of concentrations [2–10 g/L] of açaí was tested at optimal L. innocua

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growth temperature (37 °C) (Rowan and Anderson 1998; FSAI 2005) to determine the

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Minimum Inhibitory Concentration (MIC) and the range of non-inhibitory doses that modify

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bacterial kinetic behavior at this temperature. As a second step, the effect of 3 non-inhibitory

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açaí concentrations (3, 5, and 7 g/L) was analyzed in depth at 37, 22, and 10 °C, following the

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method described previously.

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For all the experiments, 3 separate repetitions were performed for each of the conditions tested,

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with a minimum number of 4 replicates per repetition.

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2.3.

Total phenolic content determination

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ACCEPTED MANUSCRIPT The Total Phenolic Content (TPC) of samples with açaí was determined spectrophotometrically

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according to the Folin–Ciocalteu colorimetric method (Singleton and Rossi 1965). Gallic acid

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calibration standards with concentrations of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 1 g/L

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were prepared. Three mL of sodium carbonate solution (20 g/L) (Sigma-Aldrich® Co., LLC,

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Missouri, USA) and 100 µL of Folin-Ciocalteu reagent (1:1 (v/v)) (Sigma-Aldrich® Co., LLC,

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Missouri, USA) were added to an aliquot of 100 µL from each gallic acid standard or sample

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tube. The mixture was vortexed and allowed to stand at room temperature, in the dark, for 1 h.

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Absorbance was then measured at 750 nm, using a Lan Optics Model PG1800

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spectrophotometer (Labolan, Navarra, Spain). The results were expressed as mg of Gallic Acid

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Equivalents (GAE) per L. 2.4.

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Modeling of Listeria innocua growth in the presence of non-inhibitory doses of açaí

Growth curves obtained at non-inhibitory açaí concentrations (2, 3, 5, 7, and 8 g/L) were fitted

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to the modified Gompertz model (Gibson et al. 1988) (Eq. (1)), in accordance with previous

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studies by other authors (Koutsoumanis et al. 1998; Valbuena et al. 2008; Gupta et al. 2012;

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Pina-Pérez et al. 2013), modeling the antimicrobial activity of compounds derived from

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vegetable origins.

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logଵ଴ሺܰ௧ ሻ = ‫ ܣ‬+ ‫ × ܥ‬eିୣ

(1)

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where Nt represents the number of microorganisms at time t (cfu/mL); A the lower asymptote

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value (log10 cfu/mL); C the difference between the curve asymptotes (log10 cfu/mL); B the

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relative growth rate when t = M ((log10 cfu/mL)/h); and M the elapsed time until the maximum

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growth rate is reached (h).

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Fits were performed by nonlinear regression, using the Marquardt algorithm to determine the

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value of the model parameters by minimizing the residual sum of squares (Zwietering et al.

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1990). A, B, C, and M were then used to calculate the lag phase duration (λ; h) and the

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maximum growth rate (µmax; (log10 cfu/mL)/h) reached by L. innocua in each of the scenarios

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studied (Eqs. (2) and (3)) (Gibson et al. 1988; McMeekin et al. 1993).

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ߣ = ‫ ܯ‬− ቀ஻ቁ +

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ߤ௠௔௫ =

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୪୭୥భబ ேబ ି஺ ఓ೘ೌೣ

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The goodness of the fits was evaluated by calculating the corrected determination coefficient

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(corrected R2) and the Mean Square Error (MSE) associated with each of them (Saucedo-Reyes

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et al. 2009). The data analysis was performed using Statgraphics® Centurion XV software

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(Statpoint Technologies, Inc., Virginia, USA).

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3.

Results and discussion 3.1.

MIC determination at optimal growth temperature (37 °C)

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L. innocua was inoculated in TSB with different açaí concentrations (0, 2, 3, 5, 7, 8, and 10 g/L)

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to assess açaí MIC at optimum growth temperature (37 °C) (Rowan and Anderson 1998; FSAI

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2005). Figure 1 presents the growth and inactivation curves obtained. Furthermore, Table 1

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shows the N0, Nf, and λ values obtained as a function of açaí concentration. According to these

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results, the concentration of 10 g/L proved to be the MIC for L. innocua growth in TSB at 37

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°C, and it was a bactericidal concentration capable of achieving inactivation of nearly 3 log10

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cycles after 18 h of incubation, and non-detection of viables after 24 h of incubation. Based on

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this value and according to previous works, açaí seems less effective than other antimicrobial

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food additives such as carvacrol or citral. Under similar conditions, the MIC of those

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compounds for L. innocua and L. monocytogenes is lower (≤ 2,22 g/ L) (Kim et al. 1995;

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Veldhuizen et al. 2007; Gutiérrez et al. 2009; Belda-Galbis et al. 2013, 2014), as expected

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taking into account that both are highly pure compounds. Nevertheless, the use of açaí as an

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ACCEPTED MANUSCRIPT antimicrobial preservative could be more convenient considering consumer’s preference for

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high quality fresh (or fresh-like) foods, free from synthetic additives, where açaí could be added

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with no legal restrictions, not only as a preservative but also as a nutritional and functional

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ingredient.

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Taking into account the TPC of the samples tested, which ranged from 363.40 ± 20.07 to

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3221.25 ± 23.80 mg of GAE/L as a function of açaí concentration (Table 2), the TPC of the

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MIC obtained (10 g/L) was 2154.91 ± 126.10 mg of GAE/L. On the basis of this value, the MIC

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obtained is in the range defined by Wen et al. (2003) (1600−2600 mg/L) when they studied the

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antilisterial activity of phenolic acids, such as caffeic and ferulic acid, against various L.

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monocytogenes strains in TSB at 30 °C. In the literature there are studies that show that açaí

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contains not only flavonoids and lignans, but also vanillic acid, syringic acid, p-hydroxybenzoic

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acid, protocatechuic acid, gallic acid, ferulic acid, caffeic acid, chlorogenic acid, p-coumaric

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acid, and ellagic acid (del Pozo-Insfran et al. 2006; Pacheco-Palencia et al. 2008; Rojano et al.

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2011). They could all be responsible for the antimicrobial potential of the açaí extract studied.

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At non-inhibitory doses, L. innocua growth was dependent on açaí concentration. In general

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terms, the higher the açaí concentration, the higher the value of λ. This influence of the

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concentration on λ has also been observed by other researchers evaluating the antimicrobial

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potential of other vegetable products (Ferrer et al. 2009; Pina-Pérez et al. 2012).

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Açaí effect on Listeria innocua growth kinetics

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L. innocua growth curves were obtained at three different temperatures (37, 22, and 10 °C), in

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TSB supplemented with açaí at concentrations below the MIC (3, 5, and 7 g/L) (Figure 2). The

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growth curves obtained were fitted to the modified Gompertz equation with corrected R2 values

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ranging from 0.98 to 0.99 and MSE values ranging from 0.01 to 0.05. From the fits, the kinetic

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parameters λ and µ max were also calculated (Table 3).

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Regardless of the açaí concentration, L. innocua growth in TSB was dependent on incubation

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temperature. Decreasing the temperature in the range studied prolonged λ and reduced µ max. 8

ACCEPTED MANUSCRIPT These results have a similar tendency to those previously reported by other authors evaluating

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the effect of temperature on bacterial growth (Gospavic et al. 2008; Pina-Pérez et al. 2012).

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According to the results obtained (Table 3), reducing the temperature from 37 to 10 °C led to a

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10-fold increase in λ and a 6-fold decrease in µ max, in the absence of açaí.

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Regardless of temperature, addition of açaí to the culture broth at the concentrations studied (3,

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5, and 7 g/L) did not affect µ max (Table 3). However, it was observed that the higher the açaí

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concentration, the higher the value of λ. This means that under isothermal conditions the

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presence of açaí could prolong lag phase duration, whereas the maximum growth rate reached in

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log phase would remain unchanged. In particular, incubation in the presence of 7 g/L of açaí

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multiplied λ by 15, 7, and 4 at 37, 22, and 10 °C, respectively. This implies that if a cold chain

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failure occurs (temperatures between 10 and 22 °C) the use of 7 g/L of açaí would increase the

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time without growth up to a maximum of 31 (± 2.24) h.

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The relationship between the natural logarithm of λ (ln λ) and the concentration of açaí (g/L) as

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a function of temperature is presented in Figure 3. As can be seen in this figure, there is a linear

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relationship between the two variables, with r2 ≥ 0.95. The value of the slope of the curves

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obtained also demonstrates that the effect of varying the açaí concentration on the value of λ is

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greater at high temperatures; açaí proved to be more effective at 37 °C than at 22 and 10 °C.

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Perhaps temperature alters the antimicrobial potential of açaí, although it is also possible that L.

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innocua may be more sensitive to changes in concentration when it is incubated at optimum

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growth temperature. When bacteria grow at low temperatures, they alter the composition of

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their membranes to increase their cold tolerance; these changes might also increase their

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resistance to açaí, (i) because it has been shown that adaptation to a particular stress can

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generate resistance to other stresses (Wesche et al. 2009), and especially (ii) because

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polyphenols are antimicrobials that act on the membrane (Cowan 1999; Daglia 2012).

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Conclusion

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ACCEPTED MANUSCRIPT The microbiological stability of minimally processed foods is essential to ensure quality and

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safety during shelf life. The use of preservatives with antimicrobial properties can avoid the

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proliferation of spoilage and/or pathogenic microorganisms after processing if a cold chain

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failure occurs, or if any other situation promotes or facilitates microbial growth during storage.

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In this context, the present study provides the first mathematical characterization and

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quantification of the effect of açaí concentration on survival and growth of L. innocua, as a non-

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pathogenic surrogate for L. monocytogenes, in the presence of various açaí concentrations [3–7

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g/L], at various temperatures [37–10 °C].

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The results obtained show that at 37 °C açaí can control or inhibit growth of L. innocua,

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depending on the concentration. In this case, 10 g/L was the MIC and was also a concentration

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capable of inactivating almost 3 log cycles after 18 h of incubation, and of producing non-

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detection of viables after 24 h of incubation in the presence of açaí. Moreover, at lower doses it

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was observed that açaí was capable of slowing bacterial growth and prolonging the latent phase

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to a maximum value of 31.01 (± 2.24) h in the presence of 7 g/L of açaí at 10 °C.

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Consequently, açaí could be included in the formulation of minimally processed foods, not only

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for its nutritional quality but also for its preservative potential, taking into account that it could

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be an effective additional control measure to ensure microbiological safety, even at slightly

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abusive refrigeration temperatures (10 °C).

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Acknowledgments

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Clara Miracle Belda-Galbis holds a JAE-Predoctoral fellowship granted by CSIC in 2010. This

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study was carried out with FEDER funds and with funds from the Interministerial Commission

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for Science and Innovation projects AGL2010-22206-C02-01 ALI and AGL2013-48993-C2-2-

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6.

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Alonso, J. R. (2012). El fruto de asaí (Euterpe oleracea) como antioxidante. Revista de

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ACCEPTED MANUSCRIPT Figure captions

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Fig. 1. Listeria innocua growth and inactivation in the presence of açaí (g/L).

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Fig. 2. Listeria innocua growth curves according to açaí concentration (g/L) at different

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temperatures. The Gompertz model is presented fitting experimental data points.

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Fig. 3. Relationship between the natural logarithm of lag time (ln λ) and the concentration of

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açaí (g/L) at the temperatures studied.

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ACCEPTED MANUSCRIPT Table 1. Listeria innocua initial concentration (N0), final concentration (Nf) and lag phase

Açaí (g/L)

N0 (log10 cfu/mL)

Nf (log10 cfu/mL)

λ (h)

0

5.62 ± 0.06

9.38 ± 0.04a

00.83 ± 0.08

2

5.65 ± 0.05

9.13 ± 0.01

a

01.20 ± 0.04

2

5.64 ± 0.02

9.26 ± 0.11a

02.34 ± 0.24

5

5.57 ± 0.06

9.16 ± 0.04a

5.54 ± 0.01

9.22 ± 0.04

a a

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duration (λ), at 37 °C, as a function of açaí concentration.

7

5.54 ± 0.01

9.24 ± 0.03

10

5.61 ± 0.06

2.78 ± 0.06b

12.35 ± 0.23 11.20 ± 0.17 11,20 -0,17

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Value obtained after 24 h of incubation; b Value obtained after 18 h of incubation

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05.35 ± 0.68

ACCEPTED MANUSCRIPT Table 2. Total phenolic content (TPC) of the different açaí concentration samples tested in the study.

2

363.40 ± 20.07

3

726.80 ± 63.20

5

1209.21 ± 101.30

7

1533.60 ± 120.30

8

1623.00 ± 136.32

10

2154.91 ± 126.10

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TPC (mg of GAEa/L)

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Gallic Acid Equivalents

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a

Açaí (g/L)

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Tabla 3. Lag time (λ) and maximum growth rate (µmax) reached by Listeria innocua according to incubation conditions. λ (h) 37 °C

22 °C

10 °C

0

00.82 ± 0.07

02.12 ± 0.59

08.12 ± 1.37

3

02.35 ± 0.22

06.01 ± 0.81

16.86 ± 3.34

5

05.35 ± 0.71

10.57 ± 0.21

30.38 ± 0.76

7

12.32 ± 0.26

13.99 ± 2.97

31.01 ± 2.24

37 °C

22 °C

10 °C

0.65 ± 0.03

0.30 ± 0.00

0.11 ± 0.01

0.51 ± 0.03

0.24 ± 0.03

0.18 ± 0.03

0.53 ± 0.03

0.25 ± 0.03

0.11 ± 0.03

0.47 ± 0.03

0.30 ± 0.03

0.14 ± 0.03

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(g/L)

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µ max ((log10 cfu/mL)/h)

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Asaí

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0

6

2

5

3

4

5

3

7

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log10 cfu/mL

8

8

2

10

1 0 4

8

12 t (h)

16

20

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0 3

7

5

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log10 cfu/mL

9

7

6

37 °C

5 2

4

6

8

10

12 t (h)

14

16

log10 cfu/mL

9

8

7

5

22

2

4

6

8

10

12

14 16 t (h)

0 3 5 7

22 °C 18

20

22

24

26

28

AC C log10 cfu/mL

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30

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0

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0 3

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5 7

6

10 °C

5 0

6

12

18

24

30

36 42 t (h)

48

54

60

66

72

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4

22 °C 10 °C

3

ln λ

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ln λ 37 = 2.18 × açaí + 2.13 (r2 = 0.95)

1

ln λ 22 = 2.94 × açaí + 0.79 (r2 = 0.99) ln λ 10 = 4.06 × açaí − 0.30 (r2 = 0.98)

0

0.2

-1

0.4

0.8

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Açaí concentration (g/L)

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ACCEPTED MANUSCRIPT Highlights

►Açaí antimicrobial effect was mathematically assessed, from a kinetic point of view. ►The total phenolic content of samples tested was determined spectrophotometrically. ►Açaí was able to inhibit and slow bacterial growth, in a dose-dependent manner ► At non-

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minimally processed foods for its preservative potential.

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inhibitory doses, increasing açaí concentration increased lag time. ►Açaí could be added to