Antimicrobial Activity of Panax ginseng and Traditional Antimicrobials on Pathogenic and Spoilage Microorganisms in Fresh-Cut Mangoes and Oranges

Antimicrobial Activity of Panax ginseng and Traditional Antimicrobials on Pathogenic and Spoilage Microorganisms in Fresh-Cut Mangoes and Oranges

Rosa Raybaudi-Massilia*

Institute of Food Science and Technology, Central University of Venezuela, Caracas-Venezuela. Postal code 1041A, Postal zip 47097.

Patrizia DAmore

Institute of Food Science and Technology, Central University of Venezuela, Caracas-Venezuela. Postal code 1041A, Postal zip 47097.

Abstract

The effectiveness of Panax ginseng (PG) combined with potassium sorbate (PS) and/or malic (MA) and citric (CA) acids against Salmonella enterica ser. Saintpaul, Escherichia coli O157:H7 and native microflora on fresh-cut mangoes and oranges was evaluated. A combination of PG 3% (w/v), MA 1% (w/v), PS 0.05% (w/v) achieved to reduce by more than

2.5 and 1.0 logs CFU/g Salmonella Saintpaul and E. coli O157:H7 populations, respectively, on fresh-cut mangoes and oranges just after added. Significant reductions (p<0.05) by more than 4 and 2 log CFU/g of Salmonella and E. coli O157:H7 populations, respectively, during 21 days of storage at 5ºC in fresh- cut mango and orange were also achieved. The native microflora growth was delayed with this combination of antimicrobials; and microbiological shelf-life of fresh-cut mangoes and oranges was extended by more than 20 days. Sensorial attributes of fresh-cut mango were not affected by the addition of antimicrobials.

Keywords: Fresh-cut fruits, antimicrobials, Salmonella;

E. coli O157:H7; orange; mango; Panax ginseng.

1. Introduction

In recent years, the consumption of fresh-cut fruits has significantly increased as consequence of consumers demand toward fresh-like, high quality and healthy products [1]. In addition, there are scientific evidences demonstrating consumption of raw fruits and vegetables can help to prevent several degenerative diseases such as cardiovascular problems and some cancers [2]. However, these new consumption patterns may also increase the risk of microbial diseases to the consumers, if those fresh and fresh-cut fruits are improperly handled, processed or stored [3]; because

these products can result in foods that may carry some microorganisms, including pathogenic bacteria such as Salmonella spp. and E. coli O157:H7 [4, 5].

Balla and Farkas [6] mentioned that the shelf-life of fresh-cut fruits is limited by deteriorative processes such as browning, softening and microbial decay. Therefore, minimal processing of fresh products to control, reduce or inactivate spoilage and pathogenic microorganisms without significantly affecting their physicochemical and nutritionals attributes is still required.

The use of natural compounds from plant origin as plant extracts, essential oils and spices as substitutes of traditional antimicrobials for controlling the growth of pathogenic and spoilage microorganisms in fresh-cut fruits have recently awakened the interest of the researchers and consumers. Several in vitro studies about the atimicrobial activity of some natural substances such as Ginkgo biloba, Aloe vera, Panax ginseng and grape seed extract have been carried out [7-12]. However, studies about their applications on fresh product such as fresh-cut fruits have not been reported.

Among them, the Panax ginseng (PG) is one of the best-known Chinese medicinal herbs and it has been widely used in China for thousands of years. Several studies have indicated that this herb cans positively inuence the cardiovascular, nervous, endocrine and immune systems [9]. In addition, it may possess some anti-cancer, anti-inammation and anti-aging effects [13]. Empirically, PG has been used as a supplement to cure fatigue (energizing), to control the blood pressure and to accelerate the small intestine transit [14].

The main objective of this research was to determine the antimicrobial activity of PG as natural plant compound alone or in combination with other antimicrobial agents such as potassium sorbate, malic and citric acids, to control Salmonella enterica ser.

Saintpaul and Escherichia coli O157:H7 in fresh-cut oranges and mangoes at time 0 day and during 21 days of storage at 5°C. In addition, microbiological shelf- life and sensory attributes of fresh-cut mangoes and oranges with antimicrobials added were also evaluated.

2. Materials and methods

1. Fruits

   Whole mangoes (Mangifera indica L.) and oranges (Citrus sinensis L.) at commercial ripeness were acquired from a distributer of tropical fruits placed in Caracas, Venezuela. A characterization of the fruits was made following the Comisión Venezolana de Normas Industriales (COVENIN) 1151-77 and 924- 83 [15, 16]. Titratable acidity, expressed as g citric or malic acid/100g fruit, was determined as follow: 400 g of fresh-cut fruit was homogenized in a stomacher Lab-blender type 80 (Model BA 6020, London, UK); then 300 g of this was added to a volumetric flask of 2000 ml and diluted with distilled water (approximately 800 ml). Afterwards, this solution is boiled by 1 h and then cooled. The remains fraction was adjusted with distilled water up to 2000 ml and then filtered. From this solution, an aliquot of 25 ml is diluted with distilled water up to 100 ml and then is titrated with 0,1 N sodium hydroxide, using 0,3 ml phenolphthalein solution, as indicator. The pH (Microprocessor pH-meter model 211, Hanna Instruments, Cluj, Romania), soluble solids, expressed as ºBrix (Digital Refractometer PR-101, Atago Co., Ltd., Tokyo, Japan), color (ColorFlex Spectrocolorimeter model 45°/0°, HunterLab, Virginia, USA) and firmness (Texture Analyzer TA.XT2i, Stable Micro Systems, Godalming, UK) were also evaluated (Table 1).

   Table 1. Physical and chemical characteristic of the fresh-cut mangoes (Mangifera indica L.) and oranges (Citrus sinensis L.)

   Parameters *

2. Microorganisms and culture preparation

   Strains of Salmonella enterica ser. Saintpaul (CVCM 488 isolated from bean sprouts) and Escherichia coli O157:H7 (CVCM 442 / ATCC 35150 isolated from human feces from hemorrhagic colitis) were supplied by the Centro Venezolano de Colecciones de Microorganismos (CVCM) of the Institute of Experimental Biology of the Central University of Venezuela, Caracas-Venezuela for this study. Strains of Salmonella Saintpaul and E. coli O157:H7 were grown in tryptone soy broth (TSB) (Himedia, Mumbai, India) and TSB plus yeast extract (Himedia) at 0.6%, respectively. Those cultures were incubated at 37ºC for 18h without agitation to obtain cell in early stationary growth phase. These conditions were obtained from growth curves previously made in the Laboratory (data not shown). The maximum population reached by both microorganisms in the growth medium was approximately 109 Colony Forming Unit (CFU)/ml

3. Antimicrobial and stabilizing agents and preparation

   Powder of Panax ginseng (PG) (ArcoIris Laboratorio Naturista, Maracay, Venezuela), Potasium sorbate (PS) (Scharlau Chemie S.A., Barcelona, Spain), DL-malic acid (MA) (Scharlau Chemie S.A.) and citric acid (CA) (Scharlau Chemie S.A.) were used as antimicrobial agents; whereas, calcium lactate (CL) (Sigma-Aldrich, Saint Louis, Missouri, USA) was used as stabilizing agent of firmness. Powders of each compound (PG, PS, MA, CA, CL) were individually prepared in sterilized warming water (35-40ºC) and mixed by 10 min using a magnetic stirrer, and then sterilized in cold through Millipore filtration (0.45 µm) to obtaining free-microorganisms solutions. For that, different concentrations of each solution were prepared as suggested by RaybaudiMassilia et al. [17] in the following way: PG (0%, 1%, 2%, 3%; w/v), PS (0%,

   0.05%; w/v), MA (0%, 0.5%; w/v), CA (0%, 0.5%;

   w/v) and CL (0, 0.75%; w/v).

   Fresh- cut fruit

   Titratable acidity

   pH (g MA or CA /

   Soluble solids

   Firmness (g)

4. Fruit processing and inoculation

   100g fruit) (°Brix)

   Mango 4.02 ± 0.36 0.10 ± 0.21ª 12.03 ± 0.61 5.64 ± 0.12

   Orange 3.52 ± 0.02 0.90 ± 0.25b 11.10 ± 0.17 15.51 ± 1.37

   *pH, titrable acidity, soluble solid and firmness are mean of four determinations ± standard deviation; MA: malic acid; CA: citric acid; aValues expressed as g MA/100g fruit; bValues expressed as g CA/100g fruit.

   Mangos and oranges were sanitized by immersion in sodium hypochlorite solution (300 ppm); then washed with potable water and finally dried with absorbent paper. Mangoes were cut in slices of 2 cm of thick with a knife of stainless steel and then cut in pieces of 2 cm of wide x 2 cm of height with a rectangular hollow instrument of stainless steel; whereas oranges were cut in slices of 1 cm of thick

   approximately with a knife of stainless steel. Fresh-cut mangoes and oranges were dipped during 1 min into a solution containing PG (0%, 1.5%, 2%, or 3% (w/v)),

   and/or MA / CA (0 or 1% (w/v)), and/or PS (0 or 0.05% (w/v)), and/or CL (0 or 0.75% (w/v)) following a multilevel factorial design (Table 2). Afterwards, fresh-cut fruits were naturally wrung out with a plastic strainer for 2 min. Fifty grams of mango or orange pieces were placed into the polypropylene trays of 173×129×35 mm and separately inoculated with 500 µl of each culture of pathogenic microorganism on the fresh-cut fruit surface using a micropipette to obtain a final concentration of approximately 107 CFU/g. The samples inoculated were then dried at room temperature (23 ± 3ºC) by 30 min. The trays were then sealed with a plastic film (water vapor permeability of

   142.86 fmol s1.m2.kPa1 at 38°C and 90% HR; oxygen permeability of 52.38 fmol s1.m2.kPa1 at 23°C and 0% HR; and carbon dioxide permeability of

   Table 2. Multilevel factorial design* of antimicrobials used to observe the effect on populations of Salmonella Saintpaul and E. coli O157:H7 inoculated on fresh-cut mangoes and oranges

   Antimicrobialsa Microorganismsb PS MA / CL PG SS EC

   CA

   0.05 1 0.75 3.0 X1 X1

   0.05 1 0.75 2.0 X2 X2

   0.05 1 0.75 1.5 X3 X3

   0.05 1 0.75 0.0 X4 X4

   0.05 0 0.75 3.0 X5 X5

   0.05 0 0.75 2.0 X6 X6

   0.05 0 0.75 1.5 X7 X7

   0.05 0 0.75 0.0 X8 X8

   0.00 1 0.75 3.0 X9 X9

   0.00 1 0.75 2.0 X10 X10

   2.38 fmol s1.m2.kPa1 at 23°C and 0% HR) using a

   0.00 1 0.75 1.5 X11

   X11

   thermo-sealing machine ILPRA Food Pack Basic model FP400 V/G (Vigenovo, Italy). The more effective combination of antimicrobials (PG at 3% (w/v), PS at 0.05% (w/v), and MA at 0.5% (w/v)), and firmness stabilizing (CL at 0.75% (w/v)) obtained from multilevel factorial design was used to study the behavior of both pathogenic microorganisms throughout the storage at 5ºC by 21 days.

5. Recovery and enumeration of viable cells.

   A recovery for 20 min in phosphate buffer (pH 7.2) and enumeration of cells by spread plate method in selective agars of Hektoen (Himedia) (for Salmonella Saintpaul) and McConkey Sorbitol (Himedia) (for E. coli O157:H7) was made just 1h after processed the pieces of mango and orange inoculated, and after 3, 7, 14, 18 and 21 days of storage at 5ºC. The plates were incubated at 37ºC by 24h for enumeration. Experiments were carried out twice and counts were made in duplicated (n=4), and reported as log CFU/g. The recovery time (20 min) was selected according to the generation time of each microorganism from growth curves previously made in the laboratory (data not shown).

6. Effect of antimicrobials on the behavior of the native microflora in fresh-cut mango and orange

   The more effective combination of antimicrobials (PG at 3% (w/v), PS at 0.05% (w/v), and MA at 0.5% (w/v)), and firmness stabilizing (CL at 0.75% (w/v)) for inactivating populations of pathogenic

   0.00 1 0.75 0.0 X12 X12

   0.05 0 0.00 3.0 X13 X13

   0.05 0 0.00 2.0 X14 X14

   0.05 0 0.00 1.5 X15 X15

   0.05 0 0.00 0.0 X16 X16

   0.00 1 0.00 3.0 X17 X17

   0.00 1 0.00 2.0 X18 X18

   0.00 1 0.00 1.5 X19 X19

   0.00 1 0.00 0.0 X20 X20

   0.00 0 0.00 3.0 X21 X21

   0.00 0 0.00 2.0 X22 X22

   0.00 0 0.00 1.5 X23 X23

   0.00 0 0.00 0.0 X24 X24

   *Based on statistic analysis run in Statgraphics Centurion XVI. PS: potassium sorbate, PG: Panax ginseng, MA: malic acid, CA: citric acid, CL: calcium lactate; SS: Salmonella Saintpaul; EC: E. coli O157:H7. aValues are applied concentrations (%) of antimicrobials. bValues (X1, X2, X23, X24) are microbial reductions (log UFC/g). The experiment was carried out two times in duplicate (n = 4).

   microorganisms was used to study the behavior of the native microflora in fresh-cut mangoes and oranges. Counts of native mesophilic, psychrotrophic, yeast and mould populations in fresh cut mangoes and oranges treated or not with the optimum combination of antimicrobials were performed at 0, 3, 7, 14, 18 and 21 days of storage at 5ºC. Counts of mesophilic and psychrotrophic populations were made according to the ISO 4833:1991[18] guideline using Plate Count Agar (PCA) (Himedia) by the pour plate method. The plates of psychrotrophic microorganisms were incubated at 5ºC for 10-14 days; whereas mesophilic populations were incubated at 35ºC for 48h. Counts of yeast and

   mould populations were made according to the ISO 7954:1987 [19] guideline using Chloramphenicol Glucose Agar (CGA) (Himedia) by the spread plate method. The plates of molds and yeasts were incubated

   considered for psychrotrophic and yeast and mold populations [24, 25].

   ln log 107 1

   at room temperature for 3-5 days. The experiment was carried out twice and counts were made in duplicated

   =

   2.7182

   Eq. 2

   (n=4), and expressed as log CFU/g.

7. Sensory evaluation

   Fresh-cut mangoes and oranges dipped in a solution containing PG (3% w/v), MA (1% w/v), PS (0.05% w/v) and CL (0.75% w/v) or distilled water only (as control) were used to carry out the sensory analyses as suggested by Raybaudi-Massilia et al. [20]. An affective proof was applied to 30 volunteers panelist aged among 20 and 50 years, who frequently like and eat mangoes and oranges. The samples were coded with three-digit numbers and randomly given to the panelists for scoring acceptability of flavor, color, taste, firmness and global characteristics on a structured 10-cm hedonic scale labeled from extremely unpleasant (0) to extremely pleasant (10). In addition, potable water and pieces of non- salted cracker were provided to panelists for eliminating the residual taste between samples.

8. Predictive model

   Growth of mesophilic, psychrotrophic, yeasts and molds populations in non-inoculated fresh-cut mangoes and oranges were modeled according to the Gompertzs equation modified by Zwietering et al.

   [21] (Equation 1) using the statistic package Statgraphics Centurion XVI (StatPoint Technologies, Inc., Virginia, USA):

   Y = k + A.exp {-exp [(µmax .e /A)(- t) + 1]} Eq. 1 where, Y, is the current number of microorganisms

   present in the fresh-cut fruit; k, is the initial level of microorganisms (log CFU/g); A, is the difference in log (CFU/g) of microorganisms found between t = 0 days (initial population) and the maximum population density achieved at the stationary phase; µmax, is the maximum growth rate (log (CFU/g)/day); , is the lag phase time (days); t, is the time (days); and e, is a constant of 2.7182 value.

   The microbiological shelf-life (SL) was calculated as suggested by Lanciotti et al. [22], considering as maximum limit of mesophilic aerobic total count at expiry date 107 CFU/g according to the Spanish regulation for hygienic processig, distribution and commerce of prepared meals [23]. This limit was also

   where, SL is the shelf-life time; , is the count of microorganisms (log CFU/g) for a given time; k, is the microorganisms initial count estimated by the model (log CFU/g); A, is the maximum microorganism growth attained at the stationary phase (log10 CFU/g),

   µmax, is the maximal growth rate (log (CFU/g)/day);

   , is the lag time (days); and t, is the storage time (days).

9. Statistical analysis

A multilevel factorial analysis of variance (ANOVA) was carried out to evaluate significant (p<0.05) effects of PG at 0%, 1%, 2% and 3% (w/v), with or without PS at 0% and 0.05% (w/v), MA or CA at 0% and 1% (w/v) and/or CL at 0% and 1.5% (w/v) just after added, on the inactivation of Salmonella Saintpaul and E. coli O157:H7 separately inoculated on fresh-cut mangoes and oranges. Likewise, significant (p<0.05) differences on the microbial reductions of Salmonella Saintpaul and E. coli O157:H7 obtained during storage (0, 3, 7, 14, 18 and

21 days) and between treatments (with or without antimicrobials), in addition to sensory attributes among samples treated or not with antimicrobials were also analyzed. A multiple range tests, using the Fishers LSD method, were then applied to determine which levels of each factor were significantly (p< 0.05) different. All experiments were carried out twice and microbial counts were made in duplicate; therefore, means and standard deviations of 4 measurements were calculated for each treatment. All the analyses were made using the statistic package Statgraphics Centurion XVI (StatPoint Technologies).

3. Results and Discussion

Analytical characteristics such as pH, titratable acidity, soluble solids and firmness were measured to offer detailed information about fresh-cut mangoes and oranges used in this study (Table 1). The quality of fresh-cut fruits depends directly on the quality of the raw material and others factors such as firmness, size, variety, and ripeness at processing [26]. The pH value and titratable acidity (defined as content of organic acid by gram) are an indicative of the microflora that can to be present in the food. In spite of than fresh-cut

mangoes have a relative low pH (4.0±0.4), these produce may support the survival and growth of Salmonella Saintpaul and E. coli O157:H7 populations. In such sense, Strawn and Danyluk [27] reported that fresh-cut mangoes (pH 4.2) may be potential vectors for E. coli O157:H7 and Salmonella spp. transmission, because they could survive by 28 days at 4 °C on this produce. Likewise, Pao et al. [28] indicated that E. coli O157:H7 and Salmonella spp. could stay alive on peeled oranges (pH 3.8) during 14 days at 4ºC. The firmness and soluble solids are others important parameters, because these indicate the ripeness state of fruit. Martín-Belloso et al. [26] indicated that ripening have an important influence on sensorial featured related to flavor and texture of fruit. The values obtained in this experiment are corresponding to commercial or intermediate ripeness. Texture fewer

Enteritidis, Escherichia coli O157:H7 and Listeria monocytogenes by agar well diffusion assay, have been suppressed or inactivated by the addition of PG extracts.

Malic acid was more effective alone as antimicrobial against Salmonella Saintpaul than PG, PS and CA; whereas E. coli O157:H7 was more sensible to PG (3% w/v) or PS. When antimicrobials were combined a higher antimicrobial effect against Salmonella Saintpaul and E. coli O157:H7 in fresh-cut mangoes and oranges was observed (Table 3).

Table 3. Microbial reductions of Salmonella Saintpaul and E. coli O157:H7 inoculated on fresh- cut mangoes and oranges treated with antimicrobials just after added

firm, is an indicative that enzymes are degrading the cell wall of vegetative cells, and as consequence, a

Treatment

Salmonella Saintpaul*

(log CFU/g)

E. coli O157:H7*

(log CFU/g)

higher amount of nutrients is available for spoilage and/or pathogenic microorganisms. On the other hand, a higher amount of soluble solids would indicate a higher available of sugars in the fruit.

Significant (p<0.05) reductions of Salmonella Saintpaul and E. coli O157:H7 populations on fresh- cut mangoes and oranges treated with PG at 1% (w/v) in dipping treatment was observed; being greater the antimicrobial effect when higher concentrations of this compound were used (Table 3). Tan and Vanitha [9] reported that compounds of ginseng such as acidic polysaccharides (uronic acids), pananotin, panaxagin and quinqueginsin may induce hemagglutination, leading to bacterial cytotoxicity and acting on genetic material. Others studies have demonstrated that ginsenosides, main active constituent of ginseng, could mostly be responsible of the antimicrobial effect [29]. Ginsenosides are amphiphilic in nature, and have the ability to intercalate into the plasma membrane. This leads to changes in membrane uidity, and thus affects membrane function. The same authors suggested that ginsenosides directly interact with specic membrane proteins. They are lipid-soluble signaling molecules, which can traverse the plasma membrane and initiate effects on genetic material.

Studies about antimicrobial effects from PG on fresh-cut fruits or real food systems have not been reported; nonetheless, in vitro studies have pointed out that E. coli and Salmonella Typhimurium had different sensibilities to PG obtained from two ways of extraction (methanol or ethylacetate) [14]. Tan and Vanitha [9] and Lee et al. [30] indicated that Staphylococcus aureus, and Helicobacter pylori by the induced hemagglutination and macrophage stimulation method, respectively; and Bacillus cereus, Salmonella

Fresh-cut Fresh-cut Fresh-cut Fresh-cut

Mangoes Oranges Mangoes Oranges

PG (1%) 0.68 ± 0.02a 0.68 ± 0.71a 1.25 ± 0.05bc 0.97 ± 0.05a

PG (2%) 0.95 ± 0,48a 1.64 ± 0,03b 1.27 ± 0,03bc 1.02 ± 0.05ab

PG (3%) 1.04 ± 0.29b 1.78 ± 0.00c 1.29 ± 0.05bc 1.13 ± 0.05cd

MA (1%) 2.18 ± 0.28d 2.24 ± 0.16de 1.12 ± 0.05a 1.00 ± 0.05ab

CA (1%) 0.99 ± 0.03b 2.35 ± 0.00e 1.08 ± 0.03a 0.93 ± 0.04a

PS (0.05%) 0.39 ± 0.51a 0.92 ± 0.01a 1.22 ± 0.02b 1.18 ± 0.01d

PG (3%) + MA 2.24 ± 0.00d 2.73 ± 0.09f 1,21 ± 0,03b 1.05 ± 0,00b

PG (3%) + CA 1.58 ± 0.23c 2.19 ± 0.00d 1.20 ± 0,03ab 1.14 ± 0,02c

PG (3%) + PS 1.96 ± 0.56c 1.65 ± 0.01b 1.30 ± 0,03c 1.19 ± 0,05cd PG (3%) + PS + MA 2,51 ± 0,36d 2.74 ± 0,13f 1.30 ± 0,05c 1.01 ± 0,08abc PG (3%) + PS + CA 2.37 ± 0,49d 2.28 ± 0,01d 1.24 ± 0,03bc 0.97 ± 0,01a

PG: Panax ginseng, MA: malic acid, CA: citric acid, PS: potassium sorbate. *Values are mean of two determinations

± standard deviation in duplicate (n = 4). Different letters in a same column indicate significant difference (p<0.05) among treatments by microorganism and fresh-cut fruit.

Combinations of PG (3% w/v) with PS and MA or PG (3% w/v) with MA were more effective for achieving the higher reductions of Salmonella Saintpaul in the two fresh-cut fruits. Populations of E. coli O157:H7 were more sensible when combinations of PG (3% w/v) with PS and MA or PG (3% w/v) alone were applied on both fresh-cut fruits. In general, populations of Salmonella Saintpaul were more sensible to antimicrobials than populations of E. coli O157:H7 under same conditions. This fact may be attributed to the sensibility or resistance of each strain to the kind of antimicrobial and medium [17]. In addition, Lin et al. [31] indicated that some strains of enterohemorrhagic E. coli can create several mechanisms of resistance under acid stress conditions and induce mechanisms of protection, which may also serve as barrier against some antimicrobial substances.

With antimicrobials Without antimicrobials

With antimicrobials Without antimicrobials

7.0

6.0

Log10 CFU/g

Log10 CFU/g

5.0

4.0

3.0

2.0

1.0

0.0

Ab

Ba

Ba/p>

Aa Bb Cb

Ca

Db Da

Ea Eb

(a)

Fa Fb

7.0

6.0

Log10 CFU/g

Log10 CFU/g

5.0

4.0

3.0

2.0

1.0

Ab

Aa Bb Bb Ba

Cb

Ca

Da

(a)

Db

Ea Fa Ea

0 3 7 14 18 21

Storage time (days)

0.0

0 3 7 14 18 21

Storage time (days)

7.0

6.0

Ab Ab Bb Aa Aa

(b)

AaCb Db Eb

8.0

7.0

Ab Bb Bb

(b)

Log10 CFU/g

Log10 CFU/g

5.0

4.0

3.0

2.0

1.0

0.0

Aa

Ba

Ca

6.0

Log10 CFU/g

Log10 CFU/g

5.0

4.0

3.0

2.0

1.0

0.0

Aa Aa

Cb Db Db Ba Ca BDa Da

0 3 7 14 18 21

Storage time (days)

Figure 1. Behavior of (a) Salmonella enterica ser. Saintpaul and (b) E. coli O157:H7 populations in fresh-cut mango treated or not with Panax ginseng (3% w/v), malic acid (1% w/v), potassium sorbate (0.05% w/v) and calcium lactate (0.75% w/v) during storage by 21 days at 5ºC. Values are the mean of two determinations ± standard deviation in duplicated (n = 4). Capital letters indicate significant differences (p<0.05) between microbial reductions for each microorganism with respect to storage time. Lower-case letters indicate significant differences (p<0.05) between treatments by day

Malic acid was generally more effective than CA against both pathogenic microorganisms in both fresh- cut fruits (Table 3). It could be explained by the molecular size of each acid. Eswaranandam et al. [32] reported that smaller undissociated molecules of MA

0 3 7 14 18 21

Storage time (days)

Figure 2. Behavior of (a) Salmonella enterica ser. Saintpaul and (b) E. coli O157:H7 populations in fresh-cut oranges treated or not with Panax ginseng (3% w/v), malic acid (1% w/v), potassium sorbate (0.05% w/v) and calcium lactate (0.75% w/v) during storage by 21 days at 5ºC. Values are the mean of two determinations ± standard deviation in duplicated (n = 4). Capital letters indicate significant differences (p<0.05) between microbial reductions for each microorganism with respect to storage time. Lower-case letters indicate significant differences (p<0.05) between treatments by day

(134.09 Dalton) may enter more easily into the bacterial cells in comparison with CA (192.13 Dalton), with a higher molecular size, which may not effectively enter toward inside of the cell. Raybaudi- Massilia et al. [33] reported that MA can pass through

water-filled channels formed by transmembrane proteins (porins) embedded into the lipid bilayer that permit hydrophilic transport, and produced damage in the cell cytoplasm of pathogens without apparent changes in the cell membrane.

From these results, PG (3%), MA (1%), PS (0.05%) and calcium lactate (0.75%) were selected as the optimum combination for studying the behavior of

both pathogenic microorganisms on fresh-cut mangoes and oranges during 21 days at 5ºC (Figures 1 and 2). Significant reductions (p<0.05) of Salmonella Saintpaul and E. coli O157:H7 populations in fresh-cut mangoes and oranges treated or not with the optimum combination were found (Figures 1 and 2). Both pathogenic populations were decreasing through the storage time in both fresh-cut fruits, irrespective of the

Mesophilic Psychrotrophic Molds Yeasts

Mesophilic Psychrotrophic Molds Yeasts

Microbial growth (log10 CFU/g)

Microbial growth (log10 CFU/g)

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0

Fresh-cut mango treated (a)

Fresh-cut mango treated (a)

0 3 6 9 12 15 18 21

Storage time (days)

8.0

Microbial growth (log10 CFU/g)

Microbial growth (log10 CFU/g)

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0

Fresh-cut mango untreated (b)

Fresh-cut mango untreated (b)

0 3 6 9 12 15 18 21

Storage time (days)

Microbial growth (log10 CFU/g)

Microbial growth (log10 CFU/g)

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0

Fresh-cut orange treated (c)

Fresh-cut orange treated (c)

0 3 6 9 12 15 18 21

Storage time (days)

8.0

Microbial growth (log10 CFU/g)

Microbial growth (log10 CFU/g)

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0

Fresh-cut orange untreated (d)

Fresh-cut orange untreated (d)

0 3 6 9 12 15 18 21

Storage time (days)

Figure 3. Behavior of the mesophilic, psychrotrophic, molds and yeast populations in mango (a, b) and orange (c, d) treated or not with a combination of Panax ginseng (3% w/v), malic acid (1% w/v), potassium sorbate (0.05% w/v) and calcium lactate (0.75%) at 5ºC. Solid symbols are mean of four determinations.

Lines are values modeled by the Gompertzs equation

treatment applied (with or without antimicrobials); however, higher reductions in those treated samples were found. On the other hand, populations of Salmonella Saintpaul were more sensitive to the inactivation than populations of E. coli O157:H7 during storage time at 5ºC (Figures 1 and 2).

The observed reductions in untreated fresh-cut fruits throughout storage time could be attributed to the storage temperature (5ºC) and/or the high acidity of fruits tested. In this sense, Liao and Sapers [34] and Strawn and Danyluk [27] reported a slight reduction of Salmonella spp. on fresh-cut apples and mangoes stored at 8ºC and 4ºC after 3 and 7 days of storage, respectively. Similar results were reported by Raybaudi-Massilia et al. [20, 35], who indicated that storage temperature (5ºC) was the main factor that caused reductions of the populations of Salmonella Enteritidis and E. coli O157:H7 in fresh-cut apples and pears dipped in distilled water by 30 days. Alegre et al.

1. reported a decreasing of E. coli O157:H7 and Salmonella spp. on processed minimally peaches during 14 days of storage at 5ºC. Fisher and Golden

2. indicated that E. coli O157:H7 was able to survive and slightly reduce its population during 18 days at 4ºC on ground Golden Delicious, Red Delicious, Rome and Winesap. All these results to indicate that populations of E. coli O157:H7 and Salmonella spp. on fresh-cut fruits could be reduced under conditions of refrigerated

storage (3-5ºC), or by the high amount of naturally occurring organic acids in these fruits. In the last case, a decrease in cytoplasm pH of the cell caused by the internalization of the undissociated organic acid molecules throughout storage time could explain the inactivation of those microorganisms studied. Lou and Yousef [38] indicated that the antimicrobial action of organic acids is attributed to cytoplasm acidification. The antimicrobial compounds added into dipping treatments exerted a significant (p<0.05) bactericidal effect on the populations of E. coli O157:H7 and Salmonella Saintpaul in fresh-cut mangoes and oranges in comparison with the control samples (Figures 1 and 2). An initial inactivation (0 day) of both populations of pathogenic microorganisms about 1 log CFU/g in fresh-cut mangoes and oranges was observed. The antimicrobial effects of PS, CL and MA on pathogenic microorganisms in fresh-cut fruits have been previously reported by Raybaudi-Massilia et al. [5]. Davidson and Taylor [39] indicated that one of the primary targets of sorbic acid in vegetative cells seem to be the cytoplasmic membrane by inhibiting amino acid uptake, affecting the proton motive force through nutrient depletion. Likewise, Alakomi et al. [40] reported that lactic acid can cause permeabilization of the outer membrane of the gram-negative bacteria such as E. coli O157:H7 and Salmonella Typhimurium.

Table 4. Gompertzs parameters to describe the growth of mesophilic, psychrophilic, yeass and molds populations and to predict the microbiological shelf-life of non-inoculated fresh-cut mangoes and oranges treated with different antimicrobials and a firmness stabilizer

Fresh- cut

Dipping 2 K A

µmax

Shelf-life

fruit Population

condition R

(log CFU/g)

(log CFU/g)

(Days)

(log [CFU/g]/day)

(days)

Mesophilic Psychrophilic Molds

Control (water)

99.42 3.92±0.08a 2,51±0.16a 6,44±0.45a 0,19±0.02a > 21

96.81 4,89±0.16a 2,28±0.20a 3,31±0.60a 0,32±0.07a 12,59

97.22 4,14±0.10a 1,50±0.14a 4,24±0.68a 0,18±0.04a > 21

Yeast 97.34 4,68±0.08a 2,25±0.19a 14,03±0.50a 0,67±0.21a > 21

Mango

Mesophilic PG (3%) 98.94 3.25±0.11b 2.76±0.14a 4.59±0.39b 0.41±0.07b > 21

Psychrophilic Molds

Yeast Mesophilic Psychrophilic

Molds

MA (1%)

PS (0.05%)

CL (0.75%)

Control (water)

99.19 4.28±0.08b 3.57±0.26b 10.21±0.56b 0.31±0.04a 19,94

98.89 3.00±0.05b 2.28±0.17b 14.95±0.35b 0.42±0.05b > 21

98.32 2.05±0.39b 5.66±0.28b 8.55±1.43b 0.28±0.04b > 21

99.92 4.61±0.17a 2.77±0.24a 3,535±0.75a 0,13±0.00a > 21

99.19 5.93±0.03a 3.41±1.60a 17.92±3.60a 0.17±0.02a > 21

95.28 3.92±0.06a 0.99±0.15a 9.68±1.32a 0.10±0.03a >21

Yeast 98.82 4.02±0.17a 4.49±1.06a 11.32±1.81a 0.20±0.02a > 21

Orange

Mesophilic PG (3%) 98.04 4.20±0.01b 3.32±0.81a 12.53±1.68b 0.19±0.03b > 21

Psychrophilic Molds

Yeast

MA (1%)

PS (0.05%)

CL (0.75%)

83.78 4.76±0.50b 2.64±3.18a 12.06±9.68a 0.11±0.04a > 21

96.99 3.05±0.10b 2.00±0.15b 6.38±0.58b 1.38±1.43a > 21

99.98 3.95±0.01a 3.13±0.02b 7.76±0.07b 0.33±0.01b > 21

K= microorganisms initial count estimated by the Gompertzs model (log CFU/g); A= maximum microorganism growth attained at the stationary phase (log CFU/g); µmax= maximal growth rate (log [CFU/g]/day); = Lag time (days). Different lower-case letters (a, b) indicate significant (p < 0.05) statistically differences between dipping conditions by parameter and fresh-cut fruit.

This optimum combination of antimicrobials was also used to study the microbiological shelf-life of fresh-cut mangoes and oranges during 21 days of storage at 5ºC. In the Figure 3 is shown the behavior of mesophilic, psychrotrophic, and mold and yeast populations in presence or not of antimicrobial substances applied by dipping treatment on fresh-cut mangoes and oranges stored at 5ºC. These curves were

4a). The antimicrobials added in fresh-cut mango did not affect significantly (p<0.05) the sensory attributes.

(a)

Color 10

8

6

modeled by the Gompertzs equation, and parameters describing their microbial growth are shown in the Table 4.

Significant (p< 0.05) statistically differences among the initial microbial counts (k values) from treated and untreated fresh-cut mangoes and oranges stored at 5ºC were found, with the exception of yeasts

Global 4

2

0

Taste

Flavor

Firmness

in fresh-cut oranges, where initial populations were not affected (Table 4). These results to indicate that k values of spoilage microorganisms in both fresh-cut fruits was significantly (p<0.05) affected by the combination of antimicrobials just after added; where the k values of treated fresh-cut mangoes and oranges

(b)

Untreated

Color 10

8

6

Treated

were lower than in those without treatment (Table 4). The lag phase of psychrotrophic and molds populations in treated fresh-cut mangoes was delayed in comparison with control samples; whereas the lag phase of mesophilic populations in treated fresh-cut oranges was also prolonged (see values in Table 4). Likewise, the maximal microbial growth rate (µmax values) of mesophilic and mold populations in treated fresh-cut mangoes was delayed in comparison with

Global 4

2

0

Taste Untreated

Flavor

Firmness

Treated

those untreated samples. In the same way, mesophilic and yeast populations in fresh-cut oranges had higher values of the maximal growth rate than control sample. The shelf-life of treated and untreated fresh-cut mangoes was limited by psychrotrophic populations. However, the shelf-life of treated fresh-cut mangoes was extended by more than 7 days in comparison with untreated samples (Table 4). On the other hand, the shelf-life of treated or untreated fresh-cut oranges was not limited by any microorganisms during the 21 days of the experiment. Similar results have been reported by Raybaudi-Massilia et al. [20, 41], who evaluated the shelf-life extension of fresh-cut Fuji apples and Flor de Invierno pears using natural antimicrobial substances, among them, MA (2.5%, w/v) as dipping treatment.

Sensory evaluations indicated that significant statistically differences (p<0.05) among the values assigned by the panelist to flavor, taste and global acceptance on treated and untreated fresh-cut oranges were found (Figure 4b); nonetheless, treated samples were accepted by panelists. On the other hand, no significant (p>0.05) differences between treated and untreated fresh-cut mangoes were observed (Figure

Figure 4. Sensory attributes of fresh-cut mangoes

(a) and oranges (b) treated or not with a combination of Panax ginseng (3% w/v), malic acid (1% w/v), potasium sorbate (0.05% w/v) and calcium lactate (0.75% w/v). Values are mean of 30 determinations

4. Conclusions

A combination of PG (3% w/v), MA (1% w/v), PS (0.05% w/v) and CL (0.75% w/v) was effective to reduce populations of Salmonella Saintpaul and E. coli O157:H7, as well as to delay the growth of the native microflora in fresh-cut mangoes and oranges. In addition, fresh-cut mangoes with antimicrobials added had a good acceptation for the panelists. This method can ensure the safety and quality of fresh-cut fruits and represents an alternative technology to physical treatments; in addition to its feasibility for food industry. Further studies using hurdle technologies are needed for reducing the negative impacts of these compounds on some kind of fresh-cut product such as orange.

5. Acknowledgments

This study was supported by the Council of Scientific and Humanistic Development of the Central University of Venezuela, Caracas-Venezuela, through the Project Nº PI 03-00-7025-2007.

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