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Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food borne pathogens and their antioxidant properties
LWT - Food Science and Technology xxx (2015) 1e6
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Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food borne pathogens and their antioxidant properties € € ven, S¸eniz Karabıyıklı*, Kader Tokatlı, Nilgün Oncül Aslıhan Demirdo Gaziosmanpas¸a University, Faculty of Engineering and Natural Science, Food Engineering Department, 60000 Tasliçiftlik, Tokat, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 January 2015 Received in revised form 24 March 2015 Accepted 25 March 2015 Available online xxx
Anthocyanins, known for their antioxidant characteristics, also have antimicrobial effects. Antimicrobial, antioxidant and some physicochemical properties of red cabbage (RC) and sour cherry pomace (SCP) anthocyanin extracts were investigated. Conventional (CE) and ultrasonic extraction (UE) methods were used. The antioxidant activities of samples extracted by UE were higher than those extracted by CE. Antimicrobial effects of the extracts were determined by the detection of minimum inhibitory concentration (MIC) values for Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella Typhimurium and Bacillus cereus. The extracts were inoculated (6 and 3 log CFU/mL) with five different pathogens, separately. All the extracts have antimicrobial effects on the tested pathogens and the results ranged depending on the concentration of the extracts and inoculation dose of the pathogen. The extraction method did not affect the inhibitive characteristics of the extract, and it was concluded that the antimicrobial effects of the extracts were mainly dependent on the anthocyanin content of the materials. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Red cabbage Sour cherry pomace Anthocyanin Antimicrobial MIC
1. Introduction There is an increasing demand for natural, safe and functional foods in the last decade. Consumers are interested in safe foods which have positive contributions to health, but contain no chemical additives or preservatives. However, keeping the food safety stable until the expiration date is not possible without the addition of any preservatives or application of thermal processing, which causes deterioration of functional ingredients. In this view, plant extracts, which could be used for natural colorant, antioxidant, flavoring or antimicrobial agents, are good alternatives for the food industry. Polyphenol rich plants especially are gaining popularity as a good source for anthocyanins. Anthocyanins are bioactive compounds present in many fruits, vegetables and their products. They are a sub-group within the flavonoids, characterized by a C6eC3eC6-skeleton (Patras, Brunton, O'Donnellb, & Tiwari, 2010). Antioxidant and antimicrobial properties of anthocyanins were
* Corresponding author. Gaziosmanpas¸a University, Faculty of Engineering and Natural Sciences, Department of Food Engineering, 60100, Tokat, Turkey. Tel.: þ90 356 2521616x2899; fax: þ90 356 2521729. €ven), E-mail addresses: [email protected] (A. Demirdo [email protected], [email protected] (S¸. Karabıyıklı), kader. € [email protected] (K. Tokatlı), [email protected] (N. Oncül).
^ te , Sylvain, & Lacroix, studied by several researchers (Caillet, Co ~es, 2012; Martin et al., 2012; Da Silva, Maquiaveli, & Magalha 2012; Oliveira et al., 2013; Park, Biswas, Phillips, & Chen, 2011; Rockenbach et al., 2011; Schved, Henis, & Juven, 1994; SmithPalmer, Stewart, & Fyfe, 1998). Anthocyanins could fight against chronic diseases, such as neuronal and cardiovascular illnesses and diabetes, among others (Konczak & Zhang, 2004). Anthocyanins may offer anti-inflammatory, anti-viral, and anti-cancer benefits (Ali, Masud, & Abbasib, 2011; Basu, Rhone, & Lyons, 2010; Cassidy et al., 2011; Faria et al., 2010; Ghosh & Konishi, 2007; Prior et al., 2008). Also, they have anti-carcinogenic effects because of their effective protection against oxidative damage by acting as an antioxidant. Antioxidant mechanisms of anthocyanins also make them available to extend the shelf life of industrial food products. The anthocyanin composition of red cabbage is very complex and the dominant structures are: cyanidin-3,5-diglucoside and cyanidin-3-sophoroside-5-glycoside acylated with sinapic acid, ferulic acid, p-coumaric acid, caffeic acid or malonic acid (Dyrby, Westergaard, & Stapelfeldt, 2001). Turkey is one of the biggest sour cherry cultivators in the world (FAOSTAT, 2010) and the pomace of the juice processing is a potential and substantial waste for the food industry. Four types of anthocyanin compounds were determined in sour cherries: cyanidin-3-glucosylrutinoside, cyanidin-3-sophoroside, cyanidin-3-rutinoside and cyanidin-3-
http://dx.doi.org/10.1016/j.lwt.2015.03.101 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101
€ven et al. / LWT - Food Science and Technology xxx (2015) 1e6 A. Demirdo
2
glucoside (Chandra, Rana, & Li, 2001; Kim, Heo, Kim, Yang, & Lee, 2005). Food borne illnesses are still a major problem all over the world, even in developed countries. Outbreaks of human infections associated with food consumption increased in frequency during the past decade (Beuchat, 2002; CDC, 2014). It was reported that 6 to 81 million cases of illnesses, and up to 9000 deaths, annually are attributed to food borne pathogens in the USA (Alzoreky & Nakahara, 2003). Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella Typhimurium and Bacillus cereus are the most common food borne pathogens (Beuchat, 1996; CDC, 2013). Various chemical antimicrobials have been used to prevent food borne infections/intoxications in the food industry. However, chemical preservatives are discountenanced nowadays as a result of increasing awareness of the consumers on the adverse effects of chemical preservatives on human health. The numbers of the studies on natural alternatives are increasing as a natural consequence of requirement by the food industry. New, available, natural and effective alternatives are required to satisfy the consumers and to supply their demand. The objective of this study was to investigate the antimicrobial, antioxidant and physicochemical properties of sour cherry pomace and red cabbage anthocyanin extracts, which were extracted by conventional and ultrasonic extraction methods. 2. Materials and methods 2.1. Materials Whole red cabbages (RC) and sour cherry pomace (SCP) were used as raw material. The red cabbages were obtained from BafraSamsun/TURKEY and sour cherry pomace was purchased from Dimes Concentrated Fruit Juice Company (Tokat, TURKEY). They were stored at þ1 C maximum for 48 h before processing. 2.2. Processing methods 2.2.1. Extraction of red cabbage and sour cherry pomace anthocyanins Sorting, washing, cutting and chopping operations were carried out for red cabbages. Red cabbages were chopped (Moulinex FP 519G-750 W) at 500 rev/min under standardized conditions determined by pre-trials. Sour cherry pomace was used after separation of stones and stems. The extraction process was carried out using solideliquid extraction method. 1% formic acid was used to acidify the environment during the extractions. An ethanol/water/ formic acid mixture was used as the solvent in all extraction applications. Extractions were carried out with conventional and ultrasonic methods. The raw material amount that was determined by pre-trials was mixed with extraction solvent (red cabbage 1/3; m/v and sour cherry pomace 1/15; m/v). The mixture was homogenized at the fourth speed position for 45 s (Ultra-Turrax IkaWerke, Staufen, Germany). Conventional and ultrasonic extractions were applied after the homogenization process. The obtained mixture was filtered (Whatman no:1, 125 mm) under a vacuum (Milipore, WP6122050, Germany). The extraction solvent was evaporated in a rotary evaporator at 50 C. Single extraction step was used in all samples and for all methods. 2.2.2. Conventional and ultrasonic extractions Conventional extractions (CE) were done by using a water bath (Memmert-WB22) whereas ultrasonic extractions (UE) were carried out using an ultrasound bath operating at 37 kHz (ElmasonicS100H). Temperatures were controlled with a digital thermometer
(Shaanxi Taurus, China) during extractions to stabilize the desired temperature. Extraction conditions of RC anthocyanins were optimized by Response Surface Method (RSM). A Box-Behnken design was used in designing the CE and UE treatments of three variables with six center points (Design Expert 7.0.0 STAT-EASE, 2005). Extraction conditions of SCP anthocyanins were determined by pre-trials. After production of anthocyanin extracts, all samples were stored at 80 C to prevent the deterioration and interaction between the compounds of extracts until analyses. All samples were held at þ4 C to thaw for a night before analyzing. The soluble solid content of the extracts was adjusted based on the natural soluble solid content values of the materials before the analyses (6 Bx for RC and 12 Bx for SCP). 2.3. Methods of analysis 2.3.1. Physicochemical analysis Total anthocyanin contents (TAC) of samples were detected by pH-differential method using two buffer systems: potassium chloride buffer, pH 1.0 (0.025 M) and sodium acetate buffer, pH 4.5 (0.4 M). TAC of samples were determined at 535 nm and (mg cyanidin-3-glucoside/100 mL of extract) were calculated with molar absorptivity of cyanidin-3-glucoside (26.900) (Glassgen, Wray, Dieter, Metzger, & Seitz, 1992). Total phenolic content (TPC) was detected at 760 nm using the Folin-Ciocalteu reagent, diluted 10 fold before use (Sigma Chemical Co., St. Louis, MO) with gallic acid (3,4,5-trihydroxybenzoic acid) as the standard and measured after reaction (2 h) (Franke, Chless, Silveria, & Robensam, 2004). Antioxidant activities of the samples were determined by the ABTS [2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] assay (Re et al., 1999). Soluble solid contents (SSC) of samples were measured with a refractometer at 20 C (RFM 330, U.K.) (AOAC, 1995). Total titrable acidity (TA) was determined by means of a potentiometric titration of the acidity of the samples by placing 10 g of sample into 90 mL of deionized water. After filtration, 10 mL filtrate was titrated up to pH 8.1 (pH meter, WTW InoLab, Weilheim, Germany) with 0.1 N NaOH. The results were expressed as g/100 mL (citric acid) (AOAC, 1995). pH values of samples were determined by pH-meter at 20 C (AOAC, 1995). Water activity (aw) values of the samples were measured by AquaLab Model Series 3 TE, USA (AOAC, 1980). 2.3.2. Microbiological analysis The microflora of the extracts was controlled before MIC analyses by enumeration of total mesophilic aerobic bacteria (TMAB, FDA-BAM online, 2001a) and yeast & mold (Y&M, FDA-BAM online, 2001b). The safety of the extracts were checked out by E. coli (FDABAM online, 2013), S. aureus (ISO 6888, 2004), L. monocytogenes (ISO 11290, 1996), Salmonella spp. (FDA-BAM online, 2014) and B. cereus (FDA-BAM online, 2012) analyses. The antimicrobial effects of the RC and SCP extracts were tested against some food borne pathogens. E. coli (ATCC 25922), S. aureus (ATCC 25923), L. monocytogenes (ATCC 19115), Salmonella Typhimurium (ATCC 14028) and B. cereus (ATCC 10876) were used as test microorganisms. Two different inoculum doses were applied: low (~3 log CFU/mL) and high (~6 log CFU/mL) to detect the responses of the RC and SCP extracts for different contamination levels. Each of the test cultures were grown at 37 C for 18e24 h in Brain Heart Infusion Broth (BHI, pH 7.4 ± 0.2, LabM-LAB049), and then diluted with 0.1% sterile peptone water (PW, pH 6.3 ± 0.2, Oxoid-L37) to achieve a final inoculum of 6.0 log CFU/mL (high inoculum dose) and 3.0 log CFU/mL (low inoculum dose), appropriately. The MIC value of the each extract was determined by modification of the method described by LaBombardi, Sotos, Allen, and Sullivan (2008).
€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101
€ven et al. / LWT - Food Science and Technology xxx (2015) 1e6 A. Demirdo
200 mL of extract was placed into the first well of a 96-well microtiter panel. 100 mL BHI Broth was placed into each of the wells from 2nd to 12th in the row. 100 mL of the extract in the first well was taken and placed into the 2nd well and mixed with BHI Broth. 100 mL was taken from this mix in the 2nd well and placed into the 3rd well. Serial dilutions were made from the 2nd well to the 11th well by this method described shortly. 100 mL of the mixture was taken from the 11th well and was discarded. The 12th well which contains no extract was used as a positive control to detect the microbial growth. The final concentrations of extracts in the wells were; 1:1 (100%), 1:2 (50%), 1:4 (25%), 1:8 (12.50%), 1:16 (6.25%), 1:32 (3.13%), 1:64 (1.56%), 1:128 (0.78%), 1:256 (0.39%), 1:512 (0.20%), 1:1024 (0.10%), and the control (0%). After dilution, 100 mL of test culture was inoculated into each well from 1st to 12th. This procedure was repeated for each extract, each test microorganism and each inoculum dose, separately. The MIC plates were incubated at 37 C for 24e48 h and the survival of the test microorganism was checked by plating on Brain Heart Infusion Agar (BHI, pH 7.4 ± 0.2, LabM-LAB048). The petri dishes were incubated at 37 C for 18e24 h then the results were recorded. Although, the microflora of the extracts were controlled to detect the presence of competitive microorganisms, the initial microflora of the extracts were analyzed by surface plating on BHI Agar for each treatment as a negative control. Three replicate trials with 2 parallels were carried out for each experiment. 2.4. Statistical methods The results were statistically analyzed by ANOVA using the software SPSS 13 (SPSS Inc., Chicago, IL, USA) with the Duncan test to evaluate differences between treatments at p < 0.05. Each experiment was repeated at least three times. Means and standard deviations of the results were calculated for physicochemical analyses. 3. Results and discussion Extraction conditions of RC anthocyanins were optimized by RSM, and time, temperature and ethanol concentration were selected as independent variables in both methods (UE and CE) in order to optimize extraction conditions. Total anthocyanin contents were determined after CE and UE applications. The response selection was determined according to the production conditions to provide maximum anthocyanin content. The effects of extraction time (5e75 min), temperature (40e80 C) and ethanol concentration (5e75%) (independent variables) were investigated to evaluate anthocyanin content (response) of RC extracts. The analysis of variance showed that the adjusted second order models were significantly fitted to the experimental data, as indicated by the regression model F values of 27.495 (p < 0.001) for CE and 8.85 (p < 0.001) for UE. The extraction time, temperature and ethanol concentration were found to be significantly important for anthocyanin content at 95% confidence interval by ANOVA. Lack of fit of experimental data was not significant (p > 0.05) for both models (CE and UE). The coefficient of variation for the CE model was 5.07%, and it was 10.07% for the UE model. The models of CE and UE showed an adequate precision of 13.886 and 10.724, respectively. The determination coefficient (R2) for CE model was 0.9687 and it was 0.9087 for UE, while the adjusted determination coefficient (Adjusted R2) values were 0.9334 and 0.8960, respectively. The solutions were obtained for the optimum covering criteria by applying the desirability function method. Optimum extraction conditions were: 75 min, 40 C and 42.39% ethanol concentration for CE and UE. The results indicated that the low temperature and long application time with moderate ethanol concentration could
3
increase extraction of anthocyanins. The desirability values for CE and UE were 94.5% and 89.5%, respectively. The detailed optimization data was not declared in this study and it will be presented in further studies. The results of pre-trials for SCP indicated that the low temperature (40 C) and short application time (40 min and 60 min) with moderate ethanol concentration (40%) could increase the extraction of anthocyanins for CE and UE methods. Physicochemical characteristics of the RC extracts are shown in Table 1. TAC, TPC and antioxidant activity are important parameters for these kinds of plant extracts. The highest TAC and TPC contents were found in CE samples, while the highest antioxidant activity value was found in UE samples. TAC values for CE and UE groups were found as 593 ± 0.9 mg/L and 546 ± 0.6 mg/L, respectively. TPC was found in CE sample as 16,320 ± 5.24 mg/L, while it was 15,470 ± 7.43 mg/L in UE groups. Antioxidant activity of UE samples was found as 41.27 ± 0.24 mM Troloks/mL and it was 36.25 ± 0.32 mM Troloks/mL for CE samples. High antioxidant activity values of UE samples can be explained by the extraction of other antioxidants such as vitamins (C, B6, K). The difference between TAC, TPC and antioxidant activity of CE and UE samples was statistically significant (p < 0.05). The pH values of the samples were 3.53 and the TA was determined as 0.98 g/100 g and 1.045 g/ 100 g for UE and CE groups, respectively (p > 0.05). The difference in aw (0.156 for CE and 0.1719 for UE) of extracts was statistically significant (p < 0.05). The results of physicochemical analyses of the SCP extracts, produced by CE and UE methods, are shown in Table 2. There was no statistically significant difference between the TAC values of SCP extracts (p > 0.05). Lower TPC (453.27 ± 7.42 mg/L) and antioxidant activity (59.61 ± 1.24 mM Troloks/mL) values were determined for 40 min at 40 C with 40% ethanol concentration in CE groups. Antioxidant activity values of UE samples were found as 106.80 ± 0.71 mM Troloks/mL (60 min at 40 C with 40% ethanol concentration) and 105.87 ± 0.20 mM Troloks/mL (40 min at 40 C with 40% ethanol concentration). High antioxidant activity values of samples can be explained by extraction of other antioxidants such as phenolics, organic acids and vitamins etc. TA values were between 6.06 and 8.80 g/100 g and pH values were between 2.37 and 2.44. There was no statistically significant difference between the pH values (p > 0.05) and aw values in the same extraction conditions for SCP extracts (p > 0.05). Ghafoor, Choı, Jeon, and Jo (2009) determined maximum anthocyanin content as 2.28 mg/mL from grape seeds by UE (52.35% ethanol, 55.13 C, 29.49 min). The UE conditions for anthocyanin extraction from red raspberry were optimized (solvent/sample: 4/1, 200 s, 400 W) by Chen et al. (2007) and the anthocyanin amount in extract was calculated as 78.13%. Solvent concentration and extraction time are important for anthocyanin extraction from plant materials (Wang, Sun, Cao, Tian, & Li, 2008). The use of UE due to the ultrasound wave breaks the cells of the vegetal matrix and it leads to release contents of the cells into the extraction medium (Vinatoru et al., 1997). Also, solideliquid extraction is a mass transport phenomenon in which solids contained in a matrix migrate into a solvent that is brought into contact with the matrix (Ghafoor et al., 2009). This phenomenon can be enhanced with changes in diffusion coefficients induced by ultrasound and extraction temperature (Corrales, Garcia, Butz, & Tauscher, 2009). Naturally existing microflora of the extracts were controlled by enumeration of TMAB, Y&M and detection of the viability of test pathogens at the beginning of each treatment and no viable cells were detected. The antimicrobial content, low pH environment of the extracts and the conditions of the extraction resulted in microbiologically safe extracts.
€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101
€ven et al. / LWT - Food Science and Technology xxx (2015) 1e6 A. Demirdo
4
Table 1 Physicochemical characteristics of the red cabbage extracts. Parameter
Conventional extraction (75 min/40 C/42.39% ethanol)
Total anthocyanin (mg/L) Total phenolic (mg/L) Antioxidant activity (mM Troloks/mL) Total titrable acidity (g/100 g) pH Water activity
593 16,320 36.25 1.04 3.53 0.156
± ± ± ± ± ±
Ultrasonic extraction (75 min/40 C/42.39% ethanol)
0.9a 5.24a 0.32a 0.2a 0a 0.001a
546 15,470 41.27 0.98 3.53 0.171
± ± ± ± ± ±
0.6b 7.43b 0.24b 0.2a 0a 0.002b
Results are the means ± SD (n ¼ 3); statistically significant difference shown levels a, b (P 0.05).
Table 2 Physicochemical characteristics of the sour cherry pomace extracts. Parameter
Conventional extraction
Total anthocyanin (mg/L) Total phenolic (mg/L) Antioxidant activity (mM Troloks/mL) Total titrable acidity (g/100 g) pH Water activity
Ultrasonic extraction
(60 min/40 C/40% ethanol)
(40 min/40 C/40% ethanol)
(60 min/40 C/40% ethanol)
a
36.01 ± 2.38 493.09 ± 10.8b 95.55 ± 2.15b
a
35.08 ± 1.06 453.27 ± 7.42a 59.61 ± 1.24a
a
(40 min/40 C/40% ethanol)
36.90 ± 0.87 488.21 ± 6.7b 106.80 ± 0.71d
38.20 ± 1.20a 493.84 ± 5.12b 105.87 ± 0.20c
7.68 ± 0.13c 2.43 ± 0.07a 0.356 ± 0.01a
6.06 ± 0.22a 2.44 ± 0.07a 0.471 ± 0.02b
8.80 ± 0.09d 2.41 ± 0.07a 0.364 ± 0.01a
7.45 ± 0.16b 2.37 ± 0.07a 0.433 ± 0.02b
Results are the means ± SD (n ¼ 3); statistically significant difference shown levels a, b (P 0.05).
There were no significant differences between the results of the two different extraction methods on the inhibition of the tested pathogens. However, SCP extracts inhibited the tested pathogens at lower concentrations than the RC extracts (Tables 3 and 4). The MIC results of the SCP were between 1:8 and 1:32 while they were between 1:1 and 1:2 for RC extracts. On the other hand, there were no significant differences between the responses of the pathogens. Both extracts have similar antimicrobial effects on the test microorganisms and the cell wall structures, or sporulation mechanisms of the microorganisms, did not affect the MIC results. The results showed that both RC and SCP extracts inhibited the growth of the tested microorganisms for both inoculum doses (6 and 3 log CFU/ mL). The pH value was increasing and the concentration of the antimicrobial content of the extract decreased from the first well to the last one as a result of MIC test principles. So, the antimicrobial effects of the extracts mainly depend on rich phenolic content. Additionally, the dilution of the extracts to adjust the desired SSC caused dilution of the antimicrobial content. Antimicrobial effects of some products containing pomegranate extracts on three Streptococci strains (mutans ATCC 25175, sanguis ATCC 10577 and mitis ATCC 9811) were investigated and the MIC values ranged from 1:1 (100%) to 1:1024 (0.04875%) (Vasconcelos et al., 2006). MIC of polyphenol-rich sour pomegranate sauce (itself, not extracts) to inhibit S. aureus (ATCC 25923) and E. coli O157:H7 ranged between 1:8 (6.35%)e1:256 (0.195%) and 1:8
(6.35%)e1:128 (0.39%), respectively (Kıs¸la & Karabıyıklı, 2013). The antimicrobial effect of the pomegranate correlated with its phenolic content as in the present study. MICs of seven synthetic phenolic compounds against S. aureus, B. cereus and Pseudomonas rrez-Larraínzar, Rúa, de Arriaga, fluorescens were studied by Gutie del Valle, and García-Armesto (2013) and the MIC values ranged between 100 mg/mL and >1600 mg/mL according to the tested pathogen and tested phenolic. It was declared that B. cereus was generally resistant for the doses inhibit S. aureus. However, there was no significant difference between the resistances of the tested pathogens in present study and it was concluded as the combine effect of all phenolic compounds in the extracts instead of testing them one by one. The antimicrobial effect of cranberry juice, cranberry extracts, apolar phenolic compounds and anthocyanins on Enterococcus faecium, E. coli O157:H7, E. coli, L. monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium and S. aureus was investigated. L. monocytogenes, E. coli O157:H7 and Salmonella Typhimurium were more resistant to the antibacterial activity of the cranberry extracts. The results indicated that the cranberry extract containing apolar phenolic compounds were not the most efficient at inhibiting the growth of the tested pathogens. However, cranberry extract containing more water-soluble phenolic compounds, low-molecular-weight phenolic acids, and flavonoids had ^te et al., 2011). showed the greatest antibacterial properties (Co Perumalla and Hettiarachchy (2011) reviewed the antimicrobial
Table 3 MIC test results of the red cabbage extracts. Test microorganism
Inoculum dose (log CFU/mL)
Conventional extraction (75 min/40 C/42.39% ethanol)
Listeria monocytogenes Staphylococcus aureus Escherichia coli Salmonella Typhimurium Bacillus cereus
3 6 3 6 3 6 3 6 3 6
1:2 1:1 1:2 1:1 1:2 1:1 1:2 1:2 1:2 1:2
a
(50%)a (100%) (50%) (100%) (50%) (100%) (50%) (50%) (50%) (50%)
Ultrasonic extraction (75 min/40 C/42.39% ethanol) 1:2 1:1 1:1 1:1 1:2 1:2 1:2 1:1 1:2 1:1
(50%) (100%) (100%) (100%) (50%) (50%) (50%) (100%) (50%) (100%)
% Concentration is given in the parentheses.
€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101
€ven et al. / LWT - Food Science and Technology xxx (2015) 1e6 A. Demirdo
5
Table 4 MIC test results of the sour cherry pomace extracts. Test microorganism Inoculum dose (log CFU/mL) Conventional extraction
Ultrasonic extraction
(60 min/40 C/40% ethanol) (40 min/40 C/40% ethanol) (60 min/40 C/40% ethanol) (40 min/40 C/40% ethanol) Listeria monocytogenes Staphylococcus aureus Escherichia coli Salmonella Typhimurium Bacillus cereus a
3 6 3 6 3 6 3 6 3 6
1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:32 1:16
(6.25%)a (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (3.125%) (6.25%)
1:16 1:16 1:16 1:16 1:16 1:8 1:16 1:16 1:32 1:16
(6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (12.5%) (6.25%) (6.25%) (3.125%) (6.25%)
1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:32
(6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (3.125%)
1:16 1:16 1:16 1:32 1:32 1:16 1:16 1:16 1:32 1:32
(6.25%) (6.25%) (6.25%) (3.125%) (3.125%) (6.25%) (6.25%) (6.25%) (3.125%) (3.125%)
% Concentration is given in the parentheses.
effect of grape seed extracts on food borne microorganisms (L. monocytogenes, Salmonella Typhimurium, S. aureus, B. cereus, Enterobacter sakazakii, E. coli O157:H7, Aeromonas hydrophila) and the antimicrobial effect of the extracts was demonstrated with their phenolic compounds. 4. Conclusion Ultrasound is a powerful tool which can efficiently improve the extraction performance of anthocyanins. However, UE did not increase the effectiveness of the antimicrobial activity. The single extraction step was applied to both CE and UE, but the amount of the extracts could be increased by several extraction steps. However, it should be considered that the single extraction step will be required for industrial applications and results of this study will have significant contributions. The SSC of the RC and SCP extracts were diluted to achieve the real SSC values of the food source for modeling real food system. It caused the dilution of the antimicrobial content too. The antimicrobial effect of the extracts on tested pathogens could be increased by using concentrated extracts. As a conclusion, these results indicate that RC and SCP extracts are available as natural antimicrobial agents to prevent food borne outbreaks related to E. coli, S. aureus, L. monocytogenes, Salmonella Typhimurium and B. cereus. When the increasing demand of the consumers for natural food additives/preservatives is concerned, these kinds of natural antimicrobial agents would be good alternatives for the food industry to produce safe and natural food products. On the other hand, the addition of plant based anthocyanins extracts in food formulations could improve the functionality of them. However, further studies designed to model real food environment are needed to detect their inhibition value for contaminated food products. Acknowledgments € an and Handan The authors wish to thank to Kenan Ozdo g an for the valuable contributions of them. The physicoAydog chemical analysis of this study was supported by Gaziosmanpas¸a University, Scientific Research Projects, 2012/110 (Tokat, Turkey). References Ali, S., Masud, T., & Abbasib, K. S. (2011). Physico-chemical characteristics of apricot (Prunus armeniaca L.) grown in northern areas of Pakistan. Scientia Horticulturae, 130, 386e392. Alzoreky, N. S., & Nakahara, K. (2003). Antibacterial activity of extracts from some edible plants commonly consumed in Asia. International Journal Food Microbiology, 80, 223e230. AOAC. (1995). Official methods of analysis of AOAC International (16th ed.). Basu, A., Rhone, M., & Lyons, T. J. (2010). Berries: emerging impact on cardiovascular health. Nutrition Reviews, 68, 168e177.
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€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101
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Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food borne pathogens and their antioxidant properties € € ven, S¸eniz Karabıyıklı*, Kader Tokatlı, Nilgün Oncül Aslıhan Demirdo Gaziosmanpas¸a University, Faculty of Engineering and Natural Science, Food Engineering Department, 60000 Tasliçiftlik, Tokat, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 January 2015 Received in revised form 24 March 2015 Accepted 25 March 2015 Available online xxx
Anthocyanins, known for their antioxidant characteristics, also have antimicrobial effects. Antimicrobial, antioxidant and some physicochemical properties of red cabbage (RC) and sour cherry pomace (SCP) anthocyanin extracts were investigated. Conventional (CE) and ultrasonic extraction (UE) methods were used. The antioxidant activities of samples extracted by UE were higher than those extracted by CE. Antimicrobial effects of the extracts were determined by the detection of minimum inhibitory concentration (MIC) values for Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella Typhimurium and Bacillus cereus. The extracts were inoculated (6 and 3 log CFU/mL) with five different pathogens, separately. All the extracts have antimicrobial effects on the tested pathogens and the results ranged depending on the concentration of the extracts and inoculation dose of the pathogen. The extraction method did not affect the inhibitive characteristics of the extract, and it was concluded that the antimicrobial effects of the extracts were mainly dependent on the anthocyanin content of the materials. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Red cabbage Sour cherry pomace Anthocyanin Antimicrobial MIC
1. Introduction There is an increasing demand for natural, safe and functional foods in the last decade. Consumers are interested in safe foods which have positive contributions to health, but contain no chemical additives or preservatives. However, keeping the food safety stable until the expiration date is not possible without the addition of any preservatives or application of thermal processing, which causes deterioration of functional ingredients. In this view, plant extracts, which could be used for natural colorant, antioxidant, flavoring or antimicrobial agents, are good alternatives for the food industry. Polyphenol rich plants especially are gaining popularity as a good source for anthocyanins. Anthocyanins are bioactive compounds present in many fruits, vegetables and their products. They are a sub-group within the flavonoids, characterized by a C6eC3eC6-skeleton (Patras, Brunton, O'Donnellb, & Tiwari, 2010). Antioxidant and antimicrobial properties of anthocyanins were
* Corresponding author. Gaziosmanpas¸a University, Faculty of Engineering and Natural Sciences, Department of Food Engineering, 60100, Tokat, Turkey. Tel.: þ90 356 2521616x2899; fax: þ90 356 2521729. €ven), E-mail addresses: [email protected] (A. Demirdo [email protected], [email protected] (S¸. Karabıyıklı), kader. € [email protected] (K. Tokatlı), [email protected] (N. Oncül).
^ te , Sylvain, & Lacroix, studied by several researchers (Caillet, Co ~es, 2012; Martin et al., 2012; Da Silva, Maquiaveli, & Magalha 2012; Oliveira et al., 2013; Park, Biswas, Phillips, & Chen, 2011; Rockenbach et al., 2011; Schved, Henis, & Juven, 1994; SmithPalmer, Stewart, & Fyfe, 1998). Anthocyanins could fight against chronic diseases, such as neuronal and cardiovascular illnesses and diabetes, among others (Konczak & Zhang, 2004). Anthocyanins may offer anti-inflammatory, anti-viral, and anti-cancer benefits (Ali, Masud, & Abbasib, 2011; Basu, Rhone, & Lyons, 2010; Cassidy et al., 2011; Faria et al., 2010; Ghosh & Konishi, 2007; Prior et al., 2008). Also, they have anti-carcinogenic effects because of their effective protection against oxidative damage by acting as an antioxidant. Antioxidant mechanisms of anthocyanins also make them available to extend the shelf life of industrial food products. The anthocyanin composition of red cabbage is very complex and the dominant structures are: cyanidin-3,5-diglucoside and cyanidin-3-sophoroside-5-glycoside acylated with sinapic acid, ferulic acid, p-coumaric acid, caffeic acid or malonic acid (Dyrby, Westergaard, & Stapelfeldt, 2001). Turkey is one of the biggest sour cherry cultivators in the world (FAOSTAT, 2010) and the pomace of the juice processing is a potential and substantial waste for the food industry. Four types of anthocyanin compounds were determined in sour cherries: cyanidin-3-glucosylrutinoside, cyanidin-3-sophoroside, cyanidin-3-rutinoside and cyanidin-3-
http://dx.doi.org/10.1016/j.lwt.2015.03.101 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101
€ven et al. / LWT - Food Science and Technology xxx (2015) 1e6 A. Demirdo
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glucoside (Chandra, Rana, & Li, 2001; Kim, Heo, Kim, Yang, & Lee, 2005). Food borne illnesses are still a major problem all over the world, even in developed countries. Outbreaks of human infections associated with food consumption increased in frequency during the past decade (Beuchat, 2002; CDC, 2014). It was reported that 6 to 81 million cases of illnesses, and up to 9000 deaths, annually are attributed to food borne pathogens in the USA (Alzoreky & Nakahara, 2003). Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella Typhimurium and Bacillus cereus are the most common food borne pathogens (Beuchat, 1996; CDC, 2013). Various chemical antimicrobials have been used to prevent food borne infections/intoxications in the food industry. However, chemical preservatives are discountenanced nowadays as a result of increasing awareness of the consumers on the adverse effects of chemical preservatives on human health. The numbers of the studies on natural alternatives are increasing as a natural consequence of requirement by the food industry. New, available, natural and effective alternatives are required to satisfy the consumers and to supply their demand. The objective of this study was to investigate the antimicrobial, antioxidant and physicochemical properties of sour cherry pomace and red cabbage anthocyanin extracts, which were extracted by conventional and ultrasonic extraction methods. 2. Materials and methods 2.1. Materials Whole red cabbages (RC) and sour cherry pomace (SCP) were used as raw material. The red cabbages were obtained from BafraSamsun/TURKEY and sour cherry pomace was purchased from Dimes Concentrated Fruit Juice Company (Tokat, TURKEY). They were stored at þ1 C maximum for 48 h before processing. 2.2. Processing methods 2.2.1. Extraction of red cabbage and sour cherry pomace anthocyanins Sorting, washing, cutting and chopping operations were carried out for red cabbages. Red cabbages were chopped (Moulinex FP 519G-750 W) at 500 rev/min under standardized conditions determined by pre-trials. Sour cherry pomace was used after separation of stones and stems. The extraction process was carried out using solideliquid extraction method. 1% formic acid was used to acidify the environment during the extractions. An ethanol/water/ formic acid mixture was used as the solvent in all extraction applications. Extractions were carried out with conventional and ultrasonic methods. The raw material amount that was determined by pre-trials was mixed with extraction solvent (red cabbage 1/3; m/v and sour cherry pomace 1/15; m/v). The mixture was homogenized at the fourth speed position for 45 s (Ultra-Turrax IkaWerke, Staufen, Germany). Conventional and ultrasonic extractions were applied after the homogenization process. The obtained mixture was filtered (Whatman no:1, 125 mm) under a vacuum (Milipore, WP6122050, Germany). The extraction solvent was evaporated in a rotary evaporator at 50 C. Single extraction step was used in all samples and for all methods. 2.2.2. Conventional and ultrasonic extractions Conventional extractions (CE) were done by using a water bath (Memmert-WB22) whereas ultrasonic extractions (UE) were carried out using an ultrasound bath operating at 37 kHz (ElmasonicS100H). Temperatures were controlled with a digital thermometer
(Shaanxi Taurus, China) during extractions to stabilize the desired temperature. Extraction conditions of RC anthocyanins were optimized by Response Surface Method (RSM). A Box-Behnken design was used in designing the CE and UE treatments of three variables with six center points (Design Expert 7.0.0 STAT-EASE, 2005). Extraction conditions of SCP anthocyanins were determined by pre-trials. After production of anthocyanin extracts, all samples were stored at 80 C to prevent the deterioration and interaction between the compounds of extracts until analyses. All samples were held at þ4 C to thaw for a night before analyzing. The soluble solid content of the extracts was adjusted based on the natural soluble solid content values of the materials before the analyses (6 Bx for RC and 12 Bx for SCP). 2.3. Methods of analysis 2.3.1. Physicochemical analysis Total anthocyanin contents (TAC) of samples were detected by pH-differential method using two buffer systems: potassium chloride buffer, pH 1.0 (0.025 M) and sodium acetate buffer, pH 4.5 (0.4 M). TAC of samples were determined at 535 nm and (mg cyanidin-3-glucoside/100 mL of extract) were calculated with molar absorptivity of cyanidin-3-glucoside (26.900) (Glassgen, Wray, Dieter, Metzger, & Seitz, 1992). Total phenolic content (TPC) was detected at 760 nm using the Folin-Ciocalteu reagent, diluted 10 fold before use (Sigma Chemical Co., St. Louis, MO) with gallic acid (3,4,5-trihydroxybenzoic acid) as the standard and measured after reaction (2 h) (Franke, Chless, Silveria, & Robensam, 2004). Antioxidant activities of the samples were determined by the ABTS [2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] assay (Re et al., 1999). Soluble solid contents (SSC) of samples were measured with a refractometer at 20 C (RFM 330, U.K.) (AOAC, 1995). Total titrable acidity (TA) was determined by means of a potentiometric titration of the acidity of the samples by placing 10 g of sample into 90 mL of deionized water. After filtration, 10 mL filtrate was titrated up to pH 8.1 (pH meter, WTW InoLab, Weilheim, Germany) with 0.1 N NaOH. The results were expressed as g/100 mL (citric acid) (AOAC, 1995). pH values of samples were determined by pH-meter at 20 C (AOAC, 1995). Water activity (aw) values of the samples were measured by AquaLab Model Series 3 TE, USA (AOAC, 1980). 2.3.2. Microbiological analysis The microflora of the extracts was controlled before MIC analyses by enumeration of total mesophilic aerobic bacteria (TMAB, FDA-BAM online, 2001a) and yeast & mold (Y&M, FDA-BAM online, 2001b). The safety of the extracts were checked out by E. coli (FDABAM online, 2013), S. aureus (ISO 6888, 2004), L. monocytogenes (ISO 11290, 1996), Salmonella spp. (FDA-BAM online, 2014) and B. cereus (FDA-BAM online, 2012) analyses. The antimicrobial effects of the RC and SCP extracts were tested against some food borne pathogens. E. coli (ATCC 25922), S. aureus (ATCC 25923), L. monocytogenes (ATCC 19115), Salmonella Typhimurium (ATCC 14028) and B. cereus (ATCC 10876) were used as test microorganisms. Two different inoculum doses were applied: low (~3 log CFU/mL) and high (~6 log CFU/mL) to detect the responses of the RC and SCP extracts for different contamination levels. Each of the test cultures were grown at 37 C for 18e24 h in Brain Heart Infusion Broth (BHI, pH 7.4 ± 0.2, LabM-LAB049), and then diluted with 0.1% sterile peptone water (PW, pH 6.3 ± 0.2, Oxoid-L37) to achieve a final inoculum of 6.0 log CFU/mL (high inoculum dose) and 3.0 log CFU/mL (low inoculum dose), appropriately. The MIC value of the each extract was determined by modification of the method described by LaBombardi, Sotos, Allen, and Sullivan (2008).
€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101
€ven et al. / LWT - Food Science and Technology xxx (2015) 1e6 A. Demirdo
200 mL of extract was placed into the first well of a 96-well microtiter panel. 100 mL BHI Broth was placed into each of the wells from 2nd to 12th in the row. 100 mL of the extract in the first well was taken and placed into the 2nd well and mixed with BHI Broth. 100 mL was taken from this mix in the 2nd well and placed into the 3rd well. Serial dilutions were made from the 2nd well to the 11th well by this method described shortly. 100 mL of the mixture was taken from the 11th well and was discarded. The 12th well which contains no extract was used as a positive control to detect the microbial growth. The final concentrations of extracts in the wells were; 1:1 (100%), 1:2 (50%), 1:4 (25%), 1:8 (12.50%), 1:16 (6.25%), 1:32 (3.13%), 1:64 (1.56%), 1:128 (0.78%), 1:256 (0.39%), 1:512 (0.20%), 1:1024 (0.10%), and the control (0%). After dilution, 100 mL of test culture was inoculated into each well from 1st to 12th. This procedure was repeated for each extract, each test microorganism and each inoculum dose, separately. The MIC plates were incubated at 37 C for 24e48 h and the survival of the test microorganism was checked by plating on Brain Heart Infusion Agar (BHI, pH 7.4 ± 0.2, LabM-LAB048). The petri dishes were incubated at 37 C for 18e24 h then the results were recorded. Although, the microflora of the extracts were controlled to detect the presence of competitive microorganisms, the initial microflora of the extracts were analyzed by surface plating on BHI Agar for each treatment as a negative control. Three replicate trials with 2 parallels were carried out for each experiment. 2.4. Statistical methods The results were statistically analyzed by ANOVA using the software SPSS 13 (SPSS Inc., Chicago, IL, USA) with the Duncan test to evaluate differences between treatments at p < 0.05. Each experiment was repeated at least three times. Means and standard deviations of the results were calculated for physicochemical analyses. 3. Results and discussion Extraction conditions of RC anthocyanins were optimized by RSM, and time, temperature and ethanol concentration were selected as independent variables in both methods (UE and CE) in order to optimize extraction conditions. Total anthocyanin contents were determined after CE and UE applications. The response selection was determined according to the production conditions to provide maximum anthocyanin content. The effects of extraction time (5e75 min), temperature (40e80 C) and ethanol concentration (5e75%) (independent variables) were investigated to evaluate anthocyanin content (response) of RC extracts. The analysis of variance showed that the adjusted second order models were significantly fitted to the experimental data, as indicated by the regression model F values of 27.495 (p < 0.001) for CE and 8.85 (p < 0.001) for UE. The extraction time, temperature and ethanol concentration were found to be significantly important for anthocyanin content at 95% confidence interval by ANOVA. Lack of fit of experimental data was not significant (p > 0.05) for both models (CE and UE). The coefficient of variation for the CE model was 5.07%, and it was 10.07% for the UE model. The models of CE and UE showed an adequate precision of 13.886 and 10.724, respectively. The determination coefficient (R2) for CE model was 0.9687 and it was 0.9087 for UE, while the adjusted determination coefficient (Adjusted R2) values were 0.9334 and 0.8960, respectively. The solutions were obtained for the optimum covering criteria by applying the desirability function method. Optimum extraction conditions were: 75 min, 40 C and 42.39% ethanol concentration for CE and UE. The results indicated that the low temperature and long application time with moderate ethanol concentration could
3
increase extraction of anthocyanins. The desirability values for CE and UE were 94.5% and 89.5%, respectively. The detailed optimization data was not declared in this study and it will be presented in further studies. The results of pre-trials for SCP indicated that the low temperature (40 C) and short application time (40 min and 60 min) with moderate ethanol concentration (40%) could increase the extraction of anthocyanins for CE and UE methods. Physicochemical characteristics of the RC extracts are shown in Table 1. TAC, TPC and antioxidant activity are important parameters for these kinds of plant extracts. The highest TAC and TPC contents were found in CE samples, while the highest antioxidant activity value was found in UE samples. TAC values for CE and UE groups were found as 593 ± 0.9 mg/L and 546 ± 0.6 mg/L, respectively. TPC was found in CE sample as 16,320 ± 5.24 mg/L, while it was 15,470 ± 7.43 mg/L in UE groups. Antioxidant activity of UE samples was found as 41.27 ± 0.24 mM Troloks/mL and it was 36.25 ± 0.32 mM Troloks/mL for CE samples. High antioxidant activity values of UE samples can be explained by the extraction of other antioxidants such as vitamins (C, B6, K). The difference between TAC, TPC and antioxidant activity of CE and UE samples was statistically significant (p < 0.05). The pH values of the samples were 3.53 and the TA was determined as 0.98 g/100 g and 1.045 g/ 100 g for UE and CE groups, respectively (p > 0.05). The difference in aw (0.156 for CE and 0.1719 for UE) of extracts was statistically significant (p < 0.05). The results of physicochemical analyses of the SCP extracts, produced by CE and UE methods, are shown in Table 2. There was no statistically significant difference between the TAC values of SCP extracts (p > 0.05). Lower TPC (453.27 ± 7.42 mg/L) and antioxidant activity (59.61 ± 1.24 mM Troloks/mL) values were determined for 40 min at 40 C with 40% ethanol concentration in CE groups. Antioxidant activity values of UE samples were found as 106.80 ± 0.71 mM Troloks/mL (60 min at 40 C with 40% ethanol concentration) and 105.87 ± 0.20 mM Troloks/mL (40 min at 40 C with 40% ethanol concentration). High antioxidant activity values of samples can be explained by extraction of other antioxidants such as phenolics, organic acids and vitamins etc. TA values were between 6.06 and 8.80 g/100 g and pH values were between 2.37 and 2.44. There was no statistically significant difference between the pH values (p > 0.05) and aw values in the same extraction conditions for SCP extracts (p > 0.05). Ghafoor, Choı, Jeon, and Jo (2009) determined maximum anthocyanin content as 2.28 mg/mL from grape seeds by UE (52.35% ethanol, 55.13 C, 29.49 min). The UE conditions for anthocyanin extraction from red raspberry were optimized (solvent/sample: 4/1, 200 s, 400 W) by Chen et al. (2007) and the anthocyanin amount in extract was calculated as 78.13%. Solvent concentration and extraction time are important for anthocyanin extraction from plant materials (Wang, Sun, Cao, Tian, & Li, 2008). The use of UE due to the ultrasound wave breaks the cells of the vegetal matrix and it leads to release contents of the cells into the extraction medium (Vinatoru et al., 1997). Also, solideliquid extraction is a mass transport phenomenon in which solids contained in a matrix migrate into a solvent that is brought into contact with the matrix (Ghafoor et al., 2009). This phenomenon can be enhanced with changes in diffusion coefficients induced by ultrasound and extraction temperature (Corrales, Garcia, Butz, & Tauscher, 2009). Naturally existing microflora of the extracts were controlled by enumeration of TMAB, Y&M and detection of the viability of test pathogens at the beginning of each treatment and no viable cells were detected. The antimicrobial content, low pH environment of the extracts and the conditions of the extraction resulted in microbiologically safe extracts.
€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101
€ven et al. / LWT - Food Science and Technology xxx (2015) 1e6 A. Demirdo
4
Table 1 Physicochemical characteristics of the red cabbage extracts. Parameter
Conventional extraction (75 min/40 C/42.39% ethanol)
Total anthocyanin (mg/L) Total phenolic (mg/L) Antioxidant activity (mM Troloks/mL) Total titrable acidity (g/100 g) pH Water activity
593 16,320 36.25 1.04 3.53 0.156
± ± ± ± ± ±
Ultrasonic extraction (75 min/40 C/42.39% ethanol)
0.9a 5.24a 0.32a 0.2a 0a 0.001a
546 15,470 41.27 0.98 3.53 0.171
± ± ± ± ± ±
0.6b 7.43b 0.24b 0.2a 0a 0.002b
Results are the means ± SD (n ¼ 3); statistically significant difference shown levels a, b (P 0.05).
Table 2 Physicochemical characteristics of the sour cherry pomace extracts. Parameter
Conventional extraction
Total anthocyanin (mg/L) Total phenolic (mg/L) Antioxidant activity (mM Troloks/mL) Total titrable acidity (g/100 g) pH Water activity
Ultrasonic extraction
(60 min/40 C/40% ethanol)
(40 min/40 C/40% ethanol)
(60 min/40 C/40% ethanol)
a
36.01 ± 2.38 493.09 ± 10.8b 95.55 ± 2.15b
a
35.08 ± 1.06 453.27 ± 7.42a 59.61 ± 1.24a
a
(40 min/40 C/40% ethanol)
36.90 ± 0.87 488.21 ± 6.7b 106.80 ± 0.71d
38.20 ± 1.20a 493.84 ± 5.12b 105.87 ± 0.20c
7.68 ± 0.13c 2.43 ± 0.07a 0.356 ± 0.01a
6.06 ± 0.22a 2.44 ± 0.07a 0.471 ± 0.02b
8.80 ± 0.09d 2.41 ± 0.07a 0.364 ± 0.01a
7.45 ± 0.16b 2.37 ± 0.07a 0.433 ± 0.02b
Results are the means ± SD (n ¼ 3); statistically significant difference shown levels a, b (P 0.05).
There were no significant differences between the results of the two different extraction methods on the inhibition of the tested pathogens. However, SCP extracts inhibited the tested pathogens at lower concentrations than the RC extracts (Tables 3 and 4). The MIC results of the SCP were between 1:8 and 1:32 while they were between 1:1 and 1:2 for RC extracts. On the other hand, there were no significant differences between the responses of the pathogens. Both extracts have similar antimicrobial effects on the test microorganisms and the cell wall structures, or sporulation mechanisms of the microorganisms, did not affect the MIC results. The results showed that both RC and SCP extracts inhibited the growth of the tested microorganisms for both inoculum doses (6 and 3 log CFU/ mL). The pH value was increasing and the concentration of the antimicrobial content of the extract decreased from the first well to the last one as a result of MIC test principles. So, the antimicrobial effects of the extracts mainly depend on rich phenolic content. Additionally, the dilution of the extracts to adjust the desired SSC caused dilution of the antimicrobial content. Antimicrobial effects of some products containing pomegranate extracts on three Streptococci strains (mutans ATCC 25175, sanguis ATCC 10577 and mitis ATCC 9811) were investigated and the MIC values ranged from 1:1 (100%) to 1:1024 (0.04875%) (Vasconcelos et al., 2006). MIC of polyphenol-rich sour pomegranate sauce (itself, not extracts) to inhibit S. aureus (ATCC 25923) and E. coli O157:H7 ranged between 1:8 (6.35%)e1:256 (0.195%) and 1:8
(6.35%)e1:128 (0.39%), respectively (Kıs¸la & Karabıyıklı, 2013). The antimicrobial effect of the pomegranate correlated with its phenolic content as in the present study. MICs of seven synthetic phenolic compounds against S. aureus, B. cereus and Pseudomonas rrez-Larraínzar, Rúa, de Arriaga, fluorescens were studied by Gutie del Valle, and García-Armesto (2013) and the MIC values ranged between 100 mg/mL and >1600 mg/mL according to the tested pathogen and tested phenolic. It was declared that B. cereus was generally resistant for the doses inhibit S. aureus. However, there was no significant difference between the resistances of the tested pathogens in present study and it was concluded as the combine effect of all phenolic compounds in the extracts instead of testing them one by one. The antimicrobial effect of cranberry juice, cranberry extracts, apolar phenolic compounds and anthocyanins on Enterococcus faecium, E. coli O157:H7, E. coli, L. monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium and S. aureus was investigated. L. monocytogenes, E. coli O157:H7 and Salmonella Typhimurium were more resistant to the antibacterial activity of the cranberry extracts. The results indicated that the cranberry extract containing apolar phenolic compounds were not the most efficient at inhibiting the growth of the tested pathogens. However, cranberry extract containing more water-soluble phenolic compounds, low-molecular-weight phenolic acids, and flavonoids had ^te et al., 2011). showed the greatest antibacterial properties (Co Perumalla and Hettiarachchy (2011) reviewed the antimicrobial
Table 3 MIC test results of the red cabbage extracts. Test microorganism
Inoculum dose (log CFU/mL)
Conventional extraction (75 min/40 C/42.39% ethanol)
Listeria monocytogenes Staphylococcus aureus Escherichia coli Salmonella Typhimurium Bacillus cereus
3 6 3 6 3 6 3 6 3 6
1:2 1:1 1:2 1:1 1:2 1:1 1:2 1:2 1:2 1:2
a
(50%)a (100%) (50%) (100%) (50%) (100%) (50%) (50%) (50%) (50%)
Ultrasonic extraction (75 min/40 C/42.39% ethanol) 1:2 1:1 1:1 1:1 1:2 1:2 1:2 1:1 1:2 1:1
(50%) (100%) (100%) (100%) (50%) (50%) (50%) (100%) (50%) (100%)
% Concentration is given in the parentheses.
€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101
€ven et al. / LWT - Food Science and Technology xxx (2015) 1e6 A. Demirdo
5
Table 4 MIC test results of the sour cherry pomace extracts. Test microorganism Inoculum dose (log CFU/mL) Conventional extraction
Ultrasonic extraction
(60 min/40 C/40% ethanol) (40 min/40 C/40% ethanol) (60 min/40 C/40% ethanol) (40 min/40 C/40% ethanol) Listeria monocytogenes Staphylococcus aureus Escherichia coli Salmonella Typhimurium Bacillus cereus a
3 6 3 6 3 6 3 6 3 6
1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:32 1:16
(6.25%)a (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (3.125%) (6.25%)
1:16 1:16 1:16 1:16 1:16 1:8 1:16 1:16 1:32 1:16
(6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (12.5%) (6.25%) (6.25%) (3.125%) (6.25%)
1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:16 1:32
(6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (6.25%) (3.125%)
1:16 1:16 1:16 1:32 1:32 1:16 1:16 1:16 1:32 1:32
(6.25%) (6.25%) (6.25%) (3.125%) (3.125%) (6.25%) (6.25%) (6.25%) (3.125%) (3.125%)
% Concentration is given in the parentheses.
effect of grape seed extracts on food borne microorganisms (L. monocytogenes, Salmonella Typhimurium, S. aureus, B. cereus, Enterobacter sakazakii, E. coli O157:H7, Aeromonas hydrophila) and the antimicrobial effect of the extracts was demonstrated with their phenolic compounds. 4. Conclusion Ultrasound is a powerful tool which can efficiently improve the extraction performance of anthocyanins. However, UE did not increase the effectiveness of the antimicrobial activity. The single extraction step was applied to both CE and UE, but the amount of the extracts could be increased by several extraction steps. However, it should be considered that the single extraction step will be required for industrial applications and results of this study will have significant contributions. The SSC of the RC and SCP extracts were diluted to achieve the real SSC values of the food source for modeling real food system. It caused the dilution of the antimicrobial content too. The antimicrobial effect of the extracts on tested pathogens could be increased by using concentrated extracts. As a conclusion, these results indicate that RC and SCP extracts are available as natural antimicrobial agents to prevent food borne outbreaks related to E. coli, S. aureus, L. monocytogenes, Salmonella Typhimurium and B. cereus. When the increasing demand of the consumers for natural food additives/preservatives is concerned, these kinds of natural antimicrobial agents would be good alternatives for the food industry to produce safe and natural food products. On the other hand, the addition of plant based anthocyanins extracts in food formulations could improve the functionality of them. However, further studies designed to model real food environment are needed to detect their inhibition value for contaminated food products. Acknowledgments € an and Handan The authors wish to thank to Kenan Ozdo g an for the valuable contributions of them. The physicoAydog chemical analysis of this study was supported by Gaziosmanpas¸a University, Scientific Research Projects, 2012/110 (Tokat, Turkey). References Ali, S., Masud, T., & Abbasib, K. S. (2011). Physico-chemical characteristics of apricot (Prunus armeniaca L.) grown in northern areas of Pakistan. Scientia Horticulturae, 130, 386e392. Alzoreky, N. S., & Nakahara, K. (2003). Antibacterial activity of extracts from some edible plants commonly consumed in Asia. International Journal Food Microbiology, 80, 223e230. AOAC. (1995). Official methods of analysis of AOAC International (16th ed.). Basu, A., Rhone, M., & Lyons, T. J. (2010). Berries: emerging impact on cardiovascular health. Nutrition Reviews, 68, 168e177.
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€ ven, A., et al., Inhibitory effects of red cabbage and sour cherry pomace anthocyanin extracts on food Please cite this article in press as: Demirdo borne pathogens and their antioxidant properties, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.101