2017 Volume 23 Issue 2 Pages 169-179
This study was conducted to characterize the bacteriostatic activities of aqueous garlic bulb- (GBE), ethanolic lemon peel- (ELPE), and ethanolic orange peel (EOPE) extracts against Escherichia coli K-12, E. coli O157:H7, Salmonella enterica serovars, Listeria monocytogenes, and Staphylococcus aureus in a tomato-based meat sauce. The maximum permissible supplementation (MPS) levels of GBE, ELPE, and EOPE in the meat sauce formulation determined through sensory evaluations were 6.0%, 3.0%, and 3.0%, respectively. However, the MPS values of ELPE and EOPE did not inhibit any of the test organisms in the subsequent antibacterial assays, with minimum inhibitory concentration (MIC) values of 6.0 – 9.0% (ELPE) and 7.5 – 10.0% (EOPE). On the other hand, GBE had an MIC values range of 1.0 – 2.5%. Thus, the study also determined the minimum inhibitory combination (MICo) values of GBE+ELPE and GBE+EOPE. Results showed that co-supplementations of the test extracts resulted in MICo values of 3.0% GBE +3.0% ELPE and 3.0% GBE +3.0% EOPE. Supplementation of the meat sauce with these MICo resulted in products with similar consumer acceptability scores as the non-supplemented control. Supplementation of inoculated meat sauce with the MICo of the test extracts exhibited bacteriostatic effect on selected pathogens in time-temperature abused (31°C, 8 h.) meat sauce.
The Philippine foodborne disease outbreaks summarized by Azanza (2006) involving cooked foods has been attributed to deviations in Good Hygienic Practices such as, poor quality ingredients, improper food storage temperature, inadequate cooking and post-cooking contaminations through the use of contaminated utensils and equipment. Majority of the reported outbreaks of foodborne illnesses from 1995 to 2004 involved meat-based home-cooked meals, particularly spaghetti with tomato-based meat sauce. The most common bacterial causative agents associated with different food products include Salmonella enterica serovars, staphylococcal enterotoxins, Vibrio parahaemolyticus, and Escherichia coli.
The occurrences of outbreaks due to contaminated foods may be addressed through the use of additives that can inhibit the proliferation of disease-causing microorganisms in the matrix. Chemical additives are used to preserve foods based on the efficacy of such compounds to treat diseases of humans, animals and plants (Forsythe, 2000; Jay, 2000). Traditional food additives that are Generally Regarded as Safe (GRAS) are chemically synthesized, and are used at levels stipulated in the General Standards for Food Additives of the Codex Alimentarius (Food and Agriculture Organization [FAO] and World Health Organization [WHO], 2006). There is however, an increasing concern on the possible long-term ill-effects of additives that deter consumers from patronizing such products; and consequently prompted for studies that look into possible alternatives. Consumer perception that chemically synthesized food antibacterials may be associated with toxicological problems has resulted in increased pressure on the food industry to use naturally occurring compounds as food additives (Lanciotti et al., 2003)
The plant based compound allicin, extracted from garlic, is probably the most investigated natural alternative food additive that has been shown to inhibit foodborne microorganisms in vitro (Alorainy, 2011; Ankri & Mirelman, 1999). Furthermore, citrus fruits have been demonstrated to contain potent antimicrobial agents against bacteria and fungi (Dubey et al., 2011). Several studies on the antimicrobial activities of plant parts of orange (Citrus sinensis) and lemon (Citrus limon) have been reported. The work by Penecilla and Magno (2011) demonstrated lemon and orange peel extracted from different solvents to possess antibacterial properties against food spoilage bacteria. Most studies on the efficacies of natural antibacterial agents are however, conducted in vitro, in laboratory media at levels that do not take into account possible effects of supplementation on the sensory characteristics of food products (Board & Gould, 1991; Shelef et al., 1984). In most studies, in order to achieve the efficacy exhibited by an additive in vitro, an increase in the supplementation level is necessary in real food systems. Such increase may lead to the alteration of the consumer acceptability of the supplemented product (Kabara & Eklund, 1991; Vigil et al., 2005).
This study aimed to determine the antibacterial activities of garlic bulb-, lemon peel-, and orange peel extracts against common foodborne organisms such as Escherichia coli O157:H7, Salmonella enterica serovars, Listeria monocytogenes, and Staphylococcus aureus in vitro and in a tomato meat sauce system. Specifically, the study aimed to establish the maximum permissible supplementation (MPS) levels of garlic bulb-, lemon peel, and orange peel extracts in a spaghetti food system through sensory evaluations. The study also determined the minimum inhibitory concentrations (MIC) and minimum inhibitory combinations (MICo) of these extracts against the test pathogens. The effects of these extracts on selected resistant pathogens artificially inoculated in the meat sauce under time and temperature abuse (subjected to temperature range optimal for microbial growth for prolonged period of time) was also determined.
Preparation of garlic bulb extract (GBE) The GBE was prepared following the method reported by Durairaj et al. (2009) with slight modifications. Fresh garlic bulbs were purchased from a local supermarket in Quezon City, Philippines. Three kilograms of garlic bulbs were manually desegmented into cloves, peeled and soaked in sodium hypochlorite solution (150 mg/L) with occasional agitation for 3 min. The disinfected garlic bulbs were rinsed with excess sterile distilled water twice and were homogenized using a pre-sanitized colloidal mill (Kolloldtechnik, Germany). The homogenized mixture was sequentially aseptically filtered through sterile cheesecloth and filter paper. The extract was collected in sterile amber bottle and was stored at 4°C until used in the study.
Preparation of ethanolic extracts orange and lemon peel extracts (EOPE and ELPE) The ethanolic extraction of lemon and orange peels was done based from the method described by Cui et al. (2010) with slight modifications. Fifty grams each of lemon and orange peels were obtained from the fresh fruits bought from a local supermarket in Quezon City. The peels were washed thoroughly in running water and were allowed to dry before comminuting with sterile scissors. The comminuted peels were suspended in 9 parts of 90% ethanol (Ajax Finechem) in sterile amber bottles for 48 h at 4°C. The residues of the peels were separated from the extract by aseptically passing the suspensions through two layers of sterile filter paper. The ELPE and ethanolic orange peel extract EOPE were collected separately in new sterile amber bottles and were stored at 4°C until used.
Determination of Maximum Permissible Supplementation (MPS) levels of extracts The MPS levels of the individual extracts were determined through a series of sensory evaluations. To narrow down the supplementation levels for testing, initial focus group discussions (FGD) were conducted involving n = 4 to 10 participants. Results of the FGDs showed that up to 6.0, 3.0 and 3.0% vol/vol ratio of GBE, ELPE, and EOPE, could be respectively added to spaghetti with tomato-based meat sauce (Luna, 2005), without significantly affecting the sensory characteristics of the product.
To validate the results obtained in the FGDs, the consumer acceptability of spaghetti with meat sauce with varying amounts of the extracts were determined. In the tests, the maximum levels tested were based on the initial findings established in the FGDs. This study adapted the spaghetti with meat sauce formulation reported by Luna (2005) with slight modifications. The formulation was consisted of 1:1 (wt/wt) meat sauce-to-pasta ratio. The meat sauce was consisted of tomato sauce, ground beef, and water at 10:5:3 (wt/wt/vol) ratio. The spaghetti sauce was prepared following the steps in the adapted procedure. The extracts were incorporated in freshly cooked spaghetti sauce (vol/vol) before mixing with the noodles. For GBE-supplemented spaghetti with meat sauce, 5.0 and 6.0% supplementation levels were tested. On the other hand, for ELPE and EOPE, 0.75, 1.50, 2.25, and 3.0% supplementation levels were tested. In the consumer acceptability tests, samples were evaluated by a n = 25-member panel for overall acceptability, aroma and flavor and using a 9-point Hedonic Scale. The Hedonic Scale is as follows: 9 – like extremely, 8 – like very much, 7 – like moderately, 6 – like slightly, 5 – neither like nor dislike, 4 – dislike slightly, 3- dislike moderately, 2 – dislike very much, and 1- dislike extremely. Members of the panel were also asked whether they could detect undesirable, off-flavor and off-odor in the samples. Sensory evaluations were conducted in duplicate and were held in the sensorium of the Pilot Food Plant of the College of Home Economics, University of the Philippines, Diliman. The MPS values were determined as the maximum amount of plant extracts that can be added to the meat sauce without resulting in consumer rejection of sensory attributes.
Microbial cultures and culture maintenance The study used a number of Gram-positive and Gram-negative foodborne bacteria as test organisms. Five strains of E. coli O157:H7 namely DT-66, CR-3, MN-28, MY-29 and HCIPH 96055 were tested individually. The first four strains were originally from The National Food Research Institute, Tsukuba, Japan; while the last strain was originally from Hiroshima City Institute of Public Health, Hiroshima City, Japan. The test E. coli K-12 (IFO 3301) was originally from the Institute of Fermentation, Osaka, Japan. The test Salmonella enterica included serovars Enteritidis (HCIPH B11), Infantis (C-269) and Montevideo. The last two Salmonella serovars were environment isolates originally maintained at the Laboratory of Food Microbiology and Hygiene, Hiroshima University (HU), Japan. The tested Methicillin-Resistant Staphylococcus aureus (MRSA) was similarly from HU. Finally, the test Listeria monocytogenes included serovars 4b (HCIPH AS-1) and 1/2c (HCIPH M24-1). In the preparation of the working cultures, two stabs from each of the refrigerated stock cultures were activated by transferring into 3-mL sterile nutrient broths (NB) (HiMedia) and incubating at 37°C for 24 h. Working stock cultures were prepared by obtaining loop inocula from each of the activated cultures, followed by transferring into 2-mL nutrient agar (NA) slants (HiMedia) and incubating at 37°C for 24 h. The working culture slants were subsequently stored at 4°C and were used within two weeks after preparation.
NB transfers of microbial cultures was carried out twice prior to being used in the determination of antibacterial activities of the test extracts. From the working slants, one stab each was transferred into individual 3-mL sterile NB and incubated at 37°C for 24 h. A loop inoculum was obtained from each of the 24-h enriched NB, transferred into new 3-mL sterile NB and incubated at 37°C for 24 h. In this study, only cultures subjected to this aforementioned consecutive activation and enrichment procedures were used in the antibacterial activity determinations.
Determination of Minimum Inhibitory Concentrations (MIC) of plant extracts The determinations of the minimum inhibitory concentrations of GBE, EOPE and ELPE against the test organisms were done based from the method described by Cui et al. (2010) with slight modifications. Freshly sterilized nutrient agars (115°C, 15 min) were prepared and tempered at 55°C prior to addition of the increasing extract concentrations. The extract-nutrient agar plates were prepared by replacing equal volumes of water in the original nutrient agar formulation with the respective extract concentrations. The supplemented NA were poured into sterile petri plates and were allowed to solidify at room temperature for 2 h prior to microbial inoculation. One loop inoculum of cells from each of the enriched NB cultures was streaked onto the corresponding extract-nutrient agar plates. Control plates were also prepared by inoculating one loop inoculum of cells from each of the enriched NB cultures in non-supplemented NA plates, and on NA plates that were supplemented with different amounts of ethanol, which corresponded to the introduced amounts of EOPE and ELPE. The inoculated plates were stored at 37°C and were observed after 24 and 48 h. All experiments were conducted in 2 independent runs, with 2 replications per run.
The antibacterial activities of the extracts were assessed by observing the colony formations or growth densities along the streaked agar region. In this study antibacterial activity was described as ‘non-inhibiting’ (+++), when the streaked inoculum exhibited full growth; ‘weakly inhibiting’ (++), when uncountable, distinct colonies emerged from the streaked region; ‘strongly inhibiting’ (+), when the streaked inoculum exhibited only a few, distinct and countable colonies; and ‘completely inhibiting’ (−), when the streaked inoculum did not exhibit growth. The MIC was determined based on the lowest concentration of extract, which resulted in complete inhibition in nutrient agar as shown in Figure 1.
Guide for interpreting antimicrobial activity of supplemented plant extracts in nutrient agar medium. In this study, the supplemented extract concentration was considered non-inhibiting when the streaked inoculum exhibited full growth (+ + +) after 24 h incubation, as demonstrated in (d, e, f, g, and h). The extract concentration was considered weakly inhibiting when uncountable distinct colonies emerged (+ +) from the streaked region, as shown in (b). Supplementation was considered strongly inhibiting when few, distinct, countable colonies emerged (+) from the streaked region, as shown in (c). When growth was not observed (−), the supplementation level was considered completely inhibiting. The minimum inhibitory concentration was determined as the lowest concentration of extract supplemented to the growth medium, which resulted in complete inhibition of the test organism. This figure shows different bacteria streaked onto NA plate supplemented with a fixed extract concentration. Differences in the growth of some streaked bacteria signify differences in resistance to the extract.
Determination of Minimum Inhibitory Combinations (MICo) of extracts The combined inhibitory effects of GBE+EOPE and GBE+ELPE were determined using the same method performed in the determination of the individual MICs. Various levels of GBE (1.0 to 3.0%) were co-supplemented with EOPE (3.0 to 10.0%) or ELPE (3.0 to 9.0%), and similarly tested against all organisms on NA. Plates containing only distilled water and ethanol were similarly streaked with all test organisms to serve as control. For each test organism, the MICo is the smallest vol/vol combination of GBE+EOPE or GBE+ELPE that resulted in complete growth inhibition (Figure 1).
Acceptability of spaghetti with meat sauce with GBE+EOPE and GBE+ELPE The established MICo values for GBE+EOPE and GBE+ELPE were finally applied to the same spaghetti with tomato based meat sauce. The evaluations of the general acceptability, as well as the acceptability of the aroma and flavor of the supplemented spaghetti with meat sauce were conducted by an n = 25-member panel; following previously described protocol. The evaluations were done in duplicate.
Validation of bacteriostatic effects of GBE+EOPE and GBE+ELPE in meat sauce To validate whether the established MICo values for the combinations GBE+EOPE and GBE+ELPE exhibit bacteriostatic activity in the real food system, the study supplemented meat sauces with the extract combinations at MICo levels. Only the previously identified most resistant strains/species were used as challenge organisms. Prior to artificial inoculations, each of the resistant test organisms was subjected to the previously described activation and enrichment cycle. Cells were harvested from each of the enriched cultures by spinning at 4000 rpm for 15 min. The supernatant liquid was decanted and the pellets were resuspended in 0.1% peptone (HiMedia) by brief vortex mixing. For test species that included multiple strains or serovars, a cocktail of inoculum was prepared. The resuspended pellets of the same species were combined in a sterile tube and vortex mixed prior to introduction to the meat sauce.
The meat sauce was prepared following previously described protocol (Luna, 2005). Prior to inoculation, 25 g aliquots of the meat sauce were dispensed in separate media bottles and thereafter subjected to sterilization by moist heat at 121°C for 15 min under 15 psi pressure. The sterilized sauce was allowed to equilibrate to ambient temperature (31°C) prior to introduction of 6 – 8 log cfu/mL of the challenge organisms. The inoculated meat sauces were then allowed to stand at ambient conditions for 8 h. Changes in the inoculated populations were monitored every 2 h. To determine the population of the challenge organism, one media bottle containing 25 g sauce was diluted with 225 mL 0.1% peptone water. The diluted system was further subjected to serial 10-fold dilution prior to surface plating 0.1 mL onto NA plates. Emerging colonies were enumerated after 24 h incubation at 35°C.
Statistical analyses Data obtained from sensory evaluations were subjected to Single-factor Analysis of Variance (ANOVA) in the General Linear Model Procedure of the SAS analytical software version 8.0 (SAS Institute, Inc., Cary, NC). Post-hoc analysis of significant difference among the samples was done using Duncan's Range Multiple Range Test (DMRT).
Consumer acceptability of spaghetti with meat sauce with individual plant extracts The plant extract supplemented spaghetti with meat sauce was evaluated by a consumer-type panel, the members of which was untrained, female students, aged 18 to 25 yr, and mostly consume pasta with meat sauce at least twice a month. The sample supplemented with 5.0% GBE was given the highest rating for the overall acceptability (Table 1). However, the score given to the sample supplemented with 6.0% GBE was not significantly different. In addition, panel members were able to detect a spicy but tolerable aftertaste from both supplemented samples. Allicin is the source of the familiar garlic odor, which is produced when the enzyme alliinase reacts with an odorless sulfur-containing amino acid alliin upon crushing of garlic (Brown, 2000). Crushed garlic imparts a stronger garlic taste than when sliced since the former has more exposed surface area to release the flavors from volatile oils. Garlic, together with onions, scallions, and leeks, is a common spice used to enhance flavors of many dishes (Greeley, 2009). Consequently, this study deemed 6.0% as the MPS level of supplementation for GBE tested in the subsequent antibacterial assays.
| Supplementation Levels | Consumer Acceptability Hedonic Scores1 per Quality Attribute | ||
|---|---|---|---|
| General Acceptability | Aroma Acceptability | Flavor Acceptability | |
| Garlic Bulb Extract | |||
| 0.00% | 6.16 ± 1.06a | 6.64 ± 1.23a | 6.14 ± 1.09a |
| 5.00% | 6.30 ± 1.06a | 6.70 ± 1.16a | 5.90 ± 1.52a |
| 6.00% | 6.00 ± 1.76a | 6.80 ± 1.69a | 6.00 ± 2.16a |
| Lemon Peel Extract | |||
| 0.00% | 6.30 ± 1.43ab | 6.26 ± 1.27a | 6.22 ± 1.37abc |
| 0.75% | 6.66 ± 1.33a | 6.24 ± 1.19a | 6.62 ± 1.41a |
| 1.50% | 6.42 ± 1.13ab | 6.50 ± 1.33a | 6.48 ± 1.18ab |
| 2.25% | 6.08 ± 1.12b | 6.38 ± 1.31a | 6.04 ± 1.21bc |
| 3.00% | 6.08 ± 1.12b | 6.22 ± 1.06b | 5.88 ± 1.47c |
| Orange Peel Extract | |||
| 0.00% | 6.54 ± 0.97a | 6.68 ± 0.89a | 6.50 ± 0.99a |
| 0.75% | 6.36 ± 0.92ab | 6.54 ± 0.93ab | 6.12 ± 1.17ab |
| 1.50% | 6.34 ± 0.92ab | 6.58 ± 0.78ab | 6.10 ± 1.28ab |
| 2.25% | 6.16 ± 0.74b | 6.50 ± 0.91ab | 5.94 ± 1.32b |
| 3.00% | 6.10 ± 0.73b | 6.22 ± 1.06b | 5.82 ± 0.85b |
The highest rating for the overall acceptability was given to the spaghetti sample supplemented with 0.75% ELPE. It should however be noted, that the overall acceptability score given to the sample with 3.0% ELPE was not significantly different from the non-supplemented control. Furthermore, the overall acceptability of all samples were given similar Hedonic ratings of ‘like slightly.’ It was also noted that sample supplemented with 3.0% ELPE had a significantly lower score for flavor acceptability among all tested supplementation levels, but the score was similarly not significantly different from the non-supplemented control. On the other hand, spaghetti sample not supplemented with EOPE was given the highest rating for overall acceptability. Spaghetti sample supplemented with 3.0% EOPE had a score significantly lower from all the tested supplementation levels, but the Hedonic ratings of all samples were in ‘like slightly.’
Lemon peel contains volatile oils, hesperidin, pectin, calcium oxalate and bitter substances. The volatile oils comprise of limonene, citral and other aromatic compounds like geranyl acetate and terpineol. These compounds are responsible for the strong, fragrant and aromatic odor as well as the aromatic and bitter taste of lemon peels (Kokate et al., 2008). With increased levels of ELPE supplementation, the panel detected increased aromatic and bitter characteristics in the supplemented samples, which could have resulted in decreased acceptability ratings. This study deemed 3.0% as an appropriate level of supplementation to be tested in the subsequent antibacterial assays. On the other hand, orange peels contain essential oils that are among the most important and most widely used flavoring ingredients in food, beverage, and other products. These essential oils consist primarily of D-Limonene and with remaining odorous constituents namely n-decylic aldehyde, citral, d-linalool, n-nonyl alcohol and traces of esters of formic, acetic, caprylic and capric acid (Remington, 2006). Orange peel oil produces the sweet and fresh top note with fruity aldehydic character in orange peel (Berger, 2007). Meanwhile the mild bitter note is due to the glycosidal compounds aurantiamarin and aurantimaric acid (Kokate et al., 2008). Decrease in the acceptability ratings was observed with increased levels of EOPE supplementation as spaghetti samples similarly acquired increased bitter compounds from EOPE detected by the test panel. However, this study still deemed 3.0% as an appropriate level of supplementation to be used in the subsequent antibacterial assays since the acceptability was not significantly different from all of the samples supplemented with lower concentrations of EOPE.
Individual antibacterial activities of GBE, ELPE, and EOPE against test organisms After the determination of the MPS levels of GBE, ELPE and EOPE in a spaghetti formulation, the individual MIC values of the extracts were determined in NA. Results (Tables 2–4) showed that GBE inhibited the various organisms with the addition of 1.0 to 2.5% extract. Complete inhibition of all test organisms at 2.5% level of supplementation was observed. The strains of E. coli and serovars of L. monocytogenes were found least resistant, S. aureus exhibited moderate resistance, while Salmonella enterica serovars were found most resistant to GBE. These results are different from those reported by Jabar & Al-Mossawi (2007), and Ankri & Mirelman (1999) where S. aureus exhibited less resistance against garlic extract than E. coli. Such difference may be attributed to the strain-dependent variation in the resistance of microorganisms towards antibacterial agents. Moreover, the MICs obtained for the tested strains of E. coli O157:H7 were close to the MIC of 1.56% (wt/vol) previously reported by Alorainy (2011). Ankri & Mirelman (1999) suggested that the wide spectrum antibacterial effects of allicin is due to the multiple inhibitory effects the compound may have on various thiol-dependent enzymatic systems. Furthermore, Feldberg et al. (1988) explained that the mechanism of action of allicin is through inhibition of RNA synthesis. In their work, DNA and protein syntheses in the bacterial cell were delayed and partially inhibited by allicin, while RNA syntheses were immediately and totally inhibited. Comparing the greatest MIC determined in this study (2.5%) and the MPS of GBE (6.0%), the supplementation can therefore be adjusted to levels between 2.5 and 6.0% to ensure safety against any of the tested organisms, without the significant alterations in the consumer acceptability of the sensory characteristics of the test spaghetti with meat sauce.
| Extract (%) | Antimicrobial Activities per microbial strain per species1 | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| E. coli | E. coli O157:H7 | Salmonella enterica | L. monocytogenes | S. aureus | |||||||||
| K-12 | |||||||||||||
| Lemon | Garlic | ||||||||||||
| Peel | Bulb | IFO | DT66 | CR3 | MN28 | MY29 | HCIPH | Enteritidis | Infantis | Montevideo | 1/2c | 4b | MRSA |
| 0.0 | 1.0 | +++ | −a | −a | −a | −a | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| 1.5 | −a | − | − | − | − | −a | +++ | ++ | +++ | −a | −a | ++ | |
| 2.0 | − | − | − | − | − | − | ++ | −a | +++ | − | − | −a | |
| 2.5 | − | − | − | − | − | − | −a | − | −a | − | − | − | |
| 3.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 3.0 | 0.0 | 0.0 | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| 1.0 | − | +++ | −b | - | +++ | +++ | −b | − | +++ | +++ | ++ | − | |
| 2.0 | +++ | −b | − | +++ | −b | −b | − | +++ | +++ | −b | +++ | +++ | |
| 3.0 | −b | − | − | −b | − | − | − | −b | −b | − | −b | −b | |
| 4.0 | 0.0 | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| 1.0 | +++ | −b | − | −b | +++ | +++ | −b | −b | − | +++ | −b | +++ | |
| 2.0 | +++ | − | +++ | − | −b | −b | − | − | +++ | +++ | − | −b | |
| 3.0 | −b | − | −b | − | − | − | − | − | −b | −b | − | − | |
| 5.0 | 0.0 | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| 1.0 | −b | −b | ++ | +++ | ++ | − | −b | −b | −b | +++ | ++ | −b | |
| 1.5 | − | − | −b | −b | −b | +++ | − | − | − | +++ | +++ | − | |
| 2.0 | − | − | − | − | − | −b | − | − | − | −b | −b | − | |
| 3.0 | − | − | − | − | − | − | − | − | − | − | − | ||
| 7.0 | 0.0 | −a | −a | −a | −a | −a | −a | −a | +++ | −a | −a | −a | +++ |
| 1.0 | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | |
| 2.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 3.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 9.0 | 0.0 | − | − | − | − | − | − | − | −a | − | − | − | −a |
| 1.0 | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | |
| 2.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 3.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| Extract (%) | Antimicrobial Activities per microbial strain per species1 | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| E. coli | E. coli O157:H7 | Salmonella enterica | L. monocytogenes | S. aureus | |||||||||
| K-12 | |||||||||||||
| Orange | Garlic | ||||||||||||
| Peel | Bulb | IFO | DT66 | CR3 | MN28 | MY29 | HCIPH | Enteritidis | Infantis | Montevideo | 1/2c | 4b | MRSA |
| 0.0 | 1.0 | +++ | −a | −a | −a | −a | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| 1.5 | −a | − | − | − | − | −a | +++ | ++ | +++ | −a | −a | ++ | |
| 2.0 | − | − | − | − | − | − | ++ | −a | +++ | − | − | −a | |
| 2.5 | − | − | − | − | − | − | −a | − | −a | − | − | − | |
| 3.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 3.0 | 0.0 | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| 1.0 | −b | ++ | +++ | +++ | +++ | +++ | ++ | +++ | +++ | +++ | ++ | +++ | |
| 2.0 | − | −b | −b | −b | −b | −b | −b | −b | −b | +++ | +++ | −b | |
| 3.0 | − | − | − | − | − | − | − | − | − | −b | −b | − | |
| 4.0 | 0.0 | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| 1.0 | −b | +++ | −b | +++ | +++ | −b | +++ | +++ | +++ | −b | +++ | +++ | |
| 2.0 | − | −b | − | −b | −b | − | −b | −b | −b | − | −b | −b | |
| 3.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 5.0 | 0.0 | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| 1.0 | +++ | +++ | +++ | ++ | +++ | −b | +++ | −b | +++ | −b | −b | +++ | |
| 2.0 | −b | −b | −b | +++ | −b | − | −b | − | −b | − | − | −b | |
| 3.0 | − | − | − | −b | − | − | − | − | − | − | − | − | |
| 6.0 | 0.0 | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ | +++ |
| 1.0 | ++ | −b | +++ | −b | −b | −b | +++ | − | +++ | −b | −b | ++ | |
| 1.5 | −b | − | −b | − | − | − | +++ | − | −b | − | − | −b | |
| 2.0 | − | − | − | − | − | − | −b | −b | − | − | − | − | |
| 3.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 8.0 | 0.0 | +++ | +++ | +++ | +++ | +++ | −a | −a | −a | −a | +++ | −a | +++ |
| 1.0 | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | |
| 2.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 3.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 10.0 | 0.0 | −a | −a | −a | −a | −a | − | − | − | − | −a | − | −a |
| 1.0 | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | −b | |
| 2.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| 3.0 | − | − | − | − | − | − | − | − | − | − | − | − | |
| Microorganisms | Lemon Peel Extract1 MIC (%) | Garlic MIC when combined with Lemon (%) | Orange Peel Extract1 MIC (%) | Garlic MIC when combined with Orange (%) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Ethanolic Lemon Peel Extract (%) | Ethanolic Orange Peel Extract (%) | ||||||||||||||
| 0.02 | 3.0 | 4.0 | 5.0 | 7.0 | 9.0 | 0.02 | 3.0 | 4.0 | 5.0 | 6.0 | 8.0 | 10.0 | |||
| E. coli K-12 | |||||||||||||||
| IFO | 6.0 | 1.5 | 3.0 | 3.0 | 1.0 | 1.0 | 1.0 | 9.0 | 1.5 | 1.0 | 1.0 | 2.0 | 1.5 | 1.0 | 1.0 |
| E. coli O157:H7 | |||||||||||||||
| DT-66 | 7.0 | 1.0 | 2.0 | 1.0 | 1.0 | 1.0 | 1.0 | 8.5 | 1.0 | 2.0 | 2.0 | 2.0 | 1.0 | 1.0 | 1.0 |
| CR-3 | 6.5 | 1.0 | 1.0 | 3.0 | 1.5 | 1.0 | 1.0 | 8.5 | 1.0 | 2.0 | 1.0 | 2.0 | 1.5 | 1.0 | 1.0 |
| MN-28 | 7.0 | 1.0 | 3.0 | 1.0 | 1.5 | 1.0 | 1.0 | 8.5 | 1.0 | 2.0 | 2.0 | 3.0 | 1.0 | 1.0 | 1.0 |
| MN-29 | 7.0 | 1.0 | 2.0 | 2.0 | 1.5 | 1.0 | 1.0 | 9.0 | 1.0 | 2.0 | 2.0 | 2.0 | 1.0 | 1.0 | 1.0 |
| HCIPH | 7.0 | 1.5 | 2.0 | 2.0 | 2.0 | 1.0 | 1.0 | 7.5 | 1.5 | 2.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
| S. enterica | |||||||||||||||
| Enteritidis | 7.0 | 2.5 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 7.5 | 2.5 | 2.0 | 2.0 | 2.0 | 2.0 | 1.0 | 1.0 |
| Infantis | 8.0 | 2.0 | 3.0 | 1.0 | 1.0 | 1.0 | 1.0 | 8.0 | 2.0 | 2.0 | 2.0 | 1.0 | 2.0 | 1.0 | 1.0 |
| Montevideo | 7.0 | 2.5 | 3.0 | 3.0 | 1.0 | 1.0 | 1.0 | 8.0 | 2.5 | 2.0 | 2.0 | 2.0 | 1.5 | 1.0 | 1.0 |
| L. monocytogenes | |||||||||||||||
| 1/2c | 7.0 | 1.5 | 2.0 | 3.0 | 2.0 | 1.0 | 1.0 | 9.5 | 1.5 | 3.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
| 4b | 7.0 | 1.5 | 3.0 | 1.0 | 2.0 | 1.0 | 1.0 | 8.0 | 1.5 | 3.0 | 2.0 | 1.0 | 1.0 | 1.0 | 1.0 |
| S. aureus | |||||||||||||||
| MRSA | 9.0 | 2.0 | 3.0 | 2.0 | 1.0 | 1.0 | 1.0 | 10.0 | 2.0 | 2.0 | 2.0 | 2.0 | 1.5 | 1.0 | 1.0 |
The observed results for ELPE and EOPE were however, different. At the MPS level of 3.0%, both extracts did not exhibit antibacterial effect against any of the tested organisms. Hence, to determine the MIC of the two extracts, higher supplementation levels in NA were tested. Results showed that ELPE inhibited the various organisms at supplementation range of 6.0 to 9.0%. Complete inhibition of all test organisms at 9.0% level of supplementation was observed. MRSA was most resistant to ELPE while strains of E. coli and L. monocytogenes serovars were least resistant. These results agree with those of Ashok Kumar et al. (2011), which reported that E. coli was less resistant towards ELPE than S. aureus. Dhanavede et al. (2011) specifically identified coumarin and tetrazene as the antibacterial compounds with broad spectrum activities in lemon peels through gas chromatography–mass spectrometry. Maxwell (1993) explained that the mechanism of action of coumarins is through inhibition of bacterial enzyme DNA gyrase. The compound binds to the B subunit of gyrase and inhibits DNA supercoiling by blocking the ATPase activity. Hardy & Cozzarelli (2003) explained that gyrase is primarily involved in supporting nascent chain elongation during replication of the microbial genome. It removes the positive supercoils caused by DNA replication and helps maintain the steady-state balance of negative supercoiling of the bacterial DNA.
Furthermore, results showed that EOPE inhibited the various bacteria at supplementation rates of 7.5 to 10%. Complete inhibition of all test organisms at 10.0% level of supplementation was observed. The test MRSA was most resistant to EOPE while Salmonella enterica serovars were most susceptible. These results also agree with those of Ashok Kumar et al. (2011) who reported S. aureus as more resistant towards EOPE than E. coli. D-Limonene is the main compound responsible for the antibacterial activity of orange peels. Limonene belongs to the cyclic monoterpene hydrocarbon family, which accumulates in the microbial plasma membrane, causing a loss of membrane integrity and dissipation of the proton motive force (Sikkema et al., 1994). Studies on E. coli inactivation by other terpenes and terpenoids have demonstrated the occurrence of sub-lethal injury in the outer and cytoplasmic membranes, implying membrane disruption as a mechanism of inactivation by these compounds (Espina et al., 2013).
Based from the data obtained, the study cannot generalize on the relative resistance of Gram-positive and Gram-negative bacteria however, previous studies explained that Gram-negative bacteria are generally more resistant than Gram-positive bacteria to actions by antibacterials obtained from plants due to the presence of lipopolysaccharide outer membrane in Gram-negative bacterium, preventing the diffusion of the hydrophobic compounds (Tajkarimi & Ibrahim, 2012). Furthermore, differences in susceptibility of bacterial strains from previous studies may be attributed to strain-to-strain variations of the microorganisms towards antibacterial agents. Furthermore, individual strains of a species may demonstrate genotypes and phenotypes different from the type strain hence development and detection of antibiotic resistance by specific bacterial strains can be expected (Vigil et al., 2005).
Combined antibacterial activities of GBE+ELPE and GBE+EOPE Based on the earlier results (Tables 2–4), the MIC values established for lemon- and orange peel extracts were higher than the MPS levels in the test spaghetti formulation. The MIC values are meaningless in terms of their utility, as supplementation of the test extracts at their respective MIC values will result in unacceptable products. Hence the study explored the possibility of combining the more strongly inhibiting GBE with ELPE or EOPE. Results showed varying inhibitory activities against the test organisms upon GBE+ELPE co-supplementation. The MIC of ELPE against the test organisms decreased with increasing co-supplementation with GBE. Without GBE, strains of E. coli O157:H7 were inhibited by ELPE at levels ≥6.5%. With 3.0% GBE co-supplementation, the MIC values for E. coli O157:H7 strains decreased to 3.0%. Similarly, MRSA was inhibited by ELPE alone at 9.0%. With 3.0% GBE co-supplementation, the ELPE MIC for MRSA decreased to 3.0%. In Table 2, it can be noted that the MIC of ELPE was reduced to 3.0% in all test organisms with 3% co-supplementation of GBE.
Varying inhibitory activities were likewise observed against the test organisms upon GBE+EOPE co-supplementation. In the absence of GBE, E. coli K-12 was inhibited by EOPE at 9.0% level of addition. With 3.0% GBE, the EOPE MIC for E. coli K-12 decreased to 3.0%. Similarly, serovars of Salmonella enterica were inhibited by EOPE without GBE co-supplementation at levels above ≥7.5%. With 3.0% GBE co-supplementation, the EOPE MIC values for Salmonella enterica serovars also decreased to 3.0%. Hence for both GBE+ELPE and GBE+EOPE co-supplementation, the MICo value of (3.0%+3.0%) was established. Supplementing the tested tomato-based meat sauce with any of these co-supplementation levels may potentially improve the safety of the product against any of the tested organisms. Vigil et al., 2005 explained that combined antibacterial agents act against specific species of a mixed microflora or on different metabolic elements within similar species or strains hence the use of combined antibacterial agents theoretically provide greater spectrum of activity, with increased antibacterial action against microorganisms. Furthermore it is worth noting that in Table 4 some levels of ELPE and EOPE supplementation, the GBE MIC increased suggesting possible antagonistic interaction of the extracts with respect to their combined effects on the test organisms. This interaction was not investigated in this current work, and should be looked into in future studies.
GBE+ELPE and GBE+EOPE co-supplementations in tomato based-meat sauce The effects of the established co-supplementation levels on the sensory attributes of spaghetti with meat sauce was similarly determined to validate the utility of the MICo values in real food systems. The consumer acceptability tests were conducted, employing an n = 25-member panel. Members of the panel were untrained and majority were female students between the ages 18 and 25, and mostly consumes pasta with meat sauce at least twice a month. Table 5 summarizes the results of the consumer acceptability tests on the acceptability of the test spaghetti formulation with (3.0% GBE, 3.0% ELPE) or (3.0% GBE, 3.0% EOPE) co-supplementation.
| Quality Attributes | Hedonic Ratings for Extract Co-supplementations1 | ||
|---|---|---|---|
| Control | Garlic Bulb-Lemon Peel | Garlic Bulb-Orange Peel | |
| Overall Acceptability | 5.98 ± 1.30a | 5.74 ± 1.44ab | 5.36 ± 1.47b |
| Aroma | 5.98 ± 1.35a | 6.14 ± 1.36a | 6.14 ± 1.32a |
| Flavor | 5.88 ± 1.41a | 5.50 ± 1.56ab | 5.10 ± 1.62b |
The highest rating for the overall acceptability was given to the non-supplemented control. However, the score given to the sample supplemented with garlic bulb-lemon peel extract was not significantly different. Spaghetti sample supplemented with GBE+EOPE had a score significantly lower from all the tested samples. It should however be noted that the overall acceptability score given to this sample was not significantly different from the sample supplemented with GBE+ELPE. On the contrary, all spaghetti samples were given aroma acceptability scores that were not significantly different. The non-supplemented sample and sample supplemented with GBE+ELPE were given scores for flavor acceptability that were not significantly different. Spaghetti sample supplemented with GBE+EOPE had a score significantly lower from all the tested samples. It was also noted that the score given to the flavor acceptability of the sample with GBE+EOPE was not significantly different from the sample supplemented with GBE+ELPE.
Aside from the effects of GBE+ELPE and GBE+EOPE co-supplementation on the sensory attributes of the spaghetti with meat sauce, the established MICo values for these co-supplementation were validated in artificially inoculated meat sauce. Specifically, GBE+ELPE was tested against a cocktail of the Gram-negative S. enterica serovars and against Gram-positive MRSA. The GBE+EOPE co-supplementation was validated against a cocktail of the Gram-negative E. coli O157:H7 strains, and against MRSA. Figure 2 presents the combined antibacterial efficacies of (3.0% GBE, 3.0% ELPE) and (3.0% GBE, 3.0% EOPE) co-supplementations against these previously identified most resistant microorganisms. The co-supplementation of GBE+ELPE resulted in a general decrease in the inoculated Salmonella enterica serovars (Figure 2a), reducing the initial population by as much as 2.0 log cycles by the end of 6 h; although an increase of >1.0 log cycle was observed 8 h post incubation. The GBE+ELPE (Figure 2b) similarly exhibited reduction in the inoculated MRSA population by >1.0 log after 2 h of incubation. On the other hand, the combination of GBE+EOPE resulted in non-remarkable change in the initial population of E. coli O157:H7 strains and of MRSA (Figure 2c–d). A slight reduction in the MRSA population of about 1.0 log cycle was observed 8 h post incubation.
Combined antimicrobial efficacies of 3.0% garlic bulb + 3.0% lemon peel extracts (a, b); and 3.0% garlic bulb + 3.0% orange peel extracts (c, d) against foodborne bacteria in spaghetti meat sauce.
These results suggest that the tested combinations of garlic bulb-lemon peel- and garlic bulb-orange peel extract co-supplementations exhibited bacteriostatic activities against the tested foodborne bacteria in the spaghetti food system. Bacteriostatic activity is characterized by the stationary phase of growth of microorganisms until efficiency of the bacteriostatic agent diminishes or the agent itself is removed (Pankey & Sabath, 2004). Furthermore, the co-supplementations did not result in substantial inactivation, but inhibited the microorganisms from growing at an abuse temperature of 31°C for 8 h. Co-supplementations with the tested extracts could therefore, provide additional margins of safety against the tested bacteria should the test food system be subjected to time-temperature abuse. Pasta dishes and food products containing meat are among the foods that have been identified to have significant risk of becoming vectors of foodborne illnesses when exposed to the temperature danger zone (5 – 60°C) for >4 h (Food Safety Information Council, 2003, New South Wales Food Authority, 2011).
This study demonstrated the importance of establishing the effect of plant extract supplementation on the sensory attribute of a food system prior to determination of antibacterial actions against pertinent microorganisms. This way, the established plant extract supplementation regimens for food shall be meaningful and can be directly used in food product formulation and development. This work also established individual and combinatorial plant extract inhibitory concentrations against foodborne organisms commonly linked to infections. While the established MICo values were able to inhibit the identified resistant microorganisms in a real food matrix, further work could still be done to improve the consumer acceptability of the supplemented product.
