Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Evaluation of sodium benzoate and licorice (Glycyrrhiza glabra) root extract as heat-sensitizing additives against Escherichia coli O157:H7 in mildly heated young coconut liquid endosperm A.A. Gabriel and S.K.P. Salazar Department of Food Science and Nutrition, College of Home Economics, University of the Philippines Diliman, Quezon City, Philippines
Significance and Impact of the Study: Fruit juice products have been linked to outbreaks of microbial infection, where unpasteurized products were proven vectors of diseases. Processors often opt not to apply heat process to juice products as the preservation technique often compromises the sensorial quality. This work evaluated two common additives for their heat-sensitizing effects against E. coli O157:H7 in coconut liquid endosperm, the results of which may serve as baseline information to smalland medium-scale processors, and researchers in the establishment of mild heat process schedule for the test commodity and other similar products.
Keywords beverages, Escherichia coli (all potentially pathogenic types), food preservation, food safety. Correspondence Alonzo A. Gabriel, Department of Food Science and Nutrition, College of Home Economics, University of the Philippines, Teodora Alonso Hall, A. Ma. Regidor Street, Diliman Campus, 1101 Quezon City, Philippines. E-mail: [email protected] 2014/0302: received 13 February 2014, revised 18 March 2014 and accepted 24 March 2014 doi:10.1111/lam.12257
Abstract This study evaluated the use of sodium benzoate (SB) and licorice root extract (LRE) as heat-sensitizing additives against Escherichia coli O157:H7 in mildly heated young coconut liquid endosperm. Consumer acceptance scoring showed that maximum permissible supplementation (MPS) levels for SB and LRE were at 300 and 250 ppm, respectively. The MPS values were considered in the generation of a 2-factor rotatable central composite design for the tested SB and LRE concentration combinations. Liquid endosperm with various SB and LRE supplementation combinations was inoculated with E. coli O157:H7 and heated to 55°C. The susceptibility of the cells towards heating was expressed in terms of the decimal reduction time (D55). Response surface analysis showed that only the individual linear effect of benzoate significantly influenced D55 value, where increasing supplementation level resulted in increasing susceptibility. The results reported could serve as baseline information in further investigating other additives that could be used as heat-sensitizing agents against pathogens in heat-labile food systems.
Introduction Young coconut (Cocos nucifera Linn.) liquid endospermbased beverages are indigenous products of tropical countries. In the Philippines, these beverages are commonly sold sweetened with sugar and added with macerated coconut meat (Gabriel et al. 2009). The potential of coconut beverages has been recently increased in foreign markets due to the perceived health benefits (Vanzi 2002; Aguiba 2005). Coconut liquid endosperm beverages contain dietary fibres, essential amino acids, vitamins and
minerals (Santoso et al. 1996; Coconut Development Board 2005). These beverages have also been reported to be effective in reducing urinary stones (Macalalag and Macalalag 1987) and can potentially be used for parenteral nutrition (Rao et al. 1972). The Yahoo! Southeast Asian Newsroom (2012) reported that in the first quarter of 2011, Philippine coconut water beverage exports to the Netherlands and Australia grew by almost 6- and 4-folds, respectively. In the first quarter of 2012, there was a 4-fold increase in the consumption of coconut water-based beverages in North America, equivalent to USD 39 million.
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However, its high nutritional value also makes coconut liquid endosperm highly susceptible to microbial spoilage (Del Rosario and Malijan 1983). According to Woodroof (1970), young coconut liquid endosperm is sterile when inside the shell. Despite the absence of reports of outbreaks involving the consumption of coconut liquid endosperm beverages, the highly manual nature of preparation of the commodity makes it susceptible to pathogen contaminations. Reddy et al. (2005) reported that microbial contaminations of as much as 60 log per ml may be introduced to the liquid endosperm when collected by the traditional manual methods. Contaminations could occur during the preparation of the beverage, specifically during the manual opening of the drupes, scraping and maceration of the solid endosperm, dilution and sweetening (Gubuan 2006). Moreover, a number of pathogenic bacteria have been isolated and reported to grow in coconut liquid endosperm (Leite et al. 2000; Hoffmann et al. 2002; Melo et al. 2003; Walter et al. 2009). Despite the risks of microbial food-borne illnesses, many manufacturers still produce unpasteurized beverage due to the high consumer demand for healthy and fresh or less processed products (Sloan 2010) and the negative influence of heat treatment on the fruit beverage sensory and nutritional qualities. Such limitation may be addressed by reducing the severity of a heat process schedule by reducing treatment temperature. The desired lethality may still be achieved with the application of other physical or chemical antimicrobial agents. This concept of simultaneous application of mild antimicrobial factors to achieve a desired process lethality is the basis of hurdle technology (Leistner and Gould 2002). The Federal Register (2001) explained that a safe process schedule should be based on the susceptibility of an identified resistant pathogen. Gabriel and Arellano (2014) compared the heat inactivation characteristics of acidadapted and nonadapted Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes in young coconut liquid endosperm. Results showed that non-acid-adapted E. coli O157:H7 was most resistant and thus an appropriate target organism for the establishment of a thermal pasteurization process. This study was conducted to evaluate the use of sodium benzoate (SB) and licorice root extract (LRE) as heat-sensitizing additives against the previously established resistant E. coli O157:H7 physiological state in mildly heated (55°C) young coconut liquid endosperm. According to the Food and Agriculture Organization and the World Health Organization Codex Alimentarius (FAO and WHO 2014), SB is generally recognized as safe (GRAS) with a maximum permissible level of 01% for fruit juices (as benzoic acid, may be in combination with sorbates, subject to national legislation of the importing country). Another emerging natural preser140
vative is licorice (Glycyrrhica glabra) root extract, which has found commercial use as a food additive in Japan, and is similarly listed as GRAS by the United States Food and Drug Administration (USFDA) (Shibata 2000; Fukai et al. 2003; Hu et al. 2011). For nonalcoholic beverages, licorice extract may be added up to 01% as flavour enhancer, flavouring agent and surface-active agent (USFDA 2014). As supplementation of the endosperm with these additives may significantly alter the consumer acceptability of the product, the maximum permissible supplementation (MPS) levels for SB and LRE were determined through consumer acceptability scoring using a 7-point Hedonics Scale affective sensory evaluation tool. The MPS values were used in the establishment of a set of different combinations of SB–LRE concentrations tested. The heat-sensitizing efficacies of SB, LRE and SB–LRE combinations were then characterized and quantified. The results of this work may be applied in the establishment of mild heat process schedules for coconut endosperm-based beverages. Results and discussion SB and LRE MPS levels The MPS levels for SB and LRE in young coconut liquid endosperm with SB and LRE were determined through sensory evaluation that involved an untrained consumer panel. As in any food product development, it is important to establish the influence of an additive to the sensory attributes of the supplemented product, to ensure consumer acceptance. Moreover, establishment of the supplementation limits narrowed down the operable region, defined by the ranges of additive concentrations that this study worked on. Results showed (Fig. 1) that the acceptability scores did not vary widely across SB-supplemented liquid endosperms, ranging from 482 (like slightly) for 800 ppm SB supplementation to 581 (like moderately) for 300 ppm SB supplementation. The liquid endosperm supplemented with 300 ppm SB had the greatest overall acceptability score, with a Hedonic descriptive equivalence close to like moderately. Furthermore, this acceptability score is not significantly different from, and even greater than that of the control, nonsupplemented sample. Similarly, overall acceptability scores across liquid endosperm samples supplemented with LRE did not vary widely, ranging from 490 (300, 650 ppm) to 579 (250 ppm). Supplementation with LRE of 250 ppm resulted in the liquid endosperm sample with the greatest overall acceptability score, with a Hedonic descriptive equivalence close to like moderately. This acceptability score is also not significantly different from and slightly greater than that of the control sample. This study there-
Letters in Applied Microbiology 59, 139--146 © 2014 The Society for Applied Microbiology
Heat sensitization of E. coli with additives
A.A. Gabriel and S.K.P. Salazar
(a) 7
a
abc
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abc
abc
abc c
Hedonic score
6 5 4 3 2 1 0
100
200
300
400
500
600
700
800
Sodium benzoate concentrations (ppm) (b) 7
ab
a
abc
abc
abc
abc
300 400 450 250 350 500 Licorice root extract concentrations (ppm)
600
c
abc bc
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Figure 1 General acceptability scores of young coconut liquid endosperm supplemented with various levels of sodium benzoate (a) and licorice root extract (b). Hedonic Scores 1: Dislike Very Much; 2: Dislike Moderately; 3: Dislike Slightly; 4: Neither Like Nor Dislike; 5: Like Slightly; 6: Like Moderately; 7: Like Very Much. Values followed by the same letter are not significantly different (p>0.05).
Hedonic score
6 5 4 3 2 1 0
200
fore determined 300 and 250 ppm as the MPS levels for SB and LRE, respectively. Hence, these values were used as upper limits in the rotatable central composite design of experiment summarized in Table 1. Influences of SB and LRE supplementations on the heat resistance of Escherichia coli O157:H7 This study utilized non-acid-adapted E. coli O157:H7 cells as challenge organism for the establishment of heat inactivation in the supplemented young coconut liquid endosperm. The choice of challenge organism species and physiological state was based on the results previously reported by Gabriel and Arellano (2014). Results showed that the tested supplementation combinations significantly influenced the thermal inactivation rates of the test organism (Table 1). The D55 values ranged from 802 (4393 ppm SB, 21339 ppm LRE) to 2400 min
650
(000 ppm SB, 12500 ppm LRE). The highest calculated D55 was slightly greater than that reported by Gabriel and Arellano (2014) of 232 min for the same organisms, with the same physiological state, in nonsupplemented young coconut liquid endosperm. To determine and quantify the effects of the additives on the D55 values of the test organism, the data obtained were subjected to response surface analysis using the freeware Design Expert 8.0. In the analysis, the software fitted the data into a quadratic equation that determined the individual linear, individual quadratic and interactive influences of the additives to the D55 values. Results showed that the calculated D55 values fitted significantly (P < 005) into a linear model (Eqn 1) without significant lack of fit. The response surface presented in Fig. 2 demonstrates that at any level of licorice supplementation, the D55 value of the test organism decreased with increasing level of SB supplementation. A similar trend was observed
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Table 1 Supplemented sodium benzoate–licorice root extract combinations with corresponding D55 values of Escherichia coli O157:H7 in heated young coconut liquid endosperm (pH = 49, SS, °Brix = 46) Coded combinations
Actual combinations
Combinations
SB (ppm)
LRE (ppm)
SB (ppm)
LRE (ppm)
D55 (min)*
1 2† 3 4 5 6 7 8 9 10†
1 0 +1 +1 1 a 0 0 +a 0
1 0 +1 1 +1 0 +a a 0 0
4393 15000 25607 25607 4393 000 15000 15000 30000 15000
3661 12500 21339 3661 21339 12500 25000 000 12500 12500
2272 167 ab 1087 026 de 0867 194 e 1316 402 cd 0802 084 e 2400 298 a 1506 372 c 2012 308 b 1104 121 de 1643 059 c
*Resulting D55 value is the average of 4 trials (n = 4) obtained from 2 independently conducted experiments. Values followed by the same letter are not significantly different (P > 005). †Center points replicated as per rotatable central composite design of experiment.
30 24
·1
23
·3
21
12
·6
19
D55 (min)
18
17 ·9
6
·2
16
0
14·4 12·8 11·0
9·3
7·6
50 100 SB 150 (p 200 pm 250 ) 300 0
200
250
150 100 50
LRE
m)
(pp
Figure 2 Response surface plot illustrating the simultaneous influences of sodium benzoate (SB) and licorice root extract (LRE) on the decimal reduction times of Escherichia coli O157:H7 at 55°C (D55) in young coconut liquid endosperm.
in D55 values as licorice supplementation increased at any SB supplementation level. Despite such observations, response surface analyses showed that only the tested SB supplementations affected significant heat sensitization of the test organisms. Furthermore, the curvilinear and interactive effects of the supplements were found not to significantly affect D55 values. D55 C ¼ 24 7766 ð0 032083SBÞ ð0 037258LREÞ ð1Þ Thermal processing is an effective method of inactivating spoilage and pathogenic micro-organisms in food 142
(Donahue et al. 2004). However, high-temperature processing has deteriorative effects on the nutritional and sensory qualities of products. To preserve food quality attributes, combinations of mild processing conditions such as relatively low-temperature heating and additive supplementations that sensitize micro-organisms to heat are being applied to food, which is the basis of hurdle technology (Leistner 2000). Combined preservation techniques or multiple hurdle technology is one of the emerging strategies for minimal food processing (Leistner and Gorris 1995; Rastogi et al. 2000; Bazhal et al. 2001; McKenzie et al. 2014) developed to respond to the consumer demands for safe, healthy, fresh and minimally processed products (Senorans et al. 2003). Individual and combinations of nonthermal beverage processing techniques such as UV light, high pressure and addition of antimicrobials are currently under investigation for efficacy to achieve 5-log reductions in pertinent pathogens without losing nutritional value or desired sensory characteristics of unpasteurized juice (USFDA, 2000; McNamee et al. 2010; Palgan et al. 2011; Walkling-Ribeiro et al. 2011; McKenzie et al. 2014). However, combining low-heat processing with heat-sensitizing additives may still be most practical because heat treatment is considered one of the simplest and affordable means of processing food (Bazhal et al. 2003). The observed efficacy of SB in sensitizing the test organism towards heat was similarly reported by a number of studies against spoilage-causing micro-organisms in fruit-based beverages (Veiga and Madeira-Lopes 2000; Walker and Phillips 2008; Bevilacqua et al. 2012). Young and Foegding (1993) explained that although no definite theory has yet been proposed to explain the antimicrobial activity of SB, one hypothesis is related to the high lipid solubility of the undissociated benzoic acid, which allows
Letters in Applied Microbiology 59, 139--146 © 2014 The Society for Applied Microbiology
Heat sensitization of E. coli with additives
A.A. Gabriel and S.K.P. Salazar
it to accumulate on the cell membranes or on various structures and surfaces of the bacterial cell, thus accumulating protons and anions within the cell. This decreases the proton motive force and interferes with the bacterial metabolism. Furthermore, Gabriel and Pineda (2014) also reported that individually, LRE did not have significant heat-sensitizing activity towards E. coli O157:H7 in coconut liquid endosperm. However, the root extract’s interaction with vanillin was able to significantly reduce the D55 values of the pathogen. On the other hand, the phenolic components of the crude LRE may possibly be responsible for the antimicrobial activity. Tsukiyama et al., (2002) isolated licochalcone A from licorice extract and reported its ability to inhibit the growth of Bacillus subtilis. It was also found to inhibit oxygen consumption of Micrococcus luteus, and the site of inhibition was thought to be between CoQ and cytochrome C in the bacterial respiratory transport chain. To sum up, the results obtained from this study underscore the efficacy of sodium benzoate as a heat-sensitizing additive against E. coli O157:H7 in a heat-sensitive product such as young coconut liquid endosperm. The study also emphasized the importance of evaluating antimicrobial additives based on supplementation levels that result in products with acceptable sensory quality. In this study, only the individual influences of the tested additives on the sensory quality of the coconut liquid endosperm were tested. The combined influences of the additives on the sensory attributes must be determined together with the application of a calculated process schedule with the recommended order of lethality against the test pathogen. This should be evaluated in a separate work. The results reported could serve as baseline information in further investigating other additives that could be used as heat-sensitizing agents against pathogens in heat-labile food systems. Materials and methods Determination of maximum permissible supplementation (MPS) levels for SB and LRE Licorice root extract preparation The dehydrated licorice roots used in this study were the gifts from Dr Haiying Cui of the School of Food and Biological Engineering, Jiangsu University, China. The roots were obtained from a traditional medicine shop in Dailan, China, sealed in a plastic bag and stored at room temperature until used. The roots were washed with tap water and chopped into small pieces. The extraction was performed by mixing 10:90 (w/v) ratio of licorice roots with 95% ethanol (RTC Chemical Ltd., Quezon City, Philippines) for 48 h at room temperature. The suspension was then filtered through a Whatman No. 41 paper (ash-
less, 20–25 lm), after which the filtrate was concentrated using a rotary evaporator (RE-200 Bibby Sterilin; Bibby Sterilin, Ltd., Staffordshire, UK) at 40°C for 8 h. The concentrated extract was then placed in amber bottles and stored at 4°C until used. Liquid endosperm preparation and physicochemical analysis The liquid endosperm samples were obtained from young coconuts procured from Barangay Krus na Ligas, University of the Philippines, Diliman, Quezon City. The young coconuts used in the study had a maturity of approximately 8 months. The liquid endosperm was obtained by making successive cuts on the mesocarp until a portion in the endocarp is severed to create an opening for the liquid to be aspirated out of the drupes. Prior to SB and LRE supplementation and sensory evaluations, the total soluble solids (°Brix) and pH of the liquid endosperm were ,respectively, measured using a hand-held refractometer (Atago Co. Ltd., Tokyo, Japan) and a Cyberscan 500 pH meter (Eutech Instruments, Singapore) calibrated with pH 70 and pH 40 standard solutions, respectively. Only liquid endosperm samples with pH and °Brix values falling within established ranges (Gabriel and Arellano 2014) were used in the inactivation studies. Consumer acceptance scoring of supplemented coconut endosperm The MPS for SB and LRE was individually determined by adding increasing amounts of each of the additives to coconut liquid endosperm and presenting the samples to a 50member consumer panel for acceptance scoring. The SB supplementation levels tested were 0, 100, 200, 300, 400, 500, 600, 700, and 800 ppm, while LRE supplementations of 0, 200, 250, 300, 350, 400, 450, 500, 600 and 650 ppm were evaluated. These values were based on initial focus group discussions conducted by the investigators (data not presented). A 7-point Hedonic Scale was used by the panellists in evaluating overall acceptability of the supplemented samples. The MPS is the highest level of SB or LRE supplementation that was rated by the panel members with the highest acceptability scores. The determined MPS values were used in the determination of SB–LRE combinations tested in the subsequent heat inactivation studies. Thermal inactivation of Escherichia coli in SB- and LREsupplemented coconut endosperm Design of experiment To elucidate the individual and interactive influences of SB and LRE on the heat inactivation of E. coli O157:H7, various combinations of SB–LRE supplementations were tested. The combinations were determined through a 2-factor rotatable central composite design of experiment
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(CCRD) using Design Expert 8.0 software (Stat-Ease Inc., Minneapolis, MN) (Table 1). In the CCRD, the lowest supplementation levels (a) for SB and LRE were similarly set to 0 ppm. On the other hand, the highest levels (+a) corresponded to the MPS previously determined in the consumer acceptability scoring. The CCRD was composed of 4 high–low and low–high SB and LRE concentration combinations with coded values of (+1, 1) and (1, +1), respectively; 4 intermediate–very high/very low (0, a) and very high/very low–intermediate (a, 0) combinations; and a duplicated combination set to intermediate–intermediate (0, 0) setting. The software determined the actual supplementation of SB and LRE levels in ppm as summarized in Table 1. Test organism propagation and composite inoculum preparation The test organisms included five strains of E. coli O157: H7. The tested serovars included the HCIPH 96055 strain, which was obtained from the Hiroshima City Institute of Public Health, Hiroshima City, Japan. The serovars MY29, DT-66, MN-28 and CR-3 were obtained from the National Food Research Institute, Tsukuba, Japan. Refrigerated stock culture in slants were activated in nutrient broth tubes (NB; Eiken Chemical Co. Ltd., Tokyo, Japan) and purified onto nutrient agar (NA; Eiken Chemical Co. Ltd.). Colonies from purified cultures were transferred and propagated onto NA slants, and working cultures were stored at 4°C until used. Propagation and growth incubation conditions were at 37 °C for 24 h. Prior to inactivation studies, loop inocula were obtained from each working cultures and propagated in separate 1 ml NB. Aliquots of 01 ml were then obtained and subjected to another round of propagation in 10 ml NB. To prepare the composite inoculum, 1-ml aliquots were obtained from the 10-ml cultures and combined in a sterile tube before vortex mixing for 1 min. One millilitre was obtained from the cocktail of E. coli O157:H7 cultures and transferred into a microcentrifuge tube. The cells were harvested by spinning at 4000 rpm for 20 min using a bench-top centrifuge (Model 800; Xiangshui Fada Medical Apparatus Factory, Jiangsu, China). The supernatant was decanted and resuspended by vortex mixing in 1-ml filter-sterilized liquid endosperm. The cells were acclimatized in filter-sterilized liquid coconut endosperm for not longer than 15 min. Young coconut liquid endosperm suspending medium preparation The young coconut liquid endosperm used as suspending medium for the heat inactivation studies was similarly obtained from Barangay Krus na Ligas, University of the Philippines, Diliman, Quezon City. The liquid endosperm 144
was extracted from the drupes following the previously described procedures. Upon arrival in the laboratory, the samples were subjected to filter-sterilization using a microfiltration set-up (Advantec; Toyo Roshi Kaisha Ltd., Tokyo, Japan) with a mixed cellulose ester filter paper with 045 lm pore size (Advantec; Toyo Roshi Kaisha Ltd.). Physicochemical properties were similarly characterized. Filter-sterilized liquid endosperm samples were placed in sterile amber bottles and stored at 4°C until used in the inactivation studies. Inoculation and heat inactivation of Escherichia coli O157: H7 For the heat inactivation studies, 99-ml filter-sterilized young coconut liquid endosperm supplemented with different concentrations of sodium benzoate and LRE in glass test tubes (24 mm i.d.) was heated to 55°C on a water bath (Citenco Co. Ltd., Somerset, UK). The medium temperature was measured by inserting a thermometer through the cold point of a control tube. When the cold point temperature reached 55°C, 01-ml aliquots of the acclimatized cell suspension were pipetted into each of the tubes. The inoculated tubes were constantly, manually agitated throughout the heating period that lasted for as long as 90 min. After heat treatments, tubes were immediately immersed into and kept in an ice bath until survivor enumerations. All thermal inactivation studies were conducted in two runs, with two replications per run. Survivor enumerations and D55 value determination Surviving cells were enumerated from each of the thermally treated tubes by conducting 10-fold serial dilutions using sterile 01% peptone water (PW; HiMedia, Mumbai, India) and surface plating onto presolidified NA plates. The plates were incubated at 37°C for 24 h. Colonies were enumerated and cell populations were reported as log cfu per ml. The survival curves were generated by plotting the enumerated population vs heating time using a freeware program DMFit 2.1 (Institute of Food Research, Reading, UK), based on the models established by Baranyi and Roberts (1994). The freeware was able to calculate the death rates of the test organism in each of the tested suspending medium. The heat resistance of E. coli O157:H7 was reported in terms of the decimal reduction times at 55°C (D55), which was equivalent to the negative inverse of the calculated death rates. The D55 value is equivalent to the number of unit time of exposure to 55°C heating that will result in the reduction in the initial population of E. coli O157:H7 in the young coconut liquid endosperm by 1 log cycle (90%). In this study, D55 values were only calculated from inactivation curves that traversed >3 log cycles and coefficients of determination (R2) values >090.
Letters in Applied Microbiology 59, 139--146 © 2014 The Society for Applied Microbiology
Heat sensitization of E. coli with additives
A.A. Gabriel and S.K.P. Salazar
Statistical analyses The data obtained from all independently replicated experiments were subjected to single-factor analysis of variance (ANOVA) using the general linear model procedure (PROC GLM) of the SAS Statistical Software System 9.1 (SAS Institute, Inc., Cary, NC). The Duncan’s multiple range test (DMRT) was also used as post hoc determination of significant differences at P < 005. The efficacies of the tested additives on the heat resistance of E. coli O157:H7 were characterized through response surface methodology using the software program Design Expert 8.0 (Stat-Ease, Inc.). Acknowledgements This project was supported by the Office of the Vice Chancellor for Research and Development of the University of the Philippines, Diliman, under the PhD Incentive Award (PhDIA 111123). The authors also extend their gratitude to Hiroyuki Nakano and Haying Cui of Hiroshima University for providing the test organisms and licorice roots. Romelia Arellano, Jennifer Karen Pineda and Mely Maceda are also being acknowledged for their help in conducting the experiments. Conflict of Interest No conflict of interest declared. References Aguiba, M. (2005) Coconut juice market seen big. Manila Bulletin. Available from: http://www.highbeam.com/doc/ 1G1-133347674.html (accessed December 2010). Baranyi, J. and Roberts, T.A. (1994) A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294. Bazhal, M.I., Lebovka, N.I. and Vorobiev, E.I. (2001) Pulsed electric field treatment of apple tissue during compression for juice extraction. J Engg 50, 129–139. Bazhal, M.I., Ngadi, M.O., Raghavan, G.S.V. and Smith, J.P. (2003) Minimal processing of foods using hurdle technologies. Available from: http://www.engr.usask.ca/ societies/csae/PapersCSAE2003/CSAE03-342.pdf (accessed March 2014). Bevilacqua, A., Campaniello, D., Sinigaglia, M., Ciccarone, C. and Corbo, M.R. (2012) Sodium benzoate and citrus increase the effect of homogenization towards spores of Fusarium oxysporum in pineapple juice. Food Cont 28, 199–204. Coconut Development Board (2005) Tender coconut water. Available from: http://coconutboard.nic.in/tendnutr.htm (accessed March 2011).
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Heat sensitization of E. coli with additives
A.A. Gabriel and S.K.P. Salazar
Leistner, L. and Gould, G.W. (2002) Hurdle Technologies: Combinations, Treatments for Food Stability, Safety and Quality. New York: Springer Inc. Leite, C.C., de Assis, P.N., da Silva, M.O., Sant’Anna, M.E.B. and de Santana, L.R.R. (2000) Avaliacao microbiologica da agua de coco produzida e comercializada na cidade de Salvador (Microbiological evaluation of coconut water produced and sold in the city of Salvador). Hig Aliment 14, 64–66. Macalalag, E.V. and Macalalag, A.L. (1987) Bukolysis: young coconut water renoclysis for urinary stone dissolution. Int Surg 72, 247. McKenzie, K., Maclean, M., Timoshkin, I.V., MacGregor, S.J. and Anderson, J.G. (2014) Enhanced inactivation of Escherichia coli and Listeria monocytogenes by exposure to 405 nm light under sub-lethal temperature, salt and acid stress conditions. Int J Food Microbiol 170, 91–98. McNamee, C., Noci, F., Cronin, D.A., Lyng, J.G. and Scannell, A.G.M. (2010) PEF based hurdle strategy to control Pichia fermentans, Listeria innocua and Escherichia coli K12 in orange juice. Int J Food Microbiol 138, 13–18. Melo, N.P.M., Cardonha, A.M.S. and Oliveira, A.C.F. (2003) Qualidade micro- biol ogica das aguas de coco envasadas e comercializadas na cidade de Natal (Microbiological quality of bottled coconut water marketed in Natal). Hig Aliment 17, 113–114. Palgan, I., Caminiti, I.M., Mu~ noz, A., Noci, F., Whyte, P., Morgan, D.J., Cronin, D.A. and Lyng, J.G. (2011) Combined effect of selected non-thermal technologies on Escherichia coli and Pichia fermentans inactivation in an apple and cranberry juice blend and on product shelf life. Int J Food Microbiol 151, 1–6. Rao, P.S., Rao, S.R., Kumar, S.V., Murthy, K.J. and Dussey, P. (1972) Intravenous administration of coconut water. JASSOC Physic India 20, 235–239. Rastogi, N.K., Angersbach, A. and Knorr, D. (2000) Evaluation of mass transfer mechanism during osmotic treatment of plant materials. J Food Sci 65, 1016–1019. Reddy, K.V., Das, M. and Das, S.K. (2005) Filtration resistances in non-thermal sterilization of green coconut water. Res Note J Food Engg 69, 381–385. Santoso, U., Kubo, K., Ota, T., Tadokoro, T. and Maekawa, A. (1996) Nutrient composition of kopyor coconuts (Cocos nucifera L.). Food Chem 57, 299–304. Senorans, F.J., Ruiz-Rodriguez, A., Ibanez, E., Tabera, J. and Reglero, G. (2003) Countercurrent super-critical fluid extraction and fractionation of alcoholic beverages. J Agric Food Chem 49, 1895–1899. Shibata, S. (2000) A drug over the millennia: pharmacognosy, chemistry and pharmacology of licorice. Yakug Zas 120, 849–862.
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Sloan, A.E. (2010) Top ten functional food trends. Food Technol 64, 22–41. Tsukiyama, R., Katsura, H., Tokuriki, N. and Kobayashi, M. (2002) Antibacterial activity of Licochalcone A against spore-forming bacteria. Antimicrob Agents Chemother 46, 1226–1230. United States Food and Drug Administration [USFDA] (2000) Kinetics of Microbial Inactivation for Alternative Food Processing Technologies Executive Summary. Center for Food Safety and Applied Nutrition. Available from: http:// www.cfsan. fda.gov/-commift-exec.html. (accessed June 2012). United States Food and Drug Administration [USFDA] (2014) Licorice and licorice derivatives. Subpart B: Listing of specific substances affirmed as GRAS. Code of Federal Regulations, Title 21, Vol 3. 21 CFR184.1408. Available from: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/ cfcfr/ (accessed March 2014). Vanzi, S.J. (2002) Buko Juice goes big time. Philippine Headline News Online. Available from: http://www. newsflash.org/2002/12/sb/sb002551.htm (accessed January 2011). Veiga, A. and Madeira-Lopes, A. (2000) Effects of weak acid preservatives on the growth and thermal death of yeast Pichia membranifaciens in a commercial apple juice. Int J Food Microbiol 56, 145–151. Walker, M. and Phillips, C.A. (2008) The effect of preservatives on Alicyclobacillus acidoterrestris and Propionibacterium cyclohexanicum in fruit juice. Food Cont 9, 974–981. Walkling-Ribeiro, M., Rodrıguez-Gonzalez, O., Jayaram, S. and Griffiths, M.W. (2011) Microbial inactivation and shelf life comparison of ‘cold’ hurdle processing with pulsed electric fields and microfiltration, and conventional thermal pasteurization in skim milk. Int J Food Microbiol 144, 379– 386. Walter, E.H.M., Kabuki, D.Y., Esper, L.M.R., Sant’Ana, A.S. and Kuaye, A.Y. (2009) Modelling the growth of Listeria monocytogenes in fresh green coconut (Cocos nucifera L.) water. Food Microbiol 26, 653–657. Woodroof, J.G. (1970) Coconuts: Production, Processing, Products. Westport, CT: The AVI Publishing Co., Inc. Yahoo! Southeast Asian Newsroom (2012) PH coco exports up, thanks to coco juice craze. Available from: http://ph. news.yahoo.com/ph-coco-exports-up– thanks-to-coco-juice-craze.html (accessed June 2012). Young, K.M. and Foegding, P.M. (1993) Acetic, lactic and citric acids and pH inhibition of Listeria monocytogenes Scott A and the effect on intracellular pH. J Appl Bacteriol 75, 515–520.
Letters in Applied Microbiology 59, 139--146 © 2014 The Society for Applied Microbiology
ORIGINAL ARTICLE
Evaluation of sodium benzoate and licorice (Glycyrrhiza glabra) root extract as heat-sensitizing additives against Escherichia coli O157:H7 in mildly heated young coconut liquid endosperm A.A. Gabriel and S.K.P. Salazar Department of Food Science and Nutrition, College of Home Economics, University of the Philippines Diliman, Quezon City, Philippines
Significance and Impact of the Study: Fruit juice products have been linked to outbreaks of microbial infection, where unpasteurized products were proven vectors of diseases. Processors often opt not to apply heat process to juice products as the preservation technique often compromises the sensorial quality. This work evaluated two common additives for their heat-sensitizing effects against E. coli O157:H7 in coconut liquid endosperm, the results of which may serve as baseline information to smalland medium-scale processors, and researchers in the establishment of mild heat process schedule for the test commodity and other similar products.
Keywords beverages, Escherichia coli (all potentially pathogenic types), food preservation, food safety. Correspondence Alonzo A. Gabriel, Department of Food Science and Nutrition, College of Home Economics, University of the Philippines, Teodora Alonso Hall, A. Ma. Regidor Street, Diliman Campus, 1101 Quezon City, Philippines. E-mail: [email protected] 2014/0302: received 13 February 2014, revised 18 March 2014 and accepted 24 March 2014 doi:10.1111/lam.12257
Abstract This study evaluated the use of sodium benzoate (SB) and licorice root extract (LRE) as heat-sensitizing additives against Escherichia coli O157:H7 in mildly heated young coconut liquid endosperm. Consumer acceptance scoring showed that maximum permissible supplementation (MPS) levels for SB and LRE were at 300 and 250 ppm, respectively. The MPS values were considered in the generation of a 2-factor rotatable central composite design for the tested SB and LRE concentration combinations. Liquid endosperm with various SB and LRE supplementation combinations was inoculated with E. coli O157:H7 and heated to 55°C. The susceptibility of the cells towards heating was expressed in terms of the decimal reduction time (D55). Response surface analysis showed that only the individual linear effect of benzoate significantly influenced D55 value, where increasing supplementation level resulted in increasing susceptibility. The results reported could serve as baseline information in further investigating other additives that could be used as heat-sensitizing agents against pathogens in heat-labile food systems.
Introduction Young coconut (Cocos nucifera Linn.) liquid endospermbased beverages are indigenous products of tropical countries. In the Philippines, these beverages are commonly sold sweetened with sugar and added with macerated coconut meat (Gabriel et al. 2009). The potential of coconut beverages has been recently increased in foreign markets due to the perceived health benefits (Vanzi 2002; Aguiba 2005). Coconut liquid endosperm beverages contain dietary fibres, essential amino acids, vitamins and
minerals (Santoso et al. 1996; Coconut Development Board 2005). These beverages have also been reported to be effective in reducing urinary stones (Macalalag and Macalalag 1987) and can potentially be used for parenteral nutrition (Rao et al. 1972). The Yahoo! Southeast Asian Newsroom (2012) reported that in the first quarter of 2011, Philippine coconut water beverage exports to the Netherlands and Australia grew by almost 6- and 4-folds, respectively. In the first quarter of 2012, there was a 4-fold increase in the consumption of coconut water-based beverages in North America, equivalent to USD 39 million.
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However, its high nutritional value also makes coconut liquid endosperm highly susceptible to microbial spoilage (Del Rosario and Malijan 1983). According to Woodroof (1970), young coconut liquid endosperm is sterile when inside the shell. Despite the absence of reports of outbreaks involving the consumption of coconut liquid endosperm beverages, the highly manual nature of preparation of the commodity makes it susceptible to pathogen contaminations. Reddy et al. (2005) reported that microbial contaminations of as much as 60 log per ml may be introduced to the liquid endosperm when collected by the traditional manual methods. Contaminations could occur during the preparation of the beverage, specifically during the manual opening of the drupes, scraping and maceration of the solid endosperm, dilution and sweetening (Gubuan 2006). Moreover, a number of pathogenic bacteria have been isolated and reported to grow in coconut liquid endosperm (Leite et al. 2000; Hoffmann et al. 2002; Melo et al. 2003; Walter et al. 2009). Despite the risks of microbial food-borne illnesses, many manufacturers still produce unpasteurized beverage due to the high consumer demand for healthy and fresh or less processed products (Sloan 2010) and the negative influence of heat treatment on the fruit beverage sensory and nutritional qualities. Such limitation may be addressed by reducing the severity of a heat process schedule by reducing treatment temperature. The desired lethality may still be achieved with the application of other physical or chemical antimicrobial agents. This concept of simultaneous application of mild antimicrobial factors to achieve a desired process lethality is the basis of hurdle technology (Leistner and Gould 2002). The Federal Register (2001) explained that a safe process schedule should be based on the susceptibility of an identified resistant pathogen. Gabriel and Arellano (2014) compared the heat inactivation characteristics of acidadapted and nonadapted Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes in young coconut liquid endosperm. Results showed that non-acid-adapted E. coli O157:H7 was most resistant and thus an appropriate target organism for the establishment of a thermal pasteurization process. This study was conducted to evaluate the use of sodium benzoate (SB) and licorice root extract (LRE) as heat-sensitizing additives against the previously established resistant E. coli O157:H7 physiological state in mildly heated (55°C) young coconut liquid endosperm. According to the Food and Agriculture Organization and the World Health Organization Codex Alimentarius (FAO and WHO 2014), SB is generally recognized as safe (GRAS) with a maximum permissible level of 01% for fruit juices (as benzoic acid, may be in combination with sorbates, subject to national legislation of the importing country). Another emerging natural preser140
vative is licorice (Glycyrrhica glabra) root extract, which has found commercial use as a food additive in Japan, and is similarly listed as GRAS by the United States Food and Drug Administration (USFDA) (Shibata 2000; Fukai et al. 2003; Hu et al. 2011). For nonalcoholic beverages, licorice extract may be added up to 01% as flavour enhancer, flavouring agent and surface-active agent (USFDA 2014). As supplementation of the endosperm with these additives may significantly alter the consumer acceptability of the product, the maximum permissible supplementation (MPS) levels for SB and LRE were determined through consumer acceptability scoring using a 7-point Hedonics Scale affective sensory evaluation tool. The MPS values were used in the establishment of a set of different combinations of SB–LRE concentrations tested. The heat-sensitizing efficacies of SB, LRE and SB–LRE combinations were then characterized and quantified. The results of this work may be applied in the establishment of mild heat process schedules for coconut endosperm-based beverages. Results and discussion SB and LRE MPS levels The MPS levels for SB and LRE in young coconut liquid endosperm with SB and LRE were determined through sensory evaluation that involved an untrained consumer panel. As in any food product development, it is important to establish the influence of an additive to the sensory attributes of the supplemented product, to ensure consumer acceptance. Moreover, establishment of the supplementation limits narrowed down the operable region, defined by the ranges of additive concentrations that this study worked on. Results showed (Fig. 1) that the acceptability scores did not vary widely across SB-supplemented liquid endosperms, ranging from 482 (like slightly) for 800 ppm SB supplementation to 581 (like moderately) for 300 ppm SB supplementation. The liquid endosperm supplemented with 300 ppm SB had the greatest overall acceptability score, with a Hedonic descriptive equivalence close to like moderately. Furthermore, this acceptability score is not significantly different from, and even greater than that of the control, nonsupplemented sample. Similarly, overall acceptability scores across liquid endosperm samples supplemented with LRE did not vary widely, ranging from 490 (300, 650 ppm) to 579 (250 ppm). Supplementation with LRE of 250 ppm resulted in the liquid endosperm sample with the greatest overall acceptability score, with a Hedonic descriptive equivalence close to like moderately. This acceptability score is also not significantly different from and slightly greater than that of the control sample. This study there-
Letters in Applied Microbiology 59, 139--146 © 2014 The Society for Applied Microbiology
Heat sensitization of E. coli with additives
A.A. Gabriel and S.K.P. Salazar
(a) 7
a
abc
ab
bc
bc
abc
abc
abc c
Hedonic score
6 5 4 3 2 1 0
100
200
300
400
500
600
700
800
Sodium benzoate concentrations (ppm) (b) 7
ab
a
abc
abc
abc
abc
300 400 450 250 350 500 Licorice root extract concentrations (ppm)
600
c
abc bc
c
Figure 1 General acceptability scores of young coconut liquid endosperm supplemented with various levels of sodium benzoate (a) and licorice root extract (b). Hedonic Scores 1: Dislike Very Much; 2: Dislike Moderately; 3: Dislike Slightly; 4: Neither Like Nor Dislike; 5: Like Slightly; 6: Like Moderately; 7: Like Very Much. Values followed by the same letter are not significantly different (p>0.05).
Hedonic score
6 5 4 3 2 1 0
200
fore determined 300 and 250 ppm as the MPS levels for SB and LRE, respectively. Hence, these values were used as upper limits in the rotatable central composite design of experiment summarized in Table 1. Influences of SB and LRE supplementations on the heat resistance of Escherichia coli O157:H7 This study utilized non-acid-adapted E. coli O157:H7 cells as challenge organism for the establishment of heat inactivation in the supplemented young coconut liquid endosperm. The choice of challenge organism species and physiological state was based on the results previously reported by Gabriel and Arellano (2014). Results showed that the tested supplementation combinations significantly influenced the thermal inactivation rates of the test organism (Table 1). The D55 values ranged from 802 (4393 ppm SB, 21339 ppm LRE) to 2400 min
650
(000 ppm SB, 12500 ppm LRE). The highest calculated D55 was slightly greater than that reported by Gabriel and Arellano (2014) of 232 min for the same organisms, with the same physiological state, in nonsupplemented young coconut liquid endosperm. To determine and quantify the effects of the additives on the D55 values of the test organism, the data obtained were subjected to response surface analysis using the freeware Design Expert 8.0. In the analysis, the software fitted the data into a quadratic equation that determined the individual linear, individual quadratic and interactive influences of the additives to the D55 values. Results showed that the calculated D55 values fitted significantly (P < 005) into a linear model (Eqn 1) without significant lack of fit. The response surface presented in Fig. 2 demonstrates that at any level of licorice supplementation, the D55 value of the test organism decreased with increasing level of SB supplementation. A similar trend was observed
Letters in Applied Microbiology 59, 139--146 © 2014 The Society for Applied Microbiology
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Heat sensitization of E. coli with additives
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Table 1 Supplemented sodium benzoate–licorice root extract combinations with corresponding D55 values of Escherichia coli O157:H7 in heated young coconut liquid endosperm (pH = 49, SS, °Brix = 46) Coded combinations
Actual combinations
Combinations
SB (ppm)
LRE (ppm)
SB (ppm)
LRE (ppm)
D55 (min)*
1 2† 3 4 5 6 7 8 9 10†
1 0 +1 +1 1 a 0 0 +a 0
1 0 +1 1 +1 0 +a a 0 0
4393 15000 25607 25607 4393 000 15000 15000 30000 15000
3661 12500 21339 3661 21339 12500 25000 000 12500 12500
2272 167 ab 1087 026 de 0867 194 e 1316 402 cd 0802 084 e 2400 298 a 1506 372 c 2012 308 b 1104 121 de 1643 059 c
*Resulting D55 value is the average of 4 trials (n = 4) obtained from 2 independently conducted experiments. Values followed by the same letter are not significantly different (P > 005). †Center points replicated as per rotatable central composite design of experiment.
30 24
·1
23
·3
21
12
·6
19
D55 (min)
18
17 ·9
6
·2
16
0
14·4 12·8 11·0
9·3
7·6
50 100 SB 150 (p 200 pm 250 ) 300 0
200
250
150 100 50
LRE
m)
(pp
Figure 2 Response surface plot illustrating the simultaneous influences of sodium benzoate (SB) and licorice root extract (LRE) on the decimal reduction times of Escherichia coli O157:H7 at 55°C (D55) in young coconut liquid endosperm.
in D55 values as licorice supplementation increased at any SB supplementation level. Despite such observations, response surface analyses showed that only the tested SB supplementations affected significant heat sensitization of the test organisms. Furthermore, the curvilinear and interactive effects of the supplements were found not to significantly affect D55 values. D55 C ¼ 24 7766 ð0 032083SBÞ ð0 037258LREÞ ð1Þ Thermal processing is an effective method of inactivating spoilage and pathogenic micro-organisms in food 142
(Donahue et al. 2004). However, high-temperature processing has deteriorative effects on the nutritional and sensory qualities of products. To preserve food quality attributes, combinations of mild processing conditions such as relatively low-temperature heating and additive supplementations that sensitize micro-organisms to heat are being applied to food, which is the basis of hurdle technology (Leistner 2000). Combined preservation techniques or multiple hurdle technology is one of the emerging strategies for minimal food processing (Leistner and Gorris 1995; Rastogi et al. 2000; Bazhal et al. 2001; McKenzie et al. 2014) developed to respond to the consumer demands for safe, healthy, fresh and minimally processed products (Senorans et al. 2003). Individual and combinations of nonthermal beverage processing techniques such as UV light, high pressure and addition of antimicrobials are currently under investigation for efficacy to achieve 5-log reductions in pertinent pathogens without losing nutritional value or desired sensory characteristics of unpasteurized juice (USFDA, 2000; McNamee et al. 2010; Palgan et al. 2011; Walkling-Ribeiro et al. 2011; McKenzie et al. 2014). However, combining low-heat processing with heat-sensitizing additives may still be most practical because heat treatment is considered one of the simplest and affordable means of processing food (Bazhal et al. 2003). The observed efficacy of SB in sensitizing the test organism towards heat was similarly reported by a number of studies against spoilage-causing micro-organisms in fruit-based beverages (Veiga and Madeira-Lopes 2000; Walker and Phillips 2008; Bevilacqua et al. 2012). Young and Foegding (1993) explained that although no definite theory has yet been proposed to explain the antimicrobial activity of SB, one hypothesis is related to the high lipid solubility of the undissociated benzoic acid, which allows
Letters in Applied Microbiology 59, 139--146 © 2014 The Society for Applied Microbiology
Heat sensitization of E. coli with additives
A.A. Gabriel and S.K.P. Salazar
it to accumulate on the cell membranes or on various structures and surfaces of the bacterial cell, thus accumulating protons and anions within the cell. This decreases the proton motive force and interferes with the bacterial metabolism. Furthermore, Gabriel and Pineda (2014) also reported that individually, LRE did not have significant heat-sensitizing activity towards E. coli O157:H7 in coconut liquid endosperm. However, the root extract’s interaction with vanillin was able to significantly reduce the D55 values of the pathogen. On the other hand, the phenolic components of the crude LRE may possibly be responsible for the antimicrobial activity. Tsukiyama et al., (2002) isolated licochalcone A from licorice extract and reported its ability to inhibit the growth of Bacillus subtilis. It was also found to inhibit oxygen consumption of Micrococcus luteus, and the site of inhibition was thought to be between CoQ and cytochrome C in the bacterial respiratory transport chain. To sum up, the results obtained from this study underscore the efficacy of sodium benzoate as a heat-sensitizing additive against E. coli O157:H7 in a heat-sensitive product such as young coconut liquid endosperm. The study also emphasized the importance of evaluating antimicrobial additives based on supplementation levels that result in products with acceptable sensory quality. In this study, only the individual influences of the tested additives on the sensory quality of the coconut liquid endosperm were tested. The combined influences of the additives on the sensory attributes must be determined together with the application of a calculated process schedule with the recommended order of lethality against the test pathogen. This should be evaluated in a separate work. The results reported could serve as baseline information in further investigating other additives that could be used as heat-sensitizing agents against pathogens in heat-labile food systems. Materials and methods Determination of maximum permissible supplementation (MPS) levels for SB and LRE Licorice root extract preparation The dehydrated licorice roots used in this study were the gifts from Dr Haiying Cui of the School of Food and Biological Engineering, Jiangsu University, China. The roots were obtained from a traditional medicine shop in Dailan, China, sealed in a plastic bag and stored at room temperature until used. The roots were washed with tap water and chopped into small pieces. The extraction was performed by mixing 10:90 (w/v) ratio of licorice roots with 95% ethanol (RTC Chemical Ltd., Quezon City, Philippines) for 48 h at room temperature. The suspension was then filtered through a Whatman No. 41 paper (ash-
less, 20–25 lm), after which the filtrate was concentrated using a rotary evaporator (RE-200 Bibby Sterilin; Bibby Sterilin, Ltd., Staffordshire, UK) at 40°C for 8 h. The concentrated extract was then placed in amber bottles and stored at 4°C until used. Liquid endosperm preparation and physicochemical analysis The liquid endosperm samples were obtained from young coconuts procured from Barangay Krus na Ligas, University of the Philippines, Diliman, Quezon City. The young coconuts used in the study had a maturity of approximately 8 months. The liquid endosperm was obtained by making successive cuts on the mesocarp until a portion in the endocarp is severed to create an opening for the liquid to be aspirated out of the drupes. Prior to SB and LRE supplementation and sensory evaluations, the total soluble solids (°Brix) and pH of the liquid endosperm were ,respectively, measured using a hand-held refractometer (Atago Co. Ltd., Tokyo, Japan) and a Cyberscan 500 pH meter (Eutech Instruments, Singapore) calibrated with pH 70 and pH 40 standard solutions, respectively. Only liquid endosperm samples with pH and °Brix values falling within established ranges (Gabriel and Arellano 2014) were used in the inactivation studies. Consumer acceptance scoring of supplemented coconut endosperm The MPS for SB and LRE was individually determined by adding increasing amounts of each of the additives to coconut liquid endosperm and presenting the samples to a 50member consumer panel for acceptance scoring. The SB supplementation levels tested were 0, 100, 200, 300, 400, 500, 600, 700, and 800 ppm, while LRE supplementations of 0, 200, 250, 300, 350, 400, 450, 500, 600 and 650 ppm were evaluated. These values were based on initial focus group discussions conducted by the investigators (data not presented). A 7-point Hedonic Scale was used by the panellists in evaluating overall acceptability of the supplemented samples. The MPS is the highest level of SB or LRE supplementation that was rated by the panel members with the highest acceptability scores. The determined MPS values were used in the determination of SB–LRE combinations tested in the subsequent heat inactivation studies. Thermal inactivation of Escherichia coli in SB- and LREsupplemented coconut endosperm Design of experiment To elucidate the individual and interactive influences of SB and LRE on the heat inactivation of E. coli O157:H7, various combinations of SB–LRE supplementations were tested. The combinations were determined through a 2-factor rotatable central composite design of experiment
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(CCRD) using Design Expert 8.0 software (Stat-Ease Inc., Minneapolis, MN) (Table 1). In the CCRD, the lowest supplementation levels (a) for SB and LRE were similarly set to 0 ppm. On the other hand, the highest levels (+a) corresponded to the MPS previously determined in the consumer acceptability scoring. The CCRD was composed of 4 high–low and low–high SB and LRE concentration combinations with coded values of (+1, 1) and (1, +1), respectively; 4 intermediate–very high/very low (0, a) and very high/very low–intermediate (a, 0) combinations; and a duplicated combination set to intermediate–intermediate (0, 0) setting. The software determined the actual supplementation of SB and LRE levels in ppm as summarized in Table 1. Test organism propagation and composite inoculum preparation The test organisms included five strains of E. coli O157: H7. The tested serovars included the HCIPH 96055 strain, which was obtained from the Hiroshima City Institute of Public Health, Hiroshima City, Japan. The serovars MY29, DT-66, MN-28 and CR-3 were obtained from the National Food Research Institute, Tsukuba, Japan. Refrigerated stock culture in slants were activated in nutrient broth tubes (NB; Eiken Chemical Co. Ltd., Tokyo, Japan) and purified onto nutrient agar (NA; Eiken Chemical Co. Ltd.). Colonies from purified cultures were transferred and propagated onto NA slants, and working cultures were stored at 4°C until used. Propagation and growth incubation conditions were at 37 °C for 24 h. Prior to inactivation studies, loop inocula were obtained from each working cultures and propagated in separate 1 ml NB. Aliquots of 01 ml were then obtained and subjected to another round of propagation in 10 ml NB. To prepare the composite inoculum, 1-ml aliquots were obtained from the 10-ml cultures and combined in a sterile tube before vortex mixing for 1 min. One millilitre was obtained from the cocktail of E. coli O157:H7 cultures and transferred into a microcentrifuge tube. The cells were harvested by spinning at 4000 rpm for 20 min using a bench-top centrifuge (Model 800; Xiangshui Fada Medical Apparatus Factory, Jiangsu, China). The supernatant was decanted and resuspended by vortex mixing in 1-ml filter-sterilized liquid endosperm. The cells were acclimatized in filter-sterilized liquid coconut endosperm for not longer than 15 min. Young coconut liquid endosperm suspending medium preparation The young coconut liquid endosperm used as suspending medium for the heat inactivation studies was similarly obtained from Barangay Krus na Ligas, University of the Philippines, Diliman, Quezon City. The liquid endosperm 144
was extracted from the drupes following the previously described procedures. Upon arrival in the laboratory, the samples were subjected to filter-sterilization using a microfiltration set-up (Advantec; Toyo Roshi Kaisha Ltd., Tokyo, Japan) with a mixed cellulose ester filter paper with 045 lm pore size (Advantec; Toyo Roshi Kaisha Ltd.). Physicochemical properties were similarly characterized. Filter-sterilized liquid endosperm samples were placed in sterile amber bottles and stored at 4°C until used in the inactivation studies. Inoculation and heat inactivation of Escherichia coli O157: H7 For the heat inactivation studies, 99-ml filter-sterilized young coconut liquid endosperm supplemented with different concentrations of sodium benzoate and LRE in glass test tubes (24 mm i.d.) was heated to 55°C on a water bath (Citenco Co. Ltd., Somerset, UK). The medium temperature was measured by inserting a thermometer through the cold point of a control tube. When the cold point temperature reached 55°C, 01-ml aliquots of the acclimatized cell suspension were pipetted into each of the tubes. The inoculated tubes were constantly, manually agitated throughout the heating period that lasted for as long as 90 min. After heat treatments, tubes were immediately immersed into and kept in an ice bath until survivor enumerations. All thermal inactivation studies were conducted in two runs, with two replications per run. Survivor enumerations and D55 value determination Surviving cells were enumerated from each of the thermally treated tubes by conducting 10-fold serial dilutions using sterile 01% peptone water (PW; HiMedia, Mumbai, India) and surface plating onto presolidified NA plates. The plates were incubated at 37°C for 24 h. Colonies were enumerated and cell populations were reported as log cfu per ml. The survival curves were generated by plotting the enumerated population vs heating time using a freeware program DMFit 2.1 (Institute of Food Research, Reading, UK), based on the models established by Baranyi and Roberts (1994). The freeware was able to calculate the death rates of the test organism in each of the tested suspending medium. The heat resistance of E. coli O157:H7 was reported in terms of the decimal reduction times at 55°C (D55), which was equivalent to the negative inverse of the calculated death rates. The D55 value is equivalent to the number of unit time of exposure to 55°C heating that will result in the reduction in the initial population of E. coli O157:H7 in the young coconut liquid endosperm by 1 log cycle (90%). In this study, D55 values were only calculated from inactivation curves that traversed >3 log cycles and coefficients of determination (R2) values >090.
Letters in Applied Microbiology 59, 139--146 © 2014 The Society for Applied Microbiology
Heat sensitization of E. coli with additives
A.A. Gabriel and S.K.P. Salazar
Statistical analyses The data obtained from all independently replicated experiments were subjected to single-factor analysis of variance (ANOVA) using the general linear model procedure (PROC GLM) of the SAS Statistical Software System 9.1 (SAS Institute, Inc., Cary, NC). The Duncan’s multiple range test (DMRT) was also used as post hoc determination of significant differences at P < 005. The efficacies of the tested additives on the heat resistance of E. coli O157:H7 were characterized through response surface methodology using the software program Design Expert 8.0 (Stat-Ease, Inc.). Acknowledgements This project was supported by the Office of the Vice Chancellor for Research and Development of the University of the Philippines, Diliman, under the PhD Incentive Award (PhDIA 111123). The authors also extend their gratitude to Hiroyuki Nakano and Haying Cui of Hiroshima University for providing the test organisms and licorice roots. Romelia Arellano, Jennifer Karen Pineda and Mely Maceda are also being acknowledged for their help in conducting the experiments. Conflict of Interest No conflict of interest declared. References Aguiba, M. (2005) Coconut juice market seen big. Manila Bulletin. Available from: http://www.highbeam.com/doc/ 1G1-133347674.html (accessed December 2010). Baranyi, J. and Roberts, T.A. (1994) A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294. Bazhal, M.I., Lebovka, N.I. and Vorobiev, E.I. (2001) Pulsed electric field treatment of apple tissue during compression for juice extraction. J Engg 50, 129–139. Bazhal, M.I., Ngadi, M.O., Raghavan, G.S.V. and Smith, J.P. (2003) Minimal processing of foods using hurdle technologies. Available from: http://www.engr.usask.ca/ societies/csae/PapersCSAE2003/CSAE03-342.pdf (accessed March 2014). Bevilacqua, A., Campaniello, D., Sinigaglia, M., Ciccarone, C. and Corbo, M.R. (2012) Sodium benzoate and citrus increase the effect of homogenization towards spores of Fusarium oxysporum in pineapple juice. Food Cont 28, 199–204. Coconut Development Board (2005) Tender coconut water. Available from: http://coconutboard.nic.in/tendnutr.htm (accessed March 2011).
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Heat sensitization of E. coli with additives
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Letters in Applied Microbiology 59, 139--146 © 2014 The Society for Applied Microbiology
