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Brown seaweed (Saccharina japonica) as an edible natural delivery matrix for allyl isothiocyanate inhibiting food-borne bacteria
Food Chemistry 152 (2014) 11–17
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Brown seaweed (Saccharina japonica) as an edible natural delivery matrix for allyl isothiocyanate inhibiting food-borne bacteria Evi Amelia Siahaan a, Phillip Pendleton b, Hee-Chul Woo c, Byung-Soo Chun a,⇑ a
Department of Food Science and Technology, Pukyong National University, Yongso-ro 45, Nam-Gu, Busan 608-737, Republic of Korea Center for Molecular and Materials Sciences, School of Pharmacy and Medical Sciences and Sansom Institute, University of South Australia, Adelaide, SA 5001, Australia c Department of Chemical Engineering, Pukyong National University, Sinseon-ro 365, Nam-Gu, Busan 608-739, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 5 August 2013 Received in revised form 15 November 2013 Accepted 20 November 2013 Available online 28 November 2013 Keywords: Saccharina japonica Allyl isothiocyanate Adsorption Desorption Antimicrobial activity Bacteria
a b s t r a c t The edible, brown seaweed Saccharina japonica was prepared as powder in the size range 500–900 lm for the desorption release of allyl isothiocyanate (AITC). Powders were used as raw (containing lipids) and as de-oiled, where the lipid was removed. In general, de-oiled powders adsorbed larger masses of AITC after vapour or solution contact. Mass adsorbed due to solution contact exceeded vapour contact. Larger particles adsorbed more than smaller particles. No chemical bonding between AITC and the powder surface occurred. Release from vapour deposited particles reached 70–85% available within 72 h; solution deposited reached 70–90% available at 192 h. The larger amounts of AITC adsorbed via solution deposition resulted in greater vapour-phase concentrations at 72 h for antimicrobial activity studies. No loss of activity was detected against Escherichia coli, Salmonella Typhimurium or Bacillus cereus. Only a nominal activity against Staphylococcus aureus was demonstrated. S. japonica powder could be used as an edible, natural vehicle for AITC delivery. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Due to greater consumer awareness and concern regarding synthetic chemical additives to food, the application of natural antimicrobial agents has been increasingly noticed in recent years as a novel trend for the biological preservation of foods (Holley & Patel, 2005; Marino, Bersani, & Comi, 2001; Schllinger, Geisen, & Holzapfel, 1996). Natural antimicrobials are defined as those derived from animal, plant or microbial sources. Those derived from sources such as plant oils tend to be mixtures with varying volatility. Volatile substances that do not influence processed food are preferred and, in some circumstances, regarded as safe preservatives (Jang, Hong, & Kim, 2010). One advantage of exploiting volatile preservatives is their ability to penetrate the bulk food matrix. A second advantage is the carrier materials need not necessarily directly contact the product (Appendini & Hotchkiss, 2002). Many researchers have reported the antimicrobial effect of volatile substances extracted from several plants: garlic, sage, oregano, basil, and peppermint (Avato, Tursi, Vitali, Miccolis, & Candido, 2000; Nedorostova, Kloucek, Kokoska, Stolcova, & Pulkrabek, 2009). Many volatile substances express their effects at very low concentration. As potential bactericides, their biodegradability suggests low toxic residue problems (Tripathi & Dubey, 2004).
⇑ Corresponding author. Tel.: +82 51 629 5830; fax: +82 51 629 5824. E-mail address: [email protected] (B.-S. Chun). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.11.116
Allyl isothiocyanate (AITC), a major essential oil component of cruciferous vegetables such as cabbage, broccoli and horseradish, has long been used as a pungent food-flavouring agent (Pechacek, Velišek, & Hrabcova, 1997). Several studies have shown the outstanding antimicrobial activity of vapour-phase AITC (Isshiki, Tokuoka, Mari, & Chiba, 1992; Nadarajah, Han, & Holley, 2005; Park, Barton, & Pendleton, 2012; Park, Taormina, & Beuchat, 2000). Interestingly, in addition to its antimicrobial potency, AITC has been shown to exhibit a high chemo-preventive activity inhibiting prostate cancer and metastasis (Hwang & Lee, 2006; Xiao et al., 2003; Zhang, Li, & Tang, 2005). High volatility, strong pungency, and poor water solubility are factors limiting AITC application to food systems. Its controlled release by desorption from various materials helps exploit the first and overcome the last of these limitations. Examples of AITC controlled release matrices include calcium alginate beads, cyclodextrin, maize, and mesoporous silica (Kim, Chung, Shin, Yam, & Chung, 2008; Li, Jin, & Wang, 2007; Paes, Faroni, Martins, Dhingra, & Silva, 2001; Park & Pendleton, 2012). The overall aim of the work presented here is to define the conditions for and to demonstrate that the highly regarded brown seaweed Saccharina japonica is a material suitable for desorption release of AITC. Brown seaweed is readily available in N.E. Asia, being used as a food supplement, and its bio-absorbable properties are exploited by both the health and food industries (Altenor, Ncibi, Emmanuel, & Gaspard, 2012; Silva, Cossich, Tavares, Filho, & Guirardello, 2002). The edible nature of S. japonica removes the concern for contamination in food application for the control of
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microbial growth during food transport and storage. A primary objective of this research is to assess the efficacy of known antimicrobial growth activity of AITC after it has been adsorbed then desorbed from powdered S. japonica against the selected pathogens: Escherichia coli, Salmonella Typhimurium, Bacillus cereus and Staphylococcus aureus. 2. Materials and methods 2.1. Materials Fresh S. japonica was collected from Guemil-eup, Wando-gun, Jeonnam, South Korea. The carbon dioxide (99.99%) was supplied by KOSEM, Korea. Allyl isothiocyanate of purity >98% (Sigma–Aldrich, St. Louis, MO) was used without further purification. All other reagents used were of analytical grade. 2.2. Bacterial strains The tests were performed against two Gram-positive and two Gram-negative food-borne bacteria. Gram-positive: S. aureus ATCC 6538p; B. cereus ATCC 13,061. Gram-negative: E. coli ATCC 25,922 and S. Typhimurium KCCM 11,862. For the tests tryptone soya agar was used. All strains and media were purchased from the Korean Culture Center of Micro-organisms, Republic of Korea. 2.3. Sample preparation The fresh S. japonica samples were washed with water, cut into small pieces and freeze dried for 3 days. The dried samples were ground using a mechanical blender, then sieved by mesh as 500, 710 and 900 lm maximum particle length and stored at –20 °C. These samples were classified as raw material samples. The lipid content of selected samples was extracted using supercritical carbon dioxide (SC-CO2), with the resulting powders classified as de-oiled materials. For the extraction procedure, S. japonica sample (250 g) was loaded onto a thin layer of previously SC-CO2 extracted and cleaned cotton placed at the base of a stainless steel extraction vessel (500 mL). The powder was then
covered with a second layer of cotton and the unit sealed. The extraction unit and supporting equipment is shown in Fig. 1. Supercritical CO2 was pumped at constant pressure into the extraction vessel by a high pressure pump, up to the desired pressure controlled by a back pressure regulator. The extraction was made at 50 °C and 25 MPa for 2 h. The CO2 flow rate was constant at 44.7 105 kg s1 for all extractions. Both raw and de-oiled samples were used as carriers for AITC. The samples were dried in an oven held at 30 °C, then stored in a desiccator. The sample weight was checked over a period of 24 h to ensure a constant dry weight. The sample dry weight was used as the basis for all measurements. 2.4. AITC loading and release S. japonica loading with AITC was achieved via vapour adsorption and via solution adsorption. The vapour adsorption method was carried out by placing a test tube containing liquid AITC (length: 50 mm; ID: 12 mm) inside a sealed, larger vial containing S. japonica samples. These larger vials were supported in sealed containers at 65 ± 0.05 °C to promote a vapour atmosphere. The solution adsorption of AITC by S. japonica samples was carried out by mixing the powder directly with an AITC solution at constant temperature (25 ± 0.05 °C). For both methods, the amount of AITC adsorbed by S. japonica was determined by monitoring sample weight increase with time. To determine the AITC release, by desorption, the AITC-loaded S. japonica samples were transferred to a tall-sided beaker and placed in a constant temperature water bath (25 ± 0.05 °C) where the adsorbed AITC was allowed to evaporate to the atmosphere with a constant air-flow, of known relative humidity (0.4) across the top of the beaker, promoting desorption and vapour removal. Weight losses with time were monitored to determine desorption, representing the maximum rate of desorption at this temperature. 2.5. GC analysis 2.5.1. AITC calibration Six accurately weighed solutions containing approximately 10, 50, 100, 300, 500 and 1000 ppm (as mg/L) of 98% of AITC in hexane
Fig. 1. Schematic diagram of SC-CO2 extraction.
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(HPLC grade, Burdick and Jackson, Muskegon, MI) were used as AITC standard solution concentrations to prepare an AITC concentration calibration curve. The GC analysis was recorded on a Hewlett Packard 5890 gas chromatograph using an HP INNOWax capillary column (length: 30 m; ID: 0.25 mm; film 0.25 lm) with helium carrier gas. Column temperature was ramped from 50 to 100 °C (10 °C min1) then held at 100 °C for 45 s. The inlet and flame ionisation detector temperatures were set at 160 and 250 °C, respectively. The output data as peak area for each concentration was recorded. Each concentration was measured in triplicate. 2.5.2. Determination of AITC in S. japonica Distilled water (2 mL) was added to each S. japonica sample (0.5 g) and mixed at 2500 rpm (vortex mixer, Fisher Scientific) for 1 min. Samples were held at 5 °C for 6 h, with mixing at 2 h intervals to encourage AITC desorption and its dissolution. Hexane (6 mL) was added to each sample, as a liquid–liquid mass separation agent, and mixed for 1 min. The sedimented material was allowed to stand for 30 min, mixed for 1 min and again allowed to stand unmixed for a further 30 min. An aliquot of the hexane phase (10 lL), containing AITC, was withdrawn using a gas tight Hamilton syringe (10 lL), transferred to then injected into the GC. Sample analysis was as described above. The resulting AITC solution concentration was determined via the calibration curve. 2.6. Scanning electron microscopy (SEM) An SEM equipped with energy dispersive X-ray microanalysis (Hitachi S-2400) was used to image the physical features of the raw and de-oiled samples. Dried samples of Saccharina Japonica were attached to double-sided tape on the sample stubs, then coated via gold sputter under reduced pressure. Images were obtained with an electron accelerating voltage of 200 kV and working distance of 35 mm. 2.7. Fourier-transform infrared measurements FTIR measurements were made to detect any chemical interactions between AITC and S. japonica. A series of transmission spectra were obtained as: (a) pure liquid AITC, (b) pure S. japonica, (c) S. japonica loaded with AITC via vapour adsorption, (d) S. japonica loading with AITC via solution adsorption. The spectrometer used was a Jasco V-670 (Jasco International Co., Ltd., Tokyo, Japan); spectra were measured over the range of 4000–500 cm1, with a resolution of 4 cm–1. 2.8. Antimicrobial assays 2.8.1. Cell suspension and inoculation for initial growth determination The bacteria maintained on the TSA plates were inoculated in the corresponding broth media overnight to promote their growth. The overnight microbial growth in each broth was examined by measuring its optical density at 540 nm using a UVIKON spectrophotometer. The total number of bacteria was measured using a 0.5 McFarland standard to make a uniform population of bacterial suspension. Serial dilutions of aliquots of each culture (100 lL) in the range 10–8–10–1 were prepared as sterile solutions. An initial cell count/mL, identified as colony forming units/mL (CFU/mL) was made for each colony. Samples were then spread across media plates in duplicate according to their dilution factor. 2.8.2. AITC antimicrobial tests Each plate, contained in a glass Petri dish, was exposed to AITC vapour diffused from the S. japonica samples. A control, containing no AITC, was run in parallel with each bacterium studied. Each
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bacterium test was housed in a separate vacuum desiccator (10 L) to avoid cross-contamination. The TSA plates containing the bacterial cultures were used to determine AITC effects on microbial growth. The duration of exposure was 4, 8, 12, 24, 48, and 72 h at 25 °C. The particles containing AITC were 500 lm maximum diameter. The relative humidity of the air within each desiccator was measured prior to sealing, and assumed constant at 45% throughout the test. The plates were held at 25 °C for 72 h and the extent of bacterial growth was determined by a colonycounting method. Triplicate analyses were made. 3. Results and discussion Controlled release of molecules from a carrier or vector for suppressive action by the molecules being desorbed is best achieved via two consecutive processes: (i) an initial burst of the contained molecules to produce a relatively high concentration in a relatively short time period, and (ii) a continuous but slower rate of release to maintain a suitable concentration to suppress growth of the material susceptible to the desorbed molecules. The antimicrobial AITC has been demonstrated to suppress, and in some cases destroy selected bacteria and yeast (Park et al., 2012). The broad-leafed, brown seaweed S. japonica has been used as a natural or introduced supply of food or food supplement for many years in China, Korea, and Japan (Suzuki, Furuva, & Takeuchi, 2006), and as a source of traditional medicine in China for more than 1000 years (Li, Zhang, & Song, 2005). Consequently, if such a material can be demonstrated as a matrix for the controlled release of AITC then such a system could be expected to be acceptable to appropriate national food and health authorities. 3.1. Particle characterisation The removal of the contained lipids from within the seaweed was made expecting an introduction of molecular-scale porosity, and thus a greater capacity for AITC. The SC-CO2 extraction transformed the smooth surface of the raw seaweed (Fig. 2a) to a roughened surface shown in Fig. 2b containing particulate matter ranging in diameter 1–10 lm. In a separate test, smooth, clean metal surfaces were contacted with the extracting fluid SC-CO2. After simulating the extraction conditions applied to the powdered S. japonica samples, SEM analyses of the metal surfaces at magnification similar to those for the powders in Fig. 2b showed no particles were present. This comparison suggests the roughness and particulate matter on the de-oiled S. japonica powders were created by the extraction process, and not deposited from the extracting fluid or the cotton layers holding the powder within the extraction unit. The adsorption profiles shown in Fig. 4 confirm the increased capacity of the de-oiled particles. Both GC analysis of the AITC extracted from the adsorbed phase and FTIR analysis of the adsorbed phase were made to determine if any chemical changes occurred to the molecule on contact with the S. japonica surface, and thus cause any change in antimicrobial potency. The AITC retention time during calibration was 12.69 ± 0.01 min. After contact then extraction from raw and de-oiled samples the retention time for both via vapour adsorption was 12.678 ± 0.02 min, and for both via solution adsorption was 12.77 ± 0.03 min. Although the retention time for the latter is statistically outside both ranges, it is not regarded as significantly different, with no additional peaks in the chromatogram output nor in the FTIR spectra (in Fig. 3). The quantity of AITC recovered from the solution adsorption-prepared samples was more than twice that via vapour adsorption, suggesting more was deposited. The presence of adsorbed AITC on the raw and de-oiled S. japonica samples was confirmed by FTIR. The IR spectrum shown
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Fig. 2. SEM images of the structure surface of (a) raw S. japonica; (b) de-oiled S. japonica.
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AITC desorbed (%)
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in Fig. 3(a) represents raw and de-oiled S. japonica, while spectrum (b) represents the S. japonica samples filled with AITC via vapour adsorption; spectrum (c) represents the S. japonica samples filled with AITC via solution adsorption, and (d) represents pure AITC. The bands at 2165, 2098, and 1648 cm–1 were attributed to the isothiocyanate group (AN@C@S) and the absorbance bands at 986 and 921 cm1 indicated AITC stretching frequencies from (@CAH, ACH@CH2). These bands are also shown in the spectrum (b) and (c), confirming the presence of AITC in the S. japonica samples. Since no new peaks or any significant positional changes for adsorbed phase compared with liquid AITC were found, we concluded AITC interacts with the S. japonica surface via physical adsorption due to dispersion interaction forces only.
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Time, h Fig. 4. (a) Fraction AITC adsorbed by raw and de-oiled S. japonica loaded via vapour adsorption; (b) Fraction AITC adsorbed by raw and de-oiled S. japonica loaded via solution adsorption; (c) Desorption (release) profile of AITC from raw and de-oiled S. japonica loaded via vapour adsorption method; (d) Desorption (release) profile of AITC from raw and de-oiled S. japonica loaded via solution adsorption. Fraction AITC adsorbed is equivalent to mass AITC adsorbed per unit mass dry S. japonica. Percent AITC desorbed is equivalent to the mass AITC desorbed per unit mass AITC adsorbed. For each figure: -d- 500 lm raw S. japonica; j 710 lm raw S. japonica; N 900 lm raw S. japonica; 500 lm de-oiled S. japonica; 710 lm de-oiled S. japonica; 900 lm de-oiled S. japonica.
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3.2. Adsorption and desorption profiles of AITC The time-dependent loading or adsorption profiles are shown in Fig. 4a, b as fraction AITC adsorbed which is equivalent to mass of AITC adsorbed per unit mass of dry S. japonica powder. In contrast, the desorption data in Fig. 4c, d are shown as percent desorbed, to indicate the time-dependent proportion of available AITC. The time-dependent AITC loading profiles for raw and de-oiled powders shown in Fig. 4a, b indicate a rapid rate of adsorption increase relative to the equilibrium fraction loading in the first 24 h exposure. Equilibrium was reached for each system within 60 h, independent of the particle size. The fraction filling, or uptake, for the de-oiled powder was up to 5 times that for the raw powders. The rate and equilibrium amount AITC vapour adsorbed clearly indicate the effect of residual lipid, with the raw samples adsorbing up to half that of the de-oiled samples. Difference in amount adsorbed with particle size for the de-oiled samples indicate the effect of the extraction conditions on the samples; the 900 lm diameter particles provided a greater cell volume available for vapour adsorption than 500 lm diameter particles. This observation suggests the cells may not have lost (too much) structure during SC-CO2 extraction, and thus act as reservoirs for AITC storage. The solution adsorption deposition of AITC showed no effect of particle size or lipid presence in the initial contact, up to 24 h. The raw samples showed no statistical difference in the equilibrium amounts adsorbed. This observation may be due to the hydrophobic nature of the lipid content, providing poor water–lipid solvency leading to restricted diffusion of AITC into those cells containing lipids. In contrast, the equilibrium amount adsorbed for the de-oiled samples showed a dependence on particle size, again attributed to the increased extracted cell volume available with increasing particle size. The amount of AITC adsorbed by the de-oiled powders is similar in rate of uptake to those for MCM-41 and SBA-15 porous silica samples shown by Park (Park et al., 2012), but the quantity adsorbed is approx. 50% of their amounts adsorbed. Efforts to determine the pore size distribution of the de-oiled powders were made, using nitrogen gas adsorption techniques, but these were unable to give meaningful data, suggesting pore sizes exceeded 500 nm, the approximate upper limit of pore sizes accountable by nitrogen gas adsorption (Rouquerol, Rouquerol, & Sing, 1999). By comparison, drawing on the FTIR observations that no chemical reactions occur, and the amounts adsorbed at 60 h, we suggest that the de-oiled sample pore volumes available for the AITC adsorption were also approx. 50% of those of the MCM and SBA powders. The equilibrium amount adsorbed for each collection of powders increased with maximum particle diameter. The time-dependent vapour-phase release, as desorption, was investigated to determine the fraction loaded to be released and the total ‘‘lifetime’’ of the process for subsequent antimicrobial activity retention studies. As shown in Fig. 4c, d, more than 50% of the loaded AITC was released in the first 48 h from all samples, irrespective of loading method. More than 70% of the available
AITC in the samples filled via vapour adsorption was released over the monitored 72 h period; for the solution adsorption-filled samples in Fig. 4d at 72 h, 58 ± 5% was released by the de-oiled samples and 37 ± 5% from the raw samples. The de-oiled samples also released greater quantities of AITC vapour at all subsequent times over the 172 h period monitored, rising to 85 ± 5% for the de-oiled and 72 ± 3% for the raw samples. The percentage AITC available desorbed for either raw or de-oiled material is similar within each method of deposition. We suggest this lack of difference is due to desorption (as evaporation) of the liquid-like adsorbed phase. The balance of AITC held is probably relatively strongly adsorbed to polar sites on the surface and within the structure of the S. japonica powder samples. In their analyses of pathogen growth-control by AITC desorption release from porous silica carriers, Park et al. (2012) showed that an atmosphere of AITC vapour was generated in the first 10 h of desorption, sufficient to control the growth of the various pathogens tested. Further desorption led to distinct reductions in the viable counts of each pathogen. A similar desorption profile, typically referred to as a burst in controlled release nomenclature, was observed for AITC desorption from the powdered S. japonica samples. Consequently, we suggest the required burst of vapour from either sample preparation is probably adequate for creating the initial vapour-phase atmosphere to stall bacteria growth, but the extended period and superior quantity of vapour available and released from the solution adsorption-loaded, de-oiled powders would be expected to provide greater and extended antimicrobial activity, and thus enhanced food protection. 3.3. Antimicrobial assessment As the S. japonica samples are proposed to be used as a delivery vector for AITC, it is important that no antimicrobial activity is lost during the adsorption and desorption processes from the S. japonica matrices. The four micro-organisms selected for analysis are commonly found bacteria: E. coli, S. Typhimurium, B. cereus and S. aureus. To compare the AITC activity, blank tests without AITC were conducted as control studies. The details in Fig. 4a, b indicate that the quantity of AITC adsorbed and available for bacteria growth control studies was affected by the method of loading and the particle average diameter. Clearly, vapour deposition resulted in smaller quantities of AITC adsorbed than solution adsorption, and the 500 lm maximum diameter particles adsorbed less than the 900 lm maximum diameter particles. Similar observations can be made of the percentage (available) AITC released data in Fig. 4c, d, where the de-oiled samples released consistently with time more AITC than the raw samples. Previous antimicrobial activity testing for AITC released from porous powders was made for periods up to 72 h (Park et al., 2012). An analysis of the amounts of AITC released by each system at 72 h per unit mass of dry S. japonica powder is given in Table 1. Since the sensitivity of E. coli, S. Typhimurium, and B. cereus to AITC is well established, the 500 lm particles were selected, as these were expected to provide the least amount of
Table 1 Mass AITC released per unit mass dry S. japonica powder at 72 h. Particle size, lm
Solution adsorption
Vapour adsorption
500
710
900
500
710
900
De-oiled powder % desorbed Mass released, mg/g
54.0 ± 0.58 192 ± 6.79
63.5 ± 0.54 203 ± 4.67
56.2 ± 0.64 232 ± 4.52
81.9 ± 0.62 52.4 ± 6.67
87.4 ± 0.58 81.3 ± 6.54
89.1 ± 0.67 87.3 ± 5.88
Raw powder % desorbed Mass released, mg/g
30.2 ± 0.68 94.3 ± 6.67
37.8 ± 0.58 112 ± 5.70
37.6 ± 0.64 120 ± 6.31
71.4 ± 0.68 40.0 ± 5.57
73.6 ± 0.54 47.1 ± 4.86
82.6 ± 0.62 67.7 ± 6.67
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3.3.1. Gram-negative bacteria Fig. 5a shows how the AITC vapour diffused from dried S. japonica inhibited the growth of E. coli as a function of time. Within the first 24 h of exposure, a modest reduction in counts/mL occurred due to evaporation and release of 50% of the available AITC. The increasing AITC concentration after 24 h resulted in reducing E. coli numbers, significantly reducing the counts/mL by approx. 102 for vapour released from the S. japonica samples prepared via vapour adsorption and via solution adsorption methods. The details in Fig. 5b show that the AITC vapour diffused from within dried S. japonica also inhibited the time-dependent growth of S. Typhimurium. Again, the release in the first 24 h of exposure resulted in a modest reduction in counts/mL. The increasing AITC concentration after 24 h again strongly retarded S. Typhimurium growth, reducing the counts/mL by approximately 102 for vapour-filled S. japonica samples, and by approximately 104 for the solution adsorption-loaded samples. We attribute these enhanced reductions from the solution adsorption-loaded samples to the increased amounts of AITC available compared with the vapour-loaded samples, as confirmed by the details in Fig. 4. From these observations, we conclude de-oiled S. japonica leaves reduced to dry powder and filled with liquidphase AITC provides a suitable, food-compatible carrier and bacteria growth-control system for these Gram-negative bacteria.
(a) Viable count (as log 10), CFU/mL
AITC, as the lowest concentration of AITC in each desiccator testing unit, and thus indicate a check on its potency. For consistency, the same conditions were applied to the tests for S. aureus.
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3.3.2. Gram-positive bacteria The effects of AITC vapour released from both types of loaded samples on B. cereus are shown in Fig. 5c. Exposure effects in the initial 12 h from either method of loading were not statistically different; continued exposure beyond 24 h demonstrated lethality of the vapour phase and the larger amounts available from the solution adsorption-loaded samples resulted in greater control over growth compared with the vapour-loaded samples. After 72 h exposure, bacteria counts reduced by 103 counts/mL for the vapour-loaded system and for the solution adsorption-loaded system by 104. Again, this greater reduction is attributed to the increased quantity released by the solution adsorption-loaded samples. S. aureus has long been recognised as one of the most important agents giving rise to food poisoning worldwide. It is a versatile pathogen of humans and animals and causes a wide variety of diseases ranging in severity from slight skin infection to more severe diseases such as pneumonia and septicemia (Lowy, 1998). S. aureus reportedly exhibits multiple antimicrobial resistance patterns, especially to beta-lactam antibiotics (Enright, 2003; Lowy, 2003). Fig. 5d shows the S. aureus growth response to the AITC vapour released. Exposure to either AITC delivery system saw no appreciable reduction in bacteria counts up to 24 h. After 72 h, 60% available AITC was released from the de-oiled powders (Fig. 4d) resulting in a reduction in counts by a factor of 101, considerably less than growth for B. cereus. Exposure to AITC clearly inhibited further growth of the organism when compared with that of the control, and it is possible further exposure, leading to an increased vapour-phase AITC concentration, might result in a larger reduction in counts. This result confirms the enhanced antimicrobial resistance of S. aureus to AITC compared with B. cereus. Overall, the antimicrobial activity of AITC is not impeded during or after contact with the surface or the internal structure of the brown seaweed S. japonica. The quantity of AITC adsorbed and available for bacteria growth-control is lower than porous silica systems previously investigated, however, since S. japonica is an accepted foodstuff, and its use as a vector for the delivery of AITC
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Time (h) Fig. 5. (a) Lethal effect of AITC on E. coli; (b) lethal effect of AITC on S. Typhimurium; (c) lethal effect of AITC on B. cereus; (d) lethal effect of AITC on S. aureus. For each figure: d Control; N AITC loaded S. japonica (500 lm) via vapour adsorption; 4 AITC loaded S. japonica (500 lm) via solution adsorption.
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is clearly demonstrated, it could be more readily acceptable as an addition within food packages. Before complete acceptance of AITC delivered via S. japonica powder, the issue of its pungency would need to be addressed. 4. Conclusion Raw S. japonica is readily susceptible to de-oiling via extraction with supercritical carbon dioxide. Both raw and de-oiled powders adsorb AITC from vapour- or solution-phase contact, with the latter producing larger quantities adsorbed; de-oiled powder adsorbs more than its raw counterpart. Both powder vectors provide an initial burst of vapour followed by continuous release, producing an appropriate antimicrobial atmosphere controlling growth of selected food-borne bacterial strains. Powdered, de-oiled S. japonica is a suitable, food-compatible carrier of antimicrobial agents for control over the growth of pathogenic bacteria, potentially enhancing food security and safety during storage and transportation. Acknowledgements This work was financially supported by the Ministry of Oceans and Fisheries (contract no. 20131039449). Pendleton was also supported by the Commonwealth of Australia through the Australia-Korea Foundation, which is part of the Department of Foreign Affairs and Trade. References Altenor, S., Ncibi, M. C., Emmanuel, E., & Gaspard, S. (2012). Textural characteristics, physiochemical properties and adsorption efficiencies of Carribean alga Turbinaria turbinate and its derived carbonaceous materials for water treatment application. Biochemical Engineering, 67, 35–44. Appendini, P., & Hotchkiss, J. P. (2002). Review of antimicrobial food packaging. Innovative Food Science & Emerging Technologies, 3, 113–126. Avato, P., Tursi, F., Vitali, C., Miccolis, V., & Candido, V. (2000). Allylsulfide constituents of garlic volatile oil as antimicrobial agents. Phytomedicine, 7, 239–243. Enright, M. C. (2003). The evolution of resistant pathogen-the case of MRSA. Current Opinion in Pharmacology, 3, 474–479. Holley, R. A., & Patel, D. (2005). Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiology, 22, 273–292. Hwang, E.-S., & Lee, H.-J. (2006). Allyl isothiocyanate and its n-acetylcysteine conjugate suppress metastasis via inhibitor of invasion, immigration, and matrix metalloproteinase -2/-9 activities in SK-Hep1 human hepatoma cells. Experimental Biology and Medicine, 232, 421–430. Isshiki, K., Tokuoka, K., Mari, R., & Chiba, S. (1992). Preliminary examination of allyl isothiocyanate vapor for food preservation. Bioscience, Biotechnology, and Biochemistry, 56, 1476–1477.
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Jang, M., Hong, E., & Kim, G.-H. (2010). Evaluation of antibacterial activity of 3butenyl, 4-pentenyl, 2-phenylethyl, and benzyl isothiocyanate in Brassica vegetables. Journal of Food Science, 75, 412–416. Kim, W.-T., Chung, H., Shin, I.-S., Yam, K. L., & Chung, D. (2008). Characterization of calcium alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate. Carbohydrate Polymers, 71, 566–573. Li, X., Jin, Z., & Wang, J. (2007). Complexation of allyl isothiocyanate by a- and bcyclodextrin and its controlled release characteristics. Food Chemistry, 103, 461–466. Li, N., Zhang, Q., & Song, J. (2005). Toxicological evaluation of fucoidan extracted from Laminaria japonica in Wistar rats. Food and Chemical Toxicology, 43, 421–426. Lowy, F. D. (1998). Staphylococcus aureus infection. New England Journal of Medicine, 339, 520–532. Lowy, F. D. (2003). Antimicrobial resistance: the example of Staphylococcus aureus. Journal of Clinical Investigation, 111, 1265–1273. Marino, M., Bersani, C., & Comi, G. (2001). Impedance measurement to study antimicrobial activity of essential oils from Lamiaceae and Compositae. International Journal of Food Microbiology, 67, 187–195. Nadarajah, D., Han, J. H., & Holley, R. A. (2005). Inactivation of Escherichia coli O157:H7 in packaged ground beef by allyl isothiocyanate. Food Microbiology, 99, 269–279. Nedorostova, L., Kloucek, P., Kokoska, L., Stolcova, M., & Pulkrabek, J. (2009). Antimicrobial properties of selected essential oils in vapor phase against food borne bacteria. Food Control, 20, 157–160. Paes, J. L., Faroni, L. R., Martins, M. A., Dhingra, O. D., & Silva, T. A. (2001). Diffusion and sorption of allyl isothyocyanate in the process of fumigation of maize. Storage and Processing of Agricultural Products, 15, 296–301. Park, S.-Y., Barton, M., & Pendleton, P. (2012). Controlled release of allyl isothiocyanate for bacteria growth management. Food Control, 23, 478–484. Park, S.-Y., & Pendleton, P. (2012). Mesoporous silica SBA-15 for natural antimicrobial delivery. Powder Technology, 223, 77–82. Park, C. M., Taormina, P. J., & Beuchat, L. R. (2000). Efficacy of allyl isothiocyanate in killing enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds. Food Microbiology, 56, 13–20. Pecha¯cˇek, R., Velı¯šek, J., & Hrabcova¯, H. (1997). Decomposition products of allyl isothiocyanate in aqueous solutions. Journal of Agricultural and Food Chemistry, 45, 4584–4588. Rouquerol, F., Rouquerol, J., & Sing, K. S. W. (1999). Adsorption by Powders and Porous Solids. London: Academic Press. Schllinger, U., Geisen, R., & Holzapfel, W. H. (1996). Potential of antagonistic microorganisms and bacteriocins for the biological preservation of foods. Trends in Food Sciences and Technology, 7(5), 158–164. Silva, E. A., Cossich, E. S., Tavares, C. R. G., Filho, L. C., & Guirardello, R. (2002). Modelling of copper (II) biosorption by marine alga sargassum sp. In fixed-bed column. Process Biochemistry, 38, 791–799. Suzuki, S., Furuva, K., & Takeuchi, I. (2006). Growth and annual production of the brown alga Laminaria japonica (Phaeophyta, Laminariales) introduced into the Uwa Sea in southern Japan. Journal of Experimental Marine Biology and Ecology, 339, 15–29. Tripathi, P., & Dubey, N. K. (2004). Exploitation of natural products as an alternative strategy to control postharvest fungal rotting of fruit and vegetables. Postharvest Biology and Technology, 32, 235–245. Xiao, D., Srivastava, S. K., Lew, K. L., Zeny, Y., Hershberger, P., Johnson, C. S., et al. (2003). Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits proliferation of human prostate cancer cells by causing G2/M arrest and inducing apoptosis. Carcinogenesis, 24, 891–897. Zhang, Y., Li, J., & Tany, L. (2005). Cancer-preventive isothiocyanates: dichoromous modulators of oxidative stress. Free Radical Biology & Medicine, 38, 70–77.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Brown seaweed (Saccharina japonica) as an edible natural delivery matrix for allyl isothiocyanate inhibiting food-borne bacteria Evi Amelia Siahaan a, Phillip Pendleton b, Hee-Chul Woo c, Byung-Soo Chun a,⇑ a
Department of Food Science and Technology, Pukyong National University, Yongso-ro 45, Nam-Gu, Busan 608-737, Republic of Korea Center for Molecular and Materials Sciences, School of Pharmacy and Medical Sciences and Sansom Institute, University of South Australia, Adelaide, SA 5001, Australia c Department of Chemical Engineering, Pukyong National University, Sinseon-ro 365, Nam-Gu, Busan 608-739, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 5 August 2013 Received in revised form 15 November 2013 Accepted 20 November 2013 Available online 28 November 2013 Keywords: Saccharina japonica Allyl isothiocyanate Adsorption Desorption Antimicrobial activity Bacteria
a b s t r a c t The edible, brown seaweed Saccharina japonica was prepared as powder in the size range 500–900 lm for the desorption release of allyl isothiocyanate (AITC). Powders were used as raw (containing lipids) and as de-oiled, where the lipid was removed. In general, de-oiled powders adsorbed larger masses of AITC after vapour or solution contact. Mass adsorbed due to solution contact exceeded vapour contact. Larger particles adsorbed more than smaller particles. No chemical bonding between AITC and the powder surface occurred. Release from vapour deposited particles reached 70–85% available within 72 h; solution deposited reached 70–90% available at 192 h. The larger amounts of AITC adsorbed via solution deposition resulted in greater vapour-phase concentrations at 72 h for antimicrobial activity studies. No loss of activity was detected against Escherichia coli, Salmonella Typhimurium or Bacillus cereus. Only a nominal activity against Staphylococcus aureus was demonstrated. S. japonica powder could be used as an edible, natural vehicle for AITC delivery. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Due to greater consumer awareness and concern regarding synthetic chemical additives to food, the application of natural antimicrobial agents has been increasingly noticed in recent years as a novel trend for the biological preservation of foods (Holley & Patel, 2005; Marino, Bersani, & Comi, 2001; Schllinger, Geisen, & Holzapfel, 1996). Natural antimicrobials are defined as those derived from animal, plant or microbial sources. Those derived from sources such as plant oils tend to be mixtures with varying volatility. Volatile substances that do not influence processed food are preferred and, in some circumstances, regarded as safe preservatives (Jang, Hong, & Kim, 2010). One advantage of exploiting volatile preservatives is their ability to penetrate the bulk food matrix. A second advantage is the carrier materials need not necessarily directly contact the product (Appendini & Hotchkiss, 2002). Many researchers have reported the antimicrobial effect of volatile substances extracted from several plants: garlic, sage, oregano, basil, and peppermint (Avato, Tursi, Vitali, Miccolis, & Candido, 2000; Nedorostova, Kloucek, Kokoska, Stolcova, & Pulkrabek, 2009). Many volatile substances express their effects at very low concentration. As potential bactericides, their biodegradability suggests low toxic residue problems (Tripathi & Dubey, 2004).
⇑ Corresponding author. Tel.: +82 51 629 5830; fax: +82 51 629 5824. E-mail address: [email protected] (B.-S. Chun). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.11.116
Allyl isothiocyanate (AITC), a major essential oil component of cruciferous vegetables such as cabbage, broccoli and horseradish, has long been used as a pungent food-flavouring agent (Pechacek, Velišek, & Hrabcova, 1997). Several studies have shown the outstanding antimicrobial activity of vapour-phase AITC (Isshiki, Tokuoka, Mari, & Chiba, 1992; Nadarajah, Han, & Holley, 2005; Park, Barton, & Pendleton, 2012; Park, Taormina, & Beuchat, 2000). Interestingly, in addition to its antimicrobial potency, AITC has been shown to exhibit a high chemo-preventive activity inhibiting prostate cancer and metastasis (Hwang & Lee, 2006; Xiao et al., 2003; Zhang, Li, & Tang, 2005). High volatility, strong pungency, and poor water solubility are factors limiting AITC application to food systems. Its controlled release by desorption from various materials helps exploit the first and overcome the last of these limitations. Examples of AITC controlled release matrices include calcium alginate beads, cyclodextrin, maize, and mesoporous silica (Kim, Chung, Shin, Yam, & Chung, 2008; Li, Jin, & Wang, 2007; Paes, Faroni, Martins, Dhingra, & Silva, 2001; Park & Pendleton, 2012). The overall aim of the work presented here is to define the conditions for and to demonstrate that the highly regarded brown seaweed Saccharina japonica is a material suitable for desorption release of AITC. Brown seaweed is readily available in N.E. Asia, being used as a food supplement, and its bio-absorbable properties are exploited by both the health and food industries (Altenor, Ncibi, Emmanuel, & Gaspard, 2012; Silva, Cossich, Tavares, Filho, & Guirardello, 2002). The edible nature of S. japonica removes the concern for contamination in food application for the control of
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microbial growth during food transport and storage. A primary objective of this research is to assess the efficacy of known antimicrobial growth activity of AITC after it has been adsorbed then desorbed from powdered S. japonica against the selected pathogens: Escherichia coli, Salmonella Typhimurium, Bacillus cereus and Staphylococcus aureus. 2. Materials and methods 2.1. Materials Fresh S. japonica was collected from Guemil-eup, Wando-gun, Jeonnam, South Korea. The carbon dioxide (99.99%) was supplied by KOSEM, Korea. Allyl isothiocyanate of purity >98% (Sigma–Aldrich, St. Louis, MO) was used without further purification. All other reagents used were of analytical grade. 2.2. Bacterial strains The tests were performed against two Gram-positive and two Gram-negative food-borne bacteria. Gram-positive: S. aureus ATCC 6538p; B. cereus ATCC 13,061. Gram-negative: E. coli ATCC 25,922 and S. Typhimurium KCCM 11,862. For the tests tryptone soya agar was used. All strains and media were purchased from the Korean Culture Center of Micro-organisms, Republic of Korea. 2.3. Sample preparation The fresh S. japonica samples were washed with water, cut into small pieces and freeze dried for 3 days. The dried samples were ground using a mechanical blender, then sieved by mesh as 500, 710 and 900 lm maximum particle length and stored at –20 °C. These samples were classified as raw material samples. The lipid content of selected samples was extracted using supercritical carbon dioxide (SC-CO2), with the resulting powders classified as de-oiled materials. For the extraction procedure, S. japonica sample (250 g) was loaded onto a thin layer of previously SC-CO2 extracted and cleaned cotton placed at the base of a stainless steel extraction vessel (500 mL). The powder was then
covered with a second layer of cotton and the unit sealed. The extraction unit and supporting equipment is shown in Fig. 1. Supercritical CO2 was pumped at constant pressure into the extraction vessel by a high pressure pump, up to the desired pressure controlled by a back pressure regulator. The extraction was made at 50 °C and 25 MPa for 2 h. The CO2 flow rate was constant at 44.7 105 kg s1 for all extractions. Both raw and de-oiled samples were used as carriers for AITC. The samples were dried in an oven held at 30 °C, then stored in a desiccator. The sample weight was checked over a period of 24 h to ensure a constant dry weight. The sample dry weight was used as the basis for all measurements. 2.4. AITC loading and release S. japonica loading with AITC was achieved via vapour adsorption and via solution adsorption. The vapour adsorption method was carried out by placing a test tube containing liquid AITC (length: 50 mm; ID: 12 mm) inside a sealed, larger vial containing S. japonica samples. These larger vials were supported in sealed containers at 65 ± 0.05 °C to promote a vapour atmosphere. The solution adsorption of AITC by S. japonica samples was carried out by mixing the powder directly with an AITC solution at constant temperature (25 ± 0.05 °C). For both methods, the amount of AITC adsorbed by S. japonica was determined by monitoring sample weight increase with time. To determine the AITC release, by desorption, the AITC-loaded S. japonica samples were transferred to a tall-sided beaker and placed in a constant temperature water bath (25 ± 0.05 °C) where the adsorbed AITC was allowed to evaporate to the atmosphere with a constant air-flow, of known relative humidity (0.4) across the top of the beaker, promoting desorption and vapour removal. Weight losses with time were monitored to determine desorption, representing the maximum rate of desorption at this temperature. 2.5. GC analysis 2.5.1. AITC calibration Six accurately weighed solutions containing approximately 10, 50, 100, 300, 500 and 1000 ppm (as mg/L) of 98% of AITC in hexane
Fig. 1. Schematic diagram of SC-CO2 extraction.
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(HPLC grade, Burdick and Jackson, Muskegon, MI) were used as AITC standard solution concentrations to prepare an AITC concentration calibration curve. The GC analysis was recorded on a Hewlett Packard 5890 gas chromatograph using an HP INNOWax capillary column (length: 30 m; ID: 0.25 mm; film 0.25 lm) with helium carrier gas. Column temperature was ramped from 50 to 100 °C (10 °C min1) then held at 100 °C for 45 s. The inlet and flame ionisation detector temperatures were set at 160 and 250 °C, respectively. The output data as peak area for each concentration was recorded. Each concentration was measured in triplicate. 2.5.2. Determination of AITC in S. japonica Distilled water (2 mL) was added to each S. japonica sample (0.5 g) and mixed at 2500 rpm (vortex mixer, Fisher Scientific) for 1 min. Samples were held at 5 °C for 6 h, with mixing at 2 h intervals to encourage AITC desorption and its dissolution. Hexane (6 mL) was added to each sample, as a liquid–liquid mass separation agent, and mixed for 1 min. The sedimented material was allowed to stand for 30 min, mixed for 1 min and again allowed to stand unmixed for a further 30 min. An aliquot of the hexane phase (10 lL), containing AITC, was withdrawn using a gas tight Hamilton syringe (10 lL), transferred to then injected into the GC. Sample analysis was as described above. The resulting AITC solution concentration was determined via the calibration curve. 2.6. Scanning electron microscopy (SEM) An SEM equipped with energy dispersive X-ray microanalysis (Hitachi S-2400) was used to image the physical features of the raw and de-oiled samples. Dried samples of Saccharina Japonica were attached to double-sided tape on the sample stubs, then coated via gold sputter under reduced pressure. Images were obtained with an electron accelerating voltage of 200 kV and working distance of 35 mm. 2.7. Fourier-transform infrared measurements FTIR measurements were made to detect any chemical interactions between AITC and S. japonica. A series of transmission spectra were obtained as: (a) pure liquid AITC, (b) pure S. japonica, (c) S. japonica loaded with AITC via vapour adsorption, (d) S. japonica loading with AITC via solution adsorption. The spectrometer used was a Jasco V-670 (Jasco International Co., Ltd., Tokyo, Japan); spectra were measured over the range of 4000–500 cm1, with a resolution of 4 cm–1. 2.8. Antimicrobial assays 2.8.1. Cell suspension and inoculation for initial growth determination The bacteria maintained on the TSA plates were inoculated in the corresponding broth media overnight to promote their growth. The overnight microbial growth in each broth was examined by measuring its optical density at 540 nm using a UVIKON spectrophotometer. The total number of bacteria was measured using a 0.5 McFarland standard to make a uniform population of bacterial suspension. Serial dilutions of aliquots of each culture (100 lL) in the range 10–8–10–1 were prepared as sterile solutions. An initial cell count/mL, identified as colony forming units/mL (CFU/mL) was made for each colony. Samples were then spread across media plates in duplicate according to their dilution factor. 2.8.2. AITC antimicrobial tests Each plate, contained in a glass Petri dish, was exposed to AITC vapour diffused from the S. japonica samples. A control, containing no AITC, was run in parallel with each bacterium studied. Each
13
bacterium test was housed in a separate vacuum desiccator (10 L) to avoid cross-contamination. The TSA plates containing the bacterial cultures were used to determine AITC effects on microbial growth. The duration of exposure was 4, 8, 12, 24, 48, and 72 h at 25 °C. The particles containing AITC were 500 lm maximum diameter. The relative humidity of the air within each desiccator was measured prior to sealing, and assumed constant at 45% throughout the test. The plates were held at 25 °C for 72 h and the extent of bacterial growth was determined by a colonycounting method. Triplicate analyses were made. 3. Results and discussion Controlled release of molecules from a carrier or vector for suppressive action by the molecules being desorbed is best achieved via two consecutive processes: (i) an initial burst of the contained molecules to produce a relatively high concentration in a relatively short time period, and (ii) a continuous but slower rate of release to maintain a suitable concentration to suppress growth of the material susceptible to the desorbed molecules. The antimicrobial AITC has been demonstrated to suppress, and in some cases destroy selected bacteria and yeast (Park et al., 2012). The broad-leafed, brown seaweed S. japonica has been used as a natural or introduced supply of food or food supplement for many years in China, Korea, and Japan (Suzuki, Furuva, & Takeuchi, 2006), and as a source of traditional medicine in China for more than 1000 years (Li, Zhang, & Song, 2005). Consequently, if such a material can be demonstrated as a matrix for the controlled release of AITC then such a system could be expected to be acceptable to appropriate national food and health authorities. 3.1. Particle characterisation The removal of the contained lipids from within the seaweed was made expecting an introduction of molecular-scale porosity, and thus a greater capacity for AITC. The SC-CO2 extraction transformed the smooth surface of the raw seaweed (Fig. 2a) to a roughened surface shown in Fig. 2b containing particulate matter ranging in diameter 1–10 lm. In a separate test, smooth, clean metal surfaces were contacted with the extracting fluid SC-CO2. After simulating the extraction conditions applied to the powdered S. japonica samples, SEM analyses of the metal surfaces at magnification similar to those for the powders in Fig. 2b showed no particles were present. This comparison suggests the roughness and particulate matter on the de-oiled S. japonica powders were created by the extraction process, and not deposited from the extracting fluid or the cotton layers holding the powder within the extraction unit. The adsorption profiles shown in Fig. 4 confirm the increased capacity of the de-oiled particles. Both GC analysis of the AITC extracted from the adsorbed phase and FTIR analysis of the adsorbed phase were made to determine if any chemical changes occurred to the molecule on contact with the S. japonica surface, and thus cause any change in antimicrobial potency. The AITC retention time during calibration was 12.69 ± 0.01 min. After contact then extraction from raw and de-oiled samples the retention time for both via vapour adsorption was 12.678 ± 0.02 min, and for both via solution adsorption was 12.77 ± 0.03 min. Although the retention time for the latter is statistically outside both ranges, it is not regarded as significantly different, with no additional peaks in the chromatogram output nor in the FTIR spectra (in Fig. 3). The quantity of AITC recovered from the solution adsorption-prepared samples was more than twice that via vapour adsorption, suggesting more was deposited. The presence of adsorbed AITC on the raw and de-oiled S. japonica samples was confirmed by FTIR. The IR spectrum shown
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(a)
0.12
Fraction AITC adsorbed
14
0.1 0.08 0.06 0.04 0.02 0
0
10
20
30
40
50
60
70
80
Time, h
Fraction AITC adsorbed
(b)
0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0
0
20
40
60
80
Time, h
(c)
80
AITC desorbed (%)
Transmission
(a) (b)
100 90
Fig. 2. SEM images of the structure surface of (a) raw S. japonica; (b) de-oiled S. japonica.
70 60 50 40 30 20 10
(c)
0
0
20
40
60
80
150
200
Time, h
(d)
(d)
100 90
3800
3300
2800
2300
1800
1300
800
Wavenumber, cm-1 Fig. 3. FTIR spectra of (a) S. japonica. (b) S. japonica with AITC via vapour adsorption. (c) S. japonica with AITC via solution adsorption. (d) pure AITC.
AITC desorbed (%)
80 70 60 50 40 30 20
in Fig. 3(a) represents raw and de-oiled S. japonica, while spectrum (b) represents the S. japonica samples filled with AITC via vapour adsorption; spectrum (c) represents the S. japonica samples filled with AITC via solution adsorption, and (d) represents pure AITC. The bands at 2165, 2098, and 1648 cm–1 were attributed to the isothiocyanate group (AN@C@S) and the absorbance bands at 986 and 921 cm1 indicated AITC stretching frequencies from (@CAH, ACH@CH2). These bands are also shown in the spectrum (b) and (c), confirming the presence of AITC in the S. japonica samples. Since no new peaks or any significant positional changes for adsorbed phase compared with liquid AITC were found, we concluded AITC interacts with the S. japonica surface via physical adsorption due to dispersion interaction forces only.
10 0
0
50
100
Time, h Fig. 4. (a) Fraction AITC adsorbed by raw and de-oiled S. japonica loaded via vapour adsorption; (b) Fraction AITC adsorbed by raw and de-oiled S. japonica loaded via solution adsorption; (c) Desorption (release) profile of AITC from raw and de-oiled S. japonica loaded via vapour adsorption method; (d) Desorption (release) profile of AITC from raw and de-oiled S. japonica loaded via solution adsorption. Fraction AITC adsorbed is equivalent to mass AITC adsorbed per unit mass dry S. japonica. Percent AITC desorbed is equivalent to the mass AITC desorbed per unit mass AITC adsorbed. For each figure: -d- 500 lm raw S. japonica; j 710 lm raw S. japonica; N 900 lm raw S. japonica; 500 lm de-oiled S. japonica; 710 lm de-oiled S. japonica; 900 lm de-oiled S. japonica.
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3.2. Adsorption and desorption profiles of AITC The time-dependent loading or adsorption profiles are shown in Fig. 4a, b as fraction AITC adsorbed which is equivalent to mass of AITC adsorbed per unit mass of dry S. japonica powder. In contrast, the desorption data in Fig. 4c, d are shown as percent desorbed, to indicate the time-dependent proportion of available AITC. The time-dependent AITC loading profiles for raw and de-oiled powders shown in Fig. 4a, b indicate a rapid rate of adsorption increase relative to the equilibrium fraction loading in the first 24 h exposure. Equilibrium was reached for each system within 60 h, independent of the particle size. The fraction filling, or uptake, for the de-oiled powder was up to 5 times that for the raw powders. The rate and equilibrium amount AITC vapour adsorbed clearly indicate the effect of residual lipid, with the raw samples adsorbing up to half that of the de-oiled samples. Difference in amount adsorbed with particle size for the de-oiled samples indicate the effect of the extraction conditions on the samples; the 900 lm diameter particles provided a greater cell volume available for vapour adsorption than 500 lm diameter particles. This observation suggests the cells may not have lost (too much) structure during SC-CO2 extraction, and thus act as reservoirs for AITC storage. The solution adsorption deposition of AITC showed no effect of particle size or lipid presence in the initial contact, up to 24 h. The raw samples showed no statistical difference in the equilibrium amounts adsorbed. This observation may be due to the hydrophobic nature of the lipid content, providing poor water–lipid solvency leading to restricted diffusion of AITC into those cells containing lipids. In contrast, the equilibrium amount adsorbed for the de-oiled samples showed a dependence on particle size, again attributed to the increased extracted cell volume available with increasing particle size. The amount of AITC adsorbed by the de-oiled powders is similar in rate of uptake to those for MCM-41 and SBA-15 porous silica samples shown by Park (Park et al., 2012), but the quantity adsorbed is approx. 50% of their amounts adsorbed. Efforts to determine the pore size distribution of the de-oiled powders were made, using nitrogen gas adsorption techniques, but these were unable to give meaningful data, suggesting pore sizes exceeded 500 nm, the approximate upper limit of pore sizes accountable by nitrogen gas adsorption (Rouquerol, Rouquerol, & Sing, 1999). By comparison, drawing on the FTIR observations that no chemical reactions occur, and the amounts adsorbed at 60 h, we suggest that the de-oiled sample pore volumes available for the AITC adsorption were also approx. 50% of those of the MCM and SBA powders. The equilibrium amount adsorbed for each collection of powders increased with maximum particle diameter. The time-dependent vapour-phase release, as desorption, was investigated to determine the fraction loaded to be released and the total ‘‘lifetime’’ of the process for subsequent antimicrobial activity retention studies. As shown in Fig. 4c, d, more than 50% of the loaded AITC was released in the first 48 h from all samples, irrespective of loading method. More than 70% of the available
AITC in the samples filled via vapour adsorption was released over the monitored 72 h period; for the solution adsorption-filled samples in Fig. 4d at 72 h, 58 ± 5% was released by the de-oiled samples and 37 ± 5% from the raw samples. The de-oiled samples also released greater quantities of AITC vapour at all subsequent times over the 172 h period monitored, rising to 85 ± 5% for the de-oiled and 72 ± 3% for the raw samples. The percentage AITC available desorbed for either raw or de-oiled material is similar within each method of deposition. We suggest this lack of difference is due to desorption (as evaporation) of the liquid-like adsorbed phase. The balance of AITC held is probably relatively strongly adsorbed to polar sites on the surface and within the structure of the S. japonica powder samples. In their analyses of pathogen growth-control by AITC desorption release from porous silica carriers, Park et al. (2012) showed that an atmosphere of AITC vapour was generated in the first 10 h of desorption, sufficient to control the growth of the various pathogens tested. Further desorption led to distinct reductions in the viable counts of each pathogen. A similar desorption profile, typically referred to as a burst in controlled release nomenclature, was observed for AITC desorption from the powdered S. japonica samples. Consequently, we suggest the required burst of vapour from either sample preparation is probably adequate for creating the initial vapour-phase atmosphere to stall bacteria growth, but the extended period and superior quantity of vapour available and released from the solution adsorption-loaded, de-oiled powders would be expected to provide greater and extended antimicrobial activity, and thus enhanced food protection. 3.3. Antimicrobial assessment As the S. japonica samples are proposed to be used as a delivery vector for AITC, it is important that no antimicrobial activity is lost during the adsorption and desorption processes from the S. japonica matrices. The four micro-organisms selected for analysis are commonly found bacteria: E. coli, S. Typhimurium, B. cereus and S. aureus. To compare the AITC activity, blank tests without AITC were conducted as control studies. The details in Fig. 4a, b indicate that the quantity of AITC adsorbed and available for bacteria growth control studies was affected by the method of loading and the particle average diameter. Clearly, vapour deposition resulted in smaller quantities of AITC adsorbed than solution adsorption, and the 500 lm maximum diameter particles adsorbed less than the 900 lm maximum diameter particles. Similar observations can be made of the percentage (available) AITC released data in Fig. 4c, d, where the de-oiled samples released consistently with time more AITC than the raw samples. Previous antimicrobial activity testing for AITC released from porous powders was made for periods up to 72 h (Park et al., 2012). An analysis of the amounts of AITC released by each system at 72 h per unit mass of dry S. japonica powder is given in Table 1. Since the sensitivity of E. coli, S. Typhimurium, and B. cereus to AITC is well established, the 500 lm particles were selected, as these were expected to provide the least amount of
Table 1 Mass AITC released per unit mass dry S. japonica powder at 72 h. Particle size, lm
Solution adsorption
Vapour adsorption
500
710
900
500
710
900
De-oiled powder % desorbed Mass released, mg/g
54.0 ± 0.58 192 ± 6.79
63.5 ± 0.54 203 ± 4.67
56.2 ± 0.64 232 ± 4.52
81.9 ± 0.62 52.4 ± 6.67
87.4 ± 0.58 81.3 ± 6.54
89.1 ± 0.67 87.3 ± 5.88
Raw powder % desorbed Mass released, mg/g
30.2 ± 0.68 94.3 ± 6.67
37.8 ± 0.58 112 ± 5.70
37.6 ± 0.64 120 ± 6.31
71.4 ± 0.68 40.0 ± 5.57
73.6 ± 0.54 47.1 ± 4.86
82.6 ± 0.62 67.7 ± 6.67
E.A. Siahaan et al. / Food Chemistry 152 (2014) 11–17
3.3.1. Gram-negative bacteria Fig. 5a shows how the AITC vapour diffused from dried S. japonica inhibited the growth of E. coli as a function of time. Within the first 24 h of exposure, a modest reduction in counts/mL occurred due to evaporation and release of 50% of the available AITC. The increasing AITC concentration after 24 h resulted in reducing E. coli numbers, significantly reducing the counts/mL by approx. 102 for vapour released from the S. japonica samples prepared via vapour adsorption and via solution adsorption methods. The details in Fig. 5b show that the AITC vapour diffused from within dried S. japonica also inhibited the time-dependent growth of S. Typhimurium. Again, the release in the first 24 h of exposure resulted in a modest reduction in counts/mL. The increasing AITC concentration after 24 h again strongly retarded S. Typhimurium growth, reducing the counts/mL by approximately 102 for vapour-filled S. japonica samples, and by approximately 104 for the solution adsorption-loaded samples. We attribute these enhanced reductions from the solution adsorption-loaded samples to the increased amounts of AITC available compared with the vapour-loaded samples, as confirmed by the details in Fig. 4. From these observations, we conclude de-oiled S. japonica leaves reduced to dry powder and filled with liquidphase AITC provides a suitable, food-compatible carrier and bacteria growth-control system for these Gram-negative bacteria.
(a) Viable count (as log 10), CFU/mL
AITC, as the lowest concentration of AITC in each desiccator testing unit, and thus indicate a check on its potency. For consistency, the same conditions were applied to the tests for S. aureus.
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3.3.2. Gram-positive bacteria The effects of AITC vapour released from both types of loaded samples on B. cereus are shown in Fig. 5c. Exposure effects in the initial 12 h from either method of loading were not statistically different; continued exposure beyond 24 h demonstrated lethality of the vapour phase and the larger amounts available from the solution adsorption-loaded samples resulted in greater control over growth compared with the vapour-loaded samples. After 72 h exposure, bacteria counts reduced by 103 counts/mL for the vapour-loaded system and for the solution adsorption-loaded system by 104. Again, this greater reduction is attributed to the increased quantity released by the solution adsorption-loaded samples. S. aureus has long been recognised as one of the most important agents giving rise to food poisoning worldwide. It is a versatile pathogen of humans and animals and causes a wide variety of diseases ranging in severity from slight skin infection to more severe diseases such as pneumonia and septicemia (Lowy, 1998). S. aureus reportedly exhibits multiple antimicrobial resistance patterns, especially to beta-lactam antibiotics (Enright, 2003; Lowy, 2003). Fig. 5d shows the S. aureus growth response to the AITC vapour released. Exposure to either AITC delivery system saw no appreciable reduction in bacteria counts up to 24 h. After 72 h, 60% available AITC was released from the de-oiled powders (Fig. 4d) resulting in a reduction in counts by a factor of 101, considerably less than growth for B. cereus. Exposure to AITC clearly inhibited further growth of the organism when compared with that of the control, and it is possible further exposure, leading to an increased vapour-phase AITC concentration, might result in a larger reduction in counts. This result confirms the enhanced antimicrobial resistance of S. aureus to AITC compared with B. cereus. Overall, the antimicrobial activity of AITC is not impeded during or after contact with the surface or the internal structure of the brown seaweed S. japonica. The quantity of AITC adsorbed and available for bacteria growth-control is lower than porous silica systems previously investigated, however, since S. japonica is an accepted foodstuff, and its use as a vector for the delivery of AITC
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Time (h) Fig. 5. (a) Lethal effect of AITC on E. coli; (b) lethal effect of AITC on S. Typhimurium; (c) lethal effect of AITC on B. cereus; (d) lethal effect of AITC on S. aureus. For each figure: d Control; N AITC loaded S. japonica (500 lm) via vapour adsorption; 4 AITC loaded S. japonica (500 lm) via solution adsorption.
E.A. Siahaan et al. / Food Chemistry 152 (2014) 11–17
is clearly demonstrated, it could be more readily acceptable as an addition within food packages. Before complete acceptance of AITC delivered via S. japonica powder, the issue of its pungency would need to be addressed. 4. Conclusion Raw S. japonica is readily susceptible to de-oiling via extraction with supercritical carbon dioxide. Both raw and de-oiled powders adsorb AITC from vapour- or solution-phase contact, with the latter producing larger quantities adsorbed; de-oiled powder adsorbs more than its raw counterpart. Both powder vectors provide an initial burst of vapour followed by continuous release, producing an appropriate antimicrobial atmosphere controlling growth of selected food-borne bacterial strains. Powdered, de-oiled S. japonica is a suitable, food-compatible carrier of antimicrobial agents for control over the growth of pathogenic bacteria, potentially enhancing food security and safety during storage and transportation. Acknowledgements This work was financially supported by the Ministry of Oceans and Fisheries (contract no. 20131039449). Pendleton was also supported by the Commonwealth of Australia through the Australia-Korea Foundation, which is part of the Department of Foreign Affairs and Trade. References Altenor, S., Ncibi, M. C., Emmanuel, E., & Gaspard, S. (2012). Textural characteristics, physiochemical properties and adsorption efficiencies of Carribean alga Turbinaria turbinate and its derived carbonaceous materials for water treatment application. Biochemical Engineering, 67, 35–44. Appendini, P., & Hotchkiss, J. P. (2002). Review of antimicrobial food packaging. Innovative Food Science & Emerging Technologies, 3, 113–126. Avato, P., Tursi, F., Vitali, C., Miccolis, V., & Candido, V. (2000). Allylsulfide constituents of garlic volatile oil as antimicrobial agents. Phytomedicine, 7, 239–243. Enright, M. C. (2003). The evolution of resistant pathogen-the case of MRSA. Current Opinion in Pharmacology, 3, 474–479. Holley, R. A., & Patel, D. (2005). Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiology, 22, 273–292. Hwang, E.-S., & Lee, H.-J. (2006). Allyl isothiocyanate and its n-acetylcysteine conjugate suppress metastasis via inhibitor of invasion, immigration, and matrix metalloproteinase -2/-9 activities in SK-Hep1 human hepatoma cells. Experimental Biology and Medicine, 232, 421–430. Isshiki, K., Tokuoka, K., Mari, R., & Chiba, S. (1992). Preliminary examination of allyl isothiocyanate vapor for food preservation. Bioscience, Biotechnology, and Biochemistry, 56, 1476–1477.
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