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Antimicrobial activity of nisin-adsorbed silica and corn starch powders
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 22 (2005) 93–99
www.elsevier.nl/locate/jnlabr/yfmic
Antimicrobial activity of nisin-adsorbed silica and corn starch powders P.L. Dawsona,*, L. Harmonc, A. Sotthibandhub, I.Y. Hana a
Department of Food Science and Human Nutrition, Clemson University, A203J Poole Hall, SC 29634-0371, Clemson, South Carolina, USA b South Carolina Governors School for Science and Mathematics, Hartsville, South Carolina, USA c Food Science and Nutrition, Faculty of Science, Srinakharinwirot University, Sukumvit 23, Bangkok, Thailand Received 17 November 2003; received in revised form 27 March 2004; accepted 6 April 2004
Abstract The antimicrobial peptide nisin is an effective bacterial inhibitor and has been adsorbed onto various surfaces and added to packaging films. In the present study, nisin was adsorbed onto food-grade powders then evaluated for nisin activity in a series of three experiments. Adsorbent powders used were calcined diatomaceous earth, synthetic calcium silicate, hydrate, two diatomaceous earth, and corn starch powders. Adsorption was conducted by placing the powders in agitated nisin solutions, followed by dehydration of the powder pellet after centrifugation. The dehydrated powders were then tested for inhibitory activity against either Lactobacillus plantarum or Listeria monocytogenes. Activity was measured by placing the nisin-adsorbed powders in solutions of 0.1% peptone water inoculated with one of the test bacterial strains. Cel-pure 65 adsorbed then released from 74.7% to 94.7% of the nisin activity contained in the original solution (500–800 IU/ml) in which the powders were agitated. Corn starch adsorbed then released from 45.4% to 60.5% of nisin activity when from 300 to 700 IU/ml nisin activity adsorbing solutions were utilized. One percent of nisin-absorbed Celpure 100 and Celpure 65 powders in 30 ml of log 4.5 cfu/ml L. monocytogenes inoculum reduced populations to below detection levels (o101 cfu/ml) within 24 and 6 h, respectively. Thus, nisin-adsorbed powders were highly efficient at both adsorption and release of antimicrobial activity. r 2004 Elsevier Ltd. All rights reserved. Keywords: Antimicrobial packaging; Nisin; Listeria monocytogenes; Corn starch; Adsorbents
1. Introduction Methods to deliver antimicrobials to food can vary from direct addition to incorporation into packaging materials. Nisin is considered a safe food antimicrobial and has been approved as a generally recognized as safe (GRAS) additive in pasteurized cheese spread and liquid egg in the US (FDA, 1988) to inhibit Clostridium botulinum spores. Nisin is primarily active against Gram-positive bacteria but when combined with a chelator, nisin also can inhibit growth of some Gramnegative bacteria (Stevens et al., 1991; Ray, 1992) thus nisin can potentially be effective against a broad spectrum of bacteria. In food systems, nisin effectiveness *Corresponding author. Tel.: +1-864-656-1138; fax: +1-864-6560331. E-mail address: [email protected] (P.L. Dawson). 0740-0020/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2004.04.001
can be reduced by food characteristics (Bell and DeLacy, 1987; Jung et al., 1992). The direct application of nisin to food surfaces thus will result in some loss of activity due to various food components. A method to control the release of nisin activity over a period of time would add a microbiological hurdle downstream in the food distribution chain. Han and Floros (2000) summarized the applications of antimicrobial packaging including nisin-containing packaging. Antimicrobial packaging films can provide a downstream hurdle via a controlled release scenario. There are two general categories of antimicrobial films, those in which the antimicrobial agent migrates from the film and those that the agent remains within the film material. Due to the nature of food, if the antimicrobial does not migrate from the film at least to the food surface, it will have limited effect. Nisin-containing packaging materials have had potential applications to prevent many
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foodborne pathogenic bacteria and their spores, C. botulinum, Listeria monocytogenes (Hurst, 1981; DelvesBroughton, 1990; Ray, 1992). Generally, antimicrobial packaging can be prepared by mixing of, coating with or chemical immobilization of antimicrobial agents to the packaging material. Numerous delivery systems have been evaluated using nisin including dialysis onto meat casings (Ming et al., 1997), coating onto collagen casings (Kassaify, 1998), cast protein films (Ko et al., 2001; Hoffman et al., 2001; Orr et al., 1997; Dawson et al., 1995; Padgett et al., 1998), polymer film coatings (Natrajan and Sheldon, 2000a), calcium alginate (Wan et al., 1997; Natrajan and Sheldon, 2000b; Cutter and Siragusa, 1997), methyl cellulose film coatings (Coma et al., 2001; Grower et al., 2004; Franklin et al., 2004) and heat-set biobased films (Padgett et al., 1998; Dawson et al., 1995; McCormick et al., 2004; Tereakarn et al., 2002). Polyethylene films and corn zein films were shown to reduce L. monocytogenes populations in peptone water from 8 logs (cfu/ ml) to below detectable levels (o102 cfu.ml) after 24 h (Hoffman et al., 1997, 2001) and reduce bacterial counts on chicken drumsticks (Natrajan and Sheldon, 2000a). Fluid foods may also be candidates for antimicrobial packaging as demonstrated with corn zein films impregnated with nisin. These films reduced L. monocytogenes in skim milk by 3 logs (cfu/ml) after 48 h (Orr et al., 1998) and L. plantarum in peptone water to below detection (Padgett et al., 1998, 2000). Diffusivity of nisin impregnated corn zein and wheat gluten films into water were affected by film type (wheat gluten or corn zein), environmental temperature and film-forming method (cast or heat-set) (Teerakarn et al., 2002). Cast wheat gluten film had the greatest diffusivity while the cast corn zein film had the lowest and heat-pressed wheat gluten and corn zein films did not differ in diffusivity. Cutter and Siragusa (1997) and Natrajan and Sheldon (2000b) used calcium alginate to deliver nisin to beef and chicken surfaces, respectively. Another approach to controlled nisin release is through adsorption of the peptide onto surfaces or particles that can deliver the antimicrobial activity to the desired location. A unique method to improve recovery of bacteriocins from cultures by adsorbing the bacteriocins onto cells from the producing strains (Yang et al., 1992). Ming et al. (1997) coated cellulose casings with either nisin or pediocin to inhibit the growth of L. monocytogenes on cooked meats but found that nisin was not effective. Wan et al. (1996) and Coventry et al. (1996) reported adsorbing bacteriocins, including nisin, onto ingestible silica compounds providing a simple method to harvest bacteriocin from culture supernatant and they also demonstrated the effectiveness of bacteriocin-adsorbed material against L. monocytogenes. A similar application was reported using rice hull ash and silicic acid to harvest five bacteriocins from freeze-dried
culture supernatants by Janes et al. (1998). Bowers et al. (1995) found no suppression of L. monocytogenes growth but a reduced adhesion rate of the bacterium to silica surfaces that had nisin adsorbed. In the current study, the previously described modes of delivery were combined by using silica and starch material in the powder form as a carrier of nisin. The objective of this research was to evaluate the adsorption and release of nisin activity onto and from food-grade powders.
2. Materials and methods 2.1. Absorbents Adsorbents used in this study included FilterCels (Calcined diatomaceous earth, SiO2, Celite Corporation, Lompoc, California), Micro-Cel s E (synthetic calcium silicate hydrate, CaSiO3, Celite Corporation, Lampoc, California), Celpures P65, CelpuresP1000 (Diatomaceous earth, Sigma-Aldrich, Saint Louis, Missouri), and anti set-off powder corn starch (Varn Products Company Inc., Oakland, New Jersey). FilterCels, Micro-Cel s E, Celpures P65, and CelpuresP1000 are used as filter aid agents due to their high absorption capacity, high surface area, and low bulk density. The Food and Drug Administration considers diatomaceous earth and calcium silicates to be GRAS according to title 21 code of Federal Regulation part 182.90 and 182.2227, respectively. 2.2. Nisin activity L. plantarum ATCC 1572 was the test organism used to determine nisin activity via the agar diffusion technique. L. monocytogenes ATCC 15313 was used to investigate effectiveness of nisin-adsorbed powders in a 0.1% peptone water suspension. Nisin standard solution was prepared using purified nisin (94.6% nisin by weight, Aplin and Barrett, Dorset, UK). The activity of nisin preparation is expressed in International Units (IU). The relationship of 1 g purified nisin=38 106 IU was presented by Ray (1992). Thus 1 mg of purified nisin used in this study contained 3.8 104 IU. To prepare the stock solution in HCl or water, 62.5 mg of nisin powder was mixed separately with both solvents using separate 50-ml volumetric flasks. Standard solutions (0.00028– 0.0125 mg purified nisin/ml) were prepared by dilution of the stock solution using sterile 0.02 n HCl or sterile distilled water. Standard solution for both sterile water and 0.02 n HCl were prepared and used since biological activity remaining in adsorbent powders was determined using 0.02 n HCl and migration from adsorbents was determined into a standard FDA simulants, sterile water. The standard curve dilution points were 0.00028, 0.0025, 0.0050, 0.0075, 0.0100, and 0.0125 mg
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pure nisin/ml. The R2 for standard curves in both 0.02 n HCl and sterile water were 0.99. The response of standard nisin solution on inhibiting L. plantarum was determined using the agar diffusion technique (Redl et al., 1996). A standard curve plot based on the relationship of the width of inhibition zones (mm) (Yaxis) and log of nisin concentration in IU (X-axis) was made for each experiment. Regression analysis was used to determine the standard equation, which was used to calculate the concentration of nisin in samples. 2.3. Experiment 1: Efficiency of nisin adsorption and release by five food-grade powders The procedure used to prepare bacteriocin-adsorbed powders followed the method previously described by Wan et al. (1996). In experiment 1 all 5 powders listed above were tested by adding 1.5 g of the powder to 1000 ml of distilled water containing 0.125 g of Nisaplint (Fig. 1). Nisin activity of solutions was 125 IU/ml for experiment 1. Commercial nisin (Nisaplin, 1,000,000 IU/g, Aplin & Barrett Ltd., Dorset, England) was used to prepare a 125 IU/ ml solution (Fig. 2). Silica powders or cornstarch were added to freshly made nisin solution at at 1% W/V in an individual sterile centrifuge tube and mixed using magnetic stirrers for 30 min at room temperature (22–24 C). Mixtures were then centrifuged (12,000g at 20 C for 10 min, Sorvells RC5B, refrigerated super-speed centrifuge, Dupont Instru-
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ments) to separate supernatant. The supernatant was assayed for residual nisin activity by agar diffusion technique previously described by Teerakarn et al. (2002). Solutions without adsorbents added were assayed to verify 125 IU/ml were contained in each solution. Diameters of clear or inhibitory zones were used to calculated nisin activity from the regression equation of the standard curve for nisin (Nisaplin) solution at 10–125 IU/ml. Averages of three clear zone readings were used to calculate nisin activity for each sample. After centrifugation, particles were dried in a hot air oven at 50 C for 4 h and at 30 C for 16–18 h with air circulation. The released biological activity of nisinadsorbed silica powders and cornstarch were determined by adding dried powders to sterile water at 1% (w/v) followed by testing for inhibitory effect on L. plantarum by the agar diffusion technique (Fig. 1). Adsorbed powder/sterile water mixtures were stirred at slow speed using a sterile magnetic stir rod for 20 min.Various mixing times were tested and under these conditions, release leveled off after 15–18 min thus 20 min was used to assess nisin adsorption by the powders. The mixture was sampled for biological activity using the same agar diffusion technique previously described for nisin activity. Non-nisin adsorbed powders were also dispersed in sterile water (1%, w/v) and sampled as described with no biological activity found in these control samples. The experiment was repeated three times with separate batches of materials. A randomizedcomplete block design using a general linear model was used to determine analysis of variance for treatment effects of replication and adsorbent type. The model contained these main effects and the replication by adsorbent type was used as the error term. Since adsorbent type had a significant treatment effect (pp0.05) on nisin activity, activity of adsorbent types was separated using the pdiff command of SAS (2000). 2.4. Experiment 2: Efficacy of nisin-adsorbed powders for inhibiting L. monocytogenes
Fig. 1. Diagram of the adsorption and analysis procedure used for food-grade powders.
Methods to produce nisin-absorbed powders were the same as described in experiment 1. L. monocytogenes was grown in Brain–Heart Infusion (BHI) media (Difco Laboratories, Detroit, Michigan) and incubated aerobically for 16 h at 37 C. The L. monocytogenes culture was centrifuged (20 min at 3000 rpm) and washed with 0.1% peptone. Thirty milliliter of inoculum in 0.1% peptone (104–105 CFU/ml) was then added to each of sterile glass petri dish containing 1% w/v of nisinadsorbed powder. Petri dishes were shaken on an orbital shaker at room temperature (20–24 C) at 50 rpm. Samples were taken after 0, 1, 2, 3, 6, 10, 24, and 48 h, diluted, and plated in duplicate on BHI agar. Colonies were counted after 48 h of aerobic incubation at 37 C. The experiment was replicated three times on different
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96 120
94.66 (76%) a
100
nisin activity (IU/ml)
79.23 80
(63.4%) a
71.53 67.76
(57.2%) a
(54.2%) a
60
40
20.92 (16.7%) b
20
0
Filter-cel
Microcel-E
Celpure 1000
Celpure 65
Corn starch
Fig. 2. Nisin activity for five food-grade powders that were previously exposed to a 125 IU/ml nisin solution for 20 min (with agitation). a,b Means with different superscripts differ significantly (pp0.05).
days. A randomized-complete block design using a general linear model was used to determine analysis of variance for treatment effects of replication, adsorbent type, and exposure time. The model included these main effects and the exposure time by adsorbent-type interaction with the residual interactions used as the error term. Since all factors included in the model had a significant effect (pp0.05) on the log CFU of L. monocytogenes, the standard error for each time and powder was determined using the proc-mixed procedure of SAS (2000).
compared in this experiment. A randomized-complete block design using a general linear model was used to determine analysis of variance for treatment effects of replication and adsorbent level. The model contained these main effects and the replication by nisin level interaction was used as the error term. Since the nisin level in adsorbing solutions had a significant treatment effect (pp0.05) on adsorbent nisin activity, the means for adsorbent activity was separated using the pdiff command of SAS (2000).
2.5. Experiment 3: Effect of nisin concentration in the adsorbing solution on adsorption of activity
3. Results and discussion
CS and CP65 were chosen for further investigation. CS was chosen since it has wide usage as a film-coating agent to prevent packaging film adherence. CP65 was chosen due to its performance compared to other agents in experiment 1. The objective of experiment 3 was to determine (within a concentration range) the efficiency of adsorption of powders (i.e. to what degree would an increase of nisin concentration in the adsorption solution result in an increased absorption by powders). Ranges of solution concentrations used were chosen based on preliminary tests. Nisin activity of solutions used to adsorb onto CS were 300, 400, 450, 500, 600, and 700 IU/ml and onto CP65 were 500, 600, 650, 700, and 800 IU/ml. As with experiments 1 and 2, 1.5 g of each adsorbent was added to 1000 ml of the nisin solutions then after adsorption the powders were dried. Biological activity of dried powders was determined with L. plantarum using the agar diffusion technique. This experiment was repeated three times each for CS and CP-65 using separate batches of materials on different days. CS and CP-65 were not statistically
Nisin has consistently been shown to inhibit Grampositive bacteria. While the bactericidal activity of nisin when adsorbed to a surface has been investigated, its mechanism of action has not been clearly described. However, previous research has shown that adsorbed nisin retains biological activity (Daeschel et al., 1992; Bower et al., 1995; Wan et al., 1996). The mechanism of nisin activity in solution is by integration into bacterial cell membranes resulting in pore formation (Ruhr and Sahl, 1985; Gao et al., 1991; Bruno et al., 1992). While the bactericidal mechanism of adsorbed nisin has not been determined it may be similar to its mechanism in solution and may require the release of nisin from the adsorbed surface to promote pore formation in the cell membrane. 3.1. Experiment 1: Efficiency of nisin adsorption and release by five food-grade powders Filter-Cel, CP-1000, CP-65 and corn starch did not differ in % adsorption themselves, ranging from 54.2% to 76% adsorption efficiency. Micro-Cel-E adsorbed
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and released the least nisin (16.7% efficiency) among the powders tested. These adsorption/release efficiencies are 10–100 times higher than those calculated for packaging films having nisin incorporated into their structure (Teerakarn et al., 2002) when tested under the same conditions. Structural changes occur when a peptide adsorbs onto a surface and these changes can also result in the formation of multi-layers and dimers (Liu and Hansen, 1990; Bowers et al., 1995). These structural changes can affect nisin activity and release from a surface. Wan et al. (1996) reported 100% recovery of nisin activity (125 IU/ml) from silica adsorbents including Micro-Cel and Filter-Cel however, this study also reported 13% of the activity remained in the supernatant. The 100% activity recovered from the adsorbents plus the additional 13% residual activity could be accounted for if the adsorbent itself resulted in a loss of bacterial cells. This does lead to question the accuracy of the measurement since more than 100% of the original activity was recovered using a supposedly inert silica adsorbent. The results from the present study verifies those found by Wan et al. (1996) that nisin can be adsorbed onto silica particles with a high level of recovery of biological activity. 3.2. Experiment 2: Efficacy of nisin-adsorbed powders for inhibiting L. monocytogenes Four of the five powders having been exposed to a 125 IU/ml nisin solution significantly reduced L. monocytogenes populations within 2 h (Fig. 3). Control treatments of Microcel-E and corn starch without nisin adsorption and peptone water with no powders added all increased in bacterial populations by 2–3 log CFU/ml after 24 h. Preliminary experiments showed the same effect of the other silica-based powders as was found with Microcel-E (data not shown) thus all were not included in experiment 2. Nisin-adsorbed CP-65 and CP-1000 both reduced cell counts below detection after CP-65
8
CP-1000 FilterCel
7
Corn Starch MicroCel
Log CFU/ml
6
Control
5 4 3 2 1 0 0
8
16
24 time (hours)
32
40
48
Fig. 3. Inhibitory effect of Cel-Pure 65 or corn starch adsorbed in solutions containing different nisin levels then exposed to 0.1% peptone water containing 104–5 cfu/ml of Listeria monocytogenes cells.
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10 h and no recovery of cells was detected up to 48 h. Nisin-adsorbed corn starch reduced L. monocytogenes by 4 logs after 10 h, however, the population then recovered with a>2 log outgrowth between 24 and 48 h. Filter-Cel similarly reached a maximum population reduction at 24 h (3.5 log reduction) then had a 1log growth recovery by 48 h. These nisin-adsorbed powders had a similar affect on L. monocytogenes in solution as nisin-impregnated corn zein films (Hoffman et al., 2001). However, nisin (as Nisaplin) was added at 5 times the amount (5%) to films and was exposed to 12 the volume of inoculated peptone water as powders in the current study. Furthermore, Teerakarn et al. (2002) found nisin-impregnated films released less than 10% of the nisin activity. Thus, under laboratory conditions the use of nisin-adsorbed powders to carry nisin activity is a more efficient method than incorporation into polymer films. 3.3. Experiment 3: Effect of nisin concentration in the adsorbing solution on adsorption of activity By using a range of adsorption solution concentrations of from 300 to 700 IU nisin/ml, adsorption/release efficiency of CP-65 decreased from 97.8% (500 IU/ml solutions) to 75% (650 IU/ml solutions) and of corn starch decreased from 60.5% (300 IU/ml solutions) to 45% (600 IU/ml solutions) (Tables 1 and 2). However, efficiency of adsorption was greater at lower solution concentrations for both powders. Time effects on release were not studied here, thus conclusions related to rate of activity release at shorter or longer times than the one used cannot be made. Bowers et al. (1995) found a similar trend of reduced adsorption efficiency with higher nisin-adsorbing concentrations when adsorbing nisin onto silica surface as opposed to particles. The objective of the Bowers study was to prevent L. monocytogenes adhesion (which it did) and suppress growth (which it did not) by adsorbing nisin onto silica surfaces. In Wan et al. (1996) and the current study, silica and starch particles were used as nisin carriers thus release was as important as adsorption. Wan et al. (1996) found a relatively constant adsorption/release efficiency of approximately 50% for pisciolin onto Micro-Cel when adsorption solutions containing between 107 and 108 AU/g were used. Conversely, the same study reported Filter-Cel adsorbed no more activity (a decreasing adsorption/release efficiency) from solutions containing pisciolin over the same range of activity. In the current study, the adsorption/release efficiency of corn starch was between 30% and 45% lower than CP65 when the same solutions concentrations were compared. Thus, CP-65 was a superior adsorbing/ releasing agent as was demonstrated in experiment 1. Little nisin activity was found in the supernantant of the adsorption solution after recovery of the corn starch
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Table 1 Nisin activity retained by corn starch and remaining in the supernatant after adsorption in solutions containing various levels of Nisaplins Solution nisin activity
Corn starch nisin activity
Activity remaining in supernatant after adsorption (IU/ml)
Activity not accounted for
300 400 450 500 600 700 Standard error of the mean
181.5c (60.5%) 237.0b (59.2%) 241.1b (53.6%) 257.2b (51.4%) 272.6b (45.4%) 330.5a (47.2%) 14.9
0c 0c 0c 4.4b 4.2b 8.2a 0.9
118.5 163.0 208.9 238.4 323.2 361.3
a–c Means with the same superscripts do not significantly differ (p>0.05). (%) represents the percentage of activity absorbed onto corn starch from the solution.
Table 2 Nisin activity retained by Cel-Pure 65 (CP-65) and remaining in the supernatant after adsorption in solutions containing various levels of Nisaplins Solution nisin activity
CP-65 nisin activity
Activity remaining in supernatant after adsorption (IU/ml)
Activity not accounted for
500 600 650 700 800 Standard error of the mean
489.5b (97.9%) 523.7b (87.3%) 485.3b (74.7%) 533.7b (76.2%) 658.6a (82.3%) 25.0
0 0d 19.3c 50.4b 107.3a 8.5
10.5 76.3 145.4 115.9 34.1
a–d
Means with the same superscripts do not significantly differ (p>0.05). (%) represents the percentage of activity absorbed onto CP-65 from the solution.
powder (Table 1). This was not the case with CP-65 at the higher solution concentrations. For example, at 700, 8.2 IU/ml were recovered in the corn starch supernantant compared to 50.4 IU/ml for the CP-65 supernatant at 700 IU/ml. Since less nisin activity could be accounted for in the corn starch compared to CP-65, corn starch may have more tightly bound or inactivated nisin compared to CP-65. As stated earlier, adsorption of nisin onto surfaces can alter the peptides conformation and also result in formation of nisin multi-layers and dimers (Liu and Hansen, 1990) which can reduce the biological activity of nisin. In summary, adsorption of nisin onto food-grade powders is a potential method to deliver antimicrobial activity to food systems. Direct nisin addition to food will quickly reduce bacterial numbers, however, this bactericidal activity can dissipate quickly due to interaction of nisin with food components or with bacteria. Furthermore, addition of nisin to packaging films results a significant loss of nisin activity, in the range of a 10% recovery of biological activity has been found when nisin was incorporated into protein films. If activity can be slowly released over extended periods of time, continued bacterial death could be delivered to a food surface or substrate post-processing which may be advantageous in some food applications. Concern for the development of nisin bacterial resistance is minimal
since the food is not consumed and will not re-enter the food supply. The current study found differences in adsorption and release of nisin activity by five foodgrade powders with an activity recovery as high as 96.7%. By portioning out the release of antimicrobial activity, inhibition of bacteria may be possible weeks and months after processing. Further testing of nisinadsorbed powders are needed to determine the efficacy and time release effects on food products.
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ARTICLE IN PRESS P.L. Dawson et al. / Food Microbiology 22 (2005) 93–99 Cutter, C.N., Siragusa, G.R., 1997. Growth of Brochothrix thermosphacta on beef surfaces following treatments with nisin in calcium alginate gels. Food Microbiol. 14, 425–430. Daeschel, M.A., McGuire, J., Al-Makhlafi, H., 1992. Antimicrobial activity of nisin adsorbed to hydrophilic and hydrophobic silicon surfaces. J. Food Prot. 55, 731–735. Dawson, P.L., Acton, J.C., Han, I.Y., Padgett, T., Orr, R., Larsen, T., 1995. Incorporation of antibacterial compounds into edible and biodegradable packaging films. Research and Development Activities Report for Military Food and Packaging Systems 47, 203–210. Delves-Broughton, J., 1990. Nisin and its uses as a food preservative. Food Technol. 44, 100,102,104,106,108,111–112,117. FDA, Food and Drug Administration, 1988. Nisin preparation: affirmation of GRAS status as a direct human food ingredient. Fed. Regist. 53, 11247. Franklin, N., Cooksey, K., Getty, K., 2004. Inhibition of Listeria monocytogenes no the surface on individually packaged hot dogs with a packaging film coating containing nisin. J. Food Prot. 67, 480–485. Gao, F.H., Abee, T., Konings, W.N., 1991. Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidasecontaining proteoliposomes. Appl. Environ. Microbiol. 57, 2164– 2170. Grower, J.L., Cooksey, K., Getty, K., 2004. Development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of Listeria monocytogenes. J. Food Prot. 67, 475–479. Han, A., Floros, H., 2000. Simulating migration models and determining the releasing rate of potassium sorbate from antimicrobial plastic film. Food Sci. Biotechnol. 9, 68–72. Hoffman, K.L., Dawson, P.L., Acton, J.C., Han, I.Y., Ogale, A.A., 1997. Film formation effects on nisin activity in corn zein and polyethylene films. Research and Development Activities Report for Military Food and Packaging Systems 47 (2), 203–210. Hoffman, K.L., Han, I.Y., Dawson, P.L., 2001. Antimicrobial effects of corn zein films impregnated with nisin, lauric acid, and EDTA. J. Food Prot. 64, 885–889. Hurst, A., 1981. Nisin. Adv. Appl. Microbiol. 27, 85–123. Janes, M.E., Nannapaneni, R., Proctor, A., Johnson, M.G., 1998. Rice hull ash and silicic acid adsorbents for concentration of bacteriocins. Appl. Environ. Microbiol. 64, 4403–4409. Jung, D.S., Bodyfelt, F.W., Daeschel, M.A., 1992. Influence of fat and emulsifier on the efficacy of nisin in inhibiting Listeria monocytogenes in fluid milk. J. Dairy Sci. 75, 387–393. Kassaify, Z.G., 1998. The potential use of nisin on fresh sausages and onto sausage casings. M.Sc. Thesis, Oxford University, Oxford, UK. Ko, S., Janes, M.E., Heittiarachchy, N.S., Johnson, M.G., 2001. Physical and chemical properties of edible films containing nisin and their action against Listeria monocytogenes. J. Food Sci. 66, 1006–1011. Liu, W., Hansen, J.W., 1990. Some chemical and physical properties of nisin, a small protein antibiotic produced by Lactococcus lactis. Appl. Environ. Microbiol. 56, 2551–2558. McCormick, K., Han, I.Y., Sheldon, B.W., Acton, J.C., Dawson, P.L., 2004. In package pasteurization combined with nisin-impregnated
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films to inhibit Listeria monocytogenes and Salmonella Typhimurium in turkey bologna. J. Food Sci. (In press). Ming, X., Weber, G.H., Ayres, J.W., Sandine, W.E., 1997. Bacteriocins applied to food packaging materials to inhibit Listeria monocytogenes on meats. J. Food Sci. 62, 413–415. Natrajan, N., Sheldon, B.W., 2000a. Efficacy of nisin-coated polymer films to inactivate Salmonella typhimurium on fresh broiler skin. J. Food Prot. 63, 1189–1196. Natrajan, N., Sheldon, B.W., 2000b. Inhibition of salmonella on poultry skin using protein- and polysaccharide-based films containing nisin formulation. J. Food Prot. 63, 1268–1272. Orr, R.V., Han, I.Y., Acton, J.C., Dawson, P.L., 1997. Comparison of the zone of inhibition and enumeration assay for evaluating the effectiveness of antimicrobial packaging films. In: Proceedings of the 42nd International Congress of Meat Science and Technology, American Meat Science Association, pp. 26–28. Orr, R.V., Han, I.Y., Acton, J.C., Dawson, P.L., 1998. Effect of nisin in edible protein films on Listeria monocytogenes in milk. Research and Development Activities Report for Military Food and Packaging Systems 48, 115–120. Padgett, T.R., Han, I.Y., Dawson, P.L., 1998. Incorporation of foodgrade antimicrobial compounds into biodegradable packaging films. J. Food Prot. 61, 1330–1335. Padgett, T.R., Han, I.Y., Dawson, P.L., 2000. Effect of lauric acid addition on the antimicrobial efficacy and water permeability of protein films containing nisin. J. Food Processing and Preserv. 24, 423–432. Ray, B., 1992. Nisin of Lactococcus lactis spp. lactis as a food preservative. In: Ray, B., Daeschel, M. (Eds.), Food Biopreservatives of Microbial Origin. CRC Press, Boca Raton, FL, pp. 207–264. Redl, A., Gontard, N., Guilbert, S., 1996. Determination of sorbic acid diffusivity in edible wheat gluten and lipid based films. J. Food Sci. 61, 116–120. Ruhr, E., Sahl, H.G., 1985. Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicle. Antimicrob. Agents Chemother. 27, 841–845. SAS, 2000. SAS Institute Inc. Cary. NC. Release 8.02. Proprietary Release (8.02.02 MOP012501). Stevens, K.A, Sheldon, B.W, Klapes N, A., Klaenhammer, T.R., 1991. Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria. Appl. Environ. Microbiol. 57, 3613–3615. Teerakarn, A., Hirt, D.E., Rieck, J.R., Acton, J.C., Dawson, P.L., 2002. Nisin diffusion in protein films: effects of time and temperature. J. Food Sci. 67, 3019–3025. Wan, J., Gordan, J., Hickey, M.W., Mawson, R.F., Coventry, M.J., 1996. Adsorption of bacteriocins by indigestible silica compounds. J. Appl. Bacteriol. 81, 167–173. Wan, J., Gordon, J.B., Muirhead, K., Hickey, M.W., Coventry, M.J., 1997. Incorporation of nisin in micro-particles of calcium alginate. Lett. Appl. Microbiol. 24, 153–158. Yang, R.G., Johnson, M.G., Ray, B., 1992. Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl. Environ. Microbiol. 58, 3355–3359.
www.elsevier.nl/locate/jnlabr/yfmic
Antimicrobial activity of nisin-adsorbed silica and corn starch powders P.L. Dawsona,*, L. Harmonc, A. Sotthibandhub, I.Y. Hana a
Department of Food Science and Human Nutrition, Clemson University, A203J Poole Hall, SC 29634-0371, Clemson, South Carolina, USA b South Carolina Governors School for Science and Mathematics, Hartsville, South Carolina, USA c Food Science and Nutrition, Faculty of Science, Srinakharinwirot University, Sukumvit 23, Bangkok, Thailand Received 17 November 2003; received in revised form 27 March 2004; accepted 6 April 2004
Abstract The antimicrobial peptide nisin is an effective bacterial inhibitor and has been adsorbed onto various surfaces and added to packaging films. In the present study, nisin was adsorbed onto food-grade powders then evaluated for nisin activity in a series of three experiments. Adsorbent powders used were calcined diatomaceous earth, synthetic calcium silicate, hydrate, two diatomaceous earth, and corn starch powders. Adsorption was conducted by placing the powders in agitated nisin solutions, followed by dehydration of the powder pellet after centrifugation. The dehydrated powders were then tested for inhibitory activity against either Lactobacillus plantarum or Listeria monocytogenes. Activity was measured by placing the nisin-adsorbed powders in solutions of 0.1% peptone water inoculated with one of the test bacterial strains. Cel-pure 65 adsorbed then released from 74.7% to 94.7% of the nisin activity contained in the original solution (500–800 IU/ml) in which the powders were agitated. Corn starch adsorbed then released from 45.4% to 60.5% of nisin activity when from 300 to 700 IU/ml nisin activity adsorbing solutions were utilized. One percent of nisin-absorbed Celpure 100 and Celpure 65 powders in 30 ml of log 4.5 cfu/ml L. monocytogenes inoculum reduced populations to below detection levels (o101 cfu/ml) within 24 and 6 h, respectively. Thus, nisin-adsorbed powders were highly efficient at both adsorption and release of antimicrobial activity. r 2004 Elsevier Ltd. All rights reserved. Keywords: Antimicrobial packaging; Nisin; Listeria monocytogenes; Corn starch; Adsorbents
1. Introduction Methods to deliver antimicrobials to food can vary from direct addition to incorporation into packaging materials. Nisin is considered a safe food antimicrobial and has been approved as a generally recognized as safe (GRAS) additive in pasteurized cheese spread and liquid egg in the US (FDA, 1988) to inhibit Clostridium botulinum spores. Nisin is primarily active against Gram-positive bacteria but when combined with a chelator, nisin also can inhibit growth of some Gramnegative bacteria (Stevens et al., 1991; Ray, 1992) thus nisin can potentially be effective against a broad spectrum of bacteria. In food systems, nisin effectiveness *Corresponding author. Tel.: +1-864-656-1138; fax: +1-864-6560331. E-mail address: [email protected] (P.L. Dawson). 0740-0020/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2004.04.001
can be reduced by food characteristics (Bell and DeLacy, 1987; Jung et al., 1992). The direct application of nisin to food surfaces thus will result in some loss of activity due to various food components. A method to control the release of nisin activity over a period of time would add a microbiological hurdle downstream in the food distribution chain. Han and Floros (2000) summarized the applications of antimicrobial packaging including nisin-containing packaging. Antimicrobial packaging films can provide a downstream hurdle via a controlled release scenario. There are two general categories of antimicrobial films, those in which the antimicrobial agent migrates from the film and those that the agent remains within the film material. Due to the nature of food, if the antimicrobial does not migrate from the film at least to the food surface, it will have limited effect. Nisin-containing packaging materials have had potential applications to prevent many
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foodborne pathogenic bacteria and their spores, C. botulinum, Listeria monocytogenes (Hurst, 1981; DelvesBroughton, 1990; Ray, 1992). Generally, antimicrobial packaging can be prepared by mixing of, coating with or chemical immobilization of antimicrobial agents to the packaging material. Numerous delivery systems have been evaluated using nisin including dialysis onto meat casings (Ming et al., 1997), coating onto collagen casings (Kassaify, 1998), cast protein films (Ko et al., 2001; Hoffman et al., 2001; Orr et al., 1997; Dawson et al., 1995; Padgett et al., 1998), polymer film coatings (Natrajan and Sheldon, 2000a), calcium alginate (Wan et al., 1997; Natrajan and Sheldon, 2000b; Cutter and Siragusa, 1997), methyl cellulose film coatings (Coma et al., 2001; Grower et al., 2004; Franklin et al., 2004) and heat-set biobased films (Padgett et al., 1998; Dawson et al., 1995; McCormick et al., 2004; Tereakarn et al., 2002). Polyethylene films and corn zein films were shown to reduce L. monocytogenes populations in peptone water from 8 logs (cfu/ ml) to below detectable levels (o102 cfu.ml) after 24 h (Hoffman et al., 1997, 2001) and reduce bacterial counts on chicken drumsticks (Natrajan and Sheldon, 2000a). Fluid foods may also be candidates for antimicrobial packaging as demonstrated with corn zein films impregnated with nisin. These films reduced L. monocytogenes in skim milk by 3 logs (cfu/ml) after 48 h (Orr et al., 1998) and L. plantarum in peptone water to below detection (Padgett et al., 1998, 2000). Diffusivity of nisin impregnated corn zein and wheat gluten films into water were affected by film type (wheat gluten or corn zein), environmental temperature and film-forming method (cast or heat-set) (Teerakarn et al., 2002). Cast wheat gluten film had the greatest diffusivity while the cast corn zein film had the lowest and heat-pressed wheat gluten and corn zein films did not differ in diffusivity. Cutter and Siragusa (1997) and Natrajan and Sheldon (2000b) used calcium alginate to deliver nisin to beef and chicken surfaces, respectively. Another approach to controlled nisin release is through adsorption of the peptide onto surfaces or particles that can deliver the antimicrobial activity to the desired location. A unique method to improve recovery of bacteriocins from cultures by adsorbing the bacteriocins onto cells from the producing strains (Yang et al., 1992). Ming et al. (1997) coated cellulose casings with either nisin or pediocin to inhibit the growth of L. monocytogenes on cooked meats but found that nisin was not effective. Wan et al. (1996) and Coventry et al. (1996) reported adsorbing bacteriocins, including nisin, onto ingestible silica compounds providing a simple method to harvest bacteriocin from culture supernatant and they also demonstrated the effectiveness of bacteriocin-adsorbed material against L. monocytogenes. A similar application was reported using rice hull ash and silicic acid to harvest five bacteriocins from freeze-dried
culture supernatants by Janes et al. (1998). Bowers et al. (1995) found no suppression of L. monocytogenes growth but a reduced adhesion rate of the bacterium to silica surfaces that had nisin adsorbed. In the current study, the previously described modes of delivery were combined by using silica and starch material in the powder form as a carrier of nisin. The objective of this research was to evaluate the adsorption and release of nisin activity onto and from food-grade powders.
2. Materials and methods 2.1. Absorbents Adsorbents used in this study included FilterCels (Calcined diatomaceous earth, SiO2, Celite Corporation, Lompoc, California), Micro-Cel s E (synthetic calcium silicate hydrate, CaSiO3, Celite Corporation, Lampoc, California), Celpures P65, CelpuresP1000 (Diatomaceous earth, Sigma-Aldrich, Saint Louis, Missouri), and anti set-off powder corn starch (Varn Products Company Inc., Oakland, New Jersey). FilterCels, Micro-Cel s E, Celpures P65, and CelpuresP1000 are used as filter aid agents due to their high absorption capacity, high surface area, and low bulk density. The Food and Drug Administration considers diatomaceous earth and calcium silicates to be GRAS according to title 21 code of Federal Regulation part 182.90 and 182.2227, respectively. 2.2. Nisin activity L. plantarum ATCC 1572 was the test organism used to determine nisin activity via the agar diffusion technique. L. monocytogenes ATCC 15313 was used to investigate effectiveness of nisin-adsorbed powders in a 0.1% peptone water suspension. Nisin standard solution was prepared using purified nisin (94.6% nisin by weight, Aplin and Barrett, Dorset, UK). The activity of nisin preparation is expressed in International Units (IU). The relationship of 1 g purified nisin=38 106 IU was presented by Ray (1992). Thus 1 mg of purified nisin used in this study contained 3.8 104 IU. To prepare the stock solution in HCl or water, 62.5 mg of nisin powder was mixed separately with both solvents using separate 50-ml volumetric flasks. Standard solutions (0.00028– 0.0125 mg purified nisin/ml) were prepared by dilution of the stock solution using sterile 0.02 n HCl or sterile distilled water. Standard solution for both sterile water and 0.02 n HCl were prepared and used since biological activity remaining in adsorbent powders was determined using 0.02 n HCl and migration from adsorbents was determined into a standard FDA simulants, sterile water. The standard curve dilution points were 0.00028, 0.0025, 0.0050, 0.0075, 0.0100, and 0.0125 mg
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pure nisin/ml. The R2 for standard curves in both 0.02 n HCl and sterile water were 0.99. The response of standard nisin solution on inhibiting L. plantarum was determined using the agar diffusion technique (Redl et al., 1996). A standard curve plot based on the relationship of the width of inhibition zones (mm) (Yaxis) and log of nisin concentration in IU (X-axis) was made for each experiment. Regression analysis was used to determine the standard equation, which was used to calculate the concentration of nisin in samples. 2.3. Experiment 1: Efficiency of nisin adsorption and release by five food-grade powders The procedure used to prepare bacteriocin-adsorbed powders followed the method previously described by Wan et al. (1996). In experiment 1 all 5 powders listed above were tested by adding 1.5 g of the powder to 1000 ml of distilled water containing 0.125 g of Nisaplint (Fig. 1). Nisin activity of solutions was 125 IU/ml for experiment 1. Commercial nisin (Nisaplin, 1,000,000 IU/g, Aplin & Barrett Ltd., Dorset, England) was used to prepare a 125 IU/ ml solution (Fig. 2). Silica powders or cornstarch were added to freshly made nisin solution at at 1% W/V in an individual sterile centrifuge tube and mixed using magnetic stirrers for 30 min at room temperature (22–24 C). Mixtures were then centrifuged (12,000g at 20 C for 10 min, Sorvells RC5B, refrigerated super-speed centrifuge, Dupont Instru-
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ments) to separate supernatant. The supernatant was assayed for residual nisin activity by agar diffusion technique previously described by Teerakarn et al. (2002). Solutions without adsorbents added were assayed to verify 125 IU/ml were contained in each solution. Diameters of clear or inhibitory zones were used to calculated nisin activity from the regression equation of the standard curve for nisin (Nisaplin) solution at 10–125 IU/ml. Averages of three clear zone readings were used to calculate nisin activity for each sample. After centrifugation, particles were dried in a hot air oven at 50 C for 4 h and at 30 C for 16–18 h with air circulation. The released biological activity of nisinadsorbed silica powders and cornstarch were determined by adding dried powders to sterile water at 1% (w/v) followed by testing for inhibitory effect on L. plantarum by the agar diffusion technique (Fig. 1). Adsorbed powder/sterile water mixtures were stirred at slow speed using a sterile magnetic stir rod for 20 min.Various mixing times were tested and under these conditions, release leveled off after 15–18 min thus 20 min was used to assess nisin adsorption by the powders. The mixture was sampled for biological activity using the same agar diffusion technique previously described for nisin activity. Non-nisin adsorbed powders were also dispersed in sterile water (1%, w/v) and sampled as described with no biological activity found in these control samples. The experiment was repeated three times with separate batches of materials. A randomizedcomplete block design using a general linear model was used to determine analysis of variance for treatment effects of replication and adsorbent type. The model contained these main effects and the replication by adsorbent type was used as the error term. Since adsorbent type had a significant treatment effect (pp0.05) on nisin activity, activity of adsorbent types was separated using the pdiff command of SAS (2000). 2.4. Experiment 2: Efficacy of nisin-adsorbed powders for inhibiting L. monocytogenes
Fig. 1. Diagram of the adsorption and analysis procedure used for food-grade powders.
Methods to produce nisin-absorbed powders were the same as described in experiment 1. L. monocytogenes was grown in Brain–Heart Infusion (BHI) media (Difco Laboratories, Detroit, Michigan) and incubated aerobically for 16 h at 37 C. The L. monocytogenes culture was centrifuged (20 min at 3000 rpm) and washed with 0.1% peptone. Thirty milliliter of inoculum in 0.1% peptone (104–105 CFU/ml) was then added to each of sterile glass petri dish containing 1% w/v of nisinadsorbed powder. Petri dishes were shaken on an orbital shaker at room temperature (20–24 C) at 50 rpm. Samples were taken after 0, 1, 2, 3, 6, 10, 24, and 48 h, diluted, and plated in duplicate on BHI agar. Colonies were counted after 48 h of aerobic incubation at 37 C. The experiment was replicated three times on different
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96 120
94.66 (76%) a
100
nisin activity (IU/ml)
79.23 80
(63.4%) a
71.53 67.76
(57.2%) a
(54.2%) a
60
40
20.92 (16.7%) b
20
0
Filter-cel
Microcel-E
Celpure 1000
Celpure 65
Corn starch
Fig. 2. Nisin activity for five food-grade powders that were previously exposed to a 125 IU/ml nisin solution for 20 min (with agitation). a,b Means with different superscripts differ significantly (pp0.05).
days. A randomized-complete block design using a general linear model was used to determine analysis of variance for treatment effects of replication, adsorbent type, and exposure time. The model included these main effects and the exposure time by adsorbent-type interaction with the residual interactions used as the error term. Since all factors included in the model had a significant effect (pp0.05) on the log CFU of L. monocytogenes, the standard error for each time and powder was determined using the proc-mixed procedure of SAS (2000).
compared in this experiment. A randomized-complete block design using a general linear model was used to determine analysis of variance for treatment effects of replication and adsorbent level. The model contained these main effects and the replication by nisin level interaction was used as the error term. Since the nisin level in adsorbing solutions had a significant treatment effect (pp0.05) on adsorbent nisin activity, the means for adsorbent activity was separated using the pdiff command of SAS (2000).
2.5. Experiment 3: Effect of nisin concentration in the adsorbing solution on adsorption of activity
3. Results and discussion
CS and CP65 were chosen for further investigation. CS was chosen since it has wide usage as a film-coating agent to prevent packaging film adherence. CP65 was chosen due to its performance compared to other agents in experiment 1. The objective of experiment 3 was to determine (within a concentration range) the efficiency of adsorption of powders (i.e. to what degree would an increase of nisin concentration in the adsorption solution result in an increased absorption by powders). Ranges of solution concentrations used were chosen based on preliminary tests. Nisin activity of solutions used to adsorb onto CS were 300, 400, 450, 500, 600, and 700 IU/ml and onto CP65 were 500, 600, 650, 700, and 800 IU/ml. As with experiments 1 and 2, 1.5 g of each adsorbent was added to 1000 ml of the nisin solutions then after adsorption the powders were dried. Biological activity of dried powders was determined with L. plantarum using the agar diffusion technique. This experiment was repeated three times each for CS and CP-65 using separate batches of materials on different days. CS and CP-65 were not statistically
Nisin has consistently been shown to inhibit Grampositive bacteria. While the bactericidal activity of nisin when adsorbed to a surface has been investigated, its mechanism of action has not been clearly described. However, previous research has shown that adsorbed nisin retains biological activity (Daeschel et al., 1992; Bower et al., 1995; Wan et al., 1996). The mechanism of nisin activity in solution is by integration into bacterial cell membranes resulting in pore formation (Ruhr and Sahl, 1985; Gao et al., 1991; Bruno et al., 1992). While the bactericidal mechanism of adsorbed nisin has not been determined it may be similar to its mechanism in solution and may require the release of nisin from the adsorbed surface to promote pore formation in the cell membrane. 3.1. Experiment 1: Efficiency of nisin adsorption and release by five food-grade powders Filter-Cel, CP-1000, CP-65 and corn starch did not differ in % adsorption themselves, ranging from 54.2% to 76% adsorption efficiency. Micro-Cel-E adsorbed
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and released the least nisin (16.7% efficiency) among the powders tested. These adsorption/release efficiencies are 10–100 times higher than those calculated for packaging films having nisin incorporated into their structure (Teerakarn et al., 2002) when tested under the same conditions. Structural changes occur when a peptide adsorbs onto a surface and these changes can also result in the formation of multi-layers and dimers (Liu and Hansen, 1990; Bowers et al., 1995). These structural changes can affect nisin activity and release from a surface. Wan et al. (1996) reported 100% recovery of nisin activity (125 IU/ml) from silica adsorbents including Micro-Cel and Filter-Cel however, this study also reported 13% of the activity remained in the supernatant. The 100% activity recovered from the adsorbents plus the additional 13% residual activity could be accounted for if the adsorbent itself resulted in a loss of bacterial cells. This does lead to question the accuracy of the measurement since more than 100% of the original activity was recovered using a supposedly inert silica adsorbent. The results from the present study verifies those found by Wan et al. (1996) that nisin can be adsorbed onto silica particles with a high level of recovery of biological activity. 3.2. Experiment 2: Efficacy of nisin-adsorbed powders for inhibiting L. monocytogenes Four of the five powders having been exposed to a 125 IU/ml nisin solution significantly reduced L. monocytogenes populations within 2 h (Fig. 3). Control treatments of Microcel-E and corn starch without nisin adsorption and peptone water with no powders added all increased in bacterial populations by 2–3 log CFU/ml after 24 h. Preliminary experiments showed the same effect of the other silica-based powders as was found with Microcel-E (data not shown) thus all were not included in experiment 2. Nisin-adsorbed CP-65 and CP-1000 both reduced cell counts below detection after CP-65
8
CP-1000 FilterCel
7
Corn Starch MicroCel
Log CFU/ml
6
Control
5 4 3 2 1 0 0
8
16
24 time (hours)
32
40
48
Fig. 3. Inhibitory effect of Cel-Pure 65 or corn starch adsorbed in solutions containing different nisin levels then exposed to 0.1% peptone water containing 104–5 cfu/ml of Listeria monocytogenes cells.
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10 h and no recovery of cells was detected up to 48 h. Nisin-adsorbed corn starch reduced L. monocytogenes by 4 logs after 10 h, however, the population then recovered with a>2 log outgrowth between 24 and 48 h. Filter-Cel similarly reached a maximum population reduction at 24 h (3.5 log reduction) then had a 1log growth recovery by 48 h. These nisin-adsorbed powders had a similar affect on L. monocytogenes in solution as nisin-impregnated corn zein films (Hoffman et al., 2001). However, nisin (as Nisaplin) was added at 5 times the amount (5%) to films and was exposed to 12 the volume of inoculated peptone water as powders in the current study. Furthermore, Teerakarn et al. (2002) found nisin-impregnated films released less than 10% of the nisin activity. Thus, under laboratory conditions the use of nisin-adsorbed powders to carry nisin activity is a more efficient method than incorporation into polymer films. 3.3. Experiment 3: Effect of nisin concentration in the adsorbing solution on adsorption of activity By using a range of adsorption solution concentrations of from 300 to 700 IU nisin/ml, adsorption/release efficiency of CP-65 decreased from 97.8% (500 IU/ml solutions) to 75% (650 IU/ml solutions) and of corn starch decreased from 60.5% (300 IU/ml solutions) to 45% (600 IU/ml solutions) (Tables 1 and 2). However, efficiency of adsorption was greater at lower solution concentrations for both powders. Time effects on release were not studied here, thus conclusions related to rate of activity release at shorter or longer times than the one used cannot be made. Bowers et al. (1995) found a similar trend of reduced adsorption efficiency with higher nisin-adsorbing concentrations when adsorbing nisin onto silica surface as opposed to particles. The objective of the Bowers study was to prevent L. monocytogenes adhesion (which it did) and suppress growth (which it did not) by adsorbing nisin onto silica surfaces. In Wan et al. (1996) and the current study, silica and starch particles were used as nisin carriers thus release was as important as adsorption. Wan et al. (1996) found a relatively constant adsorption/release efficiency of approximately 50% for pisciolin onto Micro-Cel when adsorption solutions containing between 107 and 108 AU/g were used. Conversely, the same study reported Filter-Cel adsorbed no more activity (a decreasing adsorption/release efficiency) from solutions containing pisciolin over the same range of activity. In the current study, the adsorption/release efficiency of corn starch was between 30% and 45% lower than CP65 when the same solutions concentrations were compared. Thus, CP-65 was a superior adsorbing/ releasing agent as was demonstrated in experiment 1. Little nisin activity was found in the supernantant of the adsorption solution after recovery of the corn starch
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Table 1 Nisin activity retained by corn starch and remaining in the supernatant after adsorption in solutions containing various levels of Nisaplins Solution nisin activity
Corn starch nisin activity
Activity remaining in supernatant after adsorption (IU/ml)
Activity not accounted for
300 400 450 500 600 700 Standard error of the mean
181.5c (60.5%) 237.0b (59.2%) 241.1b (53.6%) 257.2b (51.4%) 272.6b (45.4%) 330.5a (47.2%) 14.9
0c 0c 0c 4.4b 4.2b 8.2a 0.9
118.5 163.0 208.9 238.4 323.2 361.3
a–c Means with the same superscripts do not significantly differ (p>0.05). (%) represents the percentage of activity absorbed onto corn starch from the solution.
Table 2 Nisin activity retained by Cel-Pure 65 (CP-65) and remaining in the supernatant after adsorption in solutions containing various levels of Nisaplins Solution nisin activity
CP-65 nisin activity
Activity remaining in supernatant after adsorption (IU/ml)
Activity not accounted for
500 600 650 700 800 Standard error of the mean
489.5b (97.9%) 523.7b (87.3%) 485.3b (74.7%) 533.7b (76.2%) 658.6a (82.3%) 25.0
0 0d 19.3c 50.4b 107.3a 8.5
10.5 76.3 145.4 115.9 34.1
a–d
Means with the same superscripts do not significantly differ (p>0.05). (%) represents the percentage of activity absorbed onto CP-65 from the solution.
powder (Table 1). This was not the case with CP-65 at the higher solution concentrations. For example, at 700, 8.2 IU/ml were recovered in the corn starch supernantant compared to 50.4 IU/ml for the CP-65 supernatant at 700 IU/ml. Since less nisin activity could be accounted for in the corn starch compared to CP-65, corn starch may have more tightly bound or inactivated nisin compared to CP-65. As stated earlier, adsorption of nisin onto surfaces can alter the peptides conformation and also result in formation of nisin multi-layers and dimers (Liu and Hansen, 1990) which can reduce the biological activity of nisin. In summary, adsorption of nisin onto food-grade powders is a potential method to deliver antimicrobial activity to food systems. Direct nisin addition to food will quickly reduce bacterial numbers, however, this bactericidal activity can dissipate quickly due to interaction of nisin with food components or with bacteria. Furthermore, addition of nisin to packaging films results a significant loss of nisin activity, in the range of a 10% recovery of biological activity has been found when nisin was incorporated into protein films. If activity can be slowly released over extended periods of time, continued bacterial death could be delivered to a food surface or substrate post-processing which may be advantageous in some food applications. Concern for the development of nisin bacterial resistance is minimal
since the food is not consumed and will not re-enter the food supply. The current study found differences in adsorption and release of nisin activity by five foodgrade powders with an activity recovery as high as 96.7%. By portioning out the release of antimicrobial activity, inhibition of bacteria may be possible weeks and months after processing. Further testing of nisinadsorbed powders are needed to determine the efficacy and time release effects on food products.
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