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Inhibition of bacterial foodborne pathogens by the lactoperoxidase system in combination with monolaurin
International Journal of Food Microbiology 73 (2002) 1 – 9 www.elsevier.com/locate/ijfoodmicro
Inhibition of bacterial foodborne pathogens by the lactoperoxidase system in combination with monolaurin J.C. McLay, M.J. Kennedy, A.-L. O’Rourke, R.M. Elliot, R.S. Simmonds * Department of Microbiology, University of Otago, PO Box 56 1, Dunedin, New Zealand Received 4 April 2001; received in revised form 24 August 2001; accepted 10 September 2001
Abstract The lactoperoxidase system (LPS) and monolaurin (ML) are potential natural antimicrobial agents for use in foods. The LPS is considered to have greatest activity against Gram-negative bacteria while ML is usually considered to have greatest activity against Gram-positive bacteria. An LPS – ML combination system (utilizing lactoperoxidase (LPX) in the range 5 – 200 mg kg 1 and ML in the range 50 – 1000 ppm) inhibited growth of Escherichia coli O157:H7 and Staphylococcus aureus. Growth of S. aureus was inhibited more strongly in broth than in milk, in milk than in ground beef. A similar pattern was observed for E. coli O157:H7, though enhanced inhibition by LPS – ML systems over that obtained in comparable LPS only systems was not observed in ground beef. The inhibitory action of the LPS in combination with other lipids was also examined, with progressively weaker inhibition observed in combinations including palmitoleic acid, monopalmitolein, lauric acid, caprylic acid, and sodium lauryl sulphate. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Lactoperoxidase; Monolaurin; Escherichia coli O157:H7; Staphylococcus aureus; Natural antimicrobials
1. Introduction Food preservation procedures such as refrigeration, pasteurization, canning, modified atmosphere packaging or the incorporation of chemical preservatives in foods are used to prevent the growth of bacteria that may cause human disease or food spoilage. Escherichia coli O157:H7 and Staphylococcus aureus are recognized food pathogens. Outbreaks of food
*
Corresponding author. Tel.: +64-3-479-7478, +64-3-476-4474 (home); fax: +64-3-479-8540. E-mail address: [email protected] (R.S. Simmonds). 1 Goods to 720 Cumberland St.
poisoning due to E. coli O157:H7 have occurred following the ingestion of dry fermented salami (Tilden et al., 1996) and ground beef (Griffin and Tauxe, 1991). Disease due to S. aureus has been associated with the consumption of contaminated moist, high-protein and salty foods (Ollinger-Snyder and Matthews, 1996). Chemical preservatives such as benzoates, sorbates, nitrites and sulphites have been used effectively, but their safety is questionable (Frazier, 1988; Knekt et al., 1999). This has fuelled a search for biopreservatives that can safely be incorporated into various food products. Ideally, these biopreservatives have been generally recognized as safe (GRAS), resulting from a long history of their association with food and no recognized associated health concerns. The GRAS
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 6 9 8 - 5
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status and anti-bacterial properties of the lactoperoxidase system (LPS) and of monolaurin (ML) have resulted in numerous publications suggesting their use as natural food preservatives, and a number of patents have been filed covering their potential uses (de Wit and van Hooydonk, 1996). The LPS consists of three primary components: the lactoperoxidase enzyme (LPX), thiocyanate (SCN ), and hydrogen peroxide (H2O2). It occurs naturally in bovine milk (Reiter, 1985) and the saliva of humans and animals (Tenovuo, 1985). Although hydrogen peroxide can be added directly to the LPX-containing medium (Wolfson et al., 1994), it has been reported that a more sustained inhibitory effect is obtained if the hydrogen peroxide is generated in situ by added glucose oxidase (GOD) and glucose (Degre, 1990; Earnshaw et al., 1989). The LPS is very effective in inhibiting the growth of Gram-positive foodborne organisms, including S. aureus (Kamau et al., 1990); however, it has received most attention because of its bactericidal activity against many Gram-negative organisms, including E. coli (Bjorck et al., 1975). ML is a food-grade glycerol monoester of lauric acid approved for use as a food emulsifier by the US Food and Drug Administration (Oh and Marshall, 1992). It possesses significant antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes (Wang and Johnson, 1992) and S. aureus (Kabara et al., 1972). The effectiveness of the LPS or ML against foodborne pathogenic bacteria, used either alone or in combination with other bactericidal compounds has been examined (Blaszyk and Holley, 1998; Zapico et al., 1998). However, we are not aware of any study that examined the activity of these two compounds used in combination. A combination of the LPS and ML has the potential to inhibit the growth of a wide spectrum of bacteria. In this study the effect of the LPS and ML used either alone or in combination on the growth of S. aureus and E. coli was examined.
2. Materials and methods 2.1. Bacterial strains, media and chemicals S. aureus strain R37 was obtained from Dr. Roger Cook, the Meat Industries Research Institute
of New Zealand strain culture collection, and E. coli O157:H7 strain NCTC 12900 was obtained from Dr. Heather Brooks, the Department of Microbiology, University of Otago strain culture collection. Starter cultures were grown overnight at 37 C in Todd Hewitt Broth (THB) (Difco Laboratories, USA). All commercial media were prepared according to the manufacturers’ specifications. Lipids and the LPS components were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. Palmitoleic acid (cis-9-Hexadecanoic acid), monopalmitolein (1-monopalmitoleoyl-rac-glycerol (C16:1, [cis]-9)), lauric acid (dodecanoic acid), caprylic acid (n-octanoic acid) and monolaurin (1-monolauroylrac-glycerol (C12:0)) were dissolved in ethanol. Sodium lauryl sulphate (sodium dodecyl sulphate) was dissolved in MilliQ water. Glucose and GOD (from Aspergillus niger) were used to generate hydrogen peroxide in the LPS. Glucose, GOD, potassium thiocyanate and LPX (Tatua Biologics, Morrinsville, NZ. Purity index of A412/A280 = 0.65) were prepared in MilliQ water and sterilized using 0.2 mm porosity, 32-mm diameter SuporR AcrodiscR syringe filters (Gelman Sciences, Ann Arbor, MI, USA). 2.2. Inhibition of bacterial growth by combinations of LPS and ML Growth of E. coli O157:H7 and S. aureus in the presence of ML and the LPS were determined by measuring change in OD600 nm of cultures grown in Todd Hewitt broth (THB) at 37 C using a spectrophotometer (Spectronic 20D+, Milton Roy Company, USA). Tubes containing THB with either 0, 100 or 500 ppm ML were autoclaved at 121 C for 15 min, cooled to RT and the LPS components added as required, in the following order: cell inoculum, LPX stock (to 187.5 mg l 1), glucose (to 3.96 g l ), potassium thiocyanate (to 486.3 mg l 1) and sufficient GOD to attain an enzyme activity ratio of 1 unit GOD per 11.9 units of LPX. The enzyme activity of LPX preparations was determined by the standard ABTS (2,2V-azino-di(3Vethylbenzthiazoline sulphonic acid)) assay (Bardsley, 1985). Cell inocula were either 0, 0.05% or 1.0% (v/v) of an overnight culture of the appropriate bacterial strain. Cultures were read at 4 hourly
J.C. McLay et al. / International Journal of Food Microbiology 73 (2002) 1–9
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intervals for 48 h. Experiments were performed in triplicate.
plated onto Plate Count Agar (PCA) plates in 5-ml overlays.
2.3. Inhibition of bacterial growth by combinations of LPS and a monoglyceride, fatty acid or an ester
2.5. Effect of LPS –ML combinations on bacterial viability in milk and beef
The growth rates of E. coli O157:H7 and S. aureus in the presence of the LPS and various lipids were determined using the method described above. Lipids were added to tubes at concentrations of 0, 50, 100, 250, 500 and 1000 ppm prior to autoclaving. Tubes were inoculated with cells to a concentration of either 0 or 1 104 cfu ml 1, and the LPS components added as follows: LPX (to 213.75 mg l 1), glucose (to 11.9 g l 1) and potassium thiocyanate (to 486 mg l 1). Sufficient GOD was added to attain an enzyme activity ratio of 1 and 0.1 units of GOD per 11.9 units of LPX for tubes inoculated with E. coli O157:H7 and S. aureus, respectively. Cultures were read at 4 hourly intervals for 48 h. Experiments were performed in triplicate.
Unless otherwise described, experiments to examine the growth of E. coli O157:H7 and S. aureus in UHT (ultra heat treated) milk and ground beef were performed as for growth in THB. Experiments using E. coli O157:H7 were conducted at both 37 and 12 C. Due to a lack of growth of S. aureus at 12 C, experiments with this organism were conducted at only 37 C. UHT homogenised milk (Mainland Products, Christchurch, New Zealand) was purchased from a local supermarket as required. Topside steak was purchased from a local supermarket and ground to a 6-mm particle size. The meat was sterilized by exposure to a minimum dose of 25 kGy of gamma irradiation (Schering-Plough Animal Health, Upper Hutt, NZ). For experiments involving growth in ground beef, 25-g amounts were used in each Whirl-Pak bag. Sampling was performed by removal of 5 g of ground beef into a fresh Whirl-Pak bag and elution of the bacteria from meat particles by stomaching of the sample for 2 min in 50 ml of peptone water, dilution and plating as previously described.
2.4. Effect of LPS – ML combinations on bacterial viability in THB Whirl-Pak bags (Nasco, Fort Atkinson, WI, USA) containing 20-ml volumes of THB were heated to 65 C and ML added as appropriate. The ML was mixed into the medium by stomaching for 2 min in a Colworth Stomacher 400 (A.J. Seward, London, UK) and the bags incubated for 15 min at 65 C before cooling to RT. The bags were inoculated with bacteria to either 0 or 5 104 cfu ml 1, and the cells mixed into the media by stomaching for 2 min. As required, the LPS component stocks were added to achieve final concentrations of 12 mg l 1 for glucose and potassium thiocyanate, and in the range of 5– 200 mg l 1 for LPX. Sufficient GOD was added to attain a concentration of 1 unit of enzyme activity per 9 units of LPX activity. These levels approximate those with regulatory permission for use as a food processing aid (Degre, 1990). The bags were stomached for a further 2 min to combine the components and incubated at 37 C for 24 h. The viable count of bacteria was determined at 0, 6 and 24 h. Each bag was stomached before removal of sample, the sample diluted in peptone water and
Table 1 Inhibition of bacterial growth at 37 C in THB by combinations of the LPS and ML Bacterial strain and Time (h) to reach OD600 nm of 0.1 inoculum level ML (ppm) LPSa plus ML (ppm) (cfu ml 1) 0 100 500 0 100 500 E. coli O157:H7 6.0 105 3.0 107
3.1 1.0
S. aureus 1.6 105 8.0 106
3.5 > 48 >48 2.0 11.5 >48
a
3.1 1.0
3.1 1.0
3.5 1.0
5.3 1.2
26.5 11.5
>48 45.1
5.3 1.3
>48 >48
Concentration of the LPS was the same in each system: LPX to 187.5 mg l 1, glucose to 3.96 g l 1, potassium thiocyanate to 486.3 mg l 1 and sufficient GOD to attain an enzyme activity ratio of 1 unit to 11.9 units of LPX. Uninoculated tubes showed no growth.
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3.2. Inhibition of bacterial growth by a combination of the LPS and a monoglyceride, fatty acids or an ester
3. Results 3.1. Inhibition of bacterial growth by a combination of the LPS and ML
Significant inhibition ( P = 0.05, X2) of E. coli O157:H7 growth was observed in LPS – ML combination systems (50 – 500 ppm ML) and also in LPS – lauric acid combination systems incorporating 500 ppm or greater of lauric acid, relative to use of either the LPS or lipid alone (Table 2). Slightly enhanced but not significant inhibition of E. coli O157:H7 growth was observed in the LPS combination systems incorporating palmitoleic acid, caprylic acid and sodium lauryl sulphate relative to use of any agent alone. The sensitivity of S. aureus to the ML and palmitoleic acid concentrations used in lipid only tests meant that
Inhibition of growth by every LPS – ML combination was relative to the bacterial load imposed on the system (Table 1). Monolaurin did not inhibit the growth of E. coli O157:H7, the LPS and LPS –ML combinations slightly inhibited growth in tubes inoculated at 6.0 105 cfu ml 1. S. aureus growth in tubes inoculated at 8.0 106 cfu ml 1 was inhibited by both ML and the LPS alone. In the 100 ppm LPS – ML system, the time required to reach an OD600 nm of 0.1 was extended 33.6 h beyond that of cultures containing either ML or LPS alone.
Table 2 Inhibition of bacterial growth at 37 C in THB by combinations of the LPS and a monoglyceride, fatty acid or ester Lipid and bacterial strain
Time (h) to reach OD600
nm
of 0.1 LPSa plus Lipid (ppm)
Lipid (ppm) 0
50
100
250
500
1000
ML C12:0 E. coli O157:H7 S. aureus
4.0 7.3
4.1 >48
4.1 >48
4.1 >48
4.2 >48
7.1 >48
Monopalmitolein C16:1 E. coli O157:H7 S. aureus
4.0 7.0
4.0 20.4
4.0 24.4
4.0 24.8
4.0 28.5
Caprylic acid C8:0 E. coli O157:H7 S. aureus
4.7 7.7
4.8 12.7
5.1 10.5
4.1 8.5
Lauric acid C12:0 E. coli O157:H7 S. aureus
4.0 6.6
4.0 10.5
4.0 16.8
Palmitoleic acid C16:1 E. coli O157:H7 S. aureus
4.1 7.9
4.4 43.8
Sodium lauryl sulphate E. coli O157:H7 S. aureus
4.4 6.5
4.1 11.4
0
50
100
250
500
1000
5.2 8.5
15.7 >48
27.2 >48
>48 >48
18.6 >48
7.5 >48
4.2 40.2
5.6 9.5
6.2 >48
5.7 >48
4.7 >48
6.7 >48
5.5 >48
4.2 8.6
4.2 11.3
6.0 10.0
7.7 14.7
6.5 14.4
7.8 15.1
7.9 20.1
10.6 28.8
4.1 19.5
4.1 41.6
5.7 >48
5.2 9.3
5.1 16.5
5.3 >48
8.2 >48
>48 >48
>48 >48
4.4 >48
4.7 >48
4.4 >48
3.9 >48
5.6 11.4
6.4 >48
9.6 >48
7.3 >48
8.0 >48
5.1 >48
4.1 29.1
4.5 >48
4.1 >48
4.1 >48
4.9 9.3
8.5 12.3
6.5 16.9
8.4 >48
8.1 >48
8.7 >48
a Concentration of the LPS was the same in each system: LPX to 213.75 mg l 1, glucose to 11.9 g l 1, potassium thiocyanate to 486 mg l 1 and sufficient GOD to attain an enzyme activity ratio of 1 and 0.1 units to 11.9 units of LPX for E. coli O157:H7 and S. aureus R37, respectively. Uninoculated tubes showed no growth.
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Table 3 Inhibition of S. aureus at 37 C in THB containing the LPS and ML Concentration of the LPSa (mg kg 1 LPX) 0 5 10 25
Viable count (log10 cfu ml 0 ppm
1 b
) in THB containing ML 5 ppm
10 ppm
25 ppm
6h
24 h
6h
24 h
6h
24 h
6h
24 h
7.2 2.7 < 1.3 < 1.3
9.5 < 1.3 < 1.3 < 1.3
6.7 3.7 2.0 < 1.3
9.2 4.2 < 1.3 < 1.3
6.4 3.4 < 1.3 < 1.3
9.4 < 1.3 < 1.3 < 1.3
4.0 < 1.3 < 1.3 < 1.3
7.3 < 1.3 < 1.3 < 1.3
a
Final concentrations of glucose and potassium thiocyanate were 12 mg l 1. GOD was added to attain a concentration of 1 unit of enzyme activity per 9 units of LPX activity. b A viable count of 4.7 F 0.1 log10 cfu ml 1 was recorded for each treatment at zero time.
enhanced inhibition of growth was not observed when these lipids were used in combination with the LPS. Enhanced inhibition was obvious in the LPS combination systems containing monopalmitolein and lauric acid and to a lesser extent with caprylic acid. No enhancement of inhibition was observed for a combination of the LPS and sodium lauryl sulphate.
combinations all had inhibitory activity against S. aureus (Table 3). Inhibition of S. aureus growth in LPS –ML combination systems was greatest at 24 h and even at low concentrations of the LPS cofactors, viable counts were reduced to below countable numbers ( < 1.3 log10 cfu ml 1). However, at limiting concentrations of ML (5– 10 ppm) and LPX (5– 10 mg LPX kg 1 LPS), the inhibition of growth of S. aureus was significantly less in LPS –ML systems than in corresponding control systems.
3.3. Effect of LPS – ML combinations on bacterial viability in THB The LPS – ML combination systems containing 500 ppm ML and 100 mg LPX kg 1 LPS were significantly ( P = 0.05, X2) more inhibitory to E. coli O157:H7 growth (5.1 log10 cfu ml 1) than use of either the LPS (7.1 log10 cfu ml 1) or ML alone (8.1 log10 cfu ml 1) at 6 h. This difference was no longer apparent by 24 h. The LPS, ML, and the LPS –ML
3.4. Effect of LPS –ML combinations on bacterial viability in milk and beef After 6 h at 37 C, the LPS –ML combination systems showed enhanced inhibition of E. coli O157:H7 grown in milk where 100 mg or greater of LPX kg 1 LPS were used (Table 4), though this
Table 4 Inhibition of E. coli O157:H7 at 37 and 12 C in milk treated by a combination of the LPS and ML Concentration of the LPSa (mg kg 1 LPX) and ML (ppm)
Viable count (log10 cfu ml 6h
24 h
1 day
2 days
3 days
Untreated control LPS (50) LPS (100) LPS (200) ML (500) ML (500) + LPS (50) ML (500) + LPS (100) ML (500) + LPS (200)
8.5 7.3 5.2 4.1 8.4 7.6 4.2 1.9
9.1 9.1 8.9 9.1 8.9 9.1 9.1 8.2
7.0 4.3 4.3 < 1.3 6.1 4.2 4.3 < 1.3
9.0 7.8 3.8 < 1.3 8.3 4.2 3.2 < 1.3
9.0 8.0 4.1 < 1.3 9.0 6.1 2.3 < 1.3
Milk at 37 C
1 b
)
Milk at 12 C
a Final concentrations of glucose and potassium thiocyanate were 12 mg l 1. GOD was added to attain a concentration of 1 unit of enzyme activity per 9 units of LPX activity. b A viable count of 5.2 F 0.1 log10 cfu ml 1 was recorded for each treatment at zero time.
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difference was generally lost by 24 h. However, in ground beef the LPS –ML combination systems failed to enhance inhibition of E. coli O157:H7 over use of the LPS alone (data not shown). At 12 C in milk, LPS – ML combination systems containing 50 – 100 mg LPX kg 1 LPS showed enhanced inhibition of E. coli O157:H7 over 3 days relative to use of the LPS alone (Table 4). In all systems containing 200 mg LPX kg 1 LPS, viable counts were reduced three logs or greater below initial inoculum levels. In ground beef the viable count of E. coli O157:H7 in all the LPS systems after 3 days at 12 C had not increased significantly ( P = 0.05, X2) from initial inoculum levels and in most cases had fallen slightly (data not shown). By contrast, viable counts in the untreated control systems increased greater than one log during this period. However, as was the case for growth at 37 C, in ground beef the LPS – ML combination systems failed to enhance inhibition of E. coli O157:H7 over use of the LPS alone. Growth of S. aureus at 37 C in milk was strongly inhibited at 6 h by the LPS containing LPX at concentrations greater than 25 mg kg 1 and this inhibition was enhanced in LPS – ML combination systems Table 5 Inhibition of S. aureus at 37 C in food treated by a combination of the LPS and ML Concentration of LPSa (mg kg 1 LPX) and ML (ppm) Untreated control LPS (5) LPS (10) LPS (25) LPS (50) LPS (100) LPS (200) ML (500) ML (500) + LPS (5) ML (500) + LPS (10) ML (500) + LPS (25) ML (500) + LPS (50) ML (500) + LPS (100) ML (500) + LPS (200)
Viable count (log10 cfu ml Milk
1 b
)
Ground beef
6h
24 h
6h
24 h
7.9 7.2 6.6 6.1 4.1 4.5 4.5 7.6 5.9 5.7 4.0 < 1.3 < 1.3 < 1.3
9.2 8.1 8.3 8.3 8.8 8.9 8.9 8.9 9.2 9.2 9.1 8.8 8.8 8.9
8.2 7.6 7.7 7.2 7.4 7.1 6.3 8.1 7.4 7.5 6.4 6.3 5.3 4.6
8.4 8.4 8.2 8.5 8.9 9.1 9.2 8.2 8.4 8.5 8.3 8.5 8.5 7.8
a Final concentrations of glucose and potassium thiocyanate were 12 mg l 1. GOD was added to attain a concentration of 1 unit of enzyme activity per 9 units of LPX activity. b A viable count of 5.3 F and 4.8 F log10 cfu ml 1 was recorded for each treatment at zero time in milk and ground beef, respectively.
where the treatment reduced viable counts to below countable numbers (Table 5). The LPS – ML combinations where the LPS contained LPX at concentrations greater than 25 mg kg 1 reduced growth at 6 h in ground beef relative to the untreated control or use of either component alone. The inhibitory effect in both milk and ground beef was generally lost by 24 h.
4. Discussion In general, the sensitivity of the strains to the LPS or to ML were consistent with those reported by others (Bjorck et al., 1975; Kabara et al., 1977; Kamau et al., 1990). Our initial studies demonstrated that a combination of the LPS and ML enhanced inhibition of bacterial growth compared to use of either agent alone, and suggested that combinations were effective at inhibiting bacterial growth at relatively low concentrations of ML (Table 1). ML is considered to be the most inhibitory of lipids (Kabara et al., 1972). In fact, due to its high sensitivity, L. monocytogenes strain L45 showed no growth in any ML containing system and thus was not used further in this study (data not shown). Enhanced inhibition of E. coli O157:H7 by combinations of the LPS and ML in the absence of any observed or reported sensitivity of E. coli to ML suggested it would be wise to examine the effect of other lipids used in combination with the LPS. In order to maximise the likelihood of observing a synergistic effect between the LPS and lipids less inhibitory to bacteria than ML, the concentration of inoculant was reduced from that used in the initial studies and the concentration of the LPS components individually optimised for E. coli O157:H7 and S. aureus (data not shown). This produced a test system in which the time required for E. coli O157:H7 and S. aureus to attain an OD600 nm of 0.1 in the presence of the LPS was extended approximately 1.5 and 2.5 h, respectively, beyond those of systems containing no LPS (Table 2). For similar reasons, lipids were added at concentrations of between 50 and 1000 ppm. The synergistic inhibitory effect observed for the LPS used in combination with lipids other than ML, supported our initial observations and suggested other LPS –lipid combinations may make useful preservative agents in some circumstances. It was observed that material in tubes containing higher lipid concentrations was prone
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to precipitation, suggesting that maximum bioavailability was actually achieved at lower lipid concentrations. LPS – ML combinations were shown to be effective at inhibiting the growth of foodborne pathogens at relatively low concentrations of ML. Subsequently, we examined their effect on viable numbers of E. coli O157:H7 and S. aureus grown in foods. The LPS cofactor concentrations were reduced to levels approximating those that have regulatory permission for use as a food processing aid (Degre, 1990). As a first step, the efficacy of the low-cofactor LPS was tested against the bacteria in broth. Reduction in the LPS cofactor levels reduced inhibition of E. coli O157:H7 growth by the LPS – ML systems but had little effect on their inhibition of S. aureus (Table 3). These experiments also demonstrated that used at sufficient concentration, the effect of LPS – ML combinations was bacteriocidal and not just bacteriostatic. We believe the slight but significant decrease in inhibition of S. aureus observed in THB at limiting concentrations of the LPS and ML may be caused by a partitioning of the OSCN into ML micelles (Thomas, 1981), and in so doing reduce its bio-available concentration in the broth. ML concentrations strongly inhibitory to S. aureus in THB were found to have no inhibitory effect on S. aureus grown in milk or ground beef (Table 5). Increasing the milk in a THB-milk mixture from 0% to 100%, led to decreased inhibition of S. aureus by ML (500 ppm) from a 7 log difference in viable count between the treated system and untreated control to no significant difference at 6 h (data not shown). This was not entirely unexpected as it was previously reported that growth of L. monocytogenes was more strongly inhibited by ML in skim milk than whole milk (Wang and Johnson, 1992). We believe that as the fat content of the food increases, the ML progressively partitions into it, lowering the effective biocidal concentration (Blaszyk and Holley, 1998). This may explain why studies of the use of ML as a food preservative, particularly those involving meat products, have used ML at concentrations in the range of 3000– 5000 ppm (Kabara, 1984). Since our initial food studies indicated that the LPS in combination with ML at 500 ppm gave significantly enhanced inhibition, we adopted 500 ppm as a standard concentration of ML for use in all further food-based studies.
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A combination of the LPS and ML resulted in a synergistic inhibitory effect on both E. coli O157:H7 and S. aureus in milk that was not accounted for by a simple additive effect of the two agents (Tables 4 and 5). However, the inhibitory effect was generally lost by 24 h in systems at 37 C. Reducing the incubation temperature of the test system to 12 C produced a marked increase in inhibition of E. coli O157:H7 relative to the uninhibited control by both the LPS and LPS – ML combination systems. Most reports suggest that bacteria are killed more efficiently by ML as the temperature is increased (Venkitanarayanan et al., 1999), though others have reported that for L. monocytogenes grown in commercial media the MIC of ML decreased as the incubation temperature was lowered (Bala and Marshall, 1996). Previously, we noted that a reduced incubation temperature markedly increased the inhibition of E. coli O157:H7 by LPS in ground meat and suggested that the metabolic state of the cell was significant in terms of the effectiveness of this agent (Kennedy et al., 2000). We speculate that the ability of LPS – ML damaged cells to be repaired may be compromised under low temperature growth permissive conditions. The mechanism of inhibition of bacteria by the LPS –ML combinations is not known. The cell membrane is thought to be the primary target of each agent, and enhanced inhibition or synergy the result of a multiple attack on the membrane. The oxidation of sulfhydryl groups of enzymes and other proteins is believed to be the main effect of the LPS (Reiter, 1984; Perraudin, 1991). Lipophilic acids, including ML, are thought to disrupt membrane integrity which in turn interferes with membrane activities such as transport of amino acids, resulting in cell starvation (Kabara, 1993). Enhanced inhibition of microbial growth has been reported for the LPS and for ML in association with other chemicals including propylene glycerol (Hall and Maurer, 1986), various organic acids (Blaszyk and Holley, 1998; Venkitanarayanan et al., 1999) and nisin (Zapico et al., 1998; Mansour et al., 1999). It is interesting to speculate that the LPS, ML, and another agent used in combination may have enhanced effectiveness compared to any agent used alone or any combination of two agents. This study suggests that the mechanisms of action of the LPS in combination with ML, and with other potential synergistic partners would be a worthwhile subject for future studies.
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5. Conclusion The LPS and ML, which have antimicrobial activity against different spectra of bacteria, combine to create a novel system inhibitory to the food pathogens E. coli O157:H7, and S. aureus. The LPS –ML combination was more effective at inhibiting bacterial growth than either agent used alone. Treatments were more effective in milk than in ground beef and at storage temperatures non-permissive of rapid growth of bacteria.
Acknowledgements This work was supported in part by a grant from the Tatua Cooperative Dairy, Morrinsville, New Zealand.
References Bala, M.F.A., Marshall, D.L., 1996. Testing matrix, inoculum size, and incubation temperature affect monolaurin activity against Listeria monocytogenes. Food Microbiol. 13, 467 – 473. Bardsley, W.G., 1985. Steady-state kinetics of lactoperoxidase-catalyzed reactions. In: Pruitt, K.M., Tenovuo, J.O. (Eds.), The Lactoperoxidase System, Chemistry and Biological Significance. Marcel Dekker, New York, USA, pp. 55 – 87. Bjorck, L., Rosen, C., Marshall, V., Reiter, B., 1975. Antibacterial activity of the lactoperoxidase system in milk against pseudomonads and other gram-negative bacteria. Appl. Microbiol. 30, 199 – 204. Blaszyk, M., Holley, R.A., 1998. Interaction of monolaurin, eugenol and sodium citrate on growth of common meat spoilage and pathogenic organisms. Int. J. Food Microbiol. 39, 175 – 183. Degre, M.F., 1990. Procede de decontamination d’un produit alimentaire non liquide, en particulier de fromage, et composition a cet effect, European Patent 0 397 228 B1. de Wit, J.N., van Hooydonk, A.C.M., 1996. Structure, functions and applications of lactoperoxidase in natural antimicrobial systems. Neth. Milk Dairy J. 50, 227 – 244. Earnshaw, R.G., Banks, J.G., Defrise, D., Francotte, C., 1989. The preservation of cottage cheese by an activated lactoperoxidase system. Food Microbiol. 6, 285 – 288. Frazier, W.C., 1988. Preservation by drying. In: Frazier, W.C. (Ed.), Food Microbiology. McGraw-Hill, New York, USA, pp. 125 – 131. Griffin, P.M., Tauxe, R.V., 1991. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13, 60 – 98. Hall, M.A., Maurer, A.J., 1986. Spice extracts, lauricidin and propylene glycol as inhibitors of Clostridium botulinum in turkey frankfurter slurries. Poultry Sci. 65, 1167 – 1170.
Kabara, J.J., 1984. Inhibition of Staphylococcus aureus in a model agar-meat system by monolaurin. J. Food Saf. 6, 197 – 201. Kabara, J.J., 1993. Medium-chain fatty acids and esters. In: Davidson, P.M., Branen, A.L. (Eds.), Antimicrobials in Foods. Marcel Dekker, New York, USA, pp. 307 – 342. Kabara, J.J., Swieczkowski, D.M., Conley, A.J., Truant, J.P., 1972. Fatty acids and derivatives as antimicrobial agents. Antimicrob. Agents Chemother. 2, 23 – 28. Kabara, J.J., Vrable, R., Lie Ken Jie, M.S.F., 1977. Antimicrobial lipids: natural and synthetic fatty acids monoglycerides. Lipids 12, 753 – 759. Kamau, D.N., Doores, S., Pruitt, K.M., 1990. Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system. Appl. Environ. Microbiol. 56, 2711 – 2716. Kennedy, M., O’Rourke, A.-L., McLay, J., Simmonds, R., 2000. Use of a ground beef model to assess the effect of the lactoperoxidase system on the growth of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus in meat. Int. J. Food Microbiol. 57, 147 – 158. Knekt, P., Jarvinen, R., Dich, J., Hakulinen, T., 1999. Risk of colorectal and other gastro-intestinal cancers after exposure to nitrate, nitrite and N-nitroso compounds: a follow-up study. Int. J. Cancer 80, 852 – 856. Mansour, M., Amri, D., Bouttefroy, A., Linder, M., Milliere, J.B., 1999. Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations. J. App. Microbiol. 86, 311 – 324. Oh, D.-H., Marshall, D.L., 1992. Effect of pH on the minimum inhibitory concentration of monolaurin against Listeria monocytogenes. J. Food Prot. 55, 449 – 450. Ollinger-Snyder, P., Matthews, M.E., 1996. Food safety: review and implications for dietitians and dietetic technicians. J. Am. Dietetic Assoc. 96, 163 – 168. Perraudin, J.-P., 1991. Lactoperoxidase a natural preservative. Dairy Ind. Int. 56, 21 – 24. Reiter, B., 1984. Lactoperoxidase antibacterial system: natural occurrence, biological functions and practical applications. J. Food Prot. 47, 724 – 732. Reiter, B., 1985. The lactoperoxidase system of bovine milk. In: Pruitt, K.M., Tenovuo, J.O. (Eds.), The Lactoperoxidase System, Chemistry and Biological Significance. Marcel Dekker, New York, USA, pp. 123 – 142. Tenovuo, J.O., 1985. The peroxidase system in human secretions. In: Pruitt, K.M., Tenovuo, J.O. (Eds.), The Lactoperoxidase System, Chemistry and Biological Significance. Marcel Dekker, New York, USA, pp. 101 – 122. Thomas, E.L., 1981. Lactoperoxidase-catalyzed oxidation of thiocyanate: equilibria between oxidized forms of thiocyanate. Biochemistry 20, 3273 – 3280. Tilden, J., Young, W., McNamara, A., Custer, C., Boesel, B., Lambert-Fair, M., Majkowski, J., Vugia, D., Werner, S.B., Hollingsworth, J., Morris, J.G., 1996. A new route of transmission for Escherichia coli: infection from dry fermented salami. Am. J. Public Health 86, 1142 – 1145. Venkitanarayanan, K.S., Zhao, T., Doyle, M.P., 1999. Inactivation
J.C. McLay et al. / International Journal of Food Microbiology 73 (2002) 1–9 of Escherichia coli O157:H7 by combinations of GRAS chemicals and temperature. Food Microbiol. 16, 75 – 82. Wang, L.-L., Johnson, E.A., 1992. Inhibition of Listeria monocytogenes by fatty acids and monoglycerides. Appl. Environ. Microbiol. 58, 624 – 629. Wolfson, L.M., Sumner, S.S., Froning, G.W., 1994. Inhibition of
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Salmonella typhimurium on poultry by the lactoperoxidase system. J. Food Saf. 14, 53 – 62. Zapico, P., Medina, M., Gaya, P., Nunez, M., 1998. Synergistic effect of nisin and the lactoperoxidase system on Listeria monocytogenes in skim milk. Int. J. Food Microbiol. 40, 35 – 42.
Inhibition of bacterial foodborne pathogens by the lactoperoxidase system in combination with monolaurin J.C. McLay, M.J. Kennedy, A.-L. O’Rourke, R.M. Elliot, R.S. Simmonds * Department of Microbiology, University of Otago, PO Box 56 1, Dunedin, New Zealand Received 4 April 2001; received in revised form 24 August 2001; accepted 10 September 2001
Abstract The lactoperoxidase system (LPS) and monolaurin (ML) are potential natural antimicrobial agents for use in foods. The LPS is considered to have greatest activity against Gram-negative bacteria while ML is usually considered to have greatest activity against Gram-positive bacteria. An LPS – ML combination system (utilizing lactoperoxidase (LPX) in the range 5 – 200 mg kg 1 and ML in the range 50 – 1000 ppm) inhibited growth of Escherichia coli O157:H7 and Staphylococcus aureus. Growth of S. aureus was inhibited more strongly in broth than in milk, in milk than in ground beef. A similar pattern was observed for E. coli O157:H7, though enhanced inhibition by LPS – ML systems over that obtained in comparable LPS only systems was not observed in ground beef. The inhibitory action of the LPS in combination with other lipids was also examined, with progressively weaker inhibition observed in combinations including palmitoleic acid, monopalmitolein, lauric acid, caprylic acid, and sodium lauryl sulphate. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Lactoperoxidase; Monolaurin; Escherichia coli O157:H7; Staphylococcus aureus; Natural antimicrobials
1. Introduction Food preservation procedures such as refrigeration, pasteurization, canning, modified atmosphere packaging or the incorporation of chemical preservatives in foods are used to prevent the growth of bacteria that may cause human disease or food spoilage. Escherichia coli O157:H7 and Staphylococcus aureus are recognized food pathogens. Outbreaks of food
*
Corresponding author. Tel.: +64-3-479-7478, +64-3-476-4474 (home); fax: +64-3-479-8540. E-mail address: [email protected] (R.S. Simmonds). 1 Goods to 720 Cumberland St.
poisoning due to E. coli O157:H7 have occurred following the ingestion of dry fermented salami (Tilden et al., 1996) and ground beef (Griffin and Tauxe, 1991). Disease due to S. aureus has been associated with the consumption of contaminated moist, high-protein and salty foods (Ollinger-Snyder and Matthews, 1996). Chemical preservatives such as benzoates, sorbates, nitrites and sulphites have been used effectively, but their safety is questionable (Frazier, 1988; Knekt et al., 1999). This has fuelled a search for biopreservatives that can safely be incorporated into various food products. Ideally, these biopreservatives have been generally recognized as safe (GRAS), resulting from a long history of their association with food and no recognized associated health concerns. The GRAS
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 6 9 8 - 5
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status and anti-bacterial properties of the lactoperoxidase system (LPS) and of monolaurin (ML) have resulted in numerous publications suggesting their use as natural food preservatives, and a number of patents have been filed covering their potential uses (de Wit and van Hooydonk, 1996). The LPS consists of three primary components: the lactoperoxidase enzyme (LPX), thiocyanate (SCN ), and hydrogen peroxide (H2O2). It occurs naturally in bovine milk (Reiter, 1985) and the saliva of humans and animals (Tenovuo, 1985). Although hydrogen peroxide can be added directly to the LPX-containing medium (Wolfson et al., 1994), it has been reported that a more sustained inhibitory effect is obtained if the hydrogen peroxide is generated in situ by added glucose oxidase (GOD) and glucose (Degre, 1990; Earnshaw et al., 1989). The LPS is very effective in inhibiting the growth of Gram-positive foodborne organisms, including S. aureus (Kamau et al., 1990); however, it has received most attention because of its bactericidal activity against many Gram-negative organisms, including E. coli (Bjorck et al., 1975). ML is a food-grade glycerol monoester of lauric acid approved for use as a food emulsifier by the US Food and Drug Administration (Oh and Marshall, 1992). It possesses significant antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes (Wang and Johnson, 1992) and S. aureus (Kabara et al., 1972). The effectiveness of the LPS or ML against foodborne pathogenic bacteria, used either alone or in combination with other bactericidal compounds has been examined (Blaszyk and Holley, 1998; Zapico et al., 1998). However, we are not aware of any study that examined the activity of these two compounds used in combination. A combination of the LPS and ML has the potential to inhibit the growth of a wide spectrum of bacteria. In this study the effect of the LPS and ML used either alone or in combination on the growth of S. aureus and E. coli was examined.
2. Materials and methods 2.1. Bacterial strains, media and chemicals S. aureus strain R37 was obtained from Dr. Roger Cook, the Meat Industries Research Institute
of New Zealand strain culture collection, and E. coli O157:H7 strain NCTC 12900 was obtained from Dr. Heather Brooks, the Department of Microbiology, University of Otago strain culture collection. Starter cultures were grown overnight at 37 C in Todd Hewitt Broth (THB) (Difco Laboratories, USA). All commercial media were prepared according to the manufacturers’ specifications. Lipids and the LPS components were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. Palmitoleic acid (cis-9-Hexadecanoic acid), monopalmitolein (1-monopalmitoleoyl-rac-glycerol (C16:1, [cis]-9)), lauric acid (dodecanoic acid), caprylic acid (n-octanoic acid) and monolaurin (1-monolauroylrac-glycerol (C12:0)) were dissolved in ethanol. Sodium lauryl sulphate (sodium dodecyl sulphate) was dissolved in MilliQ water. Glucose and GOD (from Aspergillus niger) were used to generate hydrogen peroxide in the LPS. Glucose, GOD, potassium thiocyanate and LPX (Tatua Biologics, Morrinsville, NZ. Purity index of A412/A280 = 0.65) were prepared in MilliQ water and sterilized using 0.2 mm porosity, 32-mm diameter SuporR AcrodiscR syringe filters (Gelman Sciences, Ann Arbor, MI, USA). 2.2. Inhibition of bacterial growth by combinations of LPS and ML Growth of E. coli O157:H7 and S. aureus in the presence of ML and the LPS were determined by measuring change in OD600 nm of cultures grown in Todd Hewitt broth (THB) at 37 C using a spectrophotometer (Spectronic 20D+, Milton Roy Company, USA). Tubes containing THB with either 0, 100 or 500 ppm ML were autoclaved at 121 C for 15 min, cooled to RT and the LPS components added as required, in the following order: cell inoculum, LPX stock (to 187.5 mg l 1), glucose (to 3.96 g l ), potassium thiocyanate (to 486.3 mg l 1) and sufficient GOD to attain an enzyme activity ratio of 1 unit GOD per 11.9 units of LPX. The enzyme activity of LPX preparations was determined by the standard ABTS (2,2V-azino-di(3Vethylbenzthiazoline sulphonic acid)) assay (Bardsley, 1985). Cell inocula were either 0, 0.05% or 1.0% (v/v) of an overnight culture of the appropriate bacterial strain. Cultures were read at 4 hourly
J.C. McLay et al. / International Journal of Food Microbiology 73 (2002) 1–9
3
intervals for 48 h. Experiments were performed in triplicate.
plated onto Plate Count Agar (PCA) plates in 5-ml overlays.
2.3. Inhibition of bacterial growth by combinations of LPS and a monoglyceride, fatty acid or an ester
2.5. Effect of LPS –ML combinations on bacterial viability in milk and beef
The growth rates of E. coli O157:H7 and S. aureus in the presence of the LPS and various lipids were determined using the method described above. Lipids were added to tubes at concentrations of 0, 50, 100, 250, 500 and 1000 ppm prior to autoclaving. Tubes were inoculated with cells to a concentration of either 0 or 1 104 cfu ml 1, and the LPS components added as follows: LPX (to 213.75 mg l 1), glucose (to 11.9 g l 1) and potassium thiocyanate (to 486 mg l 1). Sufficient GOD was added to attain an enzyme activity ratio of 1 and 0.1 units of GOD per 11.9 units of LPX for tubes inoculated with E. coli O157:H7 and S. aureus, respectively. Cultures were read at 4 hourly intervals for 48 h. Experiments were performed in triplicate.
Unless otherwise described, experiments to examine the growth of E. coli O157:H7 and S. aureus in UHT (ultra heat treated) milk and ground beef were performed as for growth in THB. Experiments using E. coli O157:H7 were conducted at both 37 and 12 C. Due to a lack of growth of S. aureus at 12 C, experiments with this organism were conducted at only 37 C. UHT homogenised milk (Mainland Products, Christchurch, New Zealand) was purchased from a local supermarket as required. Topside steak was purchased from a local supermarket and ground to a 6-mm particle size. The meat was sterilized by exposure to a minimum dose of 25 kGy of gamma irradiation (Schering-Plough Animal Health, Upper Hutt, NZ). For experiments involving growth in ground beef, 25-g amounts were used in each Whirl-Pak bag. Sampling was performed by removal of 5 g of ground beef into a fresh Whirl-Pak bag and elution of the bacteria from meat particles by stomaching of the sample for 2 min in 50 ml of peptone water, dilution and plating as previously described.
2.4. Effect of LPS – ML combinations on bacterial viability in THB Whirl-Pak bags (Nasco, Fort Atkinson, WI, USA) containing 20-ml volumes of THB were heated to 65 C and ML added as appropriate. The ML was mixed into the medium by stomaching for 2 min in a Colworth Stomacher 400 (A.J. Seward, London, UK) and the bags incubated for 15 min at 65 C before cooling to RT. The bags were inoculated with bacteria to either 0 or 5 104 cfu ml 1, and the cells mixed into the media by stomaching for 2 min. As required, the LPS component stocks were added to achieve final concentrations of 12 mg l 1 for glucose and potassium thiocyanate, and in the range of 5– 200 mg l 1 for LPX. Sufficient GOD was added to attain a concentration of 1 unit of enzyme activity per 9 units of LPX activity. These levels approximate those with regulatory permission for use as a food processing aid (Degre, 1990). The bags were stomached for a further 2 min to combine the components and incubated at 37 C for 24 h. The viable count of bacteria was determined at 0, 6 and 24 h. Each bag was stomached before removal of sample, the sample diluted in peptone water and
Table 1 Inhibition of bacterial growth at 37 C in THB by combinations of the LPS and ML Bacterial strain and Time (h) to reach OD600 nm of 0.1 inoculum level ML (ppm) LPSa plus ML (ppm) (cfu ml 1) 0 100 500 0 100 500 E. coli O157:H7 6.0 105 3.0 107
3.1 1.0
S. aureus 1.6 105 8.0 106
3.5 > 48 >48 2.0 11.5 >48
a
3.1 1.0
3.1 1.0
3.5 1.0
5.3 1.2
26.5 11.5
>48 45.1
5.3 1.3
>48 >48
Concentration of the LPS was the same in each system: LPX to 187.5 mg l 1, glucose to 3.96 g l 1, potassium thiocyanate to 486.3 mg l 1 and sufficient GOD to attain an enzyme activity ratio of 1 unit to 11.9 units of LPX. Uninoculated tubes showed no growth.
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3.2. Inhibition of bacterial growth by a combination of the LPS and a monoglyceride, fatty acids or an ester
3. Results 3.1. Inhibition of bacterial growth by a combination of the LPS and ML
Significant inhibition ( P = 0.05, X2) of E. coli O157:H7 growth was observed in LPS – ML combination systems (50 – 500 ppm ML) and also in LPS – lauric acid combination systems incorporating 500 ppm or greater of lauric acid, relative to use of either the LPS or lipid alone (Table 2). Slightly enhanced but not significant inhibition of E. coli O157:H7 growth was observed in the LPS combination systems incorporating palmitoleic acid, caprylic acid and sodium lauryl sulphate relative to use of any agent alone. The sensitivity of S. aureus to the ML and palmitoleic acid concentrations used in lipid only tests meant that
Inhibition of growth by every LPS – ML combination was relative to the bacterial load imposed on the system (Table 1). Monolaurin did not inhibit the growth of E. coli O157:H7, the LPS and LPS –ML combinations slightly inhibited growth in tubes inoculated at 6.0 105 cfu ml 1. S. aureus growth in tubes inoculated at 8.0 106 cfu ml 1 was inhibited by both ML and the LPS alone. In the 100 ppm LPS – ML system, the time required to reach an OD600 nm of 0.1 was extended 33.6 h beyond that of cultures containing either ML or LPS alone.
Table 2 Inhibition of bacterial growth at 37 C in THB by combinations of the LPS and a monoglyceride, fatty acid or ester Lipid and bacterial strain
Time (h) to reach OD600
nm
of 0.1 LPSa plus Lipid (ppm)
Lipid (ppm) 0
50
100
250
500
1000
ML C12:0 E. coli O157:H7 S. aureus
4.0 7.3
4.1 >48
4.1 >48
4.1 >48
4.2 >48
7.1 >48
Monopalmitolein C16:1 E. coli O157:H7 S. aureus
4.0 7.0
4.0 20.4
4.0 24.4
4.0 24.8
4.0 28.5
Caprylic acid C8:0 E. coli O157:H7 S. aureus
4.7 7.7
4.8 12.7
5.1 10.5
4.1 8.5
Lauric acid C12:0 E. coli O157:H7 S. aureus
4.0 6.6
4.0 10.5
4.0 16.8
Palmitoleic acid C16:1 E. coli O157:H7 S. aureus
4.1 7.9
4.4 43.8
Sodium lauryl sulphate E. coli O157:H7 S. aureus
4.4 6.5
4.1 11.4
0
50
100
250
500
1000
5.2 8.5
15.7 >48
27.2 >48
>48 >48
18.6 >48
7.5 >48
4.2 40.2
5.6 9.5
6.2 >48
5.7 >48
4.7 >48
6.7 >48
5.5 >48
4.2 8.6
4.2 11.3
6.0 10.0
7.7 14.7
6.5 14.4
7.8 15.1
7.9 20.1
10.6 28.8
4.1 19.5
4.1 41.6
5.7 >48
5.2 9.3
5.1 16.5
5.3 >48
8.2 >48
>48 >48
>48 >48
4.4 >48
4.7 >48
4.4 >48
3.9 >48
5.6 11.4
6.4 >48
9.6 >48
7.3 >48
8.0 >48
5.1 >48
4.1 29.1
4.5 >48
4.1 >48
4.1 >48
4.9 9.3
8.5 12.3
6.5 16.9
8.4 >48
8.1 >48
8.7 >48
a Concentration of the LPS was the same in each system: LPX to 213.75 mg l 1, glucose to 11.9 g l 1, potassium thiocyanate to 486 mg l 1 and sufficient GOD to attain an enzyme activity ratio of 1 and 0.1 units to 11.9 units of LPX for E. coli O157:H7 and S. aureus R37, respectively. Uninoculated tubes showed no growth.
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Table 3 Inhibition of S. aureus at 37 C in THB containing the LPS and ML Concentration of the LPSa (mg kg 1 LPX) 0 5 10 25
Viable count (log10 cfu ml 0 ppm
1 b
) in THB containing ML 5 ppm
10 ppm
25 ppm
6h
24 h
6h
24 h
6h
24 h
6h
24 h
7.2 2.7 < 1.3 < 1.3
9.5 < 1.3 < 1.3 < 1.3
6.7 3.7 2.0 < 1.3
9.2 4.2 < 1.3 < 1.3
6.4 3.4 < 1.3 < 1.3
9.4 < 1.3 < 1.3 < 1.3
4.0 < 1.3 < 1.3 < 1.3
7.3 < 1.3 < 1.3 < 1.3
a
Final concentrations of glucose and potassium thiocyanate were 12 mg l 1. GOD was added to attain a concentration of 1 unit of enzyme activity per 9 units of LPX activity. b A viable count of 4.7 F 0.1 log10 cfu ml 1 was recorded for each treatment at zero time.
enhanced inhibition of growth was not observed when these lipids were used in combination with the LPS. Enhanced inhibition was obvious in the LPS combination systems containing monopalmitolein and lauric acid and to a lesser extent with caprylic acid. No enhancement of inhibition was observed for a combination of the LPS and sodium lauryl sulphate.
combinations all had inhibitory activity against S. aureus (Table 3). Inhibition of S. aureus growth in LPS –ML combination systems was greatest at 24 h and even at low concentrations of the LPS cofactors, viable counts were reduced to below countable numbers ( < 1.3 log10 cfu ml 1). However, at limiting concentrations of ML (5– 10 ppm) and LPX (5– 10 mg LPX kg 1 LPS), the inhibition of growth of S. aureus was significantly less in LPS –ML systems than in corresponding control systems.
3.3. Effect of LPS – ML combinations on bacterial viability in THB The LPS – ML combination systems containing 500 ppm ML and 100 mg LPX kg 1 LPS were significantly ( P = 0.05, X2) more inhibitory to E. coli O157:H7 growth (5.1 log10 cfu ml 1) than use of either the LPS (7.1 log10 cfu ml 1) or ML alone (8.1 log10 cfu ml 1) at 6 h. This difference was no longer apparent by 24 h. The LPS, ML, and the LPS –ML
3.4. Effect of LPS –ML combinations on bacterial viability in milk and beef After 6 h at 37 C, the LPS –ML combination systems showed enhanced inhibition of E. coli O157:H7 grown in milk where 100 mg or greater of LPX kg 1 LPS were used (Table 4), though this
Table 4 Inhibition of E. coli O157:H7 at 37 and 12 C in milk treated by a combination of the LPS and ML Concentration of the LPSa (mg kg 1 LPX) and ML (ppm)
Viable count (log10 cfu ml 6h
24 h
1 day
2 days
3 days
Untreated control LPS (50) LPS (100) LPS (200) ML (500) ML (500) + LPS (50) ML (500) + LPS (100) ML (500) + LPS (200)
8.5 7.3 5.2 4.1 8.4 7.6 4.2 1.9
9.1 9.1 8.9 9.1 8.9 9.1 9.1 8.2
7.0 4.3 4.3 < 1.3 6.1 4.2 4.3 < 1.3
9.0 7.8 3.8 < 1.3 8.3 4.2 3.2 < 1.3
9.0 8.0 4.1 < 1.3 9.0 6.1 2.3 < 1.3
Milk at 37 C
1 b
)
Milk at 12 C
a Final concentrations of glucose and potassium thiocyanate were 12 mg l 1. GOD was added to attain a concentration of 1 unit of enzyme activity per 9 units of LPX activity. b A viable count of 5.2 F 0.1 log10 cfu ml 1 was recorded for each treatment at zero time.
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difference was generally lost by 24 h. However, in ground beef the LPS –ML combination systems failed to enhance inhibition of E. coli O157:H7 over use of the LPS alone (data not shown). At 12 C in milk, LPS – ML combination systems containing 50 – 100 mg LPX kg 1 LPS showed enhanced inhibition of E. coli O157:H7 over 3 days relative to use of the LPS alone (Table 4). In all systems containing 200 mg LPX kg 1 LPS, viable counts were reduced three logs or greater below initial inoculum levels. In ground beef the viable count of E. coli O157:H7 in all the LPS systems after 3 days at 12 C had not increased significantly ( P = 0.05, X2) from initial inoculum levels and in most cases had fallen slightly (data not shown). By contrast, viable counts in the untreated control systems increased greater than one log during this period. However, as was the case for growth at 37 C, in ground beef the LPS – ML combination systems failed to enhance inhibition of E. coli O157:H7 over use of the LPS alone. Growth of S. aureus at 37 C in milk was strongly inhibited at 6 h by the LPS containing LPX at concentrations greater than 25 mg kg 1 and this inhibition was enhanced in LPS – ML combination systems Table 5 Inhibition of S. aureus at 37 C in food treated by a combination of the LPS and ML Concentration of LPSa (mg kg 1 LPX) and ML (ppm) Untreated control LPS (5) LPS (10) LPS (25) LPS (50) LPS (100) LPS (200) ML (500) ML (500) + LPS (5) ML (500) + LPS (10) ML (500) + LPS (25) ML (500) + LPS (50) ML (500) + LPS (100) ML (500) + LPS (200)
Viable count (log10 cfu ml Milk
1 b
)
Ground beef
6h
24 h
6h
24 h
7.9 7.2 6.6 6.1 4.1 4.5 4.5 7.6 5.9 5.7 4.0 < 1.3 < 1.3 < 1.3
9.2 8.1 8.3 8.3 8.8 8.9 8.9 8.9 9.2 9.2 9.1 8.8 8.8 8.9
8.2 7.6 7.7 7.2 7.4 7.1 6.3 8.1 7.4 7.5 6.4 6.3 5.3 4.6
8.4 8.4 8.2 8.5 8.9 9.1 9.2 8.2 8.4 8.5 8.3 8.5 8.5 7.8
a Final concentrations of glucose and potassium thiocyanate were 12 mg l 1. GOD was added to attain a concentration of 1 unit of enzyme activity per 9 units of LPX activity. b A viable count of 5.3 F and 4.8 F log10 cfu ml 1 was recorded for each treatment at zero time in milk and ground beef, respectively.
where the treatment reduced viable counts to below countable numbers (Table 5). The LPS – ML combinations where the LPS contained LPX at concentrations greater than 25 mg kg 1 reduced growth at 6 h in ground beef relative to the untreated control or use of either component alone. The inhibitory effect in both milk and ground beef was generally lost by 24 h.
4. Discussion In general, the sensitivity of the strains to the LPS or to ML were consistent with those reported by others (Bjorck et al., 1975; Kabara et al., 1977; Kamau et al., 1990). Our initial studies demonstrated that a combination of the LPS and ML enhanced inhibition of bacterial growth compared to use of either agent alone, and suggested that combinations were effective at inhibiting bacterial growth at relatively low concentrations of ML (Table 1). ML is considered to be the most inhibitory of lipids (Kabara et al., 1972). In fact, due to its high sensitivity, L. monocytogenes strain L45 showed no growth in any ML containing system and thus was not used further in this study (data not shown). Enhanced inhibition of E. coli O157:H7 by combinations of the LPS and ML in the absence of any observed or reported sensitivity of E. coli to ML suggested it would be wise to examine the effect of other lipids used in combination with the LPS. In order to maximise the likelihood of observing a synergistic effect between the LPS and lipids less inhibitory to bacteria than ML, the concentration of inoculant was reduced from that used in the initial studies and the concentration of the LPS components individually optimised for E. coli O157:H7 and S. aureus (data not shown). This produced a test system in which the time required for E. coli O157:H7 and S. aureus to attain an OD600 nm of 0.1 in the presence of the LPS was extended approximately 1.5 and 2.5 h, respectively, beyond those of systems containing no LPS (Table 2). For similar reasons, lipids were added at concentrations of between 50 and 1000 ppm. The synergistic inhibitory effect observed for the LPS used in combination with lipids other than ML, supported our initial observations and suggested other LPS –lipid combinations may make useful preservative agents in some circumstances. It was observed that material in tubes containing higher lipid concentrations was prone
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to precipitation, suggesting that maximum bioavailability was actually achieved at lower lipid concentrations. LPS – ML combinations were shown to be effective at inhibiting the growth of foodborne pathogens at relatively low concentrations of ML. Subsequently, we examined their effect on viable numbers of E. coli O157:H7 and S. aureus grown in foods. The LPS cofactor concentrations were reduced to levels approximating those that have regulatory permission for use as a food processing aid (Degre, 1990). As a first step, the efficacy of the low-cofactor LPS was tested against the bacteria in broth. Reduction in the LPS cofactor levels reduced inhibition of E. coli O157:H7 growth by the LPS – ML systems but had little effect on their inhibition of S. aureus (Table 3). These experiments also demonstrated that used at sufficient concentration, the effect of LPS – ML combinations was bacteriocidal and not just bacteriostatic. We believe the slight but significant decrease in inhibition of S. aureus observed in THB at limiting concentrations of the LPS and ML may be caused by a partitioning of the OSCN into ML micelles (Thomas, 1981), and in so doing reduce its bio-available concentration in the broth. ML concentrations strongly inhibitory to S. aureus in THB were found to have no inhibitory effect on S. aureus grown in milk or ground beef (Table 5). Increasing the milk in a THB-milk mixture from 0% to 100%, led to decreased inhibition of S. aureus by ML (500 ppm) from a 7 log difference in viable count between the treated system and untreated control to no significant difference at 6 h (data not shown). This was not entirely unexpected as it was previously reported that growth of L. monocytogenes was more strongly inhibited by ML in skim milk than whole milk (Wang and Johnson, 1992). We believe that as the fat content of the food increases, the ML progressively partitions into it, lowering the effective biocidal concentration (Blaszyk and Holley, 1998). This may explain why studies of the use of ML as a food preservative, particularly those involving meat products, have used ML at concentrations in the range of 3000– 5000 ppm (Kabara, 1984). Since our initial food studies indicated that the LPS in combination with ML at 500 ppm gave significantly enhanced inhibition, we adopted 500 ppm as a standard concentration of ML for use in all further food-based studies.
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A combination of the LPS and ML resulted in a synergistic inhibitory effect on both E. coli O157:H7 and S. aureus in milk that was not accounted for by a simple additive effect of the two agents (Tables 4 and 5). However, the inhibitory effect was generally lost by 24 h in systems at 37 C. Reducing the incubation temperature of the test system to 12 C produced a marked increase in inhibition of E. coli O157:H7 relative to the uninhibited control by both the LPS and LPS – ML combination systems. Most reports suggest that bacteria are killed more efficiently by ML as the temperature is increased (Venkitanarayanan et al., 1999), though others have reported that for L. monocytogenes grown in commercial media the MIC of ML decreased as the incubation temperature was lowered (Bala and Marshall, 1996). Previously, we noted that a reduced incubation temperature markedly increased the inhibition of E. coli O157:H7 by LPS in ground meat and suggested that the metabolic state of the cell was significant in terms of the effectiveness of this agent (Kennedy et al., 2000). We speculate that the ability of LPS – ML damaged cells to be repaired may be compromised under low temperature growth permissive conditions. The mechanism of inhibition of bacteria by the LPS –ML combinations is not known. The cell membrane is thought to be the primary target of each agent, and enhanced inhibition or synergy the result of a multiple attack on the membrane. The oxidation of sulfhydryl groups of enzymes and other proteins is believed to be the main effect of the LPS (Reiter, 1984; Perraudin, 1991). Lipophilic acids, including ML, are thought to disrupt membrane integrity which in turn interferes with membrane activities such as transport of amino acids, resulting in cell starvation (Kabara, 1993). Enhanced inhibition of microbial growth has been reported for the LPS and for ML in association with other chemicals including propylene glycerol (Hall and Maurer, 1986), various organic acids (Blaszyk and Holley, 1998; Venkitanarayanan et al., 1999) and nisin (Zapico et al., 1998; Mansour et al., 1999). It is interesting to speculate that the LPS, ML, and another agent used in combination may have enhanced effectiveness compared to any agent used alone or any combination of two agents. This study suggests that the mechanisms of action of the LPS in combination with ML, and with other potential synergistic partners would be a worthwhile subject for future studies.
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5. Conclusion The LPS and ML, which have antimicrobial activity against different spectra of bacteria, combine to create a novel system inhibitory to the food pathogens E. coli O157:H7, and S. aureus. The LPS –ML combination was more effective at inhibiting bacterial growth than either agent used alone. Treatments were more effective in milk than in ground beef and at storage temperatures non-permissive of rapid growth of bacteria.
Acknowledgements This work was supported in part by a grant from the Tatua Cooperative Dairy, Morrinsville, New Zealand.
References Bala, M.F.A., Marshall, D.L., 1996. Testing matrix, inoculum size, and incubation temperature affect monolaurin activity against Listeria monocytogenes. Food Microbiol. 13, 467 – 473. Bardsley, W.G., 1985. Steady-state kinetics of lactoperoxidase-catalyzed reactions. In: Pruitt, K.M., Tenovuo, J.O. (Eds.), The Lactoperoxidase System, Chemistry and Biological Significance. Marcel Dekker, New York, USA, pp. 55 – 87. Bjorck, L., Rosen, C., Marshall, V., Reiter, B., 1975. Antibacterial activity of the lactoperoxidase system in milk against pseudomonads and other gram-negative bacteria. Appl. Microbiol. 30, 199 – 204. Blaszyk, M., Holley, R.A., 1998. Interaction of monolaurin, eugenol and sodium citrate on growth of common meat spoilage and pathogenic organisms. Int. J. Food Microbiol. 39, 175 – 183. Degre, M.F., 1990. Procede de decontamination d’un produit alimentaire non liquide, en particulier de fromage, et composition a cet effect, European Patent 0 397 228 B1. de Wit, J.N., van Hooydonk, A.C.M., 1996. Structure, functions and applications of lactoperoxidase in natural antimicrobial systems. Neth. Milk Dairy J. 50, 227 – 244. Earnshaw, R.G., Banks, J.G., Defrise, D., Francotte, C., 1989. The preservation of cottage cheese by an activated lactoperoxidase system. Food Microbiol. 6, 285 – 288. Frazier, W.C., 1988. Preservation by drying. In: Frazier, W.C. (Ed.), Food Microbiology. McGraw-Hill, New York, USA, pp. 125 – 131. Griffin, P.M., Tauxe, R.V., 1991. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13, 60 – 98. Hall, M.A., Maurer, A.J., 1986. Spice extracts, lauricidin and propylene glycol as inhibitors of Clostridium botulinum in turkey frankfurter slurries. Poultry Sci. 65, 1167 – 1170.
Kabara, J.J., 1984. Inhibition of Staphylococcus aureus in a model agar-meat system by monolaurin. J. Food Saf. 6, 197 – 201. Kabara, J.J., 1993. Medium-chain fatty acids and esters. In: Davidson, P.M., Branen, A.L. (Eds.), Antimicrobials in Foods. Marcel Dekker, New York, USA, pp. 307 – 342. Kabara, J.J., Swieczkowski, D.M., Conley, A.J., Truant, J.P., 1972. Fatty acids and derivatives as antimicrobial agents. Antimicrob. Agents Chemother. 2, 23 – 28. Kabara, J.J., Vrable, R., Lie Ken Jie, M.S.F., 1977. Antimicrobial lipids: natural and synthetic fatty acids monoglycerides. Lipids 12, 753 – 759. Kamau, D.N., Doores, S., Pruitt, K.M., 1990. Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system. Appl. Environ. Microbiol. 56, 2711 – 2716. Kennedy, M., O’Rourke, A.-L., McLay, J., Simmonds, R., 2000. Use of a ground beef model to assess the effect of the lactoperoxidase system on the growth of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus in meat. Int. J. Food Microbiol. 57, 147 – 158. Knekt, P., Jarvinen, R., Dich, J., Hakulinen, T., 1999. Risk of colorectal and other gastro-intestinal cancers after exposure to nitrate, nitrite and N-nitroso compounds: a follow-up study. Int. J. Cancer 80, 852 – 856. Mansour, M., Amri, D., Bouttefroy, A., Linder, M., Milliere, J.B., 1999. Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations. J. App. Microbiol. 86, 311 – 324. Oh, D.-H., Marshall, D.L., 1992. Effect of pH on the minimum inhibitory concentration of monolaurin against Listeria monocytogenes. J. Food Prot. 55, 449 – 450. Ollinger-Snyder, P., Matthews, M.E., 1996. Food safety: review and implications for dietitians and dietetic technicians. J. Am. Dietetic Assoc. 96, 163 – 168. Perraudin, J.-P., 1991. Lactoperoxidase a natural preservative. Dairy Ind. Int. 56, 21 – 24. Reiter, B., 1984. Lactoperoxidase antibacterial system: natural occurrence, biological functions and practical applications. J. Food Prot. 47, 724 – 732. Reiter, B., 1985. The lactoperoxidase system of bovine milk. In: Pruitt, K.M., Tenovuo, J.O. (Eds.), The Lactoperoxidase System, Chemistry and Biological Significance. Marcel Dekker, New York, USA, pp. 123 – 142. Tenovuo, J.O., 1985. The peroxidase system in human secretions. In: Pruitt, K.M., Tenovuo, J.O. (Eds.), The Lactoperoxidase System, Chemistry and Biological Significance. Marcel Dekker, New York, USA, pp. 101 – 122. Thomas, E.L., 1981. Lactoperoxidase-catalyzed oxidation of thiocyanate: equilibria between oxidized forms of thiocyanate. Biochemistry 20, 3273 – 3280. Tilden, J., Young, W., McNamara, A., Custer, C., Boesel, B., Lambert-Fair, M., Majkowski, J., Vugia, D., Werner, S.B., Hollingsworth, J., Morris, J.G., 1996. A new route of transmission for Escherichia coli: infection from dry fermented salami. Am. J. Public Health 86, 1142 – 1145. Venkitanarayanan, K.S., Zhao, T., Doyle, M.P., 1999. Inactivation
J.C. McLay et al. / International Journal of Food Microbiology 73 (2002) 1–9 of Escherichia coli O157:H7 by combinations of GRAS chemicals and temperature. Food Microbiol. 16, 75 – 82. Wang, L.-L., Johnson, E.A., 1992. Inhibition of Listeria monocytogenes by fatty acids and monoglycerides. Appl. Environ. Microbiol. 58, 624 – 629. Wolfson, L.M., Sumner, S.S., Froning, G.W., 1994. Inhibition of
9
Salmonella typhimurium on poultry by the lactoperoxidase system. J. Food Saf. 14, 53 – 62. Zapico, P., Medina, M., Gaya, P., Nunez, M., 1998. Synergistic effect of nisin and the lactoperoxidase system on Listeria monocytogenes in skim milk. Int. J. Food Microbiol. 40, 35 – 42.