Annals of Microbiology, 58 (2) 207-212 (2008)

Addition of to inhibit monocytogenes in Toroi: a traditional food of New Zealand Mãori

Lorraine DIXON1, Andrea DONNISON2*, Colleen ROSS2, Ian R. McDONALD3

1Marae Development Manager, 172 Horahora Road Rd 2, Te Kauwhata, New Zealand; 2Climate, Land and Environment Section, AgResearch, Private Bag 3123, Hamilton, New Zealand; 3 Department of Biological Sciences, University of Waikato, Private Bag 3105 Hamilton, New Zealand

Received 28 August 2007 / Accepted 3 April 2008

Abstract - The effectiveness of nisin and sakacin A-producing sakei Lb706 to inhibit Listeria monocytoge- nes was determined in Toroi, a traditional Mãori food. Toroi is a mixture of boiled green vegetable and uncooked mussels that is typically stored in a refrigerator for 2 weeks to 2 months before being eaten. Three batches of Toroi were conta- minated with 2.4 x 103 CFU g-1 L. monocytogenes L70; a strain derived from NZ mussels, and stored at 4-6 °C. When nisin was added to Toroi at 1000 IU g-1 (final concentration) L. monocytogenes numbers declined (c. 0.5 log) but had returned to the initial level by 14 days. In contrast, addition of L. sakei Lb706 (4.6 x 107 CFU g-1 Toroi) resulted in a rapid decline in L. monocytogenes to a very low level (< 10 CFU g-1) with no increase over 2 months. However, L. sakei Lb706 was not effective for Toroi that contained 2 x 105 CFU g-1 L. monocytogenes. As Toroi prepared from satisfactory ingre- dients would be unlikely to contain more than103 CFU g-1 L. monocytogenes, addition of L. sakei Lb706 could be a useful and practical strategy to reduce a risk of listeriosis.

Key words: Toroi, Mãori food, , , nisin, Lactobacillus sakei.

INTRODUCTION filled to overflowing with vegetable juice and stored in a refrigerator, usually for two weeks to two Traditional foods are important to New Zealand’s months, and served without further processing. indigenous Mãori people, including Toroi (Tiroi) that Hudson et al. (2001) investigated the food safe- is eaten widely and featured on ceremonial occa- ty of traditional Maori foods including batches of sions. Typically Toroi consists of watercress Toroi prepared by the same method as for this (Nasturtium officinale) or puha (Sonchus spp.) com- study. These workers reported that although there bined with eel, trout or mussels, with mussels was some evidence of lactic acid fermentation dur- (Perna canaliculus) the most popular. Some or all of ing storage this process did not establish sufficient- these ingredients can be sourced from the wild. In ly well to reduce the pH to a level that would inhib- a review of wild foods that was undertaken for the it Listeria monocytogenes. In an investigation of the New Zealand Food Safety Authority, Turner et al. fate of L. monocytogenes Scott A in kimchi, a spiced (2005) listed mussels as high risk and watercress fermented vegetable product, Lee et al. (1995) and puha as medium risk foods. Toroi is prepared on found that inactivation was associated with a both a small (household) scale and to serve large decline in pH to 4.9. numbers of people and although not manufactured The success of fermentation as a bio-preserva- commercially it can be sold informally at Markets. tion technique is due to metabolites produced by Typically Toroi is prepared by boiling the vegetable lactic acid that can include bacteriocins. component for at least 30 minutes, cooling and Bacteriocins are proteineous compounds that kill or adding chopped mussel flesh: the mussels are inhibit a wide range of Gram-positive bacteria steamed briefly to open the shells but are not (Cleveland et al., 2001; Ross et al., 2002). Nisin, a cooked. This mixture is packed into jars that are peptide produced by Lactococcus lactis, is commer- cially available and an approved food preservative in many countries (Chen and Hoover, 2003). In New Zealand nisin is a permitted additive for processed -1 * Corresponding author. Phone: +64 7 838 5422; Fax: cheese to a maximum level of 500 IU nisin g +64 7 838 5117; E-mail: (Vandenbergh, 1993), and authorities have extend- [email protected] 208 L. DIXON et al.

ed its use to processed meats (Food Standards la. Listeria monocytogenes L70 was retrieved from Australia New Zealand, 2007). Nisin has been used storage (-80 °C), the purity checked by plating to to inhibit L. monocytogenes in many products Brain Heart Infusion (BHI) agar (Difco, USA) and including: milk (Bhatti et al., 2004), fresh-cut pro- Gram stain. A colony was inoculated into 10 ml BHI duce (Leverentz et al., 2003), cold-smoked salmon broth (Difco) in a screw capped Universal bottle (Nykänen et al., 2000), blue crab meat (Degnan et (capacity 30 ml). Incubation (agar and broth) was al., 1994) and fish sausage (Raju et al., 2003). for 24 h at 37 °C. To obtain the required number of Other bacteriocin-producing lactobacilli that cells for use in the contamination experiments a have been evaluated as alternatives to nisin include freshly grown culture in BHI was serially diluted in Lactobacillus sakei (sake). Different strains of L. sterile peptone saline buffer (0.1% peptone, 0.85% sakei produce different sakacins including sakacins NaCl; pH 7.0). A and P (Aymerich et al., 2000; Guyonnet etal., 2000; Katla et al., 2001). Lactobacillus sakei Lb706, Preparation of Lactobacillus sakei Lb706 inocu- originally isolated from fresh meat, produces la. Lactobacillus sakei Lb 706 was retrieved from sakacin A that is active against Gram-positive bac- storage (-80 °C), the purity checked by plating to teria (Schillinger and Lucke, 1989; Holck et al., MRS agar (Merck, Germany) and Gram stain. A 1992) and has been suggested as a protective cul- colony was inoculated to 10 ml MRS broth (Merck) ture against L. monocytogenes on meat products in a Universal bottle. Incubation (agar and broth) (Schillinger et al., 1991). Bredholt et al. (2001) was for 48 h at 30 °C in a microaerophilic atmos- reported that L. sakei THI, added at a concentration phere (5-6% O2, 10% CO2, 84-85% N2). 5 6 -1 of 10 to 10 CFU g to cooked vacuum-packed 3 -1 meats, inhibited L. monocytogenes (10 CFU g )at Preparation of nisin stock solution. Nisin stock both 8 °C and 4 °C. Inhibition has also been evalu- solution was prepared by dissolving 1 g nisin ated in seafood. For example, Aras Hisar et al. (Sigma-Aldrich Cat. No. N5764) in 10 ml of 0.02 M (2005) reported L. sakei Lb706 inhibited L. monocy- HCl. The stock solution was diluted as required in togenes for up to 10 days on vacuum-packed trout sterile dH20. (pH about 6.3) held at 4 °C. Listeria monocytogenes L70 was the cause of an Preliminary experiments. outbreak of food poisoning that was traced to New Inhibition by nisin. Two preliminary experiments Zealand smoked mussels (Hudson et al., 1992; were carried out to determine the effectiveness of Brett et al., 1998). As mussels are not cooked dur- nisin against L. monocytogenes L70. ing Toroi preparation this cold tolerant pathogen Firstly a freshly grown culture of L. monocyto- 1 could be a risk to consumers. The study reported genes was added to /2 strength BHI broth (pH 7.4) 2 -1 here evaluated the effectiveness of adding either at a target concentration of 10 CFU ml and 10 ml purified nisin or a culture of L. sakei Lb706 to inhib- portions dispensed to Universal bottles. Nisin was it the pathogenic strain of L. monocytogenes,L70 added to obtain final activity levels of 10, 100 or -1 (Buncic et al., 1995; Brett et al., 1998), in Toroi. 1000 IU g in the broth (i.e. 10 to 1000 µg nisin -1 ml ). All treatments were done in duplicate and two Universals did not receive nisin. The MATERIALS AND METHODS Universals were placed in a refrigerator at 4-6 °C for up to 72 h. Toroi was prepared using watercress and mussels Secondly inhibition of L. monocytogenes L70 purchased from retail outlets. The watercress was was evaluated in Toroi. A freshly grown culture of L. boiled for 30 min, strained and cooled. The mussels monocytogenes was added to Toroi at a target con- 2 -1 were steamed to just open the shells, shelled and centration of 10 CFU g and 20 g portions dis- the flesh chopped. The prepared ingredients were pensed to Universal bottles. Nisin was added to combined and the mixture pureed in a Waring obtain final activity levels in the Toroi of 10, 100 or -1 -1 blender to facilitate subsequent contamination with 1000 IU ml (i.e. 10 to 1000 µg nisin g ). All treat- bacterial cultures. ments were done in duplicate and two Universals did not receive nisin. The Universals were placed in Bacterial cultures. Listeria monocytogenes L70, a a refrigerator at 4-6 °C for up to 72 h. pathogenic strain isolated from New Zealand smoked mussels (Buncic et al., 1995, 1996) was Inhibition by Lactobacillus sakei Lb706. Two prelim- used to contaminate Toroi. A culture of Lactobacillus inary experiments were carried out to determine the sakei Lb706 was obtained from R. Jones potential of L. sakei Lb706 to inhibit L. monocyto- (AgResearch). These bacteria are stored at -80 °C genes L70. at AgResearch. Firstly inhibition was assessed by disc assay (based on Schillinger, 1994). Briefly, 20 µl of a Preparation of Listeria monocytogenes inocu- freshly grown culture of L. sakei Lb706 was pipetted Ann. Microbiol., 58 (2) 207-212 (2008) 209

onto a 6 mm paper disc and two impregnated discs When L. monocytogenes numbers were expect- placed on a BHI agar plate that had been inoculat- ed to be low (i.e. specifically for analysis of the ed over the entire surface with a loopful of a fresh- stored Toroi containing L. sakei Lb706) 0.1 g of ly grown culture of L. monocytogenes L70. After 18 Toroi was spread over the surface of a MOX plate h incubation at 30 °C in a microaerophilic atmos- and incubated as described above. phere (5-6% O2, 10% CO2, 84-85% N2)the diame- ter of inhibition zones was measured and results Enumeration of Lactobacillus sakei Lb706. The expressed as the average for the 2 discs. number of L. sakei in overnight cultures (grown in Secondly inhibition was assessed in Toroi. This MRS broth) was determined by spread-plating on was done by adding a freshly grown culture of L. MRS agar (1.5% agar was added to MRS broth). monocytogenes L70 to 20 g portions of Toroi in Plates were incubated in a microaerophilic atmos-

Universal bottles to achieve the following target phere (5-6% O2, 10% CO2, 84-85% N2) for 48 h at 5 3 1 -1 numbers: 10 ,10 and 10 CFU g Toroi, designated 30 °C. high, medium and low respectively. Duplicate Universals were prepared for each target contami- Analysis of data. Bacterial numbers (CFU g-1 or -1 nation level. Following this 1 ml of freshly grown ml ) were transformed to log10 values prior to sta- culture of L. sakei Lb706 was added to each tistical analysis. Differences between treatments Universal. Universals were placed in a refrigerator were assessed by ANOVA (Minitab 15, Minitab Inc, at 4-6 °C for up to 26 days. USA).

Assessment of nisin or Lactobacillus sakei Lb706 inhibition. Three separate batches of Toroi RESULTS AND DISCUSSION were prepared and each batch contaminated by addition of a freshly grown culture of L. monocyto- The effectiveness of two bacteriocins, purified nisin 3 -1 genes L70 to a target concentration of 10 CFU g and bacteriocin-producing L. sakei Lb706 against L. Toroi. Each batch was analysed prior to addition of monocytogenes L70, a pathogenic strain isolated L. monocytogenes L70 and it was found that none from New Zealand mussels (Brett et al., 1998), was contained naturally-occurring L. monocytogenes. measured in Toroi, a traditional Maori food made Immediately after preparation the pH of the Toroi from watercress and mussels. was pH 6.0. Preliminary experiments Nisin. Nisin was added to a portion from each of the Inhibition by nisin three L. monocytogenes L70 contaminated batches The sensitivity of L. monocytogenes L70 to nisin -1 to a final concentration of 1000 IU g Toroi. was confirmed in a soluble matrix, BHI broth (pH 2 -1 Duplicate bottles (containing c. 100 g) for each 7.1). The initial concentration was 1.8 x 10 CFU ml batch, together with a bottle from each batch that L. monocytogenes and the response depended on did not receive addition of nisin, were stored at 4 °C the amount of nisin added. There was no significant -1 for up to 14 days. During this time the Toroi was effect at 10 IU ml nisin, but complete inhibition at -1 -1 analysed for L. monocytogenes every three to four 100 IU ml (at 24 h) and 1000 IU ml (at 6 h). days (5 times in all). Buncic et al. (1995) reported an immediate decline in L. monocytogenes (they used a three strain mix- Lactobacillus sakei Lb706. A freshly grown culture ture that included strain L70) in BHI broth (pH 5.5) -1 of L. sakei Lb706 was added to each of the three held at 4 °C in response to 400 IU ml nisin but contaminated batches (to a target concentration of noted that numbers were restored to initial levels 7 -1 10 CFU g Toroi) and duplicate bottles (c. 100 g) after 2 weeks. 2 from each batch were stored at 4 °C for up to 2 For Toroi that was contaminated with 8.1 x 10 -1 months (58 days). During this time the Toroi was CFU L. monocytogenes g , addition of nisin at a final -1 analysed periodically for L. monocytogenes (10 level of 10 IU or 100 IU nisin g Toroi was not fol- times in all). lowed by a decline in numbers. Addition of 1000 IU -1 nisin g was more effective as there was a decline Enumeration of Listeria monocytogenes L70. of about one log in L. monocytogenes concentration Listeria monocytogenes was enumerated in after six hours but this was a short term response overnight (pure) cultures by spread-plating onto and growth had resumed after 72 h. BHI agar and in Toroi by spread-plating to MOX agar (Oxoid). BHI plates were incubated for 24 h at 37 Inhibition by Lactobacillus sakei Lb706 °C and MOX plates for 48 h at 37 °C. Black colonies To ensure that our culture of L. sakei Lb706 would on MOX agar were counted as L. monocytogenes.A inhibit L. monocytogenes L70, a disc diffusion assay 6 selection of typical black colonies was subjected to was carried out on BHI agar, in which 3.4 x 10 CFU Gram stain. L. sakei were added to each disc. Successful inhibi- 210 L. DIXON et al.

tion was demonstrated by the extensive clearing mussels are reported to have only a low level of zones (average diameter 18.5 mm) observed. proteases they do possess these enzymes (Teo and The L. monocytogenes L70 inoculum added to Sabapathy, 1990) and because mussels are not 7 -1 Toroi contained 2 x 10 CFU ml (BHI) so that (cal- cooked during Toroi preparation proteolytic degra- 5 3 culated) initial numbers were 2 x 10 (high), 2 x 10 dation of nisin cannot be discounted. Some or all of -1 (medium) and 20 (low) CFU g . As the L. sakei these factors would be expected to have contributed 7 -1 Lb706 inoculum contained 2.5 x 10 CFU ml there to the observed loss of nisin activity. Overall it does 6 -1 were 2 x 10 CFU g in the Toroi. As shown in Fig. 1 not appear that addition of nisin in amounts likely to the response depended on the relative numbers of be acceptable in New Zealand (maximum allowable -1 the two bacteria. For levels of L. monocytogenes of final level 500 IU g ) would an effective way to 3 -1 10 CFU g or less, inhibition was effective over the inhibit L. monocytogenes in Toroi. experimental period. At the highest level of L. Each of the three batches of Toroi that had been 5 -1 monocytogenes addition (2 x 10 CFU g ), although contaminated with L. monocytogenes L70 received numbers declined over 9 days, growth was re- L. sakei Lb706 at a (calculated) final concentration 7 -1 established after 23 days (Fig. 1). These results of 4.6 x 10 CFU g . As shown in Fig. 2, L. monocy- demonstrated that a high number of L. sakei Lb706 togenes had declined substantially after 3 days (for relative to that of the pathogen was required for the L. monocytogenes only treatment the average effective inhibition of L. monocytogenes. was log 3.50, SD 0.14 and for L. monocytogenes + L. sakei treatment the average was log 1.75, SD 0.99) and by 6 days there was a significant differ- ence (P < 0.05) in L. monocytogenes concentration for Toroi with L. sakei compared to that without. Over the remainder of the 58 day storage period L. monocytogenes remained at a barely detectable level. However, although the pH had declined from pH 6.0 to pH 4.0 there was sporadic recovery of L. -1 monocytogenes in low numbers (<10 CFU g )from time to time. For example on day 58 a single colony was present on each of the duplicate MOX plates from one of the batches. These sporadic colonies were associated with particles and demonstrated that although L. sakei Lb706 was highly effective in reducing L. monocytogenes L70 it did not complete- FIG. 1 - The effectiveness of addition of Lactobacillus 6 -1 ly eliminate it. sakei Lb706 (2.5 x 10 CFU g ) to Toroi against 3 Bredholt et al. (2001) reported inhibition of 10 Listeria monocytogenes L70 at three concentra- -1 tion levels. The Toroi was stored at 4-6 °C for up CFU g L. monocytogenes after addition of L. sakei 5 6 -1 to 26 days. THI added at 10 to 10 CFU g to cooked meat, with acceptable levels maintained after 28 days storage Assessment of nisin or Lactobacillus sakei at 4 °C. Our results demonstrated effective inhibi- 3 -1 Lb706 inhibition tion in Toroi contaminated with 10 CFU g L. mono- To more fully assess inhibition, three batches of Toroi were contaminated with L. monocytogenes L70 at initial concentrations that were found to 3 3 -1 range from 2.1 x 10 to 2.7 x 10 CFU g Toroi 3 -1 (average 2.4 x 10 CFU g ). Three days after addi- tion of nisin L. monocytogenes had declined by about 0.5 log but this was followed by a re-estab- lishment of growth (Fig. 2). At 14 days L. monocy- togenes concentrations were not significantly differ- ent in the Toroi batches with nisin from those with no nisin (P > 0.5). As Toroi is usually stored for about 14 days before being eaten these results indi- -1 cate that addition of nisin to Toroi at 1000 IU g could not be relied on to protect consumers against L. monocytogenes. Nisin is very soluble at acid pH but loses anti- FIG. 2 - Response of Listeria monocytogenes L70 to either bacterial activity above pH 7 (Vandenbergh, 1993) 1000 IU g-1 nisin or 4.6 x 107 CFU g-1 Lactobacillus and inactivation through adsorption to protein is sakei Lb706 in three batches of Toroi containing 3 -1 greater at higher pH (Aasen et al., 2003). Although 2.4 x 10 CFU g L. monocytogenes that were sto- red at 4 °C for up to 14 days. Ann. Microbiol., 58 (2) 207-212 (2008) 211

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