Inhibition of Listeria monocytogenes in cottage cheese manufactured with a lacticin 3147-producing starter culture

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1 Journal of Applied Microbiology 999, 86, 6 Inhibition of Listeria monocytogenes in cottage cheese manufactured with a lacticin 7-producing starter culture O. McAuliffe, C. Hill and R.P. Ross Department of Microbiology and National Food Biotechnology Centre, University College, and Department of Dairy Quality, Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland 678//98: received 8 May 998, revised September 998 and accepted September 998 O. McAULIFFE, C. HILL AND R.P. ROSS The efficacy of using a lacticin 7-producing starter as a protective culture to improve the safety of cottage cheese was investigated. This involved the manufacture of cottage cheese using Lactococcus lactis DPC68 (control) and L. lactis DPC7, a bacteriocin-producing transconjugant strain derived from DPC68. A number of Listeria monocytogenes strains, including a number of industrial isolates, were assayed for their sensitivity to lacticin 7. These strains varied considerably with respect to their sensitivity to the bacteriocin. One of the more tolerant strains, Scott A, was used in the cottage cheese study; the cheese was subsequently inoculated with approximately L. monocytogenes Scott A g. The bacteriocin concentration in the curd was measured at 6 AU ml, and bacteriocin activity could be detected throughout the week storage period. In cottage cheese samples held at C, there was at least a 99 9% reduction in the numbers of L. monocytogenes Scott A in the bacteriocincontaining cheese within d, whereas in the control cheeses, numbers remained essentially unchanged. At higher storage temperatures, the kill rate was more rapid. These results demonstrate the effectiveness of lacticin 7 as an inhibitor of L. monocytogenes in a food system where post-manufacture contamination by this organism could be problematic. INTRODUCTION There has been an increasing number of sporadic and epidemic cases of food-borne listeriosis in recent years (Schlech et al. 98; Bille 99; Broome et al. 99). The causative agent, Listeria monocytogenes, poses a serious health threat, particularly to the unborn, newborn (Kesseler and Dajani 99), and immunocompromised individuals (Azadian et al. 989). Listeria monocytogenes has been implicated in outbreaks resulting from the consumption of a number of dairy products, meat, egg products and vegetables (reviewed by Farber and Peterkin 99). The inability of refrigeration temperatures adequately to control the growth of this organism makes L. monocytogenes a significant threat as an infectious agent in foods such as freshly fermented dairy products. At present, proper sanitary procedures, heat treatment, and the avoidance of post-pasteurization and post-manufacture contamination (Donnelly and Briggs 986) are recommended Correspondence to: Colin Hill, Department of Microbiology, University College, Cork, Ireland ( c.hill@ucc.i.e). to prevent contamination of food with L. monocytogenes. However, in minimally-processed and refrigerated foods, additional strategies may be required to control the growth and survival of this pathogen. In particular, it has been shown to survive the manufacture of cottage (Ryser et al. 98), Camembert (Ryser and Marth 987a) and cheddar (Ryser and Marth 987b) cheese as well as dried milk preparations. As a result of increased consumer demand for natural foods, research into biopreservation has recently attracted much attention. Bacteriocins inhibitory to L. monocytogenes have been shown to control the growth of the organism in various food systems, e.g. nisin and pisicolin 6 in Camembert cheese (Maisnier-Patin et al. 99; Wan et al. 997), enterocin in raw milk (Rodriguez et al. 997) and lactocin 7 in ground beef (Vignolo et al. 996). In this study, the ability of lacticin 7, produced by Lactococcus lactis DPC7 (Ryan et al. 996), to control L. monocytogenes in cottage cheese was examined. Cases of listeriosis following consumption of cottage cheese are limited, but L. monocytogenes has been shown to survive well in this 999 The Society for Applied Microbiology

2 O. McAULIFFE ET AL. environment following post-manufacture contamination (Benkerroum and Sandine 988; Hicks and Lund 99; Piccinin and Shelef 99). Lacticin 7 has been found to inhibit a wide range of Gram-positive bacteria, including all the lactic acid bacteria tested and pathogens such as Listeria, staphylococcal and streptococcal species, by forming ionselective pores in target cell membranes (McAuliffe et al. 998). The bacteriocin is active over a wide ph range and has been shown to be extremely thermostable, particularly at low ph. Previously, lacticin 7-producing starter cultures have been used to control the non-starter lactic acid bacteria population in cheddar cheese (Ryan et al. 996); here, a starter culture producing the bacteriocin was used to manufacture cottage cheese which was subsequently inoculated with L. monocytogenes Scott A, and the effectiveness of lacticin 7 as a natural preservative determined. MATERIALS AND METHODS Bacterial strains and culture conditions Lactococcus lactis DPC68 and a transconjugant of this strain, L. lactis DPC7 (Ryan et al. 996; Coakley et al. 997) which produces lacticin 7, were the starter cultures used for the production of cottage cheese. All lactococci were maintained at C on M7 agar (Oxoid) supplemented with % glucose (GM7). The inoculum (%) for the cottage cheese manufacture was prepared by incubating the cultures at C for 6 h in % reconstituted skim milk (RSM). Lactococcus lactis subsp. cremoris HP was routinely used as a sensitive indicator organism to detect the presence of bacteriocin in the cheese. Lactococcus lactis subsp. lactis DPC7, the parent producer of lacticin 7, was used for the preparation of concentrated bacteriocin when required. Listeria monocytogenes Scott A and a number of the industrial Listeria isolates were maintained at C on tryptone soya agar (TSA (Oxoid) supplemented with 6% yeast extract (Difco) and grown at C in tryptone soya broth (TSB, Oxoid) with 6% yeast extract (Difco). For inoculation in the cheese, overnight cells of L. monocytogenes Scott A were pelleted by centrifugation, washed twice and resuspended in the same volume of cold phosphate-buffered saline (PBS, g l KCl; g l KH PO ;8gl NaCl; g l Na HPO ). Cells were diluted in PBS to obtain the desired inoculum level before addition to the cream dressing. All stock cultures were stored at 8 C in % glycerol. Bacteriocin preparation and determination of activity The presence of lacticin 7 in the cheese was established using the agar well diffusion assay, as described by Ryan et al. (996) using L. lactis subsp. cremoris HP as the indicator organism. Concentrated lacticin 7 was prepared from supernatant fluids of L. lactis subsp. lactis DPC7 cultured in MRS broth (Oxoid) using a % ammonium sulphate precipitation, which was then dialysed and the activity determined by critical dilution assay (Ryan et al. 996). The arbitrary units per millilitre (AU ml ) were obtained as described by Ryan et al. (996). Effect of lacticin 7 on resting cells in buffer Listeria monocytogenes strains to be tested were grown to an O.D. 6 (optical density at 6 nm) of 7 and harvested by centrifugation, washed twice and resuspended in mmol l sodium phosphate buffer, ph 7, to a concentration of approximately cfu ml. A preparation of concentrated lacticin 7 was added to the cells to a final activity of AU ml and the cells incubated at C. Samples were taken at intervals of, 6 and min, diluted, and plated on TSAYE to determine the viable cell count. Manufacture of cottage cheese Heat-treated skim milk (% solids) was inoculated at a level of % with L. lactis DPC 68 (Vat ) and L. lactis DPC 7 (Vat ). Single strength rennet was added to each vat at a level of ml l of milk within min of starter addition. The milk was incubated at C for 6 h, or until a ph of 6 7 was reached. The coagulum was cut into cm cubes and allowed to stand for min. The temperature of the curd was gradually increased over a period of 9 min to 7 C. The whey was drained to curd level and the curd washed three times with water at C, C and C, respectively, at about min intervals. Wash-water added was not chlorinated or acidified. The curd was gently stirred during the washing. Following these washing steps, all the whey was drained from the curd which was then left to stand overnight at C. Cheese from each vat was divided into two sanitized containers, one of which was inoculated with L. monocytogenes Scott A, the other acting as a control. Creaming was accomplished by adding dressing in the ratio of parts curd to part cream. The dressing was composed of % (w/v) commercially-pasteurized and homogenized cream (about % fat), % (w/v) non-fat milk and % (w/v) NaCl. The prepared Listeria cells were added to the dressing at a level of cells ml. Once the dressing was added, the cheese was left to settle and mixed periodically over h. The final numbers of Listeria in the cheese were approximately cells g cheese. The creamed cheese was stored at C, 8 C and C. Enumeration of L. monocytogenes Triplicate samples of cottage cheese ( g) were homogenized in 9 ml / strength Ringers solution (Merck), appropriately 999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 6

3 INHIBITION OF LISTERIA MONOCYTOGENES BY LACTICIN 7 diluted and plated on Listeria Selective Agar (LSA) (Oxoid). The plates were incubated at 7 C for 8 h, after which typical Listeria colonies were counted. Listeria API (biomerieux) were used to confirm the isolates. Samples were also plated on Plate Count Agar (PCA) (Oxoid) and incubated at 7 C. Pre-enrichment of samples was done in Listeria Selective Enrichment Broth (SEB) (Oxoid) as follows: g of cheese was added to ml of Listeria (SEB) and homogenized; samples were incubated at C for 7 d, and subcultured onto LSA by direct plating after, and 7 d; plates were incubated at C for up to 8 h and examined for typical Listeria colonies. RESULTS Initially, the sensitivity of a number of different strains of L. monocytogenes to lacticin 7 was examined. These strains included Scott A and a number of Listeria isolates obtained during routine environmental testing in a cheese-making plant. These isolates were confirmed as being L. monocytogenes by a combination of microscopic examination, streaking on LSA and EHA (enhanced haemolysis agar), Listeria API, and RAPD (random amplified polymorphic DNA) PCR. Inhibition of L. monocytogenes strains by lacticin 7 To investigate the effect of lacticin 7 on L. monocytogenes Scott A and some of the industrial isolates, a concentration of AU lacticin 7 ml was added to suspensions of mid-log phase cells incubated at 7 C, and the viable cell count and optical density determined. (a) (b) (c) Log cfu ml 6 (d) 9 6 Time (min) (e) Log cfu ml Time (min) Fig. Effect of lacticin 7 on resting cells of various strains of Listeria monocytogenes in buffer. (), No bacteriocin added; (ž), AU ml lacticin 7 added. (a) Scott A; (b) 8; (c) 879; (d) 96; (e) The Society for Applied Microbiology, Journal of Applied Microbiology 86, 6

4 O. McAULIFFE ET AL. At an initial cell count of approximately cfu ml, lacticin 7 reduced the viable count of L. monocytogenes Scott A by approximately log cfu ml over a period of h at ph 7 (Fig. a). In contrast, the cell count in the control sample (no bacteriocin) did not change significantly. The optical density of the cell suspension remained the same throughout the experiment (data not shown). The industrial isolate, L. monocytogenes 8, appeared to be more sensitive to lacticin 7 than Scott A (Fig. b), i.e. in a parallel experiment, no viable cells could be detected after min. Cells of L. monocytogenes 879 were no longer detected after 6 min (Fig. c), whereas 96 and 978 were of the same relative sensitivity as Scott A (Figs d, e). As a result, Scott A was chosen in subsequent spiking trials to represent one of the most tolerant Listeria tested to lacticin 7, in addition to being a proven human pathogen (Farber and Peterkin 99). Effect of lacticin 7 on the survival of L. monocytogenes Scott A during storage of cottage cheese Cottage cheeses manufactured with a lacticin 7-producing starter culture, DPC68, and a non-producing control strain, DPC7, were inoculated with L. monocytogenes Scott A at a level of cells g in the cream dressing. The cheese was stored at C for week and the survival of Listeria monitored. Examination of the cheese by direct plating onto LSA revealed that in the first h of storage, there was approximately a -log reduction in the numbers of L. monocytogenes Scott A (Fig. a). A slight increase was detected on day. However, the numbers began to decrease again in the subsequent days and on day, the Listeria numbers decreased below detection limits of cells g of cheese; Listeria were not recovered from the cheese by direct plating on day 6 or day 7. In contrast, the numbers of Listeria in the control cheese not containing lacticin 7 remained at approximately the same level as was inoculated in the cream dressing (approximately cells g, Fig. a). In both the control and test cheeses, the ph of the cheese and presence of lacticin 7 was monitored daily. The ph of the cheese after creaming was measured at, which did not change during the week storage period. Lacticin 7 was measured in the curd at a level of 6 AU ml and could be detected in the bacteriocin-containing cheese throughout the duration of storage; as expected, no bacteriocin activity was detected in the control cheese (Fig. ). Samples taken on day 7 were pre-enriched to recover any Listeria that may be stressed due to the presence of the bacteriocin and would not grow on the selective medium. Despite several attempts at recovering Listeria, no viable cells could be cultured from the cheese samples, indicating the bactericidal nature of lacticin 7 (McAuliffe et al. 998). Effect of lacticin 7 and temperature abuse on the survival of L. monocytogenes Scott A in cottage cheese Samples of cheese were also stored at 8 C and at C to examine if the presence of lacticin 7 offered protection to the cheese against pathogenic and spoilage micro-organisms in a situation where the cheese would be held above the recommended storage temperature of C. (a) (b) (c) Log cfu ml Not detected Not detected Not detected 6 7 Time (d) 6 7 Fig. Inhibition of Listeria monocytogenes Scott A in cottage cheese produced with a lacticin 7 starter culture stored at (a) C; (b) 8 C and (c) C. (), Cheese manufactured with DPC68, no bacteriocin; (ž), cheese manufactured with DPC7 producing lacticin The Society for Applied Microbiology, Journal of Applied Microbiology 86, 6 7

5 INHIBITION OF LISTERIA MONOCYTOGENES BY LACTICIN 7 Fig. Detection of the presence of lacticin 7 in cottage cheese samples on the day of manufacture and at the end of the storage period (day 7). (A) Cheese manufactured with DPC68, no bacteriocin; (B) cheese manufactured with DPC7 producing lacticin 7 It was observed that at 8 C, the initial kill rate was slightly slower than in the cheese stored at C; a fivefold reduction in cell numbers was observed in the first h in the bacteriocincontaining cheese (Fig. b). At C, however, Listeria could no longer be detected after h, but the numbers in the control cheese also decreased below detectable levels within d of storage at this temperature (Fig. c). However, at this point, the cheese had been extensively spoiled by yeasts and moulds. Plating the cheese samples on the non-selective media PCA revealed that the primary spoilage agents of the cheese at all storage temperatures were moulds. Lacticin 7 has been shown to inhibit only Gram-positive bacteria (Ryan et al. 996) and would have no protective effect against spoilage by moulds. DISCUSSION The results of this work have shown the effectiveness of using a lacticin 7-producing starter as a protective culture in a food system against L. monocytogenes. Lacticin 7 was shown to be inhibitory to a number of strains of L. monocytogenes which were assayed for their sensitivity. It was found that random strains, which were isolated from a cheesemaking plant and confirmed to be L. monocytogenes species, were as sensitive, if not more sensitive, to the bacteriocin as the human pathogen, Scott A. Therefore, Scott A was used in this trial, given that it is a proven, well-studied human pathogen. Interestingly, strains of L. monocytogenes seem to be routinely isolated from the environment of the cheese plant and the dairy equipment used therein, highlighting the risk of listeriosis from post-manufacture contaminated cheese. In this study, it was observed that numbers of deliberately inoculated L. monocytogenes Scott A decreased dramatically over a 7 day period at various different storage temperatures in the presence of lacticin 7, indicating the bactericidal nature of this broad spectrum bacteriocin. In addition, we were unable to recover any Listeria through pre-enrichment, whereas the regrowth of Listeria or the emergence of resistant isolates have been reported in cases where other bacteriocins have been used in food systems, e.g. nisin and pisicolin 6 (Wan et al. 997). In our laboratory, we found difficulty in isolating lacticin 7-resistant Listeria; no spontaneouslyresistant colonies were found for a number of Listeria strains tested by the method of Ming and Daeschel (99; Dodd 996), which is promising for the future potential use of this bacteriocin in such food systems. In the control cheese stored at C, no significant decrease in Listeria numbers was recorded. This is contradictory to the findings of Hicks and Lund (99) who showed that a decrease in the numbers of Listeria in cheese stored at C was most probably due to the increase in the numbers of lactic acid bacteria in the cheese and the subsequent decrease in ph. No decrease in ph was observed in our cheese during the 7 day storage period, possibly due to the fact that lacticin 7 would also inhibit other Gram-positive bacteria found in the cheese. Nisin has also been shown to inhibit L. monocytogenes in cottage cheese stored at C (Benkerroum and Sandine 988). It was also observed that nisin delayed the growth of other psychrotrophic organisms which result in spoilage of the cheese; non-sterilized cottage cheese samples without nisin spoiled week earlier than those with nisin. In our study, it appeared that the cheese containing lacticin 7 spoiled at the same rate as the control cheese. However, the primary spoilage agents in cottage cheese are Gram negatives, yeasts and moulds which are not inhibited either by lacticin 7 or nisin. In the previously mentioned study, nisin was incorporated in cottage cheese as a food additive, whereas the use of a bacteriocin-producing starter culture, such as L. lactis DPC7, in food fermentations may be more widely accepted than the use of pure bacteriocin preparations as it would circumvent the need for special labelling. Coakley et al. 997 demonstrated that L. lactis DPC7 produced acid at a rate similar to that of the parent strain, which is sufficient for commercial cheese manufacture. In contrast, it is known that nisin-producing lactococci are slow acid producers and have reduced proteolytic capacity, making them unsuitable as cheese starters (Lipinska 97). The effectiveness of other antimicrobials, such as citrate, sorbate, sodium and calcium lactate, as antilisterial agents in 999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 6

6 6 O. McAULIFFE ET AL. cottage cheese has also been investigated (Bradley et al. 96; Collins and Moustafa 969; Shelef 99). It was found that while some antimicrobial and antimycotic activity was observed, these agents did not exhibit antilisterial activity during storage at refrigeration temperatures. However, at elevated temperatures, the antibacterial activity of sorbates was markedly reduced. As has been demonstrated, lacticin 7 is an effective inhibitory agent at higher storage temperatures. It is probable that lacticin 7 will be best used in food in combination with agents such as antimycotics to combat potential pathogenic and spoilage organisms. We have demonstrated that this bacteriocin can offer some level of protection, but use of bacteriocins in food should not replace proper hygiene practices, especially in minimally-processed food such as cottage cheese where a potential problem exists. In conclusion, we have shown that use of a lacticin 7- producing starter culture can provide protection against Listeria in cottage cheese. ACKNOWLEDGEMENTS The authors acknowledge the support of the Non-Commisioned Food Research Programme, operated by the Irish Department of Agriculture, Food and Forestry and supported by national and European funds. REFERENCES Azadian, B.S., Finnerty, G.T. and Pearson, A.D. (989) Cheeseborne Listeria meningitis in immunocompetent patient. Lancet i,. Benkerroum, N. and Sandine, W.E. (988) Inhibitory action of nisin against Listeria monocytogenes. Journal of Dairy Science 7, 7. Bille, J. (99) Epidemiology of human listeriosis in Europe, with special reference to the Swiss outbreak. In Foodborne Listeriosis ed. Miller, A.J., Smith, L. and Somkuti, G.A. pp Society for Industrial Microbiology. New York: Elsevier Science. Bradley, R.L., Harmon, L.G. and Stine, C.M. (96) Effect of potassium sorbate on some organisms associated with cottage spoilage. 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