EFFECT OF LACTIC AND ASCORBIC ACID ON THE SURVIVAL OF LISTERIA MONOCYTOGENES IN VACUUM-PACKED BEEF

Adam Malicki¹, Szymon Brużewicz², Jerzy Molenda^1
1 Department of Food Hygiene and Consumer Safety, Agricultural University of Wrocław, Poland
² Institute of Social Problems of Health and Education, Warsaw School of Social Psychology, Poland

ABSTRACT

The purpose of present study was to evaluate the efficiency of certain organic acids against L. monocytogenes contaminating vacuum-packed meat stored under refrigeration. The experiment was performed on 140 samples of bovine omotransversarius muscle. Each sample was divided into 4 parts (L, A, M, C) and inoculated with L. monocytogenes PCM 2191. Subsequently the parts were superficially sprayed with 10% lactic (L) or 10% ascorbic acid (A) or the mixture of both the acids (M) or left as the control (C). All the samples were vacuum-packed and stored at 4±1°C for 14 days. The pH and water activity measurements and the microbiological analyses of the material studied were performed on 0, 1, 7 and 14 day of storage. The study proved the efficiency of superficially applied lactic and ascorbic acid against L. monocytogenes. The acids, used either individually or in mixture, caused the significant reduction of listeria at any point of storage. The efficiency of the acids was the most prominent after their application in mixture, which was proved by the calculated values of T-4D - time required for the reduction of L. monocytogenes by 4 log. The T-4D for both the acids applied together was more than twice shorter than for each of them used individually. Consequently, superficial treatment with the mixture of lactic and ascorbic acid seems to be efficient against the moderate contamination of beef with L. monocytogenes.

Key words: Listeria monocytogenes, organic acids, beef.

INTRODUCTION

Listeria monocytogenes becomes significant food-related threat in recent days. Although listeriosis is usually described sporadically, it may also occur as a point epidemic resulting from the ingestion of L. monocytogenes-contaminated food. The alimentary infection might reflect in bacteraemia or meningitis with the average mortality about 20% [10]. In the risk groups (pregnant women, newborns, patients with immunesuppression) however, the percentage of fatal outcomes might amount even 75% [15].

High prevalence of L. monocytogenes in environment increases the health risk of consumer. Water, waste-water, mud, soil and plants, particularly the rotten or ensilaged ones, constitute the main reservoir of that microorganism. From fodder plants and silages, Listeria monocytogenes might be transfered into the gut of slaughter animals, becoming the potential primary contamination of meat [11]. According to different authors, the prevalence of listeria in the meat of various animals ranges from 16-17% to over 60% [1, 5].

Technological processes, and particularly the gut rupture in course of post slaughter treatment, promote the secondary contamination of carcasses with listeria-containing alimentary contents. Moreover, L. monocytogenes was isolated from 8-70% of samples taken from the environment of slaughtering and meat plants [2, 6, 8, 12].

High prevalence of L. monocytogenes and the resulting health threat oblige to determine the factors limiting the growth of that pathogen in the food stuffs. Most of the data on the survival of listeria were obtained in vitro, and the potential food-related promoters or inhibitors of that bacterium were not taken into account. Listeria monocytogenes growths either under aerobic or unaerobic conditions or in the vacuum, within the temperature range between 1°C to 45°C, although some strains might survive even at -0.4°C. The cardinal values of pH are 4.39 to 9.40, but the growth of listeria is optimal at neutral or slightly alkaline pH [13].

The latter observation suggested the possible antilisterial effect of decreased pH. The mixture of lactic and ascorbic acid, applied on the surface of raw beef, reduced significantly the count of that bacterium during the refrigeration under aerobic conditions [7]. The purpose of present study was to evaluate the efficiency of the aforementioned organic acids against L. monocytogenes in vacuum-packed beef stored under refrigeration.

MATERIALS AND METHODS

The experiment was performed on 140 samples (250 g) of bovine omotransversarius muscle (m. omotransversarius) obtained directly post slaughter. Each sample was divided into 4 parts (L, A, M, C) and inoculated with the 18h culture of L. monocytogenes PCM 2191 strain, kindly provided by the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw. One hour after inoculation the elements L, A and M were superficially sprayed with: 10% solution of lactic acid (L), 10% solution of ascorbic acid (A), or the mixture of 10% lactic acid and 10% ascorbic acid in equal proportion (M). The part C was left untreated as the control.

Subsequently, all the samples were vacuum-packed into plastic bags and stored at 4±1°C for 14 consecutive days. The pH (V 628 type N 517pH-meter) and water activity (RTD-33 TH-1-NOVASINA avumeter) measurements and the microbiological analyses of the material studied were performed on 0, 1, 7 and 14 day of storage. The counts of L. monocytogenes were determined on Palcam Selective Agar (PLS, Merck, Warsaw, Poland, 30°C, 72h), the number of lactic fermentation bacteria – on MRS medium (Oxoid, Wesel, Germany, 37°C, 24h) and Enterobacteriaceae – on VRBG agar (Merck, Warsaw, Poland, 37°C, 24h). The total plate count was established according to the compulsory standard [9].

The bacterial counts were transformed into logarithms and subsequently underwent the mathematical and statistical analysis using Microsoft^® Excel 2000 and Statistica 5, Version 97 packages. Mean values were compared by means of Student t test (p<0.05). The values of T-4D, time required for the reduction of L. monocytogenes by 4 log, were figured out from the line regression analysis.

RESULTS AND DISSCUSION

The study proved that the superficial treatment with lactic and ascorbic acid is efficient against L. monocytogenes colonizing the vacuum packed raw beef stored under refrigeration. The acids studied, used individually or in mixture, caused the significant reduction of L. monocytogenes during the entire period of storage. The only exception was the stabilization of the test strain number between 1^st and 7^th day of experiment in the material treated with ascorbic acid (Fig. 1).

The calculated values of T4-D indicate that the mixture of both the acids was the most efficient against listeria (Table 1). If lactic and ascorbic acids were used together, the time required for the expected reduction of the test strain would be more than twice shorter than for each of them applied separately. The values of T-4D were however too high to provide the total elimination of L. monocytogenes from the meat with 4D contamination.

The aforementioned reduction of test listeria seems to illustrate not only the activity of the organic acids alone, but the combined effect of several antibacterial factors: 1) decreased pH, 2) refrigeration 3) vacuum storage, and 4) bacterial antagonism.

The pH decrease in the material studied resulted either from the treatment with organic acids or from the endogenous changes of meat and the growth of lactic acid bacteria. Listeria monocytogenes is relatively resistant to acidification and adapts to the decreased pH quite well [4]. The pH of the material studied significantly exceeded the value cardinal for listeria during the entire period of storage [13]. Consequently, the experiment revealed that the antibacterial action of organic acids results rather from the decrease of the intra- than the extracellular pH. Moreover, the study proved that the synergism of lactic and ascorbic acid, used in mixture, previously described for the raw beef stored under aerobic conditions [7], occurs also under the vacuum.

Used individually, lactic or ascorbic acid exhibited significantly lower antibacterial activity. It is known that listeria, as the lactic acid-synthesizing microorganism, is less susceptible to its action [4]. The ascorbic acid in turn, although known to enhance the antilisterial effect of other antibacterials, when used alone exhibited relatively weak activity against that pathogen [3].

The in vitro results [13] suggest that either the storage temperature or the vacuum packaging alone were not potent enough to limit the growth of L. monocytogenes in the present study.

The lactic microflora seemed to predominate among the antagonistic to listeria microorganisms present in the meat studied. Lactic acid generated by these microorganisms, alone does not inhibit significantly the growth of L. monocytogenes [4]. The numerous bacteriocins synthesized by lactic fermentation bacteria exhibited however the documented antilisterial action [14]. The dynamics of lactic fermentation bacteria was variable in the material studied, depending on the acid applied (Fig. 2). In case of lactic acid addition the growth of these microorganisms was dynamic, probably reflecting their insusceptibility to the increased concentrations of the substance discussed. In contrast, the treatment with ascorbic acid inhibited the lactic fermentation bacteria, as well as the treatment with the mixture of both the acids.

The total plate count in the material treated with organic acids did not change significantly during storage and was not different from the one determined in the controls (Fig. 3). Consequently, the inhibition of aerobic microflora resulted rather from the refrigeration and vacuum packaging than from the action of organic acids themselves. The inhibitory effect of lactic or ascorbic acid and their mixture on total plate count was however revealed if the beef was stored under aerobic conditions [7].

Used separately, lactic or ascorbic acid did not affect the counts of Enterobacteriaceae during the initial 24h of storage (Fig. 4). The subsequent inhibition, followed by the further reduction of the aforementioned microorganisms probably reflects rather the overgrowth of lactic fermentation bacteria than the activity of acids themselves, since the similar effects were observed for the controls. In the material treated with acid mixture, the inhibition of Enterobacteriaceae was evident from the beginning of experiment. Hence the dynamics of that bacterial family was similar as described for the beef stored under aerobic conditions [7].

CONCLUSIONS

1. Applied superficially, lactic or ascorbic acids, and particularly their mixture, change positively either the composition or the dynamics of microflora colonizing the vacuum packed beef stored under refrigeration.

2. The application of lactic and ascorbic acid in mixture seems to efficiently eliminate L. monocytogenes contamination, providing that the latter does not exceed 2D.

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