Received Date: 24/03/2017; Accepted Date: 31/03/2017; Published Date: 07/04/2017
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Cheese is a ready-to-eat food easily contaminated on the surface by undesirable microorganisms. Even if some are spoilage microorganisms, others are pathogenic such as L. monocytogenes, which have been associated with foodborne listeriosis by consumption of cheese. Here, we investigated through aging tests, the behaviour of L. monocytogenes during soft and pressed cheeses ripening and during their shelf-life. The contamination level relative to L. monocytogenes of 18 samples including fresh, soft and pressed cheeses was first assessed through microbiological analysis. Then soft and pressed cheeses manufactured from sheep raw milk, intentionally contaminated by L. monocytogenes were sampled in order to follow this pathogen behaviour during the ripening and shelf-life. The ripening period resulted in an increasing in pH and water activity. Likewise, a growth of L. monocytogenes of 0.7 log (ufc/g) and 0.6 log (ufc/g) were observed for the soft and pressed cheeses respectively. Furthermore, the storage at 4°C led to the decrease of the L. monocytogenes population without causing the total disappearance of this pathogen. Therefore, adequate hygienic practices are needed at every stage of the process to control the growth of the bacteria.
Raw milk cheeses, Soft cheese, Listeria monocytogenes, Behaviour, Farm
Listeria monocytogenes is an important gram-positive food-borne pathogen. This bacterium is able to cause listeriosis in animals and in humans populations . Foodborne listeriosis is relatively rare but is a serious disease with high fatality rates (20%– 30%) compared with other food-borne microbial pathogens [2,3]. L. monocytogenes is included in the safety criteria regulation (EC) No 2073/2005 for dairy products because of its ability to contaminate dairy products. The significance of L.monocytogenes in cheese and in samples from dairy plants has been previously reported [4-6]. This bacterium is a ubiquitous pathogen with many possible modes of entry into dairy processing facilities. Furthermore, Parisi et al.  highlighted the wide-spread presence of L. monocytogenes in cheese factories. According to that study, this bacterium can persist for long periods of time, resulting in a continuous contamination of the dairy products. This is of concern because L. monocytogenes has been shown to cause lifethreatening disease in foetuses, newborns, immune compromised people and the elderly . Moreover, after the isolation of strains with similar profile from different sampling sites, within and among cheese making plants, Spanu et al.  suggested a possible transfer of L. monocytogenes contamination along production lines and from one facility to another. The survival and growth of L. monocytogenes in a dairy environment depends on the manufacturing, ripening and storage conditions used for the cheeses, even when the cheese is stored at refrigeration temperatures [10,11]. Unlike many food borne pathogens, L. monocytogenes can survive and grow over a wide range of environmental conditions such as refrigeration temperatures, low pH and high salt concentration. This allows the pathogen to overcome food preservation and safety barriers, and pose potential risk to human health. Analytical data generated for several years show that low levels, <100 cfu/g of L. monocytogenes contamination are observed in raw milk cheeses . When the behaviour of the pathogen in a product under particular conditions is unknown, studies to gather the experimental data concerning the implicated product are recommended [11,13]. L. monocytogenes has been indicated as one of the principal pathogens of concern associated with dairy products, with particular reference to raw unpasteurized milk and its derivative products. Literature reports several food borne diseases involving L. monocytogenes and cheeses in different countries. In 2005, ten cases of listeriosis in a small area of Switzerland were due to locally made and distributed soft cheese; when, in 2006, the Czech Republic experienced one large outbreak, involving 78 patients, of whom 13 died here also, soft cheese was identified as the source. Likewise, a US multistate outbreak of listeriosis was linked to ricotta salata imported from Italia, in 2012. Concerning this incident, the cheese that cause the outbreak was produced in a plant in Apulia that processed semi-finished cheeses supplied by five plants in Sardinia [2,14-17]. Furthermore, according to the U.S. Food and Drug Administration Food Code, most cheeses are potentially hazardous foods based on pH and water activity . Nevertheless, the consumption of raw unpasteurized milk and raw milk cheeses all the word is greatly increasing [19-24]. The study of L. monocytogenes behaviour in cheeses made from raw sheep’s milk could help understand if and how this pathogen is likely to grow in these dairy products. Thus, to prevent food poisoning and avoid potential risk to human health, it is essential to identify the process steps that might be favourable to the development of L. monocytogenes, and to implement correctives actions. Here, we followed through aging tests, L. monocytogenes behaviour during cheeses ripening and during their shelf-life, in order to ensure that an initial contamination of less than 100 cfu/g at the end of the production would not exceeded the limit (≤ 100 cfu/g for food products) recommended by the European regulation at the end of shelf-life.
Cheese Level Contamination
Dairy products including sheep raw milk, soft, fresh and pressed cheeses from a Belgian dairy plant were used in this work. The contamination level of these products, relative to L. monocytogenes was first assessed through physicochemical and microbiological analyses, performed according to the NF EN ISO 11290-2/A1 (02/2005). The purpose was to select a type of cheese favourable for aging study.
Microbiological analyses were carried out as per the standard method (ISO, 1998). For this, 25 g /25 ml of cheese or raw milk, were weighed in sterile stomacher bags 400 (Led Techno) and mixed with 225 ml of sterile peptone buffer water. The samples were then dispersed by stomaching for 1 min at 230 rpm in a stomacher 400. A tenfold serial dilution was made, and from the appropriate dilution, 100 μl was spread plated onto baird barker, sabaureud or depth inoculation for the plate count agar (PCA), Violet Red Bile Lactose Agar (VRBL), Man Rogosa and Sharpe Agar (MRS). Following by the incubation at different temperatures and times, depending on the culture media and the microorganisms sought. The bacteria enumeration was performed according to the existing standards.
The principle of pH measurement is based on the difference in chemical potential existing between ions electrode glass and reference electrode (calomel-kcl) immersed in the same solution, when the water activity was measured by the dew point chilled mirror technique.
Raw Milk and Cheeses Analysis
In order to gain a precise idea of the raw milk contamination level, fifteen samples of sheep raw milk were analysed for the detection of L. monocytogenes, according to the standard NF EB ISO 11290-2/A1 (02/2005). Raw milk intentionally contaminated with L. monocytogenes was thereafter used for the soft and pressed cheeses production. These cheeses were then sampled to monitor the behavior of L. monocytogenes during maturation and shelf life, either a follow-up on the entire life cycle. In addition, lactic bacteria and mesophilic aerobic bacteria were enumerated.
The regulation of L. monocytogenes behavior in raw milk cheese could help in food poisoning prevention and to avoid potential risk to human health. As illustrated in the present study, the follow of this pathogen in cheeses reminds essential. Here, we investigated the behaviour of L. monocytogenes during soft and pressed cheeses ripening and during their shelf-life through aging tests. Table 1 summarize the results related to the physico-chemical characteristics and the contamination level of different dairy products including fresh, soft and pressed cheeses before L monocytogenes inoculation. The pH values <4.5 were observed in fresh cheese samples analyzed and L. monocytogenes grew in only two out of twelve cheese samples. The growth of this pathogen was ranged from 3.7 to 4 log cfu/g. The low pH, survival and growth of L. monocytogenes, whose minimum growth pH is around 4.3 [3,25,26]. By contrast, the enzymatic technology of soft and pressed cheeses contributes to increasing the pH ranged from 4.5 to 4.8 and 4.8 to 5.2 respectively (24 h after salting). Soft cheese with higher moisture content (0.97-0.99) may have provided a more favourable environment for microbial growth (Table 2) [27,28]. On the basis of these results, the soft and pressed cheeses were selected to study the behaviour of L. monocytogenes in cheeses, during their ripening and shelf-life.
Table 1. Physico-chemical and microbiological characteristics of fresh-cheeses.
|Products||Ages (days)||Samples numbers||Physicochemical parameters||L. monocytogene
|Fresh cheese||15||1||4.36 ± 0.02||0.982 ±0.001||Abs||9.0||9.0|
|20||5||4.30 ± 0.04||0.997 ±0.001||Abs||9.0||7.0|
|Fresh cheese «crottin frais»||24||5||4.48 ± 0.08||0.998 ±0.001||Abs||9.0||5.8|
|25||1||4.46 ± 0.05||0.963 ±0.006||Abs||9.0||8.6|
Table 2. Physico-chemical and microbiological characteristics of soft and pressed cheeses.
|Products||Ages (days)||Samples numbers||Physico-chemical parameters||L. monocytogenes(log ufc/g)||Mesophilic aerobic bacteria (log ufc/g)||Lactic bacteria (log ufc/g)|
|Soft cheese||42||5||4.66 ± 0,02||0.982 ± 0.015||Abs||9.0||7.0|
|Pressed cheese||25||1||4.46 ± 0.05||0.963 ±0.006||4||9.0||8.7|
The Contamination Rate of L. monocytogenes in Sheep Raw Milk
The contamination level of the raw milk used for the soft and pressed cheeses production was determined before its inoculation by the L. monocytogenes strain. According to the results presented in Table 3, L. monocytogenes was detected in two out of fifteen milk samples analyzed. Previously recognized sources of raw milk contamination by L. monocytogenes have been highlighted by different authors [29-31]. According to these studies, the raw milk contamination could be linked the environment, feed, cattle feces, wild life, biofilm on the milking machine or improper milking procedures. Mesophilic aerobic bacteria were isolated from all raw milk samples tested (Table 3). Applying the criteria in Recommendation EC 2073/2005, these samples were above the legal limits for the mesophilic aerobic bacteria. This high level of these bacteria may lead to an improper milking procedure. Similarly, Allonso-Calleja et al.  have isolated high (7.66 log cfu/g) counts of aerobic mesophilic bacteria from a raw goat’s milk cheese during the manufacturing and ripening processes. The increasing bacteria in raw milk sampled from dairy farms could be linked to the temperature or time, as observed by Vithanage et al. . Furthermore, survey from various countries have monitored the presence of different types of pathogens in raw milk, with prevalence levels as high as 13% for bacteria like Campylobacter jejuni and L. monocytogenes . The relative importance of the various sources of contamination not only depends on the farming practices, but may also be different for each pathogen, according to Soboleva T .
Table 3. Physicochemical and microbiologycal analysis of sheep raw milk analyses.
|N° Samples||Physicochemical parameters||Bacteria|
|pH||aw||Mesophilic aerobic bacteria log (ufc/ml)||L. monocytogenes log (cfu/ml)|
|1||6.56 ± 0.02||0.999 ± 0.012||10||Abs|
|2||6.66 ± 0.13||1.010 ± 0.023||9||Abs|
|3||6.63 ± 0.05||0.990 ± 0.001||9||1.8|
|4||6.68 ± 0.08||0.990 ± 0.009||9||Abs|
|5||6.69 ± 0.02||0.990 ± 0.005||9||Abs|
|6||6.69 ± 0.02||1.000 ± 0.001||9||Abs|
|7||6.72 ± 0.06||1.001 ± 0.007||9||Abs|
|8||6.75 ± 0.03||1.000 ± 0.001||10||1.5|
|9||6.75 ± 0.01||0.999 ± 0.005||10||Abs|
|10||6.65 ± 0.01||0.999 ± 0.007||9||Abs|
|11||6.85 ± 0.01||1.000 ± 0.019||9||Abs|
|12||6.53 ± 0.04||1.001 ± 0.001||7||Abs|
|13||6.63 ± 0.09||0.999 ± 0.007||8||Abs|
|14||6.70 ± 0.02||0.999 ± 0.006||7||Abs|
|15||6.42 ± 0.01||0.999 ± 0.004||9.6||Abs|
The Behavior of L. monocytogenes in Soft and Pressed Cheeses during the Ripening and Shelf-life
The evolution of L. monocytogenes counts has been studied in soft and pressed cheeses manufactured from sheep raw milk, intentionally contaminated by this pathogen. The monitoring was carried up for the ripening period and cold storage at 4°C. An increasing of L. monocytogenes number in soft cheese was observed during the ripening, ranging from 4.2 ± 0.1 log cfu/g to 4.9 ± 0.5 log cfu/g at the end of the ripening period, meaning an increase of 0.7 log cfu/g. This growth could be related to the favorable conditions including pH increasing at the beginning of ripening time (Table 4), that is in line with previous studies [28,36,37]. Witch showed that favorable properties include pH and aw, for growth initiation at the early stages of the cheese making process, could lead to high development of L. monocytogenes populations. Furthermore, most cheeses are potentially hazardous foods based on pH and water activity, as observed the U.S Food and Drug Administration Food Code . However, a low variability in L. monocytogenes growth in raw milk cheeses during the ripening step has been highlighted. During the cold storage (4°C), the cessation of L. monocytogenes growth in soft cheeses was observed. The population decreased from 4.9 ± 0.5 to 4.4 ± 0.2 log cfu/g, following by a stationary phase with L. monocytogenes concentration maintained about 4.4 ± 0.2 log cfu/g until the cheese shelf-life end (Table 4). The inhibition of L. monocytogenes growth by the amount of organic acid produced by the lactic bacteria can explain these results. Furthermore, the decrease in water activity during the stationary phase did not result in the disappearance of the pathogen. L. monocytogenes has survived in cheese but not multiply. It was observed a similar L. monocytogenes growth in a cheese with a flowered crust like Camembert. Table 4 also shows a stability of the mesophilic aerobic bacteria and lactic bacteria counts during the ripening phase, unlike lactic bacteria rate, which decreased from 8.1 ± 0.1 log cfu/g to 6.6 ± 0.4 log cfu/g, during the stationary step. Concerning the pressed cheese, similar results were observed, with an increasing of L. monocytogenes count from 3.3 ± 0.3 log cfu/g to 3.9 ± 0.3 log cfu/g at the end of the ripening. During the storage, the pH and water activity conditions have given rise to a reduction of L. monocytogenes rate (Table 5). In the same vein, Chatelard- Chauvin et al.  observed a significant decrease of L. monocytogenes in the uncooked pressed cheese rinds after 45 days of storage. According to them, the low aw would be responsible for this decrease.
Table 4. Evolution of physico-chemical and microbiological characteristics of soft cheese during ripening and shelf-life.
|Steps||Days||pH||aw||L. monocytogenes||Total count||Lactic bacteria|
|Ripening (12°C/95% HR)||J+24||5.64±0.18||0.983±0.000||4.2±0.1||8.4±0.5||8.1±0.1|
|Storage at 4°C||J+51||7.32±0.15||0.971±0.001||4.4±0.1||8.1±0.5||6.8±0.1|
Table 5. Evolution of physico-chemical characteristics of pressed cheese during ripening and shelf-life.
|Steps||Days||pH||aw||L. monocytogenes||Total count||Lactic bacteria|
|Ripening (12°C/95%HR)||J+30||5.69 ± 0.01||0.98 ± 0.002||3.3±0.3||9.3±0.2||8.6±0.2|
|J+37||5.75 ± 0.35||0.98 ± 0.002||3.4±0.2||8.6±0.5||7.5±0.5|
|J+44||5.75 ± 0.35||0.98 ± 0.002||3.5±0.6||8.8±0.1||7.5±0.4|
|J+51||6.04 ± 0.21||0.983 ± 0.001||3.8±0.0||8.8±0.5||7.4±0.8|
|J+58||6.28 ±0.008||0.986 ± 0.003||3.9±0.3||8.5±0.3||7.2±0.5|
|Storage at 4°C||J+65||6.33 ±0.007||0.978 ± 0.008||3.0±0.2||8.0±0.3||6.8±0.4|
|J+72||6.1 ± 0.007||0.966 ± 0.002||2.9±0.2||8.1±0.5||6.8±0.5|
|J+79||6.12 ±0.21||0.965 ± 0.001||2.8±0.5||8.9±0.1||7.5±0.2|
|J+86||6.1 ± 0.02||0.965 ± 0.001||2.6±0.4||7.9±0.3||7.2±0.5|
|J+90||6.13 ± 0.01||0.964 ± 0.001||2.5±0.5||7.9±0.3||6.4±0.2|
Soft and pressed cheeses were used for the study of L. monocytogenes behaviour in these foods during their ripening and shelf-life. The ripening period result in an increasing in pH and water activity. Likewise, a growth of L. monocytogenes of 0.7 log (ufc/g) and 0.6 log (ufc/g) were observed for the soft and pressed cheeses respectively. However, the storage at cold temperature leads to the decrease of the L. monocytogenes population without causing the total disappearance of this pathogen. To avoid any health hazards in these artisanal food products, adequate hygienic practices are needed at every stage of the process to assess and control bacteria growth.
This work was supported by DiversiFerm, a grant from the Public Service of Wallonia, Directorate-General for Agriculture and a grant from the Belgian Development Cooperation.