Behaviour of Listeria Monocytogenes in Artisanal Raw Milk Pecorino Umbro Cheese: A Microbiological Challenge Test.

In the present study, a microbiological challenge test in artificially contaminated raw milk Pecorino Umbro cheese during cheese-making was carried out. Raw ewe milk was contaminated by a suspension of particular Listeria monocytogenes strains. The number of L. monocytogenes and L. monocytogenes dynamic growth were evaluated during cheese-making and storage. A significant decrease of the viable count of L. monocytogenes was observed during ripening and L. monocytogenes viable count was below the limit of quantification during storage. The results show that the product is unable to support the growth of the pathogen.

Introduction

Listeria monocytogenes is a foodborne pathogen, which has been responsible for several outbreaks of listeriosis reported in Europe (Gambarin et al., 2012; Lunden et al., 2004). Some outbreaks have been linked to the consumption of food products of animal origin that did not undergo heat treatment; their contamination came from several farm sources (Kongo et al., 2006). In particular, the contamination of fermented dairy products made from raw milk by L. monocytogenes may be due to either the use of contaminated raw milk or post-processing contamination from environmental sources not directly linked to raw milk (Kells and Gilmour, 2004). According to Regulation No. 2073/2005 (European Commission, 2005), the tolerance level of 100 CFU g–1 of L. monocytogenes in ready-to-eat (RTE) products such as cheese was introduced in Europe and RTE were legislatively distinguished into those that support the growth of L. monocytogenes and those that do not support the growth of this species. For RTE foods that are able to support the growth of L. monocytogenes and which may pose a L. monocytogenes risk for public health, food business operators responsible for manufacturing of the product shall conduct scientific studies in order to demonstrate that the product complies with the criteria of the regulations throughout the shelf-life (Spanu et al., 2014). Nevertheless, food business operators hardly found studies in the literature that could be adopted for their own products, especially for artisanal ones (Dalzini et al., 2014).

For these reasons, the aim of the study was to evaluate the dynamic growth of L. monocytogenes during cheese-making and the storage of a traditional raw milk Pecorino Umbro cheese characterized by a shorter ripening time (40 gg) and semi-hard consistency (Branciari et al., 2014). The aim of this study was to propose a useful scientific tool for food manufacturers that produces cheese with similar characteristics, to demonstrate whether their product supports the growth of L. monocytogenes or not, according to EC Regulation No. 2073/2005 (European Commission, 2005).

Materials and Methods

Cheesemaking of Pecorino Umbro

For Pecorino Umbro cheese making, whole sheep milk was heated at 55-58°C and a lyophilised mixed-strain starter culture was added (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis subsp. lactis e/o subsp. cremoris, Lyofast MOT 0.82 CE, CO, Italy). The coagulation took place at 33-38°C with the addition of powder calf rennet, and was completed in about 40 min. Then the curd was cut in grain of 3-5 mm, and was cooked at 41-43°C. Afterwards, 2 kg-cheeses were placed into moulds and salting was carried out in brine (NaCl 25%) for 24 hours. Ripening was conducted in appropriate cells for 40 days at 12°C. The shelf-life of the cheese was 50 days at +4°C, as established by the manufacturer.

Preparation of samples

For the experiment, three batches of cheese were produced and each batch consisted in 3 experimental groups of 10 2 kg-cheese each. The first group was produced at the Institute for Experimental Veterinary Medicine of Umbria and Marche (IZSUM) and was done with contaminated milk, during the adjunct of starter, by a suspension of multi-strain mix of L. monocytogenes at approximatively 102 CFU mL–1 (CLm). This sample unit was used to evaluate the behaviour of L. monocytogenes during cheese making and storage.

The second group (CTRIZS) was produced by the food business operator and ripened/stored at the IZSUM to ensure that the same ripening condition and product evolution occurred both at manufacturer and IZSUM level. Chemical, physical and microbial analyses of the cheese were carried out to verify the ripening conditions. The unit was also used to detect and/or enumerate L. monocytogenes occurring naturally in this type of cheese.

The third group (CTR) was produced and ripened by the manufacturer in order to compare the products submitted to challenge testing (CLm) to the products that are routinely produced by the processor (CTR) and to evaluate the physicochemical and microbial characteristics of cheese.

In total, 81 cheeses (9 sample units/3 groups/3 batches) were sampled along the testing period and submitted to analytical determinations. After ripening, the cheese was maintained at 8°C for 7 days and 12°C for 42 days. These storage conditions were determined following the EURL Lm Technical Guidance Document - On shelf-life studies on L. monocytogenes in ready-to-eat foods Version 2 – November 2008.

Bacterial cultures and inoculum preparation

A multi-strain mix of L. monocytogenes (ATCC® 7644™, Lm2963/12 and Lm6976/13 from the IZSUM collection and derived from pecorino cheese) was used in the study. Each stock culture was lyophilised and kept at +4°C.

The cultures were regenerated into BHI and incubated at 37°C for 24 h. Aliquots of each culture were transferred into tubes containing BHI and incubated at 12±1°C, which is a temperature close to the storage condition of the product. The stationary phase was reached after 18-20 h. After centrifuging each strain at 2178 g at 10°C for 5 min, the supernatant was discarded and the pellet was resuspended in 10 mL of sterile physiological solution (H2O with 0.9% NaCl). Counts were confirmed by serial decimal dilution and inoculation in Agar Listeria Ottaviani (ALOA Selective Supplement, ALOA Enrichment Supplement; Biolife, Italy) plates incubated at 37°C for 24-48 h. Each of the 3 strains was combined in an equal quantity at the concentration of about 7 Log CFU g–1. Dilutions of the mixed cultures were made to obtain a concentration in the foodstuff that was similar to the concentration occurring naturally in the foodstuff. These second stationary phase subcultures were used for contamination.

Physicochemical analysis of the cheese

The physicochemical determinations were conducted at 0, 1, 4, 7, 15, 30, and 40 days of ripening and after 20 and 50 days of storage time in two unit samples of each batch (CTR and CTRIZS). The pH measurements were made using a puncture electrode probe connected to a portable pH meter (Mettler Toledo Inc., Columbus, OH, USA). The salt content was determined as described by Branciari et al. (2014). Water activity (aw) was measured at 25°C with the aw recorder AquaLab, series 3, Model TE (Decagon Devices Inc., Pullman, WA, USA).

Microbiological analysis of the cheese

Enumeration of viable lactic acid bacteria in cheese

The enumeration of lactic acid bacteria (LAB) was conducted at 0, 1, 4, 7, 15, 30, and 40 days of ripening and after 20 and 50 days of storage time in all unit samples of each batch. The analyses were performed using the Tempo System (automated quality indicator solution; bioMérieux, Mercy Etoile, France) and Tempo LAB tests (automated test for the enumeration of LAB microorganisms; bioMérieux). The Tempo System calculates the number of microorganisms according to a calculation based on the most probable number method. The Tempo LAB test has been demonstrated to obtain performance levels similar to the standard NF ISO 15214, 1998 (Jaworska et al., 2011).

Listeria monocytogenes detection and enumeration

The detection and enumeration of L. monocytogenes were conducted at 0, 1, 4, 7, 15, 30, and 40 days of ripening and after 20 and 50 days of storage time in all unit samples of each batch according to Annex I of Regulation No. 2073/2005 (European Commission, 2005), using the validated method AFNOR BIO 12/11-03/04 (AFNOR BIO, 2010) for detection and EN ISO 11290-2 (ISO, 1996), the reference method for enumeration.

Results

The physicochemical and microbial characteristics of the cheese during ripening and storage are reported in Table 1. No difference in pH, aw and NaCl were detected among batches, or between the two different sample units during the ripening and storage time. Both CTR and CTRIZS cheese samples showed a rapid decrease in pH in the early stage of ripening (first days). The decrease was about 1.2 U (from 6.40 to 5.20). During ripening pH values undergo only a slight fall reaching a final value of 5.04-5.05 for CTR and CTRIZS respectively. At the beginning of the shelf life, the average value of pH was 4.96 and 4.99 for CTR and CTRIZS respectively. At the end of shelf life the pH value slightly increased. A reduction of aw values during cheese ripening occurred gradually and the value 0.989 and 0.988 for CTR and CTRIZS, respectively, in the curd reached the values 0.960 and 0.959, respectively, at the end of ripening. During the first stage of storage, the aw value was 0.956 and 0.954 for CTR and CTRIZS, respectively, and reached values of 0.949 and 0.952, respectively, at the end of storage. The NaCl value, as expected, increased with ripening time and during storage.

Table 1.

Physicochemical and microbial characteristics in curd and Pecorino Umbro cheese during cheese ripening and storage. Data represent the average values±standard deviation of three replicates samples for three cheese-making replicates.

		Curd			Cheese ripening				Storage
		0	1	4	7	15	30	40	20	50
CTR	pH	6.40±0.10	5.20±0.02	5.19±0.01	5.24±0.17	5.19±0.08	5.18±0.09	5.04±0.13	4.96±0.16	5.05±0.13
	aw	0.989±0.003	0.985±0.002	0.982±0.006	0.973±0.010	0.977±0.002	0.965 ±0.004	0.960 ±0.004	0.956±0.004	0.949±0.008
	NaCl %	0.163±0.004	0.328±0.134	0.543±0.130	0.780±0.051	1.371±0.133	1.753±0.339	1.994±0.314	2.318±0.212	2.508±0.281
	LAB (Log10 CFU g-1)	5.90±0.69	6.57±0.68	7.30±0.60	7.83±0.27	8.28±0.52	8.43±0.15	8.64±0.33	8.28±0.07	8.20±0.17
CTRIZS	pH	6.40±0.10	5.20±0.01	5.14±0.11	5.22±0.14	5.19±0.07	5.17±0.09	5.05±0.10	4.99±0.15	5.07±0.14
	a	0.988±0.003	0.988±0.001	0.986±0.001	0.979±0.009	0.976±0.002	0.959±0.003	0.959±0.006	0.954±0.007	0.952±0.003
	NaCl %	0.156±0.008	0.383±0.127	0.637±0.028	0.876±0.107	1.331±0.167	1.873±0.215	2.052±0.297	2.288±0.180	2.452±0.229
	LAB (Log10 CFU g-1)	5.90±0.69	6.72±0.62	7.19±0.68	7.86±0.32	8.34±0.52	8.53±0.24	8.38±0.25	8.27±0.14	8.02±0.09

CTR, third group produced and ripened by the manufacturer in order to compare the products submitted to challenge testing to the products that are routinely produced by the processor; CTRIZS, second group produced by the food business operator and ripened/stored at the Institute for Experimental Veterinary Medicine of Umbria and Marche; CFU, colony forming units.

The results of the behaviour of LAB during ripening and storage performed on CTR and CTRIZS also showed no difference among batches and between CTR and CTRIZS samples. A significant increase of LAB was observed in the first two week of cheese ripening and a small decline of LAB was observed at the end of the shelf life. The results of the behaviour of L. monocytogenes are reported in Figure 1.

Figure 1.

Behaviour of Listeria monocytogenes (Log CFUg-–1) during cheese ripening and storage in Pecorino Umbro cheese (data represent the average values±standard deviation of three replicates samples for three cheesemaking replicates).

A significant decrease of 1 Log unit of the viable count of L. monocytogenes was observed before ripening. During 30 days of ripening, the average pathogen count was around 1 Log CFU g–1. After 30 days of ripening and during storage, the L. monocytogenes viable count was below the limit of quantification with the method EN ISO 112090-2. Using the method AFNOR BIO 12/11-03/04, it was possible to reveal the presence of L. monocytogenes during storage, even when it was constantly below 1 Log CFU g–1. No L. monocytogenes was found in the control sample.

Discussion

Results from the current study demonstrate the inhibitory effect of the process for L. monocytogenes growth, even though the cheese showed a pH and aw value during storage at which L. monocytogenes could grow (EUCRL, 2008). The presence of a certain % of NaCl was one method used to control the growth of L. monocytogenes, but the concentration used in the experiment is not demonstrated to limit L. monocytogenes growth (Faleiro et al., 2003). The main injury against L. monocytogenes suppressing its growth probably occurred due to the Lactic acid bacteria; several studies have shown that the inoculation of food with a strain of LAB can inhibit the growth of L. monocytogenes. Several studies show microbial antagonism and refer to the inhibition of other microorganisms by the competition for nutrients or by the production of microbial metabolites (Holzapfel, et al., 1995; Hugas, 1998; Hurst, 1973; Amezquita and Brashears, 2002; Leroy and Devuyst 2004; Skalina and Nikolajeva, 2010; Reis et al., 2012). In addition to lactic acid and other organic acids, LAB produce other metabolites with antimicrobial activity. The inhibitory activity of LAB has been displayed towards sensitive food spoilage or pathogenic bacterial strains such as Clostridium botulinum, Staphylococcus aureus, and L. monocytogenes. This inhibitory activity towards pathogens has been documented for dairy products (Benkerroum et al., 2002; Foulquie Moreno et al., 2003; Giraffa, 1995; Leroy and Devuyst 2004; Reis et al., 2012). The decrease in L. monocytogenes counts could be due to the combined inhibitory effect of pH and the activity of lactic starters. The rapid decrease in the pH values of cheeses and the high number of LAB when the level of contamination of L. monocytogenes is 102 CFU mL–1 create an unfavourable environmental condition for the pathogen.

Conclusions

In cheese-making, the use of raw milk permits the manufacture of high-value traditional artisan varieties, but brings about safety risks, e.g. the development of L. monocytogenes. The control strategy of using an appropriate starter culture in the manufacturing process of raw milk pecorino cheese, as demonstrated in the present study, may be considered as a significant hurdle that creates an unfavourable environment, causing L. monocytogenes to be reduced to a level that no longer represents a risk to the consumer.