Bacteriological Quality of Raw Milk Used for Production of a Brazilian Farmstead Raw Milk Cheese

Abstract

The objective of this study was to evaluate the bacteriological quality of raw cow's milk utilized for the production of Traditional Minas Serro cheese, a Brazilian farmstead raw milk cheese. Raw milk samples were collected from six farmstead cheese operations manufacturing raw milk cheese from cow's milk. Coliform count (CC) and Escherichia coli counts were determined using Petrifilm^™ EC plates, and Staphylococcus aureus counts were determined using Petrifilm^™ Staph Express count plates. The standard plate count (SPC) was determined using plate count agar. The somatic cell count (SCC) was determined with a DeLaval cell counter. The detection of Listeria monocytogenes was based in the ISO 11290-1 protocol. A total of 165 samples were analyzed, and the SPC was 1.85–7.88 log CFU/mL. Coliform were detected in 140 (84.8%) of the 165 samples, with counts of 1–6.39 log CFU/mL. E. coli was detected in 17 (10.3%) samples, with counts of 1–2.18 log CFU/mL. The SCC in raw milk was 10,000–1,390,000 cells per mL, with mean and geometric mean values of 247,000 and 162,181, respectively. The SCC did not differ significantly between the seasons (p>0.05), but differed between different farms (p<0.05). None of the 155 samples were positive for the presence of Listeria monocytogenes. S. aureus was isolated in 145 (94.1%) of the 154 samples, and the count was 1.47–5.03 log CFU/mL. The median of SPC, CC, and S. aureus counts differed significantly between seasons and between farms (p<0.05). Our results indicate that raw milk for production of farmstead raw milk cheese has a low incidence of L. monocytogenes and a high incidence of S. aureus, and suggest that measurements (such as SCC or SPC) may not serve as a predictor of other bacterial (including pathogenic) presence.

Introduction

T he presence of pathogens in milk products has been a concern of public health because such pathogens have contributed to several outbreaks of foodborne diseases (Denny et al., 2008; Dominguez et al., 2009; Oliver et al., 2009). Recently, Oliver et al. (2009) reviewed the prevalence of milkborne pathogens from bulk tank milk and dairy farm environments. However, there are few data regarding the microbiological quality and pathogen prevalence in raw milk used for the manufacture of artisanal cheeses (D'Amico et al., 2008b; D'Amico and Donnelly, 2010). Farmstead cheeses represent a subset of artisanal cheeses; these cheeses are manufactured with milk from the farmer's own herd or flock from the farm where the animals are raised (American Cheese Society, 2010).

Traditional raw milk cheeses have great social importance in Brazil, particularly in Minas Gerais State, as a consequence of its historical and cultural environment. Traditional Minas Serro cheese is a semi-cured, semi-hard, enzymatically coagulated artisanal raw cow's milk cheese produced in the Serro region, Minas Gerais State, Brazil (Brant et al., 2007; Pinto et al., 2009). This cheese is manufactured in the absence of any commercial starter, and the production does not include a processing step that inactivates any foodborne pathogens that may be present in the raw milk. According to Brazilian legislation, this cheese should remain under minimum ripening for 60 days (Brasil, 2000).

Previous work has demonstrated that raw milk used for the manufacturing of farmstead cheese can be of high microbiological quality with a low incidence of pathogens in the United States (D'Amico et al., 2008b; D'Amico and Donnelly, 2010) and Portugal (Kongo et al., 2006, 2008). In addition, ripening is not a critical point of control to ensure the safety of raw milk cheese (D'Amico et al., 2008a; Pinto et al., 2009). Thus, the objective of the present study was to examine the bacteriological quality and presence of milkborne pathogens in raw milk destined for the manufacturing of Traditional Minas Serro cheese, a Brazilian farmstead cheese.

Materials and Methods

Milk collection

Farms (n=6) located in the Serro region in Minas Gerais State, Brazil were chosen randomly based on their manufacturing of farmstead cheese and willingness to participate. All six farms included in this study produced milk and cheese seasonally, and manufactured raw milk cheese from the milk of cows. Raw milk samples were collected between March 2009 and July 2010 on the same day each week from the six farmstead cheese operations, for a total of 165 samples. A sample of 300 mL of raw milk was aseptically collected in sterile plastic bottles by the farmer or cheesemaker from the cheese vat following agitation, and either placed in a refrigerator prior to pickup or immediately stored on ice for transport to the laboratory. All milk samples were collected immediately before cheese manufacturing and were analyzed within 8 h of collection.

Bacteriological analysis

After homogenization, 25 mL of raw milk was aseptically collected and diluted in 225 mL of a sterile 0.9% NaCl solution, followed by manual homogenization for 2 min. Then, 10-fold dilutions were carried out with sterile 0.9% NaCl until the total dilution factor was 1:1,000,000. The standard plate count (SPC) was determined following the guidelines from the Standard Methods for the Examination Dairy Products (Laird et al., 2004), using plate count agar (Difco, Detroit, MI). The results were expressed as CFU/mL of milk. Coliform count (CC) and Escherichia coli count were determined using Petrifilm^™ E. coli/Coliform Count plates (3M Microbiology, St. Paul, MN) that were incubated for 24 h at 35°C. Colonies were counted on all plates containing 15–150 CFU, and the plates were subsequently reincubated at 35°C for an additional 24 h before repeating the count. The results were expressed as CFU per milliliter of milk. Staphylococcus aureus counts were determined using Petrifilm^™ Staph Express Count Plates (3M Microbiology, St. Paul, MN) that were incubated for 24 h at 35°C. Colonies were counted on all plates containing 15–150 CFU, and the plates were subsequently reincubated at 35°C for an additional 24 h before the count was repeated. The detection of Listeria monocytogenes was performed using the ISO 11290-1 protocol. An aliquot of 25 mL of each raw milk sample was homogenized with 225 mL of Demi-Fraser broth containing the Fraser supplement (Difco, Detroit, MI) and incubated at 35°C for 24 h. After 24 h, an aliquot of the Demi-Fraser was streaked onto a CromoCen Listeria Chromogenic Agar (ALOA Base) plate (BioCen do Brasil, São Paulo, Brazil) and incubated at 35°C for 24 h. Negative plates were reincubated for an additional 24 h. A subculture of 0.1 mL of Demi-Fraser was inoculated in 10 mL of Fraser broth and incubated at 35°C for 24 h. Then, 0.1 mL aliquots of the Fraser broth were streaked onto Chromogenic Agar plates (BioCen do Brasil, Brazil) and incubated at 35°C for 24 h. Negative plates were reincubated for an additional 24 h when no growth was observed. When appropriate, typical colonies were confirmed by API Listeria (bioMérieux S.A., France).

Somatic cell counts

The somatic cell counts (SCCs) were determined with a DeLaval cell counter DCC (DeLaval Inc., Kansas City, MO) calibrated for cow's milk. Each sample was analyzed in duplicate.

Statistical analysis

The data were log-transformed and analyzed using Sigmaplot 11 (Systat Software Inc., Chicago, IL). The level of statistical significance was set at 0.05 for all analyses. Correlations among the indicators of milk quality (using original values) were assessed using the Spearman rank correlation coefficient. The seasons were defined as summer (December to February), winter (June to August), spring (September to November), and fall (March to May). Normal distribution of data was tested according to Anderson–Darling's test, and all distributions included variables that were not found to be normally distributed. The SPC, SCC, CC, and S. aureus counts were compared for different seasons and different farms by using the Kruskal–Wallis one-way analysis of variance on ranks and the pairwise multiple comparison procedures by using Dunn's method.

Results

In the present study, trends in the bacterial and somatic cell loads in raw milk used for the production of farmstead cheese were evaluated through repeated collection of samples from six farms. A total of 165 samples were analyzed for SPC, CC, and E. coli. Distributions of the SPC, CC, and E. coli in raw milk are presented in Figures 1 and 2. The SPC was 1.85–7.88 log CFU/mL, with a mean of 4.86 log CFU/mL. Overall, 59.4% of the samples analyzed had SPC of <5 log CFU/mL; 18.8% had SPC of <4 log CFU/mL, which is the target recommended as the best practice for raw milk intended for the manufacturing of raw milk cheese (D'Amico et al., 2008b).

FIG. 1.

Relative frequency of standard plate count (SPC) in raw milk of farmstead cheeses obtained from six farmstead cheese operations manufacturing Traditional Minas Serro cheese, a Brazilian farmstead raw milk cheese.

FIG. 2.

Relative frequency of coliform count (CC), Escherichia coli and Staphylococcus aureus in raw milk of farmstead cheeses obtained from six farmstead cheese operations manufacturing Traditional Minas Serro cheese, a Brazilian farmstead raw milk cheese.

There was a seasonal variation in the SPC (p<0.05), with lowest counts detected in the winter and highest in spring (Table 1); the SPC differed significantly (p<0.05) between different farms (Table 2). Of the six farms, two had mean SPC over the legislation limit for the manufacturing of artisan cheese (Minas Gerais, 2002).

Table 1.

Seasonal Distribution of the Standard Plate Count, Coliform Count, S. aureus Count, and Somatic Cell Count in Raw Milk of Six Farmstead Cheese Operations Manufacturing Traditional Minas Serro Cheese (a Brazilian Farmstead Raw Milk Cheese)

Parameter Season	Winter (n=43)	Fall (n=55)	Spring (n=30)	Summer (n=37)	Total (n=165)
Standard plate count (log CFU/mL)	4.43¹ (0.98)²	4.83 (0.92)	5.34 (1.16)	5.03 (1.12)	4.86 (1.06)
	4.32^3,b	4.87^a,b	5.05^a	4.99^a,b	4.78
Coliform count (log CFU/mL)	2.47¹ (1.52)²	2.75 (1.62)	3.83 (1.60)	2.80 (1.26)	2.89 (1.57)
	2.83^3,b	3.03^b	3.89^a	2.89^b	3.03
S. aureus count (log CFU/mL)	2.78¹ (0.84)²	2.76 (0.94)	3.29 (1.29)	2.70 (1.02)	2.86 (1.02)
	2.71^3,b	2.77^b	3.60^a	2.71^b	2.85
				(n=26)	(n=154)
Somatic cell count (log[cells/mL])	5.20¹ (0.56)²	5.13 (0.51)	5.33 (0.25)	5.22 (0.48)	5.21 (0.49)
	5.32^3,a	5.20^a	5.36^a	5.33^a	5.31
	(n=33)	(n=50)	(n=28)	(n=36)

Arithmetic mean.

Standard deviation.

Median.

a,b

Median values followed by a different letter within the line are significantly different (p<0.05) by Dunn's method.

Table 2.

Distribution of Standard Plate Count, Coliform Count, S. aureus Count, and Somatic Cell Count in Raw Milk from Six Farmstead Cheese Operations Manufacturing Traditional Minas Serro Cheese (a Brazilian Farmstead Raw Milk Cheese)

		Farm
Parameter		A	B	C	D	E	F
Standard plate count (log CFU/mL)	N	34	32	31	15	35	18
	Mean	4.37	5.46	4.42	5.99	4.83	4.64
	Median	4.33^c	5.54^a,b	4.30^c	6.22^a	4.68^b,c	4.82^b,c
	SD	0.70	0.80	0.80	0.74	0.95	1.65
Coliform count (log CFU/mL)	N	34	32	31	15	35	18
	Mean	2.13	3.70	1.73	4.04	3.52	2.66
	Median	2.62^b	3.80^a	2.04^b	4.17a	3.42^a	2.82^a,b
	SD	1.38	1.36	1.32	1.50	0.82	1.87
S. aureus count(log CFU/mL)	N	32	30	29	14	33	16
	Mean	2.71	3.51	2.94	3.07	2.54	2.24
	Median	2.86^b	3.46^a	2.83^b	3.30^a,b	2.42^b	2.46^b
	SD	1.02	0.68	0.64	1.04	1.08	1.34
Somatic cell count (log[cells/mL])	N	31	28	27	13	32	16
	Mean	4.90	5.45	5.31	5.54	5.04	5.26
	Median	5.14^b	5.39^a	5.32a,b	5.62^a	5.04^b	5.50^a
	SD	0.58	0.25	0.26	0.38	0.31	0.73

a,b,c

Values median followed by a different letter within column are significantly different (p<0.05) by Dunn's method.

Standard deviation.

Coliform bacteria were detected in 140 (84.8%) of the 165 samples. CC was 1–6.39 log CFU/mL, with a mean of 3.40 log CFU/mL. Overall, 47.9% of samples analyzed had CC of <3 log CFU/mL. A significant positive correlation was observed between the SPC and CC (r=0.66, p<0.001). There was a seasonal variation in the CC (p<0.05), with the highest counts detected in the spring and similar counts between all other seasons (Table 1). The CC differed significantly (p<0.05) between different farms (Table 2). Distributions of CC and E. coli in raw milk are presented in Figure 2.

E. coli was detected in 17 (10.3%) samples, where the count was 1–2.18 log CFU/mL, with a mean of 1.30 log CFU/mL. Overall, 16 (9.7%) samples had counts of 1–2 log CFU/mL, and E. coli count exceeded the regulatory level of 2 log CFU/mL in only one (0.6%) sample (Minas Gerais, 2002), which was much less severe than the results reported by Nero et al. (2004), who showed E. coli counts of >2 log CFU/mL in 29.4% of the samples. No significant correlation was observed between the SPC and E. coli counts (r=0.13, p=0.092), or between the CC and E. coli counts (r=0.07, p=0.354). Although contaminated raw milk and cheese made from unpasteurized milk have been responsible for outbreaks of infection by the E. coli serotype O157:H7, published studies indicate a low prevalence (Baylis, 2009).

The distribution of the SCC in the raw milk of the six farmstead cheese is presented in Figure 3. A total of 147 samples were analyzed for their SCC. The SCC was 10,000–1,390,000 cells per mL of raw milk, with a mean and geometric mean of 247,000 and 162,181, respectively. The regulation establishes a limit of 400,000 somatic cells per mL of raw milk for the manufacturing of artisanal cheese (Minas Gerais, 2002), and 124 (84.4%) samples were <400,000 somatic cells per mL. Only two samples (1.36%) exceeded 1,000,000 cells per mL. The SCC did not differ significantly between the seasons (p>0.05), as shown in Table 1. However, the SCC differed significantly (p<0.05) between different farms (Table 2). Only one farm (16.7%) had a mean SCC of <100,000 cells per mL. Significant correlations between the SCC, SPC (r=0.24, p<0.05), and CC (r=0.22, p<0.05) were observed. The seasonal variation of SCC is not statistically significant (p>0.05) (Table 1), and a similar trend was observed for the SPC, CC, and S. aureus count.

FIG. 3.

Relative frequency of somatic cell count (SCC) in raw milk of farmstead cheeses obtained from six farmstead cheese operations manufacturing Traditional Minas Serro cheese, a Brazilian farmstead raw milk cheese.

S. aureus strains may produce staphylococcal enterotoxins, which have been responsible for food poisoning associated with cheese consumption in Brazil (Simeão do Carmo et al., 2002; Do Carmo et al., 2004). The distribution of S. aureus in raw milk is presented in Figure 2. In this study, S. aureus was detected in 145 (94.1%) of the 154 samples analyzed, with counts of 1.47–5.03 log CFU/mL and a mean value of 2.86 log CFU/mL, similar to the 91.7% observed by Arcuri et al. (2006), but higher than the prevalence detected by Fagundes et al. (2010) (7.3% of samples) and D'Amico and Donnelly (2010) (38% of samples). In Malaysia, Chye et al. (2004) detected S. aureus in 565 (60.7%) of 930 samples, with a mean count of 4.08 log CFU/mL. In Norway, Jørgensen et al. (2005) detected S. aureus in 75% of 220 samples of bulk cow's milk used to produce raw cheese, and Jakobsen et al. (2011) detected S. aureus in 47.2% in raw cow's milk samples, an incidence higher than that observed in the United States (D'Amico et al., 2008b), which was 27.4% of cow's milk samples, despite the similar contamination level of approximately 2.4 log CFU/mL.

The Minas Gerais State regulation establishes a maximum of 2 log CFU/mL for the S. aureus count. However, only 20 (13%) samples were <2 log CFU/mL; 71 (46.1%) samples were over 3 log CFU/mL. There is a positive correlation between somatic cells count (SCC) and S. aureus counts (r=0.41, p<0.001). There was a seasonal variation in the S. aureus count (p<0.05), with the highest counts detected in the spring and similar counts between all other seasons (Table 1). Moreover, the S. aureus count differed significantly (p<0.05) between different farms (Table 2).

L. monocytogenes was not detected in any of the 155 samples tested. Therefore, raw milk does not appear to be a source of contamination with L. monocytogenes in the farmstead cheesemaking evaluated. This result is consistent with previous studies (D'Amico et al., 2008b; D'Amico and Donnelly, 2010; Kongo et al., 2008; Moshtaghi and Mohamadpour, 2007; Nero et al., 2008; Oliver et al., 2005; Ortolani et al., 2010), which showed a low incidence of this pathogen associated with poor microbiological quality of raw milk.

Discussion

The SPC is often considered an important microbiological parameter for the quality of raw milk and dairy products, and our results (levels that are >5 log) are indicative of serious deficiencies in production hygiene. A count of <3.7 log CFU/mL indicates proper hygiene (D'Amico et al., 2008b).

Similar results for SPC in raw milk were obtained in other countries (Breurec et al., 2010; Chye et al., 2004; Kongo et al., 2008; Muehlherr et al., 2003; Soler et al., 1995) as well as other Brazilian regions (Arcuri et al., 2006; Nero et al., 2004, 2009; Ortolani et al., 2010). Arcuri et al. (2006) found 11 (46%) herds with SPC of <100,000 CFU/mL, whereas in the study of Nero et al. (2004), mesophilic aerobic counts were >100,000 CFU/mL in 75.7% of the samples. Kessel et al. (2004), D'Amico et al. (2008b), and D'Amico and Donnelly (2010) found different results for SPC in raw milk, and calculated that 93.1%, 96.8%, and 94% of the samples had SPC of <100,000 CFU/mL, respectively.

The mean SPC for the raw milk in our study was 4.86 log CFU/mL; this is more than 2 log lower than levels reported in Malaysia (Chye et al., 2004), is similar to the SPC reported in the United States (D'Amico and Donnelly, 2010) and in Senegal (Breurec et al., 2010) in cow's milk, and is higher than the levels reported by D'Amico et al. (2008a) in raw cow's milk utilized for farmstead cheese production.

CC regularly above 100 CFU/mL is considered an evidence of unsatisfactory production practices by some sanitarians (Chambers, 2002). In our study, CC was <1 to 6.4 log CFU/mL, that is, on average 1.5 log more than observed in the United States (D'Amico et al., 2008a) and 2 log more than reported in France (Desmasures et al., 1997). Arcuri et al. (2006) observed mean levels of CC of 0.7–4.4 log CFU/mL, and Nero et al. (2004) detected CC of >2 log CFU/mL in 80.4% of the raw milk samples. Our mean CC is similar to that reported by Jayarao and Wang (1999) in the United States, 10-fold higher than that reported by D'Amico et al. (2008b), and 1.83 log less than that observed by Chye et al. (2004).

High numbers of E. coli may be present in the milk as a consequence of mastitis, and this bacterium is responsible for several diseases of varying severity in humans. While direct links between E. coli udder infection and human disease have not been reported, a wide range of E. coli serotypes have been isolated from cow's milk, and it is probable that some of these are pathogenic for humans (Baylis, 2009). In this study, 17 (10.3%) samples were contaminated with E. coli; this result is sixfold lower than that reported in Malaysia (Chye et al., 2004).

Moreover, SCC is used as an indicator of proper management, since it has a direct relation to the microbiological and sensory quality of raw milk (Barbano et al., 2006; Ma et al., 2000). Similar results (geometric mean) were reported in raw milk from the state of Vermont in the United States (D'Amico and Donnelly, 2010), and a higher geometric mean was observed in Canada (Elmoslemany et al., 2009). The mean SCC in this study was similar to that found by Kessel et al. (2004) and Elmoslemany et al. (2009), and higher than that reported by D'Amico et al. (2008b).

S. aureus is an ubiquitous organism commonly isolated from bulk raw milk. It is one of the most common contagious pathogens infecting dairy cows and a major causative agent of mastitis. The presence of S. aureus and the possibility for production of the staphylococcal toxin represent a potential risk for the public health; thus, its presence in raw milk is a major concern for the safety and quality of traditionally produced cheeses, including raw milk cheeses (De Buyser et al., 2001; Jørgensen et al., 2005; Heidinger et al., 2009; Oliveira et al., 2011).

Although there are no Minas Gerais State standards for L. monocytogenes in raw milk, the absence of Listeria sp. was defined in 25 g of raw milk cheese. The absence of L. monocytogenes in raw milk reinforces the low prevalence of this pathogen in raw milk in Brazil. According to Nero et al. (2008), the indigenous microbiota of Brazil, especially above 5 log CFU/mL, can interfere with survival or growth of L. monocytogenes in raw milk. The antagonistic activity of lactic acid bacteria and other genera, such as Enterococcus, against L. monocytogenes has been described in dairy products (Suh and Knabel, 2001; Carvalho et al., 2006; Nero et al., 2008).

Conclusion

The results indicate that the quality of raw milk produced by local farmers was associated with a low incidence of L. monocytogenes, high levels of SPC, and a high incidence of S. aureus. Our results suggest that a single laboratory measurement, such as the SCC or SPC, may not serve as a predictor of other bacterial values, including the presence of pathogens, and that compliance with bacteriological limits does not guarantee the absence of pathogenic bacteria. Therefore, it is recommended that training and guidance, particularly on the use of proper practices of hygiene during milking and processing of raw milk, are provided to farm owners and their workers involved in these dairy farm and cheese-making operations.

Footnotes

Acknowledgments

We gratefully acknowledge the farmers for their participation. This study was supported by CNPq (IC scholarship to Julia Silva Pinheiro and Financial support Edital CNPq/MAPA/SDA no. 64/2008) and by FAPEMIG (financial support CAG APQ-01566/08 and IC scholarship to Camila A.M. de Faria).

Disclosure Statement

No competing financial interests exist.

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