Introduction

Artisanal cheeses haven been produced and consumed in different Brazilian regions [1, 2], leading to the establishment of quality and safety parameters and production guidelines by agriculture and health agencies mainly in the last two decades. Most of these laws that regulate the production and trade of Brazilian artisanal cheeses (BAC) were established after the publication of the “Technical Regulation for the Identity and Quality of Dairy Products” (in Portuguese: Regulamento Técnico de Identidade e Qualidade de Produtos Lácteos, RTIQPL) [3] and the “Industrial and Intellectual Property Law” (in Portuguese: Lei de Propriedade Industrial e Intelectual) [4], as reviewed by Araújo et al. [2] and Kamimura et al. [1]. The production of BAC is developed in more than 30 regions, and some of them are well characterized and defined. However, little is known about chemical, sensory, and microbiological composition of cheeses produced in areas that lack of formal recognition, demanding studies to characterize both these regions and the identity of these products, as well as to establish criteria and guidelines for production and sale [5, 6].

Most of BAC are produced using raw milk on farms with poor or little infrastructure. These products must be subjected to a ripening process, a step that is part of the identity and contributes to cheese safety, particularly to BAC produced with raw milk. Health status of the dairy herd, good manufacturing practices (GMP), and compliance with the ripening times are mandatory in order to meet quality and safety criteria [710]. Dominance of lactic acid bacteria (LAB) is extremely desirable in artisanal cheeses, once these microorganisms confer typical sensory characteristics and contribute to safety through the production of antimicrobial compounds [1113]. Yet, spoilage microorganisms and pathogens can cause economic losses and pose risks to consumers’ health, either by direct ingestion of the pathogen, or even by ingestion of pre-produced enterotoxins, mycotoxins, or other undesirable compounds [1416]. Considering that publication of novel laws stimulated the trade of BAC, here we aimed to map the quality and safety aspects of these products by comparing available scientific data with official microbiological criteria.

Methods

We performed a systematic review of the published literature to identify scientific data that describe the production characteristics and microbiological aspects of BAC, with a major focus on quality and safety status of these traditional products. The main search terms applied were “production of artisanal cheeses in Brazil,” “lactic microbiota of Brazilian artisanal cheeses,” “microbial interactions in Brazilian artisanal cheeses,” “microbial composition of raw milk and raw milk cheeses,” “influence of ripening on the microbiological parameters of traditional Brazilian cheeses,” “microbial ecology of Brazilian artisanal cheeses,” “microbial shifts in Brazilian artisanal cheeses throughout the ripening,” “filamentous fungi and mycotoxins in Brazilian artisanal cheeses,” “microbiological status of Brazilian artisanal cheeses,” “microbiological quality of Brazilian artisanal cheeses,” “microbiological spoilage of Brazilian dairy products,” “microbial contaminants in Brazilian artisanal cheeses,” “microbiological safety of Brazilian artisanal cheeses,” “pathogens in Brazilian artisanal cheeses,” and “milk products and foodborne diseases.”

The online research was conducted by accessing databases such as PubMed (https://www.ncbi.nlm.nih.gov/pubmed/), Web of Science (https://www.webofknowledge.com), Scielo (https://www.scielo.org/), ScienceDirect (https://www.sciencedirect.com, Scopus (https://www.scopus.com), and Google Scholar (https://www.scholar.google.com). Specific legislation related to BAC was obtained throughout the access of official platforms from Ministry of Agriculture, Livestock and Supply (in Portuguese: Ministério da Agricultura, Pecuária e Abastecimento, MAPA, http://www.agricultura.gov.br/legislacao-1), Ministry of Health (in Portuguese: Ministério da Saúde, MS, http://www.saude.gov.br/saudelegis), Official Diary of the Union (in Portuguese: Diário Oficial da União, DOU, http://www.in.gov.br), and Official Diary of the Brazilian States (various).

The references obtained were evaluated individually to select those pertinent to the research theme. The inclusion criteria were as follows: related scientific articles, books, book chapter, thesis, legislation, or official documents published in Portuguese or English from 1996 to 2020. The full texts were read and data from researches conducted using non-recognized or validated methodology were excluded. After the screening, 155 references fulfilled the inclusion criteria and were selected for the final analysis. For proper analysis, the data was organized accordingly to related topics, for example, production aspects, desirable microbiota, and microbiological criteria. To facilitate the analysis, data about quality and safety aspects of BAC collected from “research articles” were organized in tables displaying cheese types, producer region/state, sampling source, number of collected samples, methodology, and percentage of non-conformity samples. Thus, the obtained data are presented and discussed by the authors in the topics below.

Production and identity of Brazilian artisanal cheeses

Brazilian artisanal cheeses are usually produced in small farms by family labor and regional characteristics confers the peculiarities of each product [5, 8]. Cow milk is mostly used for cheese production, but the use of milk from other animal species, such as buffalo, goat, and sheep, is also observed. Each cheese type or producing state has their specifications regarding the use of the raw material [1720].

Production characteristics of the most common BAC were recently reviewed by Kamimura et al. [1]. The production process of some cheeses from Minas Gerais state, such as Cabacinha and Alagoa, is similar to that of Italian artisanal cheeses Caccio cavalo and Parmigiano Reggiano, respectively [2123], while the recipe of Kochkäse cheese was originated from Germany, being maintained by the German descendants settled in Santa Catarina state [2426]. The identification and characterization of artisanal cheeses are fundamental steps to define their identity. In this way, it is possible to determine the desirable physical-chemical, microbiological, and sensory characteristics aiming to typify and distinguish each cheese variety. Different varieties of BAC have their “Technical Regulation for the Identity and Quality” (in Portuguese: Regulamento Técnico de Identidade e Qualidade, RTIQ) approved by specific Federal or State laws. Minas Gerais state has been the pioneer in the development of comprehensive technical-scientific studies, aiming to establish and to regulate standards for the production and trade of artisanal cheeses [2]. RTIQ have been created for different artisanal cheeses [3, 18, 2729], but they neither cover all types of produced cheeses, nor determine their minimum maturation or commercialization times for all.

The “Industrial and Intellectual Property Law” [4] represented an important milestone for the definition of Geographical Indication (GI) in Brazil. GI consent is given to a product or service notoriety regarding its quality and specificity, determined by its region of origin and traditional production process [30, 31]. Currently, different Brazilian regions producing artisanal cheeses are in a progress to meet the criteria to obtain GI. Even with the actions developed by MAPA and other governmental, only three cheeses obtained GI so far: Minas Canastra and Minas Serro (both in Minas Gerais state), and Colônia de Witmarsum (Paraná state) [2].

The requirements for granting the GI consider not only identity aspects, but also quality and safety standards. Thus, the implementation of GI constitutes an important model for characterization of traditional cheeses and preservation of their traditional characteristics, adding economic-cultural values and regional development. The expansion of GI in Brazil demands partnerships among associations of rural producers, research, and extension institutions. This will enable the development of different RTIQ that will clearly and objectively determine the identity and quality standards of different types of artisanal cheeses, as well as their production characteristics.

Desirable microbiota of artisanal cheeses

Lactic acid bacteria are predominant in milk obtained in appropriate hygienic conditions, being the main desirable microbial group present in artisanal cheeses [7]. These microorganisms are mainly responsible for milk fermentation and coagulation in the initial stage of cheese production. During ripening, they are responsible for providing desirable sensory characteristics [11, 12].

Lactic acid bacteria can be characterized according to their optimal growth temperature, in mesophilic or thermophilic, and according to lactic fermentation, as homofermentative and heterofermentative. Homofermentative LAB (Pediococcus, Streptococcus, Lactococcus, Vagococcus, and some lactobacilli) produce lactic acid as the main metabolic product resulting from the glucose fermentation process. Thus, they are important starter cultures, as they acidify the milk and start the coagulation process originating the cheese and still releasing enzymatic compounds responsible for lipolysis, proteolysis, and conversion of amino acids which act in the formation of characteristic flavors. Heterofermentative LAB (Leuconostoc, Oenococcus, Weissella, Carnobacterium, Lactosphaera, and some lactobacilli) produce substances other than lactic acid, such as acetic, butyric, and propionic acids, as well as alcohols, such ethanol. The metabolism of compounds such as carbon dioxide, hydrogen peroxide, acetaldehyde, and diacetyl is common to this microbial group. The production associated with the fermentation process mentioned above results in typical sensory characteristics of each artisanal product, such as texture and flavor [13, 3235].

The pH decrease is determined by the production of organic acids and plays an important role in the conservation of artisanal cheeses, since it limits the growth of many pathogenic and spoilage bacteria [33, 35]. In addition, some LAB also harbors genes that encode production of bacteriocins, peptides with antagonistic activity against groups of microorganisms that may contaminate cheeses during the production process [3639].

Although LAB comprises a wide diversity of bacteria, Lactococcus, Streptococcus, Leuconostoc, Enterococcus, and lactobacilli represent the main genera usually identified from artisanal cheeses [5, 13, 40, 41]. Besides, a recent study based on high-throughput sequencing (HTS) identified the core microbiome of artisanal cheeses produced in eleven different geographical areas in Brazil: the genus Streptococcus was predominant in the Marajó, Minas Serro, and Minas Cerrado cheeses; Leuconostoc was prevalent in Coalho and Manteiga cheeses; and Lactococcus was dominant in Caipira, Minas Araxá, Minas Canastra, Minas Campos das Vertentes, Serrano, and Colonial cheeses [5]. Recently, other studies have also used HTS to identify microbial communities and populational dynamics in Minas Artisanal Cheeses (MAC) [4143]. Studies based on such methodologies have a major contribution because they allow the identification of microorganisms that are unable to multiply in culture mediums and conditions commonly used, microorganisms in viable but in non-culturable states or at low population levels. In addition, they easily demonstrate how geographical location and starter culture can play a role on determining the microbial signature of artisanal cheeses.

Studies carried out in different Brazilian regions are essential to characterize the microbiota of BAC. Through microbiological cultivation and identification by the 16S sRNA, Luiz et al. [44] isolated four different LAB genera in MAC from Araxá in two seasons (rainy and dry): lactobacilli, Enterococcus, Pediococcus, and Lactococcus. The authors observed the predominance of lactobacilli over the two seasons and also identified a variation in the species profile over time of maturation. Castro et al. [45] identified the prevalence of Enterococcus, Lactococcus, and lactobacilli in MAC produced in Campos das Vertentes region, but the microbiological quality of the analyzed cheeses was jeopardized by the high counts of undesirable microorganisms. In another study carried out with MAC from different microregions (Serro, Canastra, Serra do Salitre, Araxá, and Campo das Vertentes), different LAB were identified: lactobacilli, Lactococcus, Enterococcus, Weissella, Streptococcus, Pediococcus, and Leuconostoc; with predominance of lactobacilli [46], high counts of mesophilic aerobes and coagulase-positive cocci were also detected in most samples. These results were similar to those described by Martins et al. [47] in a study conducted with artisanal cheeses from Amazon region, where these same genera were identified as predominant, except for Streptococcus. Some studies conducted in Brazil still demonstrate that, in addition to the region of origin, the lactic microbiota prevalent in producing establishments may also determine the characteristics of artisanal cheeses [42, 43, 46].

In addition to LAB, there are other microbial groups that give desirable characteristics to artisanal cheeses. Several species of fungi can compose the endogenous microbiota of these products, or even be added directly to milk, or to milk curd, or incorporated into the cheeses subjected to ripening process. The chosen strains are usually acid and halotolerant and produce alkalis in the rind, due to the conversion of amino acids to ammonia (NH3) [13, 48]. These microorganisms can diffuse into the mass, making it alkaline. On the other hand, yeasts capable of fermenting lactose promote acidification in the inner portion of the cheese. The interaction between specific species of fungi and bacterial populations is important in the production of certain varieties of ripened cheeses, directly influencing the aroma, color, flavor, and texture of these products [9, 49].

In some European countries, species of fungi are used in the production of traditional cheeses. Penicillium roqueforti is intentionally incorporated into the blue Roquefort, Danablu, Gorgonzola, Stilton, and Cabrales cheeses, among others. P. camemberti, P. nalgiovense, and Debaryomyces hansenii are species that colonize the rind of white-blue cheeses, such as Camemberti and Brie. Other potentially beneficial species, such as P. brevicompactum, P. commune, Geotrichum candidum, Kluyveromyces lactis, and Yarrowia lypolytica, have already been isolated from cheeses such as Grana-type (Parmigiano Reggiano and Grana Padano), Manchego, Reblochon, Fiore Sardo, Arzúa-Ulloa, among others [48, 5055].

The fungal microbiota of artisanal cheeses can be considered an identity pattern, since they determine sensory characteristics typical to some artisanal cheeses. Specific studies carried out with MAC have enabled the isolation, identification, and characterization of several yeasts of interest in artisanal cheese production chain, especially in the microregions of Canastra [56, 57], Serro [58], and Salitre [59]. The yeast species predominant in MAC are D. hansenii, K. lactis, and K. marxianus. Isolates of these species have been studied, aiming to characterize their technological potentials, such as the production of proteases, lipases, and β-galactosities, which may enable their application as starter cultures in cheeses. Furthermore, the characteristics conferred by G. candidum, in some cheeses produced in the microregions of Serro and Canastra, have gained national and international evidence. This fungus acts as an element of valorization of these products: this microorganism gives a wrinkled texture on the surface of the cheeses during the maturation process and contributes to the development of sensory characteristics such as aroma and flavor, due to the production of specific enzymes [60].

Microbiological criteria

Milk obtained or handled with unsatisfactory hygienic conditions may present a high population of contaminating microorganisms and it certainly jeopardizes the quality of dairy products, especially those produced with raw milk. Milk contamination can occur into the udder, in case of infections [61], by microorganisms present in the teats or skin of the animals, and even after milking, depending on handling and storage conditions [7, 62]. The improvement of the milk quality is still a major challenge in Brazil, due to production, storage, and transport issues [63]. Many guidelines and laws have been developed and implemented in a systematic way over the past decades, aiming to raise the quality of raw material [64]. However, studies suggest that good hygiene practices are more applied in farms with high production; on the other hand, many small farms still have unsatisfactory hygiene and sanitary practices, with a negative impact on the quality of artisanal cheeses manufactured by these producers [6567].

Currently, the evaluation of milk identity, quality, and safety aspects in Brazil is covered by specific legislation [3, 6870]. Regarding cheeses, although there are different state regulations in force, the microbiological parameters follow the criteria established by Federal legislation [3, 27, 71]. The coverage of such parameters by state regulations would certainly individualize artisanal cheese, since state laws may be stricter.

MAPA published Ordinance no 146 (in Portuguese: Portaria) [3] that describes the RTIQPL in Brazil, including cheeses, while Resolution 12 (in Portuguese: Resolução) [68] from the National Health Surveillance Agency (in Portuguese: Agência Nacional de Vigilância Sanitária, ANVISA) approved the technical regulation on microbiological standards for food available for retail sale in Brazil: ANVISA is an official autarchy associated to MS, and it is responsible to conduct the sanitary control of products and services subjected to health surveillance, including the safety of foods available for human consumption. In December 2019, ANVISA published Resolution 331 [72] and Normative Instruction no 60 (in Portuguese: Instrução Normativa) [72], which established new minimum acceptable microbiological parameters for foods, and replaced the Resolution 12 [68] in December 2020. These mentioned laws categorize the quality and safety criteria for different types of cheese, according to their moisture content and type of processing (Table 1). Among the major changes and differences between the laws regarding cheese quality assessment, it can be noted that Resolution 12 established reference values for thermotolerant coliform, criteria that was replaced by Escherichia coli count by Normative Instruction no 60 (Table 1). About the safety aspects, Normative Instruction no 60 started to allow a count of Listeria monocytogenes up to 1 × 102 UFC per 25 g of cheese (valid for those varieties that fits the characteristics specified by law) and determined the absence of staphylococcal enterotoxins (regardless of the type of cheese). Moreover, Ordinance no 146 [3] determines the count of fungi in varieties of higher moisture cheeses (> 55%) and grated cheeses, while Normative Instruction no 60 [72] requires these analyses only for grated cheeses and cheese powder.

Table 1 Microbiological criteria established for production (Ordinance no 146, [3]) and trade (Resolution no 12, [68]; and Normative Instruction no 60, [72]) of cheeses in Brazil

The production of artisanal cheeses with raw milk is very common in Brazil and it requires strict health control of the herd, as well as the application of good hygienic-sanitary practices during milking, cheese production, and compliance with the minimum maturation time determined by specific regulations. Brazilian laws determine that artisanal cheeses recognized by the “ARTE” seal (in Portuguese: selo ARTE) must be inspected by State or District agriculture and livestock agencies, while inspection in retail and wholesale trade is the responsibility of State and District public health agencies [73, 74]. In the case of municipal public consortia, the municipal agriculture and health services are responsible for the inspection of animal origin products marketed in the circumscription of the cities that form part of the consortium, as long as their inspection agencies have obtained equivalence [74].

Microbiological quality

Hygiene indicator microorganisms are defined as groups or microbial species that, present in higher population, demonstrate that obtaining, preserving, or processing hygienic-sanitary practices are unsatisfactory. Mesophiles, enterobacteria, coliforms, and psychrotrophics are groups of microorganisms commonly researched as hygiene indicators. Species representing these groups are likely to cause problems related to milk and dairy product spoilage, and even jeopardize their safety [62]. Fundamentally, the species that compose the LAB group are considered beneficial; however, some LAB can also cause food spoilage [10], as well as some fungi species [48, 75].

Mesophiles is a quite diverse group, being fundamentally composed of microorganisms that grow at mesophilic temperatures (25 to 37 °C) and in an aerobic environment [76]. This group comprises some species that can be considered potentially pathogenic, including some of which are capable of producing spores [7678]. Within the group of psychrotrophics bacteria, the genus Pseudomonas is considered the most relevant for dairy products, presenting high spoilage potential, in addition to causing undesirable flavors and pigments in cheeses [7981].

The coliform group consists of at least 19 genera, and most of them are representatives of the Enterobacteriaceae family. Coliforms classified as thermotolerant (represented mainly by Escherichia coli) are capable of producing acid and gas from lactose at a temperature of 44 °C. This specific group is highly relevant because it suggests contamination of fecal origin [82, 83]. Early stuffing due to gas production is one of the main cheese defects associated with coliforms [10, 83]. However, there is growing concern about the use of this group as a hygiene indicator, especially in cheeses made with raw milk. Some studies suggest that coliforms may compose the natural microbiota of certain cheeses made with raw milk [8385], and some species of this group can produce lipolytic and proteolytic enzymes that contribute both desirably and undesirably to the flavor and texture of the cheeses. Hafnia alvei is an example of a species that confers desirable characteristics, mainly due to the production of aromatic compounds [83, 86].

Late stuffing consists of another defect leading to economic losses, being usually associated with ripened cheeses. This process consists of excessive formation of carbon dioxide (as result of lactose fermentation under anaerobic conditions) produced by sporulated bacteria, mainly from the genus Clostridium spp.: C. beijerinckii, C. butyricum, C. sporogenes, and C. tyrobutyricum are usually associated to this type of defect [8790].

Different species of fungi can also act in the spoilage of dairy products, producing undesirable flavors, as well as compounds harmful to health, such as mycotoxins. These microorganisms of ubiquitous nature can be found in the soil and milking parlor, being dispersed through the air in the form of spores. Currently, more than one hundred species of fungi that cause food spoilage are known, most of them from Penicillium, Aspergillus, Candida, Debaryomyces, Kluyveromyces, Yarrowia, Galactomyces, and Saccharomyces genera [48, 75].

In European countries, USA, Australia, and New Zealand, there are no specifications for coliforms counts in cheeses, being the criteria established for E. coli [9193]. This criterion was recently adopted by the novel Brazilian laws [3, 68]. Brazil, USA, and European and Oceanian countries have standards for coagulase-positive Staphylococcus (CPS) or enterotoxins produced by Staphylococcus spp., but the parameters vary according to the region and product categories. CPS counts greater than 105 CFU/g are usually associated with possible production of enterotoxins [91]. Yet high counts should be interpreted primarily as indicative of hygienic failures, not necessarily indicating the production of these toxic compounds [94, 95].

Studies conducted in Brazil aim to determine the conformity of the microbiological criteria of traditional Brazilian cheeses (Table 2). According to available studies, it is possible to identify many cases of non-compliance for coliforms and CPS in different types of artisanal cheeses, indicating hygiene failures during milking and/or production.

Table 2 Conformity of traditional cheeses produced and available for retail sale in different Brazilian regions according to criteria stablished by Ordinance no 146 [3] and Resolution no 12 [68]. Values indicate numbers of samples (%) in conformity with the established microbiological parameters

Figueiredo et al. [119] tried to correlate the seasons with microbiological quality MAC produced in the Serro region and found higher counts of thermotolerant coliforms and Staphylococcus spp. during the months with higher temperatures and rain incidence. In a study related to ripening time, Dores et al. [120] randomly collected MAC produced in eight farms from Serra da Canastra (cheeses were collected 2 days after manufacturing). Coliforms, E. coli and S. aureus counts reduced to the levels required by the legislation when the cheeses were ripened at room temperature (25 °C) for at least 22 days (in dry or rainy seasons), while the cheeses ripened at refrigerated temperature (8 °C) required up to 64 days (depending of the season) to reach acceptable count levels. The results also revealed that L. monocytogenes or Salmonella spp. were absent in all samples. Using a similar approach, Martins et al. [121] found that 17 days (at room temperature) was the minimum ripening time for artisanal cheeses from Serro region to reach the microbiological standards required by law. The results obtained by these studies indicate that Aw, pH, and sodium chloride values found in cheeses ripened at room temperatures may have influenced the control of contaminants.

A research conducted in southern Brazil evaluated the microbiological parameters along the ripening (15, 30, 45, and 60 day) of Serrano artisanal cheese [122]. The number of samples exhibiting S. aureus and total coliforms counts above 103 UFC/g did not change significantly along the ripening while a reduction was observed for E. coli. In addition, no statistical changes were found in pH and NaCl values along the period of 60 days.

Recently, a study revealed the microbiological characteristics of MAC from Serra da Canastra collected in two distinct sampling approaches [123]. The results showed that populations of hygiene indicator microorganisms (total coliforms, coagulase-positive Staphylococcus, and E. coli) in cheeses harvested from three producers registered in the state’s quality program were in accordance with the levels established by the current regulations before the minimal ripening time of 22 days. On the other hand, among the cheese samples collected from 78 local producers (after the 22 ripening) was identified a high number of non-compliance samples, regardless of registration in the quality program. According to the authors, microbial counts had no correlation to pH and Aw values.

Safety of Brazilian artisanal cheeses

Considering pathogenic bacteria or toxins, the main targets of national and international legislation for dairy products are as follows: Bacillus cereus, enterohemorrhagic E. coli [EHEC], L. monocytogenes, Salmonella, Clostridium botulinum toxins and/or C. perfringens, and staphylococcal enterotoxins [9193]. In Brazil, cheese safety is evaluated mainly by the research of Salmonella and L. monocytogenes (Table 1). Since December 2020, Normative Instruction no 60 allows L. monocytogenes counts less than 1 × 102 UFC in 25 g of commercially available cheeses (ready for consumption), in case of products not intended for infants or for special purposes [72]. Other great concern in dairy products manufactured with unpasteurized milk are the zoonotic diseases caused by Brucella spp. and Mycobacterium bovis. Therefore, farms which produce and commercialize dairy products manufactured with raw milk in Brazil must attend the requirements stablished by the National Program for the Control and Eradication of Brucellosis and Animal Tuberculosis (in Portuguese: Programa Nacional de Controle e Erradicação da Brucelose e da Tuberculose Animal, PNCEBT) [124], ensuring their herd are free of brucellosis and tuberculosis.

Pasteurization eliminates most of the vegetative bacterial forms present in milk. However, enterotoxins previously formed by S. aureus, C. botulinum toxin B, and Bacillus cereus emetic toxin (cereulide) are not destroyed by pasteurization. In the case of sporulated microorganisms (such as Clostridium spp. and B. cereus), pasteurization can induce spore germination, allowing the growth and eventual production of toxins in pasteurized milk depending on storage conditions [125]. C. perfringens and C. difficile may also be isolated from milk and its dairy products, indicating risks to consumers due to the potential production of enterotoxins, especially in ripened cheeses, since these foods provide anaerobic conditions that favor spore germination [126, 127].

Cheeses contaminated by staphylococcal enterotoxins can pose risks for consumers [14, 16]. Brazilian law determines the limit of 103 CFU/g of CPS in cheeses [3, 68], while the laws of New Zealand and Australia establish the direct search for staphylococcal enterotoxins [92, 93] and in Europe the legislation determines the search for enterotoxins in cheeses with CPS counts above 105 CFU/g [91]. The regulations that determine the absence of these enterotoxins are certainly more appropriated to assess the safety of the foods, since coagulase-negative Staphylococcus can also produce these substances [128]. Thus, Normative Instruction no 60 incorporated the research for staphylococcal enteroxins in cheeses, determining its absence in 25 g of the product, independent of CPS counts [72].

In a study that evaluated the influence of ripening time and seasons on the quality and safety of MAC from Serro region, Cardoso et al. [95] concluded that ripening was not effective in eliminating staphylococcal enterotoxins. These results indicate that the enumeration of CPS is not the most appropriate way to ensure the safety of these products, as there is not necessarily an evident relationship between the counts of these microorganisms and the presence of enterotoxins. In other independent studies, Andretta et al. [94] and Sales [129] did not detect classic staphylococcal enterotoxins in artisanal cheeses from Serro and Araxá regions, although many of the samples evaluated presented CPS counts above the parameters established by the current legislation.

Pathogenic E. coli strains have been isolated and identified in cheeses produced in different Brazilian regions. In a study with 59 samples of artisanal mozzarella cheese from Vale do Jequitinhonha, in Minas Gerais, Cardoso [130] identified that among 157 E. coli isolates, 16 presented the gene stx1 and 13 were positive for the gene eae; also, 5 isolates were multidrug-resistant. stx1 is related to type 1 Shiga-toxin (STEC) producing strains and eae encodes intimin, the protein responsible for the adhesion of enteropathogenic (EPEC) and enterohemorrhagic E. coli (EHEC) [131]. Paneto et al. [132] isolated E. coli from 48 (96.0%) samples of Minas cheeses (fresh type) produced with raw milk, which belonged to four different serogroups: O125, O111, O55, and O119. The authors identified three verotoxigenic strains (VTEC) and one enterotoxigenic strain (ETEC). Pathogenic and multidrug-resistant E. coli strains were also identified by other studies carried out with BAC [133, 134], highlighting the potential risk for public health.

Official data from epidemiological surveillance (MS) indicate that up to 66.8% of foodborne diseases do not have an identified origin in Brazil [135]. Some studies have already established the association of staphylococcal poisoning due to the consumption of contaminated cheeses, suggesting that these cases are underreported in Brazil [136]. Although there are systems for foodborne disease surveillance in the country, there are a lack of accurate information and it is estimated that only a fraction of the cases and outbreaks are reported to the authorities, which limits the understanding the real dimension and the development of preventive and control strategies [137]. To date, no outbreak of listeriosis or salmonellosis associated with the consumption of artisanal cheeses has been described in Brazil. Other microorganisms, such as B. cereus, Campylobacter spp., Coxiella burneti, Clostridium spp., and Yersinia spp., are also potential pathogens that can contaminate dairy products made with raw milk and cause serious health risks to consumers [78, 125].

In the last decades, studies performed with samples of traditional Brazilian cheeses, many whose production was artisanal, aimed to detect pathogenic bacteria (Table 3). These studies demonstrate the low occurrence of pathogens in BAC: this may be associated with the low capacity of pathogens to compete with other groups of microorganisms more abundant in the milk and production environment [36, 151]. In addition, the ripening of artisanal cheeses made with raw milk is mandatory, according to Federal and State laws in Brazil; during this process, acidification, reduced humidity and water activity, and occasional production of bacteriocins and other antimicrobial compounds configure the main modifications responsible for the inhibition of pathogenic bacteria [13, 3235, 37].

Table 3 Detection of potentially pathogenic bacteria in traditional cheeses produced and marketed in different Brazilian regions. Values indicate numbers of samples (%) that the pathogen was detected

Studies conducted with different MAC showed the importance of the ripening time in reducing the counts of spoilage microorganisms and eliminating potential pathogens [121, 144, 150, 152]. However, Carvalho et al. [142] found the ripening of Colonial cheese produced on 12 different farms (Santa Catarina State) for up to 36 days was not enough to meet the microbiological criteria required by the legislation. Similar results were found by Pontarolo et al. [143] in a research carried out with Serrano cheese in the same State, whose results suggested 14 and 28 days of ripening were not enough to guarantee total safety. The data presented in Table 3 indicate that ripening has a relevant role in the elimination of pathogens such as L. monocytogenes and Salmonella spp. However, bacteria with pathogenic potential have been identified in artisanal cheeses samples collected in retail stores.

Some species of fungi are also capable of producing toxins. These secondary metabolites can be produced by potentially spoilage genera, such as Aspergillus, Fusarium, and Penicillium. Ochratoxin A (OTA) and aflatoxin M1 (AFM1) are the mycotoxins offering the greatest risk to humans. The first, produced by Aspergillus spp. and Penicilium spp., is considered the most potent fungal toxin [153]. AFM1, produced by Aspergillus spp., is the mycotoxin most detected in cheeses. Although studies refer to cheese as an unfavorable matrix for mycotoxins production, some strains of fungi with sensory relevance, such as P. roqueforti, P. brevicompactum, P. commune, P. camembert, and P. nordicum, can produce these substances [15, 154].

Studies related to the detection of mycotoxins in BAC are scarce. Pereira et al. [155] identified AFM1 in 5% of Serrano cheese samples obtained in properties from Santa Catarina, but in concentrations below 2.5 μg/kg, in accordance with the limits established by national legislation determining the maximum tolerated limits for mycotoxins in food [156]. In a study conducted by Barroncas et al. [157], among 25 samples of different cheeses (Coalho, buffalo Coalho, mozzarella, buffalo mozzarella, and Minas frescal), no AFM1 was detected in any of the samples.

Conclusions

Specific laws have stimulated the artisanal cheese production and trade in Brazil. The quality and safety of these products made with raw milk demand application of strict hygienic practices in all steps of the production chain. Available scientific data reveals that artisanal cheeses produced and commercialized in Brazil do not fully meet the microbiological criteria indicated by current legislation. The data suggest low occurrence of pathogens; this may reflect production conditions, probably due to the low competition capacity of these pathogenic microorganisms.

Improvements in microbiological quality of BAC can be achieved especially by training small producers on hygienic procedures, providing technical guidelines and implementation of traceability systems. In addition, proper designed studies contemplating wider sampling should be developed through the joint action of agricultural agencies, research centers, and cooperatives, in order to obtain consistent scientific data allowing the development of this sector and the definition of adequate standards for production and inspection. In conclusion, this review contributed to better understanding microbiological aspects of BAC, and the data compiled by the authors highlight flaws in the hygienic practices.