Microbiological standards for food : what has changed in 18 years ?

Objective: This study aimed to show the main changes in the microbiological standards for food, occurred after the repeal of Resolution RDC No. 12, of January 2, 2001, by Resolution RDC No. 331, of December 23, 2019. Methods: A comparative study of RDC was performed to identify the modifications regarding food groups, the number of sample units to be collected, indication of the number of acceptable samples, types of microorganisms, and their tolerance limits. Results: The following main changes were observed: 1) inclusion of Research, Society and Development, v. 9, n. 11, e5639119839, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9839 2 aerobic mesophiles and enterobacteria (hygienic indicators), Escherichia coli (fecal microorganism), Cronobacter spp. (for infant foods), and microbial toxins and metabolites; 2) increased demand for tolerance limits for some food groups; and 3) the creation of specific categories for certain food groups, such as poultry meat. Conclusion: RDC No. 331, of December 23, 2019, provided more security to consumers, as it is applied to foods ready for consumption, implying greater rigor in their production, especially those easily accessible to the population, with high nutritional content and intended for groups at risks such as infants and newborns.


Introduction
Foods are naturally subject to chemical changes, by enzymatic activity, and microbiological changes, by deteriorating and/or pathogenic microorganisms. This is due to certain intrinsic characteristics of most foods, such as high-water activity, adequate pH, and balanced nutritional and chemical composition (Fellows, 2019). Under ideal conditions, enzymatic and microbial activities cause changes in color, taste, odor, texture, and nutritional value.
Nevertheless, microbial growth can also represent a series of risks to consumers, although it is influenced by and related to the infectivity and virulence potential of microorganisms, as well as to the host's pre-existing immune status and diseases (Dubugras;Pérez-Gutiérrez, 2008). These risks involve bacteria, viruses, parasites, spores, toxins, or even chemicals, which can be responsible for causing foodborne diseases.
According to estimates by the World Health Organization (WHO, 2015), foodborne diseases affect 1 in 10 people worldwide, causing about 420 thousand deaths per year, of which 1/3 in children under 5 years old. Also, norovirus, E. coli, Campylobacter, and nontyphoid Salmonella are the main etiologic agents of foodborne diseases.
Because this is a serious public health problem and seeking a way to offer the consumer a safe food, from a microbiological point of view, the legislation of a country should establish microbiological criteria from a sampling plan to indicate the acceptability of Development, v. 9, n. 11, e5639119839, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9839 4 food or batch of foods. In Brazil, these criteria were established by Resolution -RDC No. 12, of January 2, 2001(Brasil, 2001, which was revoked only in 2019 by Resolution -RDC No. 331, of December 23, 2019 -ANVISA (Brasil, 2019).
Due to this update in the current microbiological standards, we sought to address the main changes, occurred after 18 years, and assess their impacts in the search to ensure the quality of food products.

Methodological Procedures
This exploratory qualitative study consisted of analyzing RDC No. 12/2001 andNormative Instruction No. 60, of December 23, 2019, which is applied in a complementary way to the Collegiate Board Resolution -RDC No. 331, of December 23, 2019. This RDC provides for microbiological standards for food and their application and will come into force on December 23, 2020. The components of a microbiological standard include the food, the specific point of the chain at which this standard is applicable, the microorganism, the microbiological limits, and the sampling plan.
Therefore, the main changes in Resolution were analyzed regarding the contemplated food groups, the number of sample units to be collected, indication of the number of acceptable samples, types of microorganisms, and their tolerance limits. Then, based on data from the literature, we sought to discuss, critically, the implications of these changes, regarding the acceptability of a food batch or process, protection of consumers, or even as an indication of a risk assessment.

Results and Discussion
The National Health Surveillance Agency (ANVISA) regulates standards for food microbiological analysis, i.e., it establishes rules for companies, industries, and food businesses, in general, to ensure the safest possible food for consumers. The establishment of RDC No. 331, of December 23, 2019, led to significant changes in these regulations, especially regarding intolerance, even higher to pathogenic microorganisms and substances harmful to consumers.
Coliforms at 45ºC indicate the presence of pathogens in the gastrointestinal system of humans and animals, whereas E. coli/g, Enterobacteriaceae/g, and Enterococcus, although belonging to the coliform group, when analyzed individually, as required in RDC No. 331/2019, serve as a parameter to indicate sanitary control at a more specific stage of the process. The presence of Enterobacteriaceae and Enterococcus, e.g., indicates contamination after pasteurization and E. coli is an indicator of fecal contamination in processed foods. The most significant changes between these resolutions are shown in Tables and discussed throughout the text.
In the case of food groups, more specifically fruits and derivatives, the number of samples to be analyzed increased, and the number of acceptable samples and the maximum allowed value decreased (Table 1). This greater rigidity may be due to the various sources of contamination during agricultural practices, derived from fertilizer, soil, and/or irrigation water, which can carry microorganisms such as E. coli/g and Salmonella, as well as molds and yeasts, which are indicators of food deterioration. however, both are prevalent in human salmonellosis (Brasil, nd).

According to results presented by EMBRAPA (Brazilian Agricultural Research
Corporation) in 2017 (Brasil, 2017), regarding a study of the projection of Brazilian agricultural production for the next decade, the marketing of poultry in Brazil will have the highest growth over 10 years, until 2027.
Poultry is one of the most consumed meats in Brazil and worldwide, due to its high production potential and demand of the population for a healthier and more nutritious diet since it has low-fat content and a good source of proteins, in addition to its low cost. Thus, a greater rigor for this group of food is justified, which also has intrinsic characteristics conducive to the growth of pathogens, such as high-water activity. perfringens can cause consequences, ranging from diarrhea to death of the consumer. Some isolates of C. perfringens produce an enterotoxin (CPE) responsible for clinical symptoms in cases of food toxinfection (Otuki, 2010).
Bottled waters, fish, vegetables, roots, tubers, edible fungi and derivatives, and infant food had significant changes regarding their analysis, tolerance limit, and analyzed samples.
In the category '7. Fish', according to RDC No. 12/2001, three microorganisms were analyzed: Salmonella spp./25g, coagulase-positive staphylococci/g, and coliforms at 45ºC. In RDC No. 331/2019, the coliform group was replaced by E. coli, and histamine analysis (mg/Kg) was included only for fish with high histidine content such tuna, mackerel, chub mackerel, "guaraiúba", crevalle jack, yellowfin tuna, shrimp, crustaceans, among others. Research, Society and Development, v. 9, n. 11, e5639119839, 2020 (CC BY 4. Research, Society and Development, v. 9, n. 11, e5639119839, 2020 (CC BY 4.  Histidine is an amino acid that gives rise to histamine when fish after death is subjected to inappropriate conservation and storage. Under conditions favorable to microbial growth, histidine is converted to histamine, which is responsible for undesirable effects. The symptoms are similar to those of allergic reactions, such as hypotension, flushing, headache, urticaria/pruritus, palpitations/tachycardias, and other gastrointestinal problems. These effects were reported by people who consumed bonito fish (Sarda sarda) involved in outbreaks of food intoxication. S. sarda samples had a concentration of 6,407.9 mg/kg of histamine, whereas current microbiological standards indicate a maximum limit of 200 mg/kg (Takemoto et al., 2019).
Category '13. Infant Food' also deserves attention regarding the changes in resolution (Table 3). This category now includes other groups of microorganisms to be analyzed, especially formulations for infants and newborns, for which only the analysis of coliforms at Development, v. 9, n. 11, e5639119839, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.9839 45ºC was required; after such change, Salmonella, Cronobacter spp., presumptive Bacillus cereus, Enterobacteriaceae, and aerobic mesophiles were included.  There was a significant increase in the number of samples used for the analysis of Salmonella spp. (Table 3) in powdered formulations for infants and early childhood children.
Before such change, only 5 samples were required for analysis; now 60 samples are required.
Salmonella is a pathogen that, when affecting adults, can cause nausea, stomachache, and other disturbances, but in young children, the presence of this bacterium acts more aggressively, as it can cause diarrhea, meningitis, or even death (Cahill et al. , 2008).
These formulations are characterized as a medium of high nutritional value; therefore, they have a high potential for the growth of pathogenic microorganisms. Infant formulations have compounds similar to those of mother's milk, which assist in feeding the child, acting in the replacement of food, in whole or in part. The target audience of this food category are children who are still developing their immune and metabolic system, and precisely because of this immunological immaturity, the newborn becomes more vulnerable to infections, being even more exposed to possible contamination (De Mello; De Oliveira Rosa, 2017). Strapasson (2019) stated that powdered infant formulations have been related to bacteria that cause necrotizing enteritis, meningitis, meningoencephalitis, septicemia, and even death.
Given the above, the analyses of pathogens provided for in RDC No. 331/2019 are based on the concern to ensure higher food quality, aiming at protecting consumer health. To better group the categories and specify in more detail the microorganisms to be analyzed, some categories were created or included in RDC.
In addition to the changes above-mentioned, some categories were also changed, such   From the concept of "sick", considering the above-mentioned studies, it is understood that patients are in a weakened state, with low immunity, which justifies the greater rigor in the microbiological standards established in RDC No. 331, of December 23, 2019.

Final Considerations
Based on what was presented here, it was clear that RDC No. 331/2019 provides more security to consumers, as it was intended for food ready for consumers, demanding greater rigor in the production of food, especially those easily accessed by the population, with high nutritional content and intended for risk groups, such as infants and newborns. Thus, from the analysis performed here, we can conclude that ANVISA has new regulatory microbiological standards for food, with the expansion of regulatory convergence based on the main international guidelines dealing with such issue, correction of previous inconsistencies, and expansion and inclusion of new food categories, in order ensure the population's safe access to food products, until their expiration date.