Abstract

Good manufacturing practices in production facilities are necessary to control microbiological growth and prevent foodborne illnesses. Among the various foods that can be contaminated by intensive handling, meat products such as fresh sausages stand out. With this in mind, the aim of this study was to establish a microbiological profile of the fresh sausages produced in the municipality of Alegrete-RS, Brazil, as part of the SIM/SISBI inspection between 2014 and 2016 and to promote and evaluate the producing establishments in terms of compliance with good manufacturing practices. Samples were regularly taken from 10 businesses in the city and tested for thermotolerant coliform bacteria, sulphitereducing Clostridium, coagulase-positive Staphylococcus and Salmonella spp. In addition, the adequacy of good manufacturing practices was assessed and measures in the form of questionnaires, guidelines and training to improve the production chain were implemented in these companies. Over the years, significant improvements in compliance with good manufacturing practices have been observed in the production units. The microbial count of thermotolerant coliforms, sulphite-reducing Clostridium, coagulase-positive Staphylococcus and Salmonella spp. decreased significantly, indicating that the interventions contributed to improving the microbiological profile of the fresh sausage produced in the community.

Keywords:
Salmonella spp; sulphite-reducing Clostridium; coagulase-positive Staphylococcus; thermotolerant coliform bacteria; contamination

Resumo

Boas Práticas de Fabricação nas agroindústrias são necessárias para controlar a multiplicação de microrganismos e prevenir a ocorrência de doenças veiculadas por alimentos. Dentre os diversos alimentos que podem ser contaminados pela manipulação intensiva, destacam-se os produtos cárneos, como a linguiça frescal. Com base no exposto, objetivou-se avaliar o nível de contaminação de linguiças frescais produzidas no município de Alegrete (RS) no âmbito da fiscalização do SIM/SISBI entre 2014 e 2016 e correlacionar ao atendimento das Boas Práticas de Fabricação (questionários, orientações e treinamentos). Amostras regulares foram coletadas em 10 estabelecimentos da cidade e testadas para coliformes termotolerantes, Clostridium sulfito redutor, Staphylococcus coagulase positiva e Salmonella spp. A quantificação microbiana de coliformes termotolerantes, Clostridium sulfito redutor, Staphylococcus coagulase positiva e Salmonella spp. diminuiu significativamente, indicando que as intervenções contribuíram para melhorar o perfil microbiológico da linguiça frescal produzida na cidade.

Palavras-chave:
Salmonella spp; Clostridium sulfito redutor; Staphylococcus coagulase positiva; coliformes termotolerantes; contaminação

1 Introduction

The microbiological quality of food is directly related to the hygiene of the industrial environment, the raw materials, the personnel and the surface of the production line (¹1 Lehto M, Kuisma R, Määttä J, Kymäläinen H-R, Mäki M. Hygienic level and surface contamination in fresh-cut vegetable production plants. Food Control. 2011;22(3-4):469-475. http://doi.org/10.1016/j.foodcont.2010.09.029
http://doi.org/10.1016/j.foodcont.2010.0... ,²2 Lopes LC, Prestes CF, Mendes LG, De Paula M, Augusto MMM, Da Cruz WS. Boas práticas de fabricação: treinamento aplicado aos manipuladores de alimentos de restaurante universitário. Brazilian Journal of Development. 2020;6(7):49282-49289. http://doi.org.br/10.34117/bjdv6n7-540
http://doi.org.br/10.34117/bjdv6n7-540... ). If this quality is not guaranteed, food can be a carrier of pathogenic microorganisms that cause foodborne diseases (FBD), which can affect the health of consumers (³3 Melo ES, Amorim WR, Pinheiro REE, Corrêa PGN, Carvalho SMR, Santos ARSS, Barros DS, Oliveira ETAC, Mendes CA, Sousa FV. Doenças transmitidas por alimentos e principais agentes bacterianos envolvidos em surtos no Brasil: revisão. PubVet. 2018; 10:1-9. https://doi.org/10.31533/pubvet.v12n10a191.1-9
https://doi.org/10.31533/pubvet.v12n10a1... ). These diseases, which are caused by various microorganisms and their toxins following the consumption of contaminated water and/or food, lead to major public health problems as well as significant economic losses (⁴4 World Health Organization - WHO. Initiative to estimate the global burden of foodborne diseases: information and publications. Fourth formal meeting of the Foodborne Disease Burden Epidemiology Reference Group (FERG); 2014. Available from: https://apps.who.int/iris/bitstream/handle/10665/159844/9789241507950_eng.pdf
https://apps.who.int/iris/bitstream/hand... ,⁵5 Pal M, Ayele Y. Emerging Role of Foodborne Viruses in Public Health. Biomedical Research International. 2020; 5:1-4.).

In Brazil, between 2013 and 2022, the main pathogens causing FBD were Escherichia coli (32.3%), Salmonella spp (10.9%), Staphylococcus aureus (10.8%) and Bacillus cereus (6.5%). The manifestations of FBD in the body depend on the pathogen and can range from mild to severe symptoms, such as death (⁶6 Brazil. Ministry of Health. Outbreaks of Waterborne and Foodborne Diseases Report - 2023 from: < https://www.gov.br/saude/pt-br/assuntos/saude-de-a-a-z/d/dtha/publicacoes/surtos-de-doencas-de-transmissao-hidrica-ealimentar-no-brasil-informe-2023>
https://www.gov.br/saude/pt-br/assuntos/... ).

Foods that can be a source of contamination with pathogenic microorganisms include cured meat due to its intensive handling(⁷7 Pavelquesi SLS, Gomes BIBJ, Franca SR, Silva ICR, Orsi DC. Qualidade microbiológica de linguiças de frango do tipo frescal comercializadas no Distrito Federal, Brasil. Revista Brasileira de Higiene e Sanidade Animal. 2021; 15(1):1-12.). “Cured meats” are all products made from edible meat or organs that are cured, seasoned, cooked, smoked and dried and have an animal or artificial casing (⁶6 Brazil. Ministry of Health. Outbreaks of Waterborne and Foodborne Diseases Report - 2023 from: < https://www.gov.br/saude/pt-br/assuntos/saude-de-a-a-z/d/dtha/publicacoes/surtos-de-doencas-de-transmissao-hidrica-ealimentar-no-brasil-informe-2023>
https://www.gov.br/saude/pt-br/assuntos/... ). Sausage is the most produced meat product in Brazil, made from beef, pork or poultry, and may or may not be salted, aged, dried or cooked (⁸8 Carvalho CCP, Lopes Filho F, Hoffmann FL, Romanelli PF. Histórico e aspectos tecnológicos do processamento da linguiça cuiabana. Revista do Instituto Adolfo Lutz. 2010; 69(3):428-433.). Among the types of sausage, fresh sausage stands out due to its great acceptance among consumers (⁸8 Carvalho CCP, Lopes Filho F, Hoffmann FL, Romanelli PF. Histórico e aspectos tecnológicos do processamento da linguiça cuiabana. Revista do Instituto Adolfo Lutz. 2010; 69(3):428-433.

9 Araújo HL, Veiga SMOM, Silva D, Boas AFV, Silva MLR. Physical, chemical and microbiological characteristics of sausage processed with flour from barley malt bagasse. Research, Society and Development. 2021; 10(3):e22610312069. http://10.33448/rsd-v10i3.12069
http://10.33448/rsd-v10i3.12069... -¹⁰10 Corrêa LMM, Pereira JG, Pinto JPAN, Barcellos VC, Bersot LS. Behavior of Staphylococcus aureus and autochthone microbiota in fresh sausages added of sodium nitrite and stored under refrigeration. Ciência Rural. 2014; 44(10):1880-1885.).

There is a risk of microbiological contamination in fresh sausages, as they are subjected to high stress during processing and are not heat-treated. Since production requires several operations, the possibility of contamination with different types of pathogenic or harmful microorganisms increases, which may affect the quality and safety of the product (⁷7 Pavelquesi SLS, Gomes BIBJ, Franca SR, Silva ICR, Orsi DC. Qualidade microbiológica de linguiças de frango do tipo frescal comercializadas no Distrito Federal, Brasil. Revista Brasileira de Higiene e Sanidade Animal. 2021; 15(1):1-12.,⁸8 Carvalho CCP, Lopes Filho F, Hoffmann FL, Romanelli PF. Histórico e aspectos tecnológicos do processamento da linguiça cuiabana. Revista do Instituto Adolfo Lutz. 2010; 69(3):428-433.,¹¹11 Marques SC, Boari CA, Brcko CC, Nascimento AR, Piccol RH. Avaliação higiênico-sanitária de linguiças tipo frescal comercializadas nos municípios de Três Corações e Lavras-MG. Ciência Agrotécnica. 2006; 30(6):1120-1123.).

In this context, the support, guidance, control and inspection of establishments processing cured meat, such as fresh sausages, by public bodies is essential to promote improvements in the production chain and, above all, to ensure the safety of consumers of this type of product.

Based on the above, the main objective of this study was to establish a microbiological profile for the production of fresh cured meats in this municipality which were under the control of the Municipal Inspection Service for Animal Products during the 2014-2016 period ⸻ to promote and evaluate the manufacturing establishments in terms of their compliance with good manufacturing practices.

2 Materials and Methods

2.1 Selection of facilities

The present study was carried out in the municipality of Alegrete, Rio Grande do Sul, Brazil, in collaboration with the Secretariat of Agriculture and Livestock of the municipality; specifically, the Municipal Inspection Service for Animal Products (SIM-POA) and registered units of this service, which currently comprises 38 establishments. Of these registered production units, ten were randomly selected (defined by lot) to be evaluated.

After the selection of the establishments, data was collected through surveys in which the adequacy of good manufacturing practices was assessed using questionnaires with checklists (supplementary material) and microbiological analyses of fresh sausage samples between 2014 and 2016.

2.2 Inspections of manufacturing establishments

The establishments were assessed during twice-monthly inspections, using a questionnaire in the form of a checklist based on good manufacturing practices. The checklist used included the assessment of 18 points relating to raw materials, production, packaging, storage, transportation, cleaning and hygiene of the establishment and employees. After applying the questionnaires, the data obtained was converted into a percentage of adequacy and classified as excellent (100% to 90%), very good (89% to 80%), good (79% to 70%), regular (69% to 60%) or bad (less than 59%). For each inspection, measures were implemented in the form of instructions for the employees involved in the process.

2.3 Microbiological analyses

Samples of fresh sausages were taken once a month in the production and storage rooms of the establishments. The samples were taken according to a predetermined scheme in which a sausage was selected at random and a portion of it was cut up with previously sterilized knives. At the time of sampling, the samples had a temperature between 0 and 7°C. After collection, the samples were sterile-packed, stored at a temperature of 5 to 7 °C in an isothermal container with ice and sent to the laboratory for analysis.

On arrival at the laboratory, the samples were separated from their origin and coded from “1” to “10” depending on the production unit and analysed for the presence of Salmonella spp, quantification of thermotolerant coliform bacteria, coagulase-positive Staphylococcus and sulphite-reducing Clostridium.

The analysis of Salmonella spp. (presence or absence) was performed according to method ISO 6579:2002 (12). The count of thermotolerant coliform bacteria was performed according to the AFNOR method, Certificate no. 3M 01/02-09/89 (13). Coagulase-positive Staphylococcus were counted according to the method ISO 6888-1:1999(¹⁴14 International Organization and Standarization - ISO 6888-1. Microbiology of Food and Animal Feeding Stuffs. Horizontal of Method for the Enumeration of Coagulase Positive Staphylococcus (Staphylococcus aureus and Other species). Technique Using Baird-Parker Agar Medium, International Organization Standarization, Geneva, 1999.). Finally, the number of sulphite-reducing Clostridium was determined according to method ISO 15213:2003(¹⁵15 International Organization and Standarization - ISO 15213:2003. Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of sulfitereducing bacteria growing under anaerobic conditions, 2003.).

2.4 Statistical analysis

Differences between quarters of the years and between the establishments in terms of the presence of Salmonella in the samples and the number of other microorganisms were checked by subjecting them to an analysis of variance (ANOVA, one-way ANOVA) followed by a Fisher’s Least Significant Difference test (LSD test, p < 0.05).

3 Results and Discussion

3.1 Adequacy of good manufacturing practices in the establishments

Significant improvements in the adequacy of good manufacturing practices have been observed in all manufacturing plants over the years (Table 1), possibly due to the measures implemented through training. This result confirms what was reported in the study by Lopes et al. (²2 Lopes LC, Prestes CF, Mendes LG, De Paula M, Augusto MMM, Da Cruz WS. Boas práticas de fabricação: treinamento aplicado aos manipuladores de alimentos de restaurante universitário. Brazilian Journal of Development. 2020;6(7):49282-49289. http://doi.org.br/10.34117/bjdv6n7-540
http://doi.org.br/10.34117/bjdv6n7-540... ), who found an improvement of more than 60% in the good manufacturing practice index after the implementation of training for food artisans. According to Vidal-Martins et al. (¹⁶16 Vidal-Martins AMC, Bürger KP, Aguilar CEG, Gonçalves ACS, Grisólio APR, Rossi GAM. Implantação e avaliação do programa de boas práticas de manipulação em açougues do Município de São José do Rio Preto - SP. Revista Brasileira de Higiene e Sanidade Animal. 2014; 8(2): 73-86. http://dx.doi.org/10.5935/19812965.20140022
http://dx.doi.org/10.5935/19812965.20140... ), implementing good manufacturing practices in sausage factories is difficult because monitoring is not strict enough. Therefore, the training of workers in these factories was ultimately inadequate. This result is consistent with the findings of the present study, as there was not only strict supervision, but also guidance for those interested.

Other possible factors that led to the acquisition of this data were the investments made in the inspection service during this period, with the purchase of consumables and works that allowed for better training of staff and effective inspection measures. It should be noted that the improvement observed from 2014 to 2016 results in greater public health benefits for consumers who have access to a product with greater food safety.

3.2 Microbiological control of fresh mixed sausages

Improvements were also observed in the microbiological analyses, which may be related to the training of the staff and the commitment of the entire team to the application of good manufacturing practices. These results confirm the findings of Senter et al.(¹⁷17 Senter L, Rossi EM, Sardiglia CU. Avaliação da qualidade microbiológica de salames artesanais e implantação de Boas Práticas de Fabricação em uma mini-indústria. Revista Higiene Alimentar, 2010; 24:186-187.), who found a decrease in contamination of meat products after staff training. When evaluating the number of sulphite-reducing Clostridium (Table 2), a decrease in contamination of fresh mixed sausages from the city of Alegrete was found when comparing 2014 and 2016. However, there were no significant differences (p < 0.05) in the bacterial counts between the quarters and between the 10 farms studied.

In a study conducted with tested sausages, Montovani et al. (¹⁸18 Mantovani D, Corazza ML, Cardozo Filho L, Costa SC. Avaliação higiênico-sanitária de linguiças tipo frescal após inspeção sanitária realizada por órgãos federal, estadual e municipal na região noroeste do Paraná. Revista Saúde e Pesquisa. 2011; 4:357-362.) found levels of sulphite-reducing Clostridium reducer within the parameters of the legislation that we can correlate with the data found. These results can be associated with the following factors: Compliance with control standards and corrective actions when deviations are found in the checklist.

The current Brazilian legislation (Normative Instruction - IN No. 161, of July 1, 2022, of the National Health Surveillance Agency - ANVISA, of the Ministry of Health - MS of Brazil) does not establish parameters for Clostridium perfringens in fresh sausages; however, for products made from cooked meat, whether cured or not, smoked or not, dried or not, sausages or not, refrigerated or not (mortadella, sausage, ham, black pudding, pâtés, galantines) a maximum value of 10³ (3 log CFU g^-1) for Clostridium perfringens is defined. Looking at the latter parameters, only four samples showed values above the maximum permitted level.

Contamination of food by Salmonella spp. is one of the main origins of illness worldwide, caused by contaminated food, which is detrimental to public health. According to Brazilian law, the presence of Salmonella spp. in fresh sausages is not allowed (⁶6 Brazil. Ministry of Health. Outbreaks of Waterborne and Foodborne Diseases Report - 2023 from: < https://www.gov.br/saude/pt-br/assuntos/saude-de-a-a-z/d/dtha/publicacoes/surtos-de-doencas-de-transmissao-hidrica-ealimentar-no-brasil-informe-2023>
https://www.gov.br/saude/pt-br/assuntos/... ). Nevertheless, the presence of Salmonella spp. was detected in some of the samples analysed (Table 3). This result is consistent with the results of the study by Pavelquesi et al. (⁷7 Pavelquesi SLS, Gomes BIBJ, Franca SR, Silva ICR, Orsi DC. Qualidade microbiológica de linguiças de frango do tipo frescal comercializadas no Distrito Federal, Brasil. Revista Brasileira de Higiene e Sanidade Animal. 2021; 15(1):1-12.), who found the presence of the microorganism in 25% of the samples of fresh chicken sausages analysed in the Federal District, and with the study by Cabral et al. (¹⁹19 Cabral CC, Conte-Junior CA, Silva JT, Paschoalin VMF. Salmonella spp. contamination in fresh pork and chicken sausages marketed in Niterói and Rio de Janeiro, Brazil. Journal Für Verbraucherschutz Und Lebensmittelsicherheit. 2014; 9(3):243-249.), who found the presence of Salmonella spp. in 26% of the fresh chicken and pork sausages analysed.

When comparing the samples regarding the presence or absence of Salmonella spp. in the quarters and years, there were no significant differences, showing that contamination was not related to the period studied, but there was a significant difference (p < 0.05) between the production sites. As can be seen in Table 3, production site “1” was statistically different from the others, with the exception of production sites “5” and “7”.

This contamination could be related to the fact that these production facilities are less in line with good practices, necessitating more effective work to improve sanitary and hygienic conditions. Souza et al. (²⁰20 Souza M, Pinto FGS, Bona EAM, Moura AC. Qualidade higiênicosanitária e prevalência de sorovares de Salmonella em linguiças frescais produzidas artesanalmente e inspecionadas, comercializadas no oeste do Paraná, Brasil. Arquivos do Instituto Biológico. 2014;81 (2), 107-112.) found in their study that 30% of fresh sausage samples were contaminated with Salmonella spp, while Alberti and Nava (²¹21 Alberti J, Nava A. Avaliação higiênicossanitária de linguiças tipo frescal comercializadas a granel por supermercados e produzidas artesanalmente no município de Xaxim, SC. Unoesc & Ciência. 2014; 5(1):41-48.) found 67%. These authors cited the production of contaminated raw materials and/or industrialization without hygienic care as possible causes of contamination. These could also be the causes of the presence of Salmonella spp. in the samples of this study, in which more effective control is required at production establishments “1”, “5” and “7”.

The third quarter includes the cold months (July, August), which could have been one of the reasons for this difference. Among the production establishments, all samples had thermotolerant coliforms, but production establishments”3”, “9” and “10” had the lowest values and were statistically different (p < 0.05) from the others, with lower values, which can be verified in Table 4.

Thermotolerant coliforms are indicators of the hygienic and sanitary conditions of food (²²22 Damer JRS, Dill RE, Gusmão AA, Moresco TR. Contaminação de Carne Bovina Moída por Escherichia coli e Salmonella sp. Contexto Saúde. 2014; 14(26):20-27.). In this study, when comparing the quarters of the period from 2014 to 2016 (Table 4), a significant difference (p < 0.05) was found between the 3rd quarter and the other quarters, with the lowest values for these microorganisms in the third quarter.

According to Sirtoli and Comarella(²³23 Sirtoli DB, Comarella L. O papel da vigilância sanitária na prevenção das doenças transmitidas por alimentos (DTA). Revista Saúde e Desenvolvimento. 2018; 12:197-209.), food contamination is closely related to poor hand hygiene, inadequate temperatures, cross-contamination and recontamination. Among these factors, in the present study, we may add the temperature of the production areas, because in establishments “3”, “9” and “10” the ambient temperature in the production areas remained stable at values below or equal to 10 °C due to industrial cooling systems. This also confirms the data in Table 4, which show the influence of temperature, where contamination by thermotolerant coliforms decreased in the coldest quarters.

Table 5 shows the results on the number of coagulase-positive Staphylococcus.

In production establishments “5”, “7” and “8”, the number of coagulase-positive Staphylococcus differed significantly (p < 0.05) from the others and was lower than in production establishments “1”, “3” and “4”. Considering that operators are the main source of coagulase-positive Staphylococcus, factors such as the high turnover of employees and the lack of continuous training of operators in production establishments “1”, “3” and “4” could be related to these results.

Souza et al. (²⁰20 Souza M, Pinto FGS, Bona EAM, Moura AC. Qualidade higiênicosanitária e prevalência de sorovares de Salmonella em linguiças frescais produzidas artesanalmente e inspecionadas, comercializadas no oeste do Paraná, Brasil. Arquivos do Instituto Biológico. 2014;81 (2), 107-112.), in a study of controlled and artisanal fresh sausages, demonstrated the presence of coagulase-positive Staphylococcus in the samples analysed. The authors found greater contamination in artisanal sausages and associated this with excessive manipulation and lack of hygienic controls. In the present research, samples of controlled fresh sausages were analysed. It was found that the number of coagulase-positive Staphylococcus was higher in some production establishments (Table 5) than in the others, yet within the thresholds established by Brazilian legislation.

4 Conclusion

The results show that training in the agribusiness for meat products, especially fresh sausage in Alegrete, RS, Brazil, between 2014 and 2016 brought great improvements in terms of good manufacturing practices. It was found that the contamination with sulphite-reducing Clostridium decreased. The current Brazilian legislation does not establish parameters for Clostridium perfringens in fresh sausage, but for other products, a maximum count of 10³ (3 log CFU g^-1) is allowed. If we consider these last parameters, only four samples have counts above the maximum allowed. However, higher levels of coagulase-positive Staphylococcus and, especially, Salmonella spp. were detected in some production establishments in relation to others, requiring stronger intervention, retraining of staff and more effective control at all production stages. Lower levels of thermotolerant coliforms were found in cold rooms and in production establishments with effective temperature control, demonstrating the importance of adapting refrigeration to the production environment.

References

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History