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Pseudomonas Spp. and Other Psychrotrophic Pesq. Vet. Bras. 39(10):807-815, October 2019 DOI: 10.1590/1678-5150-PVB-6037 Original Article Livestock Diseases ISSN 0100-736X (Print) ISSN 1678-5150 (Online) PVB-6037 LD Pseudomonas spp. and other psychrotrophic microorganisms in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic and AprX production potential1 Pedro I. Teider Junior2, José C. Ribeiro Júnior3* , Eric H. Ossugui2, Ronaldo Tamanini2, Juliane Ribeiro2, Gislaine A. Santos2 2 and Vanerli Beloti2 , Amauri A. Alfieri ABSTRACT.- Teider Junior P.I., Ribeiro Júnior J.C., Ossugui E.H., Tamanini R., Ribeiro J., Santos G.A., Pseudomonas spp. and other psychrotrophic microorganisms Pseudomonas spp. and other psychrotrophic in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic andAlfieri AprX A.A. production& Beloti V. 2019. potential . Pesquisa Veterinária Brasileira 39(10):807-815. Instituto microorganisms in inspected and non- Nacional de Ciência e Tecnologia para a Cadeia Produtiva do Leite, Universidade Estadual de inspected Brazilian Minas Frescal cheese: Londrina, Rodovia Celso Garcia Cid PR-445 Km 380, Cx. Postal 10.011, Campus Universitário, proteolytic, lipolytic and AprX production Londrina, PR 86057-970, Brazil. E-mail: [email protected] The most consumed cheese in Brazil, Minas Frescal cheese (MFC) is highly susceptible to potential microbial contamination and clandestine production and commercialization can pose a risk to consumer health. The storage of this fresh product under refrigeration, although more appropriate, may favor the growth of spoilage psychrotrophic bacteria. The objective of this [Pseudomonas spp. e outros micro-organismos study was to quantify and compare Pseudomonas spp. and other psychrotrophic bacteria in inspected and non-inspected MFC samples, evaluate their lipolytic and proteolytic activities and e não inspecionados: potencial proteolítico, lipolítico e their metalloprotease production potentials. Twenty MFC samples were evaluated: 10 inspected psicrotróficos em queijos Minas Frescalprodução inspecionados de AprX.]. and 10 non-inspected. Counts of psychrotrophic bacteria and Pseudomonas spp., evaluation of Teider Junior P.I., Ribeiro Júnior J.C., Ossugui E.H., of alkaline metalloprotease (AprX) were assessed. The mean total psychrotrophic counts thewere proteolytic 1.07 (±2.18) and ×lipolytic 109CFU/g potential in the of inspected the isolates, samples and identification and 4.5 (±5.86) of potential × 108CFU/g producers in the V. 807- spp. 815 Pseudomonas Tamanini R., Ribeiro J., Santos G.A., Alfieri A.A. & Beloti was 6.86 (±18.6) × 105 and 2.08 (±3.65) × 106 CFU/g for the inspected and non-inspected MFC non-inspected, with no significant difference (p=0.37). The averagePseudomonas score of spp. represented 0.06% and 0.004% of psychrotrophic bacteria found in inspected and non-inspected MFC samples, respectively,respectively. with Collectively, no significant 694 psychrotrophic difference (p=0.1). strains and 47 Pseudomonas spp. were isolated, of which 59.9% and 68.1% were simultaneously proteolytic and lipolytic, respectively. Of the 470 psychrotrophs isolated from inspected and 224 from non-inspected cheese samples, 5.74% and 2.23% contained aprX, respectively, while 100 and 86.96% of the Pseudomonas spp. isolates in inspected and non-inspected cheese samples contained the gene. The production potential of AprX did not, however, determine the proteolytic activity on plates of these isolates under the conditions evaluated in this study. Of total, 65.63% of the psychrotrophs that contained aprX Pseudomonas spp., using gene were confirmedSerratia as Raoultella ornithinolyticagenus-specific PCR. PhylogeneticAcinetobacter analysis of schindleri the 16S rRNA gene of the other psychrotrophs Psychrobacterthat were potential sanguinis producers of AprXLeuconostoc identified mesenteroides them as spp. (n=7), (n=1), and (n=1) in the inspected samples and 1 Received on February 18, 2019. (n=1) and (n=1) in the non-inspected Accepted for publication on April 1, 2019. 2 Instituto Nacional de Ciência e Tecnologia para a Cadeia Produtiva do Leite (INCT-Cadeia do Leite), Universidade Estadual de Londrina (UEL), Rodovia 3 Escola de Medicina Veterinária e Zootecnia, Universidade Federal do Celso Garcia Cid PR-445 Km 380, Cx. Postal 10.011, Campus Universitário, Tocantins (UFT), BR-153 Km 112, Zona Rural, Araguaína, TO 77804-970, Londrina, PR 86057-970, Brazil. Brazil. *Corresponding author: [email protected] 807 808 Pedro I. Teider Junior et al. samples. The production conditions of Brazilian MFC of these samples, while meeting the Pseudomonas and other spoilage-related psychrotrophs. Thus, stricter hygienic measures are required during the formal production legalof this determinations, type of cheese. are not sufficient to control INDEX TERMS: Pseudomonas spp., psychrotrophic, microorganisms, Minas Frescal cheese, cheese, AprX production, alkaline metalloprotease, proteolysis, lipolysis. RESUMO.- [Pseudomonas spp. e outros micro-organismos TERMOS DE INDEXAÇÃO: Pseudomonas spp., micro-organismos, psicrotróficos em queijos Minas Frescal inspecionados e não inspecionados: potencial proteolítico, lipolítico e produção alcalina, proteólise, lipólise. de AprX.] O mais consumido no Brasil, o queijo Minas Frescal psicrotróficos, queijo, Minas Frescal, produção de AprX, metaloprotease (QMF) é altamente suscetível à contaminação microbiana e a produção e comercialização clandestina podem representar INTRODUCTION um risco para a saúde do consumidor. O armazenamento deste produto fresco sob refrigeração, embora mais apropriado, product obtained by the enzymatic coagulation of milk with Byrennet definition, and/or Brazilian other suitable Minas Frescal coagulating cheese (MFC)enzymes, is a freshwith compararpode favorecer Pseudomonas a multiplicação de bactérias psicrotróficas powdered milk, cream, milk solids, sodium chloride, and emdeteriorantes. amostras de O QMF objetivo inspecionadas deste estudo e não foi inspecionadas, quantificar e or without supplementation with specific lactic bacteria, avaliar o potencial lipolítico, spp. e proteolíticooutras bactérias e de psicrotróficas produção de (25 to 44.9% fat) cheese with very high moisture content metaloprotease alcalina. Vinte amostras de QMF foram avaliadas: calcium(not less chloride than 55.0%) (Brasil (Brasil 1997). 2004). It is classifiedThe production as a semi-fat of this 10 inspecionadas e 10 não inspecionadas. Foram avaliadas as type of cheese in Brazil is carried out formally by dairies that Pseudomonas spp., o follow the legal stipulations of the “Ministério da Agricultura, Pecuária e Abastecimento” (Brasil 1997) and mainly small milk decontagens potenciais de produtoresbactérias psicrotróficas de metaloprotease e alcalina (AprX). producers, with the objective of adding value and increases potencial proteolítico e lipolítico dos isolados e a identificação the shelf life of milk through the preparation of derivatives. de 1,07 (±2,18) × 109UFC/g nas amostras inspecionadas e The MFC is quite susceptible to microbial contamination 4,5A média (±5,86) total × 10 das8UFC/g contagens nas não de inspecionadas, bactérias psicrotróficas sem diferença foi when prepared using raw milk, or during or after processing (Carvalho et al. 2007). Moreover, it can be a source of food Pseudomonas spp. foi de pathogens (Campos et al. 2017). 6,86 (±18,6) × 105 e 2,08 (±3,65) × 106UFC/g para as amostras In addition to pathogenic microorganisms, proteolytic QMFsignificativa inspecionadas (p=0,37). e não A- inspecionadas,média de respectivamente, sem and/or lipolytic microorganisms, mainly psychrotrophs, may spp. representaram Pseudomonas interfere with the quality of the MFC, since as it is a fresh product, refrigeration is necessary for its storage. When diferença significativa (p=0,1). em amostras QMF inspecionadas e não-inspecionadas, present in the raw material, psychrotrophic bacteria reduce respectivamente.0,06% e 0,004% de Das bactérias amostras psicrotróficas inspecionadas encontradas e não the cheese improper for consumption (Murphy et al. 2016). e 47 Pseudomonas spp., dos quais 59,9% e 68,1% foram the Asindustrial the degradation yield, flavor, of and proteins aroma, resultsbeing able in non-acidto render simultaneamenteinspecionadas, foram proteolíticos isoladas e 694 lipolíticos, colônias respectivamente. psicrotróficas metabolites, protease activity confers a bitter taste and putrid inspecionados e dos 224 isolados das não inspecionadas, 5,74% eDos 2,23% 470 continhamisolados de o psicrotróficosgene aprX, respectivamente, das amostras enquantode queijo smellcoagulation, to the cheese. and greater Undesirable loss of effects casein are fragments also influenced in the 100 e 86,96% das Pseudomonas spp. isoladas em amostras byserum, the reductionrequiring 20-30of the percentintegrity more of milk milk proteins, per kilo ofdeficient cheese de queijo inspecionadas e não inspecionadas continham o potencial de expressão de AprX. Esse potencial, no entanto, não Alkaline metalloprotease (AprX), which is heat-resistant, determinou a atividade proteolítica em placas desses isolados (Samaržijais considered et theal. 2012). main microbial protease, and is encoded by nas condições avaliadas neste estudo. Do total, 65,63% dos aprX. This gene is found in various proteolytic bacteria, such aprX as Pseudomonas spp. and Serratia spp. (Dufour et al. 2008, como Pseudomonas Marchand et al. 2009, Bagliniere et al. 2013). This enzyme is psicrotróficos
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