Identification of lactic acid bacteria with anti-listeria activity. Characterization and application of a bacteriocinogenic strain in the control of Listeria monocytogenes in cheese.

The purpose of the research paper was firstly to identify bacteriocin-producing lactic acid bacteria characterizing strains with anti-listeria activity and, secondly, to characterize bacteriocin evaluating its in vitro efficiency as a natural preservative and, thirdly, to evaluate the anti-listeria effect of the bacteriocinogenic strain of Lactiplantibacillus plantarum in cheeses and produce an edible film with anti-listerial effect. Of 355 lactic acid bacteria strains tested, two were able to produce bacteriocin against Listeria monocytogenes and were identified as Lactiplantibacillus plantarum and Lactiplantibacillus pentosus. A bactericidal effect of strain QS494 (Lactiplantibacillus plantarum) was observed in the first 8 h, with a reduction of 1.7 log, using cell-free supernatant with Listeria monocytogenes, where viable cells were counted on listeria selective agar. Both strains showed good technological characteristics and were without production of virulence factors. Changes in the pH of the cell-free supernatant obtained from Lactiplantibacillus plantarum did not affect its antimicrobial activity, which remained stable after heat treatments for up to 15 min at 121°C. Inhibitory activity was also observed after 12 weeks of storage at -20°C. In the evaluation of the anti-listeria effect in cheeses, a 3 log reduction in the Listeria monocytogenes count was observed in 120 h in cheeses produced with bacteriocinogenic lactic acid bacteria, while in cheeses produced with non-bacteriocinogenic culture, we observed a 2 log increase in the count. Edible films produced with the addition of precipitate from the cell free supernatant showed an antimicrobial effect against Listeria monocytogenes. Thus, the two strains studied have technological and biosafety potential.

Pseudomonas spp. and other psychrotrophic microorganisms in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic and AprX production potential / Pseudomonas spp. e outros micro-organismos psicrotróficos em queijos Minas Frescal inspecionados e não inspecionados: potencial proteolítico, lipolítico e produção de AprX

The most consumed cheese in Brazil, Minas Frescal cheese (MFC) is highly susceptible to 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 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 their metalloprotease production potentials. Twenty MFC samples were evaluated: 10 inspected and 10 non-inspected. Counts of psychrotrophic bacteria and Pseudomonas spp., evaluation of the proteolytic and lipolytic potential of the isolates, and identification of potential producers of alkaline metalloprotease (AprX) were assessed. The mean total psychrotrophic counts were 1.07 (±2.18) × 109CFU/g in the inspected samples and 4.5 (±5.86) × 108CFU/g in the non-inspected, with no significant difference (p=0.37). The average score of Pseudomonas spp. was 6.86 (±18.6) × 105 and 2.08 (±3.65) × 106 CFU/g for the inspected and non-inspected MFC samples, respectively, with no significant difference (p=0.1). Pseudomonas spp. represented 0.06% and 0.004% of psychrotrophic bacteria found in inspected and non-inspected MFC samples, respectively. Collectively, 694 psychrotrophic strains and 47Pseudomonas 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 gene were confirmed as Pseudomonas spp., using genus-specific PCR. Phylogenetic analysis of the 16S rRNA gene of the other psychrotrophs that were potential producers of AprX identified them as Serratia spp. (n=7), Raoultella ornithinolytica (n=1), and Acinetobacter schindleri (n=1) in the inspected samples and Psychrobacter sanguinis (n=1) and Leuconostoc mesenteroides (n=1) in the non-inspected samples. The production conditions of Brazilian MFC of these samples, while meeting the legal determinations, are not sufficient to control Pseudomonas and other spoilage-related psychrotrophs. Thus, stricter hygienic measures are required during the formal production of this type of cheese.(AU)

O mais consumido no Brasil, o queijo Minas Frescal (QMF) é altamente suscetível à contaminação microbiana e a produção e comercialização clandestina podem representar um risco para a saúde do consumidor. O armazenamento deste produto fresco sob refrigeração, embora mais apropriado, pode favorecer a multiplicação de bactérias psicrotróficas deteriorantes. O objetivo deste estudo foi quantificar e comparar Pseudomonas spp. e outras bactérias psicrotróficas em amostras de QMF inspecionadas e não inspecionadas, avaliar o potencial lipolítico, proteolítico e de produção de metaloprotease alcalina. Vinte amostras de QMF foram avaliadas: 10 inspecionadas e 10 não inspecionadas. Foram avaliadas as contagens de bactérias psicrotróficas e Pseudomonas spp., o potencial proteolítico e lipolítico dos isolados e a identificação de potenciais produtores de metaloprotease alcalina (AprX). A média total das contagens de bactérias psicrotróficas foi de 1,07 (±2,18) × 109UFC/g nas amostras inspecionadas e 4,5 (±5,86) × 108UFC/g nas não inspecionadas, sem diferença significativa (p=0,37). A média de Pseudomonasspp. foi de 6,86 (±18,6) × 105 e 2,08 (±3,65) × 106UFC/g para as amostras QMF inspecionadas e não-inspecionadas, respectivamente, sem diferença significativa (p=0,1). Pseudomonas spp. representaram 0,06% e 0,004% de bactérias psicrotróficas encontradas em amostras QMF inspecionadas e não-inspecionadas, respectivamente. Das amostras inspecionadas e não inspecionadas, foram isoladas 694 colônias psicrotróficas e 47 Pseudomonasspp., dos quais 59,9% e 68,1% foram simultaneamente proteolíticos e lipolíticos, respectivamente. Dos 470 isolados de psicrotróficos das amostras de queijo inspecionados e dos 224 isolados das não inspecionadas, 5,74% e 2,23% continham o gene aprX, respectivamente, enquanto 100 e 86,96% das Pseudomonasspp. isoladas em amostras de queijo inspecionadas e não inspecionadas continham o potencial de expressão de AprX. Esse potencial, no entanto, não determinou a atividade proteolítica em placas desses isolados nas condições avaliadas neste estudo. Do total, 65,63% dos psicrotróficos que continham o gene aprX foram confirmados como Pseudomonasspp., utilizando PCR gênero-específico. A análise filogenética do gene 16S rRNA dos outros psicrotróficos que foram produtores potenciais de AprX os identificou como Serratia spp. (n=7), Raoultella ornithinolytica (n=1) e Acinetobacter schindleri (n=1) nas amostras inspecionadas e Psychrobacter sanguinis (n=1) e Leuconostoc mesenteroides (n=1) nas amostras não inspecionadas. As condições de produção do QMF dessas amostras, atendendo às determinações legais, não são suficientes para controlar a Pseudomonas e outros psicrotróficos relacionados à deterioração. Assim, medidas higiênicas mais rígidas são necessárias durante a produção formal deste tipo de queijo.(AU)

X-ray diffraction has limited applicability in investigation of milk tampering.

The aim of this work was to use X-ray diffraction to identify substances used for adulteration of raw milk and to determine if crystallographic analysis can detect extraneous substances in milk. Two unknown substances were sent anonymously by employers linked to the dairy chain, who claimed that they were added directly in milk prior to water addition by truck drivers. The samples were analyzed by X-ray diffraction and submitted to physicochemical analysis. The first substance was identified by X-ray diffraction as sodium citrate, complying with its physicochemical attributes, such as the powerful ability to decrease the freezing point. The second substance was identified by X-ray diffraction as sucrose and this result was also in agreement with its ability to increase the density, decrease the freezing point and finally, to be positive for sucrose in the resorcinol qualitative test. To evaluate if X-ray diffraction can detect extraneous substances already mixed in milk, fresh raw milk samples tampered with urea, sodium hydroxide, sodium citrate and sucrose were freeze dried and analyzed by X-ray diffraction, with no detection of any extraneous substances at any percentage. This is the first report of attempted diagnosis of extraneous substances in milk by X-ray diffraction. However, this technique can be useful only when applied to identify substances used for adulteration prior to its dilution in milk, since the amorphous nature of milk seems to be a limitation for the accurate detection of extraneous substances.

Short communication: Effect of bactofugation of raw milk on counts and microbial diversity of psychrotrophs.

Bactofugation is a centrifugal process for removing spores of microorganisms from milk, especially when it is destined for cheese making. Other microorganisms may be removed in bactofugation. This study aimed to verify the effect of milk bactofugation on the counts and microbial diversity of psychrotrophs. The raw milk was preheated (≈55°C) before being bactofuged, and samples were collected from 3 batches of milk: refrigerated raw, preheated, and bactofuged, representing the immediate conditions before and after bactofugation. The mean psychrotrophic counts of the 3 batches were 3.08 (±1.69) × 106, 193 (±232), and 20 (±26) cfu/mL, respectively. Preheating was sufficient to eliminate 99.99% of the raw milk psychrotrophs, but bactofugation further reduced 89.66% of psychrotrophs from preheated milk. Lysinibacillus fusiformis was the most frequently isolated species (45.7%) among the psychrotrophs of raw milk and, proportionally, were more frequent in preheated (37.5%) and bactofuged (60%) milk. Bacillus invictae (20%), Enterococcus faecalis (10%), and Kurthia gibsonii (10%) were also isolated from bactofuged milk. Albeit in small numbers, psychrotrophic, thermoduric, and spore-forming bacteria with known proteolytic and lipolytic activity remained in the milk after bactofugation, which apparently had no effect on a specific population of microorganisms but proportionally reduced the entire psychrotrophic microbiota of raw milk.

Efeito da bactofugação nas contagens e diversidade microbiana de psicrotróficos do leite cru / Effect of bactofugation of raw milk on counts and microbial diversity of psychrotrophs

Esse estudo teve por objetivo verificar o efeito da bactofugação do leite na quantidade e diversidade de psicrotróficos. Como o leite cru era pré-aquecido (≈55ºC) para ser bactofugado, foram coletadas amostras de três lotes de leite cru refrigerado, pré-aquecido e bactofugado a 10.000g. O pré-aquecimento foi suficiente para eliminação de 99,99% dos psicrotróficos do leite cru. A bactofugação reduziu 89,66% das contagens de psicrotróficos do leite pré-aquecido. Lysinibacillus fusiformis predominou (45, 7%) no leite cru, pré -aquecido (37,5%) e bactofugado (60%). Bacillus invictae (20%), Enterococcus faecalis (10%) e Kurthia gibsonii (10%) também foram remanescentes à bactofugação, que aparentemente não tem efeito sobre uma população específica de micro-organismos, mas reduz proporcionalmente toda a microbiota psicrotrófica do leite cru.

Resistência antimicrobiana e caracterização molecular do grupo filogenético de STEC e EPEC típicas e atípicas isoladas de queijos Minas Frescal clandestinos / Antimicrobial resistance and molecular characterization of the phylogenetic group of STEC and EPEC typical and atypical isolated from clandestines Minas Frescal cheeses

Esse estudo avaliou a resistência antimicrobiana e o grupo filogenético de Escherichia coli enteropatogênicas (EPEC) e produtoras de toxina shiga-like (STEC) em 10 amostras de queijos Minas Frescal clandestinos. A média da contagem de E. coli foi de 1,1 x 105 UFC/g. Duas (1,8%) das 111 cepas foram identificadas como EPEC (gene eaeA) sendo uma EPEC típica (gene bfpA) e outra atípica. Outras três (2,7%) foram identificadas como STEC (gene stx2). A t-EPEC foi resistente à estreptomicina e a a-EPEC à cefoxitina e ampicilina. Uma STEC foi considerada multirresistente (ampicilina, estreptomicina e tetraciclina), outra resistente à tetraciclina e outra sensível. A presença de t-EPEC, juntamente com o predomínio de cepas do grupo filogenético A (60%), confirmam a possível origem fecal humana dos isolados de E. coli nos queijos clandestinos.

Proteolytic and lipolytic potential of Pseudomonas spp. from goat and bovine raw milk / Potencial proteolítico e lipolítico de espécies de Pseudomonas do leite cru caprino e bovino

Pseudomonas, the main genus of gram-negative microorganisms isolated from milk, is psychrotrophic, biofilm-forming, and thermo-resistant deteriorating enzyme producers. The aim of this study was to quantify Pseudomonas spp. in goat's and cow's milk produced in the Paraná state, Brazil, to evaluate the deteriorating activity of the isolates at mesophilic and psychrotrophic conditions and to identify, at the species level, the isolates with alkaline metalloprotease (aprX gene) production potential. Microbiological, biochemical and molecular methods were used for isolating, confirming and identifying of isolates. The mean counts were 1.6 (±6.3)x104 and 0.89(±3)x102 CFU/mL for goat and bovine milk samples, respectively, immediately after milking. Of the Pseudomonas colonies isolated from goat milk (n=60), 91.7% showed proteolytic potential when incubated at 35°C/48 h and 80% at 7°C/10 days, and lipolytic potential was observed in 95% of the isolates incubated in mesophilic and 78.3% at refrigeration conditions. From the isolates of bovine milk (n=20), 35% showed proteolytic activity only when incubated at 35°C/48 h, and lipolytic potential was observed in 25% of the isolates incubated at 7°C/10d and 35°C/48h. It was observed that 83.3% and 25% of the isolates genetically confirmed as Pseudomonas spp. of goat and bovine milk showed the potential for alkaline metalloprotease production, with the species P. azotoformans, P. koreensis, P. gessardii, P. monteilii and P. lurida being the most frequent in goat milk and P. aeruginosa the only species identified in cow milk.(AU)

Pseudomonas é o principal gênero de micro-organismos Gram negativos isolados do leite, são psicrotróficos, formadores de biofilmes e produtores de enzimas deteriorantes termodúricas. O objetivo do presente trabalho foi quantificar Pseudomonas spp. no leite de cabras e vacas produzido no estado do Paraná, Brasil, avaliar a atividade deteriorante em temperatura mesofílica e psicrotrófica e identificar, em nível de espécie, os isolados com potencial de produção de metaloprotease alcalina (geneaprX). Foram utilizados métodos microbiológicos, bioquímicos e moleculares para isolamento, confirmação e identificação dos isolados. As contagens médias foram de 1,6 (±6,3) x 104 e 0,9 (±3) x 102 UFC/mL para as amostras de leite caprino e bovino, respectivamente. Dos isolados de Pseudomonas do leite de cabra (n=60), 91,7% demonstraram potencial proteolítico quando incubadas a 35°C/48h e 80% a 7°C/10dias e lipolíticos em 95% dos isolados incubados em mesofilia e em 78,3% dos incubados em temperatura de refrigeração. Dos isolados do leite bovino (n=20), foi verificada atividade proteolítica de 35% apenas quando incubadas a 35°C/48h e lipolítica em 25% dos isolados incubados a 7°C/10d e 35°C/48h. Foi observado que 83,3% e 25% dos isolados confirmados geneticamente como Pseudomonas spp. do leite caprino e bovino, respectivamente, apresentaram o potencial de produção de metaloprotease alcalina, sendo as espécies P. azotoformans, P. koreensis, P. gessardii, P. monteilii e P. lurida as mais frequentes no leite de cabras e P. aeruginosa a única identificada do leite de vacas.(AU)

Selection of Leuconostoc strains isolated from artisanal Serrano Catarinense cheese for use as adjuncts in cheese manufacture.

BACKGROUND: Serrano Catarinense cheese is a raw bovine milk cheese produced in the region of Santa Catarina, Brazil. Twelve representative strains of Leuconostoc isolated from 20 samples of this artisanal cheese were selected and submitted for evaluation of the acidifying, proteolytic, autolytic, aminopeptidase and lipolytic activities, NaCl and acid resistance, production of dextran and biogenic amines and antimicrobial activity. The aim was to genetically and technologically characterize the Leuconostoc strains in order to use them in mixed starter cultures for cheese manufacture. RESULTS: Leuconostoc mesenteroides subsp. mesenteroides was the species that accounted for the largest proportion of isolates of Leuconostoc genus. Two leuconostoc isolates stood out in the acidifying activity, with reduction in pH of 1.12 and 1.04 units. The isolates showed low proteolytic and autolytic activity. Most of the isolates were dextran producers, presented good resistance to the salt and pH conditions of the cheese and showed antimicrobial activity against cheese pathogen bacteria, and none of them produced biogenic amines. CONCLUSION: These results allowed the selection of five strains (UEL 04, UEL 12, UEL 18, UEL 21 and UEL 28) as good candidates for use as adjunct cultures for cheese manufacture. © 2018 Society of Chemical Industry.

Complete Genome Sequence of Streptococcus agalactiae Strain S25 Isolated from Peritoneal Liquid of Nile Tilapia.

Streptococcus agalactiae (Lancefield group B; GBS) is one of the major pathogens in fish production, especially in Nile tilapia (Oreochromis niloticus). The genomic characteristics of GBS isolated from fish must be more explored. Thus, we present here the genome of GBS S25, isolated from Nile tilapia from Brazil.

Preservatives and neutralizing substances in milk: analytical sensitivity of official specific and nonspecific tests, microbial inhibition effect, and residue persistence in milk / Sustâncias conservantes e neutralizantes no leite: sensibilidade analítica das provas oficiais específicas, provas inespecíficas, inibição microbiana e persistência de resíduos no leite

Milk fraud has been a recurring problem in Brazil; thus, it is important to know the effect of most frequently used preservatives and neutralizing substances as well as the detection capability of official tests. The objective of this study was to evaluate the analytical sensitivity of legislation-described tests and nonspecific microbial inhibition tests, and to investigate the effect of such substances on microbial growth inhibition and the persistence of detectable residues after 24/48h of refrigeration. Batches of raw milk, free from any contaminant, were divided into aliquots and mixed with different concentrations of formaldehyde, hydrogen peroxide, sodium hypochlorite, chlorine, chlorinated alkaline detergent, or sodium hydroxide. The analytical sensitivity of the official tests was 0.005%, 0.003%, and 0.013% for formaldehyde, hydrogen peroxide, and hypochlorite, respectively. Chlorine and chlorinated alkaline detergent were not detected by regulatory tests. In the tests for neutralizing substances, sodium hydroxide could not be detected when acidity was accurately neutralized. The yogurt culture test gave results similar to those obtained by official tests for the detection of specific substances. Concentrations of 0.05% of formaldehyde, 0.003% of hydrogen peroxide and 0.013% of sodium hypochlorite significantly reduced (P<0.05) the microbial counts in milk after 24 and 48h refrigeration. Formaldehyde and sodium hypochlorite remained detectable in milk after 48 and 24h of refrigeration, respectively; while hydrogen peroxide could not be detected after 24h. Official tests for the detection of milk fraud by the addition of preservatives and neutralizing substances present limitations and may be ineffective in detecting milk adulteration

Fraudes no leite têm sido um problema recorrente no Brasil, tornando importante conhecer o efeito dos conservantes e neutralizantes utilizados com maior frequência, e a capacidade de detecção das provas oficiais. O objetivo deste trabalho foi avaliar a sensibilidade analítica de provas descritas pela legislação, das provas por inibição microbiana inespecífica, o efeito dessas substâncias na inibição do crescimento microbiano e a persistência de resíduos detectáveis após 48 horas de refrigeração. Lotes de leite cru, livres de sustâncias adulterantes, foram aliquotados e adicionados de diferentes concentrações de formaldeído, peróxido de hidrogênio, hipoclorito de sódio, cloro, detergente alcalino clorado e hidróxido de sódio. A sensibilidade analítica das provas oficiais foi: 0,005% para formaldeído; 0,003% para peróxido de hidrogênio e 0,013% para hipoclorito. Cloro e detergente alcalino clorado não foram detectados pelas provas oficiais. A prova de neutralizantes não detectou hidróxido de sódio quando a acidez foi neutralizada com precisão. A prova da cultura de iogurte apresentou resultados próximos ao das provas oficiais para substâncias específicas. Concentrações de 0,05% formaldeído, 0,003% de peróxido de hidrogênio e 0,013% de hipoclorito de sódio reduziram significativamente (P<0,05) a microbiota do leite após 24 e 48 horas de refrigeração. O formaldeído e hipoclorito de sódio permaneceram detectável no leite após 48 e 24 horas de refrigeração, respectivamente e o peróxido de hidrogênio não foi detectável após 24 horas. As provas oficiais para a pesquisa de fraudes por adição de conservantes e neutralizantes ao leite apresentam limitações e podem não ser capazes de detectar adulterações no leite

Detecção de adulterações do leite pasteurizado por meio de provas oficiais / Detection of pasteurized milk adulteration through official methods

A qualidade do leite consumido é uma constante preocupação dos técnicos e autoridades ligados à área de saúde e laticínios bem como dos consumidores. Um dos problemas mais graves são as diversas fraudes que causam prejuízos econômicos, riscos à saúde dos consumidores e, às vezes, problemas para as indústrias, como a diminuição do rendimento industrial. Muitos alimentos estão sujeitos às fraudes, mas o leite é um dos mais comumente fraudados. O objetivo do presente trabalho foi determinar a presença de substâncias fraudulentas e avaliar as características físico-químicas do leite pasteurizado produzido em laticínios da região norte do Paraná. Foram avaliadas 80 amostras no período de março a junho de 2014 e realizadas provas específicas para detecção dos reconstituintes: amido, álcool, cloreto e sacarose; neutralizantes: bicarbonato e hidróxido de sódio; conservantes: cloro, hipoclorito, peróxido de hidrogênio e formaldeído. A avaliação das características físico-químicas foi realizada através das seguintes análises: densidade a 15°C, índice crioscópico, acidez titulável Dornic, estabilidade ao alizarol 72%, pH, fosfatase alcalina, peroxidase, teor de gordura, sólidos totais, sólidos não gordurosos, ureia, proteína e lactose. Foram verificadas amostras fora do padrão para as seguintes análises: gordura (12,5%), sólidos não gordurosos (5%), densidade (1,25%), crioscopia (3,75%),pH (48,75%), ureia (1,25%). Foram verificadas fraudes por adição de água e sacarose (3,75%), presença de hipoclorito (5%) e ocorrência de desnate (12,5%). As provas em conjunto podem auxiliar na detecção das fraudes mais comumente realizadas no leite pasteurizado, porém não mostra em qual segmento podem ter ocorrido. No entanto, se não detectada, não se pode assegurar a qualidade do produto visto que muitas fraudes têm sido realizadas de forma equilibrada dificultando sua detecção.

The quality of milk consumed is a constant concern of dairy industry and healthcare-related authorities, technicians as well as consumers. The most serious problems are the various frauds which cause economic losses, risks to consumer health and, sometimes, problems for industries, such as the decrease in industrial output. Many foods are subject to fraud, but milk is one of the most commonly spoofed. The purpose of this study was to determine the presence of fraudulent substances and assess the physico-chemical properties of pasteurized milk produced in dairies from the North region of Paraná. Eighty samples were evaluated in the period from March to June 2014 and carried out specific methods for detection of restoratives: starch, alcohol, chloride and sucrose; neutralizers: bicarbonate and sodium hydroxide; preservatives: chlorine, sodium hypochlorite, hydrogen peroxide and formaldehyde. The evaluation of physico-chemical characteristics was carried out through the following analyses: density at 15° C, cryoscopic index, Dornic titratable acidity, 72 alizarol stability, pH, alkaline phosphatase, peroxidase, fat, total solids, non-fat solids, urea, protein and lactose. Non-standard samples were observed for the following analyses: fat (12.5%), non-fat solids (5%), density (1.25%), cryoscopy (3.75%), pH (48.75%), urea (1.25%). Frauds were observed by addition of water and sucrose (3.75%), presence of hypochlorite (5%) and occurrence of skim milk (12.5%). The evidence together can assist in detecting fraud most commonly performed in pasteurized milk, but does not show in which thread might have occurred. However, if not detected, the quality of the product cannot be assured since many frauds have been carried out in a balanced way hindering its detection.

Evolução da qualidade microbiológica e físico-química do leite pasteurizado / Evolution of microbiological and physico-chemical quality of pasteurized milk

A qualidade do leite é definida, entre outros parâmetros, por um reduzido número de micro-organismos deteriorantes, baixa contagem de células somáticas e ausência de patógenos e resíduos químicos. Diversos trabalhos realizados em diferentes regiões do país têm enfatizado o elevado percentual de amostras fora dos padrões. O objetivo do presente trabalho foi avaliar a evolução da qualidade microbiológica e físico química do leite pasteurizado produzido no Estado do Paraná ao longo de 7 anos. Foram analisadas 457 amostras de leite Pasteurizado, sendo 104 amostras no ano de 2008, 269 amostras em 2011 e 84 amostras em 2014. As amostras foram submetidas às análises físico-químicas de crioscopia e pesquisa de enzimas fosfatase alcalina e peroxidase. Quanto às análises microbiológicas, foram realizadas contagens de coliformes a 30°C, coliformes 45°C e contagem padrão em placas. No laboratório, as análises físico químicas foram realizadas de acordo com a Instrução Normativa 68 e microbiológicas conforme a Instrução Normativa 62, ambas do Ministério da Agricultura Pecuária e Abastecimento. Os resultados demonstraram que com o passar dos anos a qualidade microbiológica do leite diminuiu, havendo um aumento de amostras fora do padrão. Quanto às enzimas fosfatase alcalina e peroxidase, a temperatura de pasteurização foi respeitada ao longo do tempo e o superaquecimento do leite foi mais frequente em 2011.As fraudes por adição de água diminuíram ou então ficaram mais sofisticadas, dificultando sua detecção.

Milk quality is defined, among other parameters, by a reduced number of spoilage microorganisms, low somatic cell count and the absence of pathogens and chemical waste. Several studies conducted in different regions of the country have emphasized the high percentage of samples not complying with the standard. The purpose of this study was to evaluate the evolution of microbiological and physicochemical qualityof pasteurized milk produced in the State of Paraná over 7 years. A total of 457 samples of pasteurized milk were analyzed, 104 samples in 2008, 269 samples in 2011 and 84 samples in 2014. The samples were subjected to physico chemical analysis of cryoscopy and enzyme search for alkaline phosphatase and peroxidase. Regarding microbiological tests, coliform counts were performed at 30°C and 45°C and countplate pattern. In the laboratory, physico chemical analysis were performed according to the Normative 68 and microbiological as normative instruction 62, both of the Brazilian Ministry of Agriculture, Livestock and Food Supply. The results showed that over the years the microbiological quality of milk decreased, with an increase of non-standard samples. For enzymes alkaline phosphatase, peroxidase, the pasteurization temperature has been observed over time and the overheating of the milk was more frequentin 2011. Fraud by addition of water in milk has either decreased or become more sophisticated, makingits detection difficult.

Acid-base balance of dairy cows and its relationship with alcoholic stability and mineral composition of milk / Equilíbrio ácido-base de vacas leiteiras e sua relação com a estabilidade alcoólica e composição mineral do leite

This study aimed to associate the occurrence of acid-base disorders with the alcoholic stability of milk from animals in the field, and to evaluate differences between the mineral composition of milk that was both stable and unstable in alcohol. The sample comprised 96 dairy cows, where the milk and blood of each corresponding animal was collected. The mineral composition of stable and unstable milk in alcohol was different and may be related to acid-base disturbances. The average amount of phosphate was lower in the milk that was unstable in alcohol, while potassium was greater. Frequency of the alcoholically unstable milk cases was higher in the cows with acid-base disturbances. Respiratory alkalosis was the disorder that was most observed.

Esse trabalho teve como objetivo associar a ocorrência de distúrbios ácido-base com a estabilidade alcoólica do leite de animais a campo, bem como avaliar diferenças entre a composição mineral de leites estáveis e instáveis ao álcool. A amostragem compreendeu 96 vacas leiteiras, das quais o leite e o sangue correspondente de cada animal foram coletados. A composição mineral entre leites estáveis e instáveis ao álcool foi diferente e também pode estar relacionada aos distúrbios ácido-básicos. A quantidade média de fosfato foi menor no leite instável ao álcool, enquanto a de potássio foi maior. A frequência de amostras de leite com instabilidade alcoólica foi maior nas vacas com distúrbios ácido-básicos. A alcalose respiratória foi o desequilíbrio mais observado.

Organophosphorus and carbamates residues in milk and feedstuff supplied to dairy cattle / Resíduos de praguicidas organofosforados e carbamatos em leite e alimentação animal de propriedades leiteiras

Considering acute and chronic toxicity effects on human and animal health caused by pesticide residues in food, this study aimed to analyze organophosphorate (OP) and carbamate (CB) in feedstuff and water destined for dairy cattle, as well as in the milk produced by these animals, through gas chromatography (GC). In the Agreste region of Pernambuco, Brazil, 30 raw milk samples and all components of the animals' diet were collected from several farms. Out of the 30 milk of milk analyzed, six (20 percent) were contaminated with OP, five (16.7 percent) with CB, and one sample with both pesticides. From 48 analyzed feed samples, 15 (31.25 percent) were contaminated with residues of OP, six (12.50 percent) with CB, and one sample was contaminated with both pesticides. Out of 16 water samples analyzed, six (37.50 percent) were contaminated with OP residues, but non with CB. In four dairy farms the pesticides detected in milk were compatible with the active principles found in water and/or foodstuff, suggesting them to be the source of contamination.

Considerando os efeitos tóxicos, agudos e crônicos, para a saúde humana e animal, causados por resíduos de praguicidas em alimentos, este trabalho teve como objetivo a identificação e quantificação por cromatografia gasosa (CG) de resíduos de praguicidas organofosforados (OF) e carbamatos (CB) no leite cru, nos componentes da alimentação e água dos animais. Foram coletadas 30 amostras de leite cru da região agreste de Pernambuco e ao mesmo tempo eram coletadas amostras de alimentação e água ofertada aos animais em lactação de cada propriedade, totalizando 109 amostras de alimentação e 38 amostras de água. Das 30 amostras de leite analisadas, seis (20 por cento) estavam contaminadas por resíduos de OF, cinco (16,7 por cento) por resíduos de CB e uma amostra por ambos os praguicidas. Das 109 amostras de alimentação ofertada aos animais coletadas, 48 foram analisadas, com 15 amostras (31,25 por cento) apresentando resíduos de OF, seis amostras (12,50 por cento) contaminadas por resíduos de CB e uma amostra positiva para ambos os praguicidas. Das 16 amostras de água analizadas, seis (37,50 por cento) estavam contaminadas por resíduos de OF e nenhuma apresentou resíduos de CB. Em quatro propriedades leiteiras os praguicidas detectados no leite foram compatíveis com o princípio ativo detectado na alimentação e/ou na água ofertada aos animais, sugerindo uma possível fonte de contaminação, mas não a única.

Interference of raw milk autochthonous microbiota on the performance of conventional methodologies for Listeria monocytogenes and Salmonella spp. detection.

Pathogen detection in foods by reliable methodologies is very important to guarantee microbiological safety. However, peculiar characteristics of certain foods, such as autochthonous microbiota, can directly influence pathogen development and detection. With the objective of verifying the performance of the official analytical methodologies for the isolation of Listeria monocytogenes and Salmonella in milk, different concentrations of these pathogens were inoculated in raw milk treatments with different levels of mesophilic aerobes, and then submitted to the traditional isolation procedures for the inoculated pathogens. Listeria monocytogenes was inoculated at the range of 0.2-5.2 log CFU/mL in treatments with 1.8-8.2 log CFU/mL. Salmonella Enteritidis was inoculated at 0.9-3.9 log CFU/mL in treatments with 3.0-8.2logCFU/mL. The results indicated that recovery was not possible or was more difficult in the treatments with high counts of mesophilic aerobes and low levels of the pathogens, indicating interference of raw milk autochthonous microbiota. This interference was more evident for L. monocytogenes, once the pathogen recovery was not possible in treatments with mesophilic aerobes up to 4.0 log CFU/mL and inoculum under 2.0 log CFU/mL. For S. Enteritidis the interference appeared to be more non-specific.

Listeria spp. associated to different levels of autochthonous microbiota in meat, meat products and processing plants

High levels of microbial contamination, commonly found in animal origin foods and food processing environments, are able to hinder the growth of pathogens in these products and interfere in the results of laboratory analyses for detection of these pathogens. With the aim of verifying the possible interference of the autochthonous microbiota encountered in meat and meat products and processing plants over the presence of Listeria spp., 443 samples, collected from 11 meat retail establishments, were submitted to microbiological analysis to determine the levels of mesophilic aerobes, total coliforms and Escherichia coli and the presence of Listeria spp., according to the methodology proposed by the USDA. The results did not show evident interference of the autochthonous microbiota over Listeria spp., once the genus was detected even in the meat, meat products and environmental samples with high levels of contamination by mesophilic aerobes and coliforms.

Altos níveis de contaminação microbiana, usualmente encontrados em alimentos de origem animal e nos ambientes de processamento, podem inibir a multiplicação de microrganismos patogênicos nesses produtos e interferir nos resultados das análises laboratoriais para o isolamento desses patógenos. Com o objetivo de verificar as possíveis interferências da microbiota autóctone encontrada na carne, produtos cárneos e plantas de processamento sobre a presença de Listeria spp., 443 amostras, coletadas em 11 estabelecimentos processadores, foram submetidas a análises microbiológicas para determinação dos níveis de contaminação por aeróbios mesófilos, coliformes totais e Escherichia coli e para verificação da presença de Listeria spp., de acordo com a metodologia proposta pelo USDA. Os resultados obtidos não mostraram uma interferência evidente da microbiota autóctone sobre Listeria spp., uma vez que esse gênero foi detectado mesmo nas amostras de carne e produtos cárneos e amostras ambientais e de superfície de equipamentos que apresentaram altos níveis de contaminação por aeróbios mesófilos e coliformes.

Características da produção leiteira da região do agreste do estado de Pernambuco, Brasil / Characteristics of the milk production of the agreste region of the state of Pernambuco, Brazil

O Agreste pernambucano é caracterizado por uma economia diversificada, com diferentes cultivos, além da pecuária de leite e de corte. Em 2004 o Estado produziu 398 milhões de litros de leite, o que representou 14,7% da produção da Região Nordeste e 1,7% da produção Nacional. Este trabalho teve como objetivo caracterizar as propriedades leiteiras da região Agreste do Estado de Pernambuco, observando condições e fatores que pudessem interferir na qualidade do leite. Foram selecionadas 41 propriedades leiteiras da região. Observou-se o predomínio das propriedades de pequeno porte, com até 25 (43,9%) animais em lactação, sendo ordenha manual em 36 (87,8%) das propriedades. Em relação às práticas de higiene na ordenha, 16 (39,0%) dos produtores lavavam os tetos antes da ordenha, mas apenas 4 (9,8%) realizavam pré-dipping e 34 (82,9%) ordenhavam na presença do bezerro. Apenas 5 (12%) das propriedades realizavam algum tipo de tratamento da água utilizada para higienização de instalações e equipamentos, mesmo a água para consumo humano era tratada em apenas 43,9% das propriedades. A refrigeração do leite era realizada por 10 (24,4%) propriedades em tanques comunitários. Quanto a sanidade do rebanho 39(95,1%) proprietários realizavam vacinação para febre aftosa e 23 (56,1%) para brucelose, enquanto 10(24,4%) realizavam teste para tuberculose. As condições de produção se revelaram precárias quanto às estruturas nas propriedades, mostrando a necessidade da melhoria das instalações e manejo dos animais e, principalmente, a implantação de Boas Práticas na ordenha, adequadas à realidade local.

The Agreste region in the state of Pernambuco is characterized by a diversified economy, with differentcultivars, besides the daily and beef farms. In 2004 the State produced 398 million liters of milk; it represented 14.7% of the Northeast Region production and 1.7% of the National production. The aim ofthis work was to characterize the milk properties of the Agreste region of Pernambuco, observingconditions and factors that could interfere with milk quality. In the survey, 41 milk farms were selected inthe region. Small milk farms were the most common found, with less than 25 (43.9%) animals in lactation,and 36 (87.8%) farms held a manual milking process. In relation to hygiene practices during milking, 16(39.0%) of the producers washed the teats before beginning, but only 4 (9.8%) performed pre-dippingand 34 (82.9%) milked in the presence the calf. Only 5 (12%) farms applied some treatment to the waterused for cleaning installations and equipment, even the water for human consumption was treated inonly 43.9% of the farms. Milk refrigeration was performed in 10 (24.4%) farms in communitarian tanks. Asfar as animal health is concerned, 39 (95.1%) owners vaccinated for food and mouth disease and 23(56.1%) for brucellosis, while 10 (24.4%) tested for tuberculosis. Regarding the farms structure theproduction conditions were precarious, showing the need of improving the structure itself and animalhandling. As well as the implementation of Good Manufacturing Practices in the milking process, properto the local reality.

Screening and enumeration of lactic acid bacteria in milk using three different culture media in Petrifilm Aerobic Count plates and conventional pour plate methodology.

This study aimed to compare Petrifilm Aerobic Count (AC) plates and the conventional pour plate methodology using de Mann-Rogosa-Sharpe (MRS), Kang-Fung (KF) and Kang-Fung-Sol (KFS) culture media for screening and enumeration of lactic acid bacteria (LAB) in milk. Suspensions of 10 LAB species in reconstituted powder skim milk and 30 raw milk samples, without experimental inoculation, were tested. For selective enumeration, all samples were previously diluted in MRS, KF and KFS broths and then plated in Petrifilm AC and conventional pour plate methodology, using the same culture media with added agar. All plates were incubated at 37 degrees C for 48 h in anaerobic conditions. Differences in the counts were observed only for raw milk samples using KFS in conventional methodology, when compared with the counts obtained from MRS and KF (P0.05). The results showed excellent correlation indexes between both methodologies using the three culture media for LAB suspensions (r=0.97 for MRS, KF and KFS). For raw milk samples, the correlation indexes were excellent (r=0.97, for MRS) and good (r=0.84 for KF, and r=0.82 for KFS), showing some interference in Petrifilm AC when supplements were added, especially lactic acid. These results indicate the possibility of using Petrifilm AC plates for enumeration of LAB in milk, even with the use of selective supplements.

Avaliação da qualidade microbiológica e dos parâmetros enzimáticosda pasteurização de leite tipo C produzido na região norte do Paraná / Microbiological quality evaluation and pasteurization enzymaticparameters in type "C" milk produced in the north of Paraná state, Brazil

O presente trabalho teve como objetivo avaliar a qualidade microbiológica e parâmetros enzimáticos dapasteurização de 80 amostras de leites pasteurizados tipo C produzidos na região norte do Paraná noperíodo de janeiro de 2004 a dezembro de 2005. Os resultados obtidos foram comparados com os padrões estabelecidos no Regulamento da Inspeção Industrial e Sanitária de Produtos de Origem Animal (RIISPOA). As análises microbiológicas realizadas foram: contagem de aeróbios mesófilos, determinaçãodo número mais provável de coliformes totais e coliformes termotolerantes. A avaliação da pasteurizaçãofoi realizada a partir da pesquisa das enzimas peroxidase e fosfatase alcalina. As amostras em desacordocom os padrões estabelecidos foram: 3,7% para aeróbios mesófilos, 30,0% para coliformes totais e 14,0%para coliformes termotolerantes. Ainda 16,2% das amostras apresentavam peroxidase negativa, indicandosuperaquecimento, o que pode causar prejuízos à composição do leite. Com os resultados obtidospodemos concluir que a qualidade microbiológica de parte das amostras foi insatisfatória.

The aim of this study was to evaluate the microbiological quality and pasteurization enzymatic parametersin 80 samples of pasteurized milk produced in the north of Paraná state - Brazil from January, 2004 toDecember, 2005. The obtained results were compared with legislation established in the Regulation ofIndustry and Sanitary Federal Inspection of Food of Animal Origin. The microbiological analyses weremesophilic aerobes enumeration, most probable number (MPN) of total coliforms and thermotolerantcoliforms. Peroxidase and alkaline phosphatase enzymes were used to evaluate the pasteurization process.Samples disagreeing with the standards were 3,7% to mesophilic aerobes, 30,0% to total coliforms and14,0% to thermotolerant coliforms. Furthermore, in 16,2% of the samples the peroxidase was not present,indicating overheating, what may affect negatively milk composition. According to the results it couldbe verified that the microbiological quality of some samples were unsatisfactory.

Listeria monocytogenes: Occurrence in beef and identification of the main contamination points in processing plants.

This study aimed to establish the occurrence of Listeria spp., especially L. monocytogenes and its main serotypes, in beef and processing plants. A total of 443 samples were obtained from equipment, installations and products from 11 meat processing establishments from Paraná state, Brazil. All samples were analyzed using USDA methodology for Listeria spp. detection, followed by species identification. The occurrence of Listeria spp. in the samples was 38.1% of which 51.4% were from equipment, 35.4% from installations and 30.2% from products. The identified species were: L. monocytogenes (12.6%), L. innocua (78.4%), L. seeligeri (1.2%), L. welshimeri (7.2%) and L. grayi (0.6%). The identified serotypes of L. monocytogenes were 1/2a and 4b. The results demonstrate the significance of equipment and installations as sources of contamination by Listeria spp. and L. monocytogenes in the processing of beef and meat products.