Study of the Oxidative and Microbiological Stability of Nitrite-Reduced, Vacuum-Packed, Refrigerated Lamb Sausages Supplemented with Red Propolis Extract.

Vacuum-packed lamb sausages with or without red propolis extract and a reduced sodium nitrite content were evaluated for oxidative and microbiological stability during storage for 21 days at 2 °C. The following treatments were evaluated: EN150 (control, base formulation (BF) + 500 mg/kg sodium erythorbate and 150 mg/kg sodium nitrite); EN75 (BF + 500 mg/kg sodium erythorbate and 75 mg/kg sodium nitrite); P1N75 (without the addition of erythorbate, BF + 1800 mg/kg propolis extract and 75 mg/kg sodium nitrite); and P2N75 (without the addition of erythorbate, BF + 3600 mg/kg propolis extract and 75 mg/kg sodium nitrite). Analyses were conducted to characterize the samples on day 0 with respect to the proximate composition (moisture, protein, fat, and ash) and sensory acceptance. Stability during refrigerated storage was evaluated on days 0, 7, 14 and 21 for the parameters pH, color profile (L*, a*, and b*), TBARs index (oxidative stability) and microbiological count of aerobic psychrotrophic microorganisms. Texture profile, cooking weight loss (WLC), peroxide index and free fatty acids were evaluated on days 0 and 21. The treatments with propolis and reduced nitrite (EN150 and P1N75) showed a red color intensity (a*) similar to the treatment with erythorbate and the same nitrite content (EN75) at the end of storage, maintaining the characteristic reddish color of the sausages. The extract slowed down lipid oxidation during storage, especially P2N75, which showed the lowest level of TBARS (0.39 mg MDA/kg) and the peroxide index (2.13 mEq g O2) on day 21. The residual nitrite value in EN75 was the lowest (p < 0.05) on day 21, showing that synthetic antioxidants are more efficient than the extract in nitrite reduction reactions. The results for the counts of psychrotrophic microorganisms showed that the extract did not have the expected antimicrobial effect on the growth of this microorganisms, and leveling the results revealed no differences (p < 0.05) between the treatments. Despite the red propolis extract not showing a significant antimicrobial improvement in lamb sausages, it can be considered a healthy option with good prospects for replacing synthetic antioxidants with a natural product.

Isolation, characterization, and application of bacteriophage cocktails for the biocontrol of Pseudomonas fluorescens group strains in whole and skimmed milk.

Pseudomonas fluorescens group strains can lead to spoilage of milk as well as loss of quality in dairy products through their heat-resistant enzymes. Phages are important alternatives for combating spoilage bacteria in food industry and used successfully in many applications. The aim of this study was the isolation and characterization of phages and to assess the efficiency of a phage cocktail in whole and skimmed milk. For this purpose, phages effective against Pseudomonas fluorescens (L23.2), Pseudomonas tolaasii (P22.1), and Pseudomonas rhodesiae (A11.1) were isolated. Their host range was found to be highly specific, and the transmission electron micrographs indicates that they belonged to Tectiviridae family. Their genome sizes were found to be vary between 38.3 and 53.5 kb. The latent periods and burst sizes were determined as 15, 10, 15 min and 91, 20, 80 PFU/infected cell for L23.2, P22.1, and A11.1, respectively. All three phages were found to be sensitive to low pH and high temperature. The effect of the phage cocktail was monitored in milk with different fat contents during storage at 4 °C for 5 days. As a result, bacterial reductions up to 4.09 and 5.29 log-units were observed for the whole and skimmed milk, respectively. Thus, the efficacy of a phage cocktail against a bacterial mixture of different P. fluorescens strains was tested in milk samples with different fat contents in accordance with real-life scenarios for the first time.

Mycosynthesis of silver nanoparticles using psychrotrophic strains of Tulasnella albida Bourdot & Galzin from the South Orkney Islands (Antarctica).

This study is the first report on mycosynthesis of silver nanoparticles (NPs) using psychrotrophic Antarctic filamentous fungi, and the first report regarding Tulasnella (Basidiomycota). In this work, the ability to synthesize silver NPs from cell free filtrates of strains of Tulasnella albida isolated from Antarctica was assessed. All fungal filtrates were capable of synthesizing silver NPs with the addition of AgNO3. UV-vis spectroscopy, TEM and SEM microscopy analyses were performed to characterize the synthesized NPs. ATR-FTIR and Micro Raman spectroscopy analyses were conducted to find functional groups responsible for the reduction of AgNO3 and to detect the presence of silver oxide on the AgNPs. Theoretical calculations of optical absorption based on core-shell Ag-Ag2O were used to characterize the experimental absorption spectra of silver NPs colloids. Spherically shaped silver NPs, typically 2-3nm in diameter, were obtained. The largest ones showed a capping shell around them, which could be associated with the formation of small silver NPs. Functional groups corresponding to amides and alcohols were detected, confirming the presence of proteins as possible intermediates in the synthesis of AgNPs. On the other hand, the Micro Raman analysis confirms the presence of silver oxide on the surface of the AgNPs. This work presents a simple procedure for the synthesis of silver NPs using a psychrotrophic organism that could be interesting for the industry.

Quality of refrigerated raw milk according to the bulk transport conditions

Technical regulations that determine milk quality standards in Brazil establish the minimum criteria that the processing industry's raw material must present. These rules can challenge the logistics sector (transport) to maintain adequate milk conservation levels. We aimed to evaluate the influence of bulk transport and storage conditions on refrigerated raw milk quality. The experiment was carried out in a dairy industry in Santa Catarina State. Information and samples of refrigerated raw milk were collected from dairy farms, from isothermal compartments of milk trucks, and from the industry's storage silo. These samples were submitted to microbiological analysis, somatic cell count(SCC), and physicochemical analysis. The results were evaluated by ANOVA and multivariate analysis (factor analysis). The transport of raw milk in bulk and the transfer of raw material to the industrial silo worsened the milk's microbiological quality (p < 0.0001) for standard plate count (SPC) and psychrotrophic microorganism count(PMC), without affecting, the physicochemical characteristics of the milk (p > 0.05). Poor hygienic conditions in milk storage rooms, in the refrigeration bulk tanks of the dairy farms, and during the transport, as well as the transport on longer routes, were related to higher SPC and psychrotrophic count while receiving milk by the dairy industry with higher temperatures was only associated with higher SPC.(AU)

Prospection of Psychrotrophic Filamentous Fungi Isolated from the High Andean Paramo Region of Northern Ecuador: Enzymatic Activity and Molecular Identification.

The isolation of filamentous fungal strains from remote habitats with extreme climatic conditions has led to the discovery of a series of enzymes with attractive properties that can be useful in various industrial applications. Among these, cold-adapted enzymes from fungi with psychrotrophic lifestyles are valuable agents in industrial processes aiming towards energy reduction. Out of eight strains isolated from soil of the paramo highlands of Ecuador, three were selected for further experimentation and identified as Cladosporium michoacanense, Cladosporium sp. (cladosporioides complex), and Didymella sp., this last being reported for the first time in this area. The secretion of seven enzymes, namely, endoglucanase, exoglucanase, ß-D-glucosidase, endo-1,4-ß-xylanase, ß-D-xylosidase, acid, and alkaline phosphatases, were analyzed under agitation and static conditions optimized for the growth period and incubation temperature. Cladosporium strains under agitation as well as incubation for 72 h mostly showed the substantial activation for endoglucanase reaching up to 4563 mU/mL and xylanase up to 3036 mU/mL. Meanwhile, other enzymatic levels varied enormously depending on growth and temperature. Didymella sp. showed the most robust activation at 8 °C for endoglucanase, ß-D-glucosidase, and xylanase, indicating an interesting profile for applications such as bioremediation and wastewater treatment processes under cold climatic conditions.

Pseudomonas fluorescens and Pseudomonas putida from refrigerated raw milk: genetic diversity and lipoproteolytic activity.

In this research communication the genetic diversity of Pseudomonas fluorescens (n = 67) and Pseudomonas putida (n = 44) isolated from refrigerated raw milk from bulk tank trucks were verified. The relationship between the genetic profile of the isolates and their lipoproteolytic potential was evaluated using skim milk agar and tributyrin agar (21°C/72 h). The lipoproteolytic potential (low or high), evaluated by the diameter of the halos (cm), was correlated with the number of milk producing properties that contributed to each sample (one sample = one bulk tank truck; 8-80 producers/sample) and the distance between the dairy properties and the processing plant (21-370 km). P. fluorescens was confirmed in all samples, while P. putida in 60% samples. For both species, two clusters (I and II) were observed, and the first one showed lower genotypic diversity and the presence of isolates with 100% similarity. P. fluorescens isolates presenting at least 70% similarity were 83.9% in Cluster I (n = 31) and 44.4% in Cluster II. In both clusters (I and II) observed in the P. fluorescens dendrogram, the occurrence of high proteolytic and lipolytic potential were equivalent. The higher the number of farms per milk sample, the greater the lipoproteolytic intensity of P. fluorescens isolates. In relation to P. putida isolates, 74% presented at least 50% similarity in Cluster I (n = 27) and only 35% in Cluster II (n = 17). The occurrence of high proteolysis linked to P. putida was proportional between both Clusters, but the occurrence of high lipolysis was greater in Cluster II. No significant association was detected between P. putida isolates and the variables studied. The results indicate the circulation of P. putida and P. fluorescens with 100% similarity in different milk producing regions. The level of genetic diversity was related only to the lipolytic capacity of P. putida.

Lytic bacteriophages UFJF_PfDIW6 and UFJF_PfSW6 prevent Pseudomonas fluorescens growth in vitro and the proteolytic-caused spoilage of raw milk during chilled storage.

In this study, P. fluorescens-infecting phages were isolated, characterized, and evaluated to their potential to control the bacterial counts and, consequently, the proteolytic spoilage of raw milk during cold storage. The UFJF_PfDIW6 and UFJF_PfSW6 phages showed titers of 9.7 and 7.6 log PFU/ml; latent period of 115 and 25 min, and burst size of 145 and 25 PFU/infected cell, respectively. They also were highly specific to the host bacterium, morphologically classified as the Podoviridae family, stable at pH 5 to 11 and were not inactivated at 63 °C or 72 °C for 30 min. These phages found to be effective against P. fluorescens, reducing bacterial count throughout the entire exponential growth phase in broth formulated with milk at both 4 °C and 10 °C. This effect on bacteria growth led to inhibition by at least 2 days in proteases production, delaying the degradation of milk proteins. When applied together in raw milk stored at 4 °C, they reduced the total bacteria, psychrotrophic, and Pseudomonas by 3 log CFU/ml. This study's findings indicate that these phages have a great potential to prevent the growth of Pseudomonas and, consequently, to retard proteolytic spoilage of raw milk during chilled storage.

Presence of aprX gene in Pseudomonas spp. from refrigerated raw milk and their proteolytic ability / Presença do gene aprX em Pseudomonas spp. isolados de leite cru refrigerado e sua capacidade proteolítica

This study aimed to determine the frequency of Pseudomonas fluorescens, P. putida, and P. aeruginosa in refrigerated raw milk; their proteolytic potential; and your association with the aprX gene. Of the 173 isolates confirmed as belonging to Pseudomonas spp., 37% were P. fluorescens, 25.4% P. putida, and none belongs to P. aeruginosa. The aprX gene was distributed proportionally between P. putida (68%) and P. fluorescens (75%), but it was not associated with low or high proteolytic potential in both species. P. putida (16) and P. fluorescens (14) isolates with no aprX gene identified also had proteolytic potential. Considering the synthesis of proteases other than AprX by the isolates under study, we concluded that P. fluorescens and P. putida represented 62.4% of the Pseudomonas genus, with high probability of having the aprX gene and proteolytic potential. However, there was no association between the deteriorating potential with the presence of the aprX gene.(AU)

O objetivo do estudo foi verificar a frequência, em leite cru refrigerado de Pseudomonas fluorescens, P. putida e P. aeruginosa, o potencial proteolítico dos isolados e sua associação com a presença do gene aprX. Dos 173 isolados confirmados como pertencente ao gênero Pseudomonas spp, 37% foram P. fluorescens, 25,4% P. putida e nenhum pertencia à espécie P. aeruginosa. A prevalência do gene aprX se distribuiu proporcionalmente entre P.putida (68%) e P.fluorescens (75%) porém não foi associada ao baixo ou alto potencial proteolítico nas duas espécies avaliadas. P. putida (16) e P. fluorescens (14) que não tiveram o gene aprX identificado também expressaram potencial proteolítico, considerando-se a síntese de outras proteases além da AprX pelos isolados estudados. Concluiu-se que Pseudomonas fluorescens e P. putida representaram 62,4 % do gênero Pseudomonas ssp., com alta probabilidade da presença do gene aprX e potencial proteolítico, porém sem associação da expressão do potencial deteriorante com a presença do gene aprX.(AU)

Influence of time and storage temperature on raw milk deteriorating microbiota / Influência do tempo e temperatura de estocagem sobre a microbiota deteriorante do leite cru

The quality of raw milk depends on initial microbial contamination and conditions of storage until industry processing. Considering the influence of time and storage temperature on raw milk microbiota, the objective of this work was to quantify and monitor the multiplication of these groups under different conditions. For this purpose, 41 samples of raw milk were collected immediately after milking, stored in the following storage conditions: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h and 7 °C/60 h and analyses of aerobic mesophilic, psychrotrophic and proteolytic psychrotrophic microorganisms. The milk samples analyzed in the study had an initial mean count of mesophilic aerobes of 5.38 Log CFU/mL at Time Zero. The milk stored at 25 °C/2 h and 35 °C/2 h kept the mesophilic aerobic counts within the limits established by the legislation (5.48 Log CFU/mL), with an increase in counts of psychrotrophic and proteolytic microorganisms. When stored at 7 °C/24 h and 7 °C/48 h, the count of mesophiles exceeded the established parameters. A significant increase in the count of proteolytic psychrotrophs and psychrotrophs was also observed during storage at 7 °C from 24 h. The results of this study indicate that the temperature of 7 °C is not suitable for the milk conservation, since it was not able to control the microbial multiplication. Thus, the results contribute to the change in milk storage temperature proposed by the new Brazilian legislation.(AU)

A qualidade do leite cru depende da contaminação microbiana inicial e das condições de armazenamento até o processamento na indústria. Considerando a influência do tempo e da temperatura de armazenamento na microbiota do leite cru, o objetivo deste trabalho foi quantificar e monitorar a multiplicação desses grupos de microrganismos sob diferentes condições. Para tanto, foram coletadas 41 amostras de leite cru imediatamente após a ordenha, armazenadas nas seguintes condições de armazenamento: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h e 7 °C/60 h para análise de microrganismos psicrotróficos, aeróbios mesófilos, psicrotróficos e proteolíticos. As amostras de leite analisadas no estudo apresentaram uma contagem média inicial de aeróbios mesófilos de 5.38 Log UFC/mL no Tempo Zero. O leite armazenado a 25 °C/2 h e 35 °C/2 h manteve as contagens aeróbias mesófilas dentro dos limites estabelecidos pela legislação (5,48 Log UFC/mL), com aumento nas contagens de microrganismos psicrotróficos e proteolíticos. Quando armazenado a 7 °C/24 h e 7 °C/48 h a contagem de mesófilos excedeu os parâmetros estabelecidos. Um aumento significativo na contagem de psicrotróficos e psicrotróficos proteolíticos também foi observado durante o armazenamento a 7 °C a partir das 24 h. Os resultados deste estudo indicam que a temperatura de 7 °C não é adequada para a conservação do leite, uma vez que não foi capaz de controlar a multiplicação microbiana. Assim, os resultados contribuem para a mudança na temperatura de armazenamento de leite proposta pela nova legislação brasileira.(AU)

Influence of time and storage temperature on raw milk deteriorating microbiota / Influência do tempo e temperatura de estocagem sobre a microbiota deteriorante do leite cru

The quality of raw milk depends on initial microbial contamination and conditions of storage until industry processing. Considering the influence of time and storage temperature on raw milk microbiota, the objective of this work was to quantify and monitor the multiplication of these groups under different conditions. For this purpose, 41 samples of raw milk were collected immediately after milking, stored in the following storage conditions: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h and 7 °C/60 h and analyses of aerobic mesophilic, psychrotrophic and proteolytic psychrotrophic microorganisms. The milk samples analyzed in the study had an initial mean count of mesophilic aerobes of 5.38 Log CFU/mL at Time Zero. The milk stored at 25 °C/2 h and 35 °C/2 h kept the mesophilic aerobic counts within the limits established by the legislation (5.48 Log CFU/mL), with an increase in counts of psychrotrophic and proteolytic microorganisms. When stored at 7 °C/24 h and 7 °C/48 h, the count of mesophiles exceeded the established parameters. A significant increase in the count of proteolytic psychrotrophs and psychrotrophs was also observed during storage at 7 °C from 24 h. The results of this study indicate that the temperature of 7 °C is not suitable for the milk conservation, since it was not able to control the microbial multiplication. Thus, the results contribute to the change in mil

A qualidade do leite cru depende da contaminação microbiana inicial e das condições de armazenamento até o processamento na indústria. Considerando a influência do tempo e da temperatura de armazenamento na microbiota do leite cru, o objetivo deste trabalho foi quantificar e monitorar a multiplicação desses grupos de microrganismos sob diferentes condições. Para tanto, foram coletadas 41 amostras de leite cru imediatamente após a ordenha, armazenadas nas seguintes condições de armazenamento: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h e 7 °C/60 h para análise de microrganismos psicrotróficos, aeróbios mesófilos, psicrotróficos e proteolíticos. As amostras de leite analisadas no estudo apresentaram uma contagem média inicial de aeróbios mesófilos de 5.38 Log UFC/mL no Tempo Zero. O leite armazenado a 25 °C/2 h e 35 °C/2 h manteve as contagens aeróbias mesófilas dentro dos limites estabelecidos pela legislação (5,48 Log UFC/mL), com aumento nas contagens de microrganismos psicrotróficos e proteolíticos. Quando armazenado a 7 °C/24 h e 7 °C/48 h a contagem de mesófilos excedeu os parâmetros estabelecidos. Um aumento significativo na contagem de psicrotróficos e psicrotróficos proteolíticos também foi observado durante o armazenamento a 7 °C a partir das 24 h. Os resultados deste estudo indicam que a temperatura de 7 °C não é adequada para a conservação do leite, uma vez que não foi

Influence of time and storage temperature on raw milk deteriorating microbiota / Influência do tempo e temperatura de estocagem sobre a microbiota deteriorante do leite cru

The quality of raw milk depends on initial microbial contamination and conditions of storage until industry processing. Considering the influence of time and storage temperature on raw milk microbiota, the objective of this work was to quantify and monitor the multiplication of these groups under different conditions. For this purpose, 41 samples of raw milk were collected immediately after milking, stored in the following storage conditions: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h and 7 °C/60 h and analyses of aerobic mesophilic, psychrotrophic and proteolytic psychrotrophic microorganisms. The milk samples analyzed in the study had an initial mean count of mesophilic aerobes of 5.38 Log CFU/mL at Time Zero. The milk stored at 25 °C/2 h and 35 °C/2 h kept the mesophilic aerobic counts within the limits established by the legislation (5.48 Log CFU/mL), with an increase in counts of psychrotrophic and proteolytic microorganisms. When stored at 7 °C/24 h and 7 °C/48 h, the count of mesophiles exceeded the established parameters. A significant increase in the count of proteolytic psychrotrophs and psychrotrophs was also observed during storage at 7 °C from 24 h. The results of this study indicate that the temperature of 7 °C is not suitable for the milk conservation, since it was not able to control the microbial multiplication. Thus, the results contribute to the change in mil

A qualidade do leite cru depende da contaminação microbiana inicial e das condições de armazenamento até o processamento na indústria. Considerando a influência do tempo e da temperatura de armazenamento na microbiota do leite cru, o objetivo deste trabalho foi quantificar e monitorar a multiplicação desses grupos de microrganismos sob diferentes condições. Para tanto, foram coletadas 41 amostras de leite cru imediatamente após a ordenha, armazenadas nas seguintes condições de armazenamento: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h e 7 °C/60 h para análise de microrganismos psicrotróficos, aeróbios mesófilos, psicrotróficos e proteolíticos. As amostras de leite analisadas no estudo apresentaram uma contagem média inicial de aeróbios mesófilos de 5.38 Log UFC/mL no Tempo Zero. O leite armazenado a 25 °C/2 h e 35 °C/2 h manteve as contagens aeróbias mesófilas dentro dos limites estabelecidos pela legislação (5,48 Log UFC/mL), com aumento nas contagens de microrganismos psicrotróficos e proteolíticos. Quando armazenado a 7 °C/24 h e 7 °C/48 h a contagem de mesófilos excedeu os parâmetros estabelecidos. Um aumento significativo na contagem de psicrotróficos e psicrotróficos proteolíticos também foi observado durante o armazenamento a 7 °C a partir das 24 h. Os resultados deste estudo indicam que a temperatura de 7 °C não é adequada para a conservação do leite, uma vez que não foi

Influence of time and storage temperature on raw milk deteriorating microbiota / Influência do tempo e temperatura de estocagem sobre a microbiota deteriorante do leite cru

The quality of raw milk depends on initial microbial contamination and conditions of storage until industry processing. Considering the influence of time and storage temperature on raw milk microbiota, the objective of this work was to quantify and monitor the multiplication of these groups under different conditions. For this purpose, 41 samples of raw milk were collected immediately after milking, stored in the following storage conditions: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h and 7 °C/60 h and analyses of aerobic mesophilic, psychrotrophic and proteolytic psychrotrophic microorganisms. The milk samples analyzed in the study had an initial mean count of mesophilic aerobes of 5.38 Log CFU/mL at Time Zero. The milk stored at 25 °C/2 h and 35 °C/2 h kept the mesophilic aerobic counts within the limits established by the legislation (5.48 Log CFU/mL), with an increase in counts of psychrotrophic and proteolytic microorganisms. When stored at 7 °C/24 h and 7 °C/48 h, the count of mesophiles exceeded the established parameters. A significant increase in the count of proteolytic psychrotrophs and psychrotrophs was also observed during storage at 7 °C from 24 h. The results of this study indicate that the temperature of 7 °C is not suitable for the milk conservation, since it was not able to control the microbial multiplication. Thus, the results contribute to the change in milk storage temperature proposed by the new Brazilian legislation.(AU)

A qualidade do leite cru depende da contaminação microbiana inicial e das condições de armazenamento até o processamento na indústria. Considerando a influência do tempo e da temperatura de armazenamento na microbiota do leite cru, o objetivo deste trabalho foi quantificar e monitorar a multiplicação desses grupos de microrganismos sob diferentes condições. Para tanto, foram coletadas 41 amostras de leite cru imediatamente após a ordenha, armazenadas nas seguintes condições de armazenamento: 25 °C/2 h; 35 °C/2 h; 7 °C/24 h; 7 °C/48 h e 7 °C/60 h para análise de microrganismos psicrotróficos, aeróbios mesófilos, psicrotróficos e proteolíticos. As amostras de leite analisadas no estudo apresentaram uma contagem média inicial de aeróbios mesófilos de 5.38 Log UFC/mL no Tempo Zero. O leite armazenado a 25 °C/2 h e 35 °C/2 h manteve as contagens aeróbias mesófilas dentro dos limites estabelecidos pela legislação (5,48 Log UFC/mL), com aumento nas contagens de microrganismos psicrotróficos e proteolíticos. Quando armazenado a 7 °C/24 h e 7 °C/48 h a contagem de mesófilos excedeu os parâmetros estabelecidos. Um aumento significativo na contagem de psicrotróficos e psicrotróficos proteolíticos também foi observado durante o armazenamento a 7 °C a partir das 24 h. Os resultados deste estudo indicam que a temperatura de 7 °C não é adequada para a conservação do leite, uma vez que não foi capaz de controlar a multiplicação microbiana. Assim, os resultados contribuem para a mudança na temperatura de armazenamento de leite proposta pela nova legislação brasileira.(AU)

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...(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...(AU)

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)

Multispecies biofilm formation by the contaminating microbiota in raw milk.

Biofilms can be formed on the surfaces of dairy processing equipment and are a potential source of product contamination. This study evaluated the diversity of multispecies biofilms formed on stainless steel (SS) due to the contaminating microbiota in raw milk. Samples of raw milk were used: one was fresh milk and the other maintained in refrigerated bulk tanks for up to 48 h. The mesophilic aerobic contamination was ∼104 CFU ml-1 in fresh milk and 106 CFU ml-1 in bulk milk. SS coupons were kept immersed in the milk at 7 ±2 °C for 10 days, and every two days, the raw milk was changed for samples of the same origin collected on the current day. After incubation for 10 days, sessile cells in the biofilm reached 105 CFU cm-2 in the presence of fresh milk, and 106 CFU cm-2 in the presence of bulk milk. The genetic diversity analysis showed that Gammaproteobacteria and Bacilli predominated in the biofilms throughout the incubation of both milk samples and these biofilms showed a reduction in diversity over time. The main classes of bacteria found in these biofilms have representatives of great importance since many of them have spoilage potential.

Effect of Alpinia zerumbet essential oil on the shelf life of tambaqui fillets during short-term refrigerator storage / Efeitos do óleo essencial de Alpinia zerumbet sobre a vida útil de filés resfriados de tambaqui

We investigated the effect of Alpinia zerumbet essential oil on the quality and shelf life of tambaqui (Colossoma macropomum) fillets stored under refrigeration (10.0 ± 0.5 °C) for 14 days. The treatments were A. zerumbet essential oil at 0.75% v v-1 (AEO 0.75%), A. zerumbet essential oil at 1.5% v v-1 (AEO 1.5%) and a control (no essential oil). The sample quality and shelf life were determined by the total psychrotrophic count (TPC) and chemical parameters (pH, total volatile basic nitrogen, centesimal composition and thiobarbituric acid reactive substances - TBARS) at zero, seven and 14 days of storage time. The TPC decreased significantly (p < 0.05) with an A. zerumbetessential oil level of 1.5% until seven days of storage. The concentration of A. zerumbet essential oil at 0.75% resulted in lower pH, TBARS, and TVBN values in comparison with the other treatment and the control. Thus, A. zerumbet essential oil was efficient in extending the shelf life of refrigerated tambaqui fillets up to approximately seven days.(AU)

Investigamos o efeito do óleo essencial de Alpinia zerumbet sobre a qualidade e vida útil de filés de tambaqui (Colossoma macropomum) armazenados sob refrigeração (10,0 ± 0,5 °C) durante 14 dias. Os tratamentos foram óleo essencial de A. zerumbet (OEA) a 0,75% v v-1 (OEA 0,75%) e 1,5% v v 1 (OEA 1,5%), além de um controle (sem OEA). A qualidade e vida útil das amostras foram determinadas por contagem psicrotrófica total (CPT) e parâmetros químicos (pH, bases nitrogenadas voláteis totais, composição centesimal e substâncias reativas ao ácido tiobarbitúrico - SRATB), nos dias zero, sete e 14 de armazenamento. O TPC diminuiu significativamente (p < 0,05) com óleo essencial de A. zerumbet a 1,5% por até sete dias de armazenamento. O tratamento contendo OEA a 0,75% resultou em menores valores de pH, SRTAB e BNV-T em comparação ao outro tratamento e ao controle. Portanto, o óleo essencial de A. zerumbet foi eficiente para prolongar a vida útil do filé de tambaqui por aproximadamente sete dias.(AU)

Effect of Alpinia zerumbet essential oil on the shelf life of tambaqui fillets during short-term refrigerator storage

We investigated the effect of Alpinia zerumbet essential oil on the quality and shelf life of tambaqui (Colossoma macropomum) fillets stored under refrigeration (10.0 ± 0.5 °C) for 14 days. The treatments were A. zerumbet essential oil at 0.75% v v-1 (AEO 0.75%), A. zerumbet essential oil at 1.5% v v-1 (AEO 1.5%) and a control (no essential oil). The sample quality and shelf life were determined by the total psychrotrophic count (TPC) and chemical parameters (pH, total volatile basic nitrogen, centesimal composition and thiobarbituric acid reactive substances - TBARS) at zero, seven and 14 days of storage time. The TPC decreased significantly (p < 0.05) with an A. zerumbetessential oil level of 1.5% until seven days of storage. The concentration of A. zerumbet essential oil at 0.75% resulted in lower pH, TBARS, and TVBN values in comparison with the other treatment and the control. Thus, A. zerumbet essential oil was efficient in extending the shelf life of refrigerated tambaqui fillets up to approximately seven days. (AU)

Pseudomonas spp. and other psychrotrophic microorganisms in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic and AprX production potential

ABSTRACT: 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.

RESUMO: 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.

Relative expression of stress-related genes during acclimation at low temperature of psychrotrophic Oenococcus oeni strains from Patagonian wine.

In the present study, we evaluated the transcriptional response of four stress-related genes in three Oenococcus oeni strains after acclimation at two different temperatures. Gene expression was analyzed at time zero and after 48 h acclimation at 18 and 21 °C. After the acclimation period cells were inoculated into sterile Pinot noir wine and MLF was followed for 25 days to investigate if different acclimation temperatures could influence cell survival and MLF performance. L-malic acid consumption, population survival, and transcriptional behavior were different upon the acclimation temperature. rmlB and hsp20 genes presented a considerable increase in their expression level when strains were acclimated at 18 °C particularly in the psychrotrophic strains UNQOe19 and UNQOe4 isolated from Patagonian Pinot noir wine in comparison with the control strain (ATCC 27310). The increase in rmlB and hsp20 expression could account for the better survival of these strains in Pinot noir in comparison with the control strain. In addition, Patagonian populations acclimated at 18 °C were able to consume a higher percentage of L-malic acid in comparison with cells acclimated at 21 °C. Our results suggest that gene expression analysis of cells acclimated at sub-optimal temperatures could benefit the selection of psychrotrophic strains aimed as starter cultures.

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)