Evaluation of biogenic amines producing potential of bacteria associated with proteinous food

@#Aim: The aim of this study was to assay for the biogenic amine-producing capacity of bacteria isolated from proteinous food. Methodology and results: Previously characterized bacterial isolates (Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae) obtained from proteinous food samples (smoked fish and yoghurt) were subjected to proteolytic analysis using nutrient agar supplemented with 0.2 g/mL casein and decarboxylase activity using nutrient broth supplemented with 0.004 g/mL amino acids (histidine, tyrosine, asparagines, leucine and lysine). Isolates that expressed proteolytic and decarboxylase activities were screened for biogenic amine producing capacity using decarboxylase broth which was supplemented with an amino acid (tyrosine). Biogenic amines obtained in this research were classified into primary amine and secondary amine based on their qualitative characteristics. Confirmatory and quantitative analysis of biogenic amines produced was done using high-performance liquid chromatography. The confirmatory screening revealed the presence of methylamine, ethylamine, putrescine, cadaverine, histamine, spermidine, phernylethylamine, spermine, agmatine, tyramine, dopamine, tryptamine, norepinephrine and serotonin respectively. Total biogenic amines produced by S. aureus was 70.12 mg/kg, K. pneumoniae (62.58 mg/kg) and E. coli (56.57 mg/kg) respectively. Conclusion, significance and impact of study: Enzymatic decarboxylation of free amino acids and other metabolic processes by the test organisms (S. aureus, E. coli and K. pneumoniae) leads to production of biogenic amines which can be used as a quality indicator in food in terms of degree of spoilage, use of non-hygienic raw material and poor manufacturing environment. Thus, effect of biogenic amines obtained in this research would be determined by individual toxicological threshold which can be extremely variable from few mg/kg in sensitive person to several hundred mg/kg in healthy person. The concentrations of each biogenic amine quantified are within the limit but their toxic effects depend on the type of amine, the presence of modulating compounds and the efficiency of an individual’s detoxification mechanism.

Clinical Efficacy of Modified Qingjin Huatan Tang on Bronchiectasis with Syndrome of Phlegm-heat Accumulating Lung at Acute Exacerbation / 中国实验方剂学杂志

<b>Objective::To observe the clinical efficacy of modified Qingjin Huatan Tang on bronchiectasis with syndrome of phlegm-heat accumulating lung at acute exacerbation and its inhibitory effect on pro-inflammatory factors and proteolytic activity. <b>Method::One hundred and thirty patients were randomly divided into control group and observation group by random number table. Patients in control group got tazobactam sodium and piperacillin sodium for injection, 3.375 g/time, 1 time/6 hours, and the types of antibiotics were regulated according to the bacterial culture results. And patients in control group also got Ambroxol Hydrochloride injection, 30 mg/time, 2 time/days, and postural drainage. In addition to the therapy of control group, patients in observation group were also given modified Qingjin Huatan Tang, 1 dose/day. Before and after treatment, symptoms and signs were scored. And levels of white blood cell count (WBC), neutrophile granulocyte (GRAN), C-reactive protein (CRP), procalcitonin (PCT) were detected. And scores of forced expiratory volume in one second (FEV₁), forced vital capacity (FVC), peak expiratory flow rate (PEFR) and BODE were graded. And levels of tumor necrosis factor-<italic>α</italic> (TNF-<italic>α</italic>), interleukin-4 (IL-4), IL-6 and IL-8 in sputum, peripheral neutrophil elastase (NE) and cathepsin G were detected. <b>Result::By rank sum test, the clinical efficacy in observation group was better than that in control group (Z=2.086, <italic>P</italic><0.05), while scores of symptoms and signs in observation group were lower than those in control group (<italic>P</italic><0.01). WBC, GRAN, CRP, PCT, airflow limitation (O), dyspnea (D), motor ability (E) score, BODE index, TNF-<italic>α</italic>, IL-4, IL-6, IL-8, plasma NE and cathepsin G were all lower than those in control group (<italic>P</italic><0.01). And levels of FEV₁, FVC, PEF and FEV₁/FVC were higher than those in control group (<italic>P</italic><0.01). <b>Conclusion::In addition to routine anti-infection and expectoration western medicine therapy, modified Qingjin Huatan Tang can be added to control symptoms and signs, alleviate the degree of illness, improve pulmonary function and the quality of life of patients, and inhibit expression of airway pro-inflammatory factor and proteolysis, with a better clinical efficacy than pure western medicine.

Proteolytic activity of excretory/secretory products of Cochliomyia hominivorax larvae (Diptera: Calliphoridae) / Atividade proteolítica de produtos de excreção/secreção de Cochliomyia hominivorax (Diptera: Calliphoridae)

The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30µg, 40µg and 50µg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.(AU)

Os perfis protéicos e a atividade proteolítica dos produtos de excreção/secreção (PE/S) das larvas de primeiro (L1), segundo (L2) e terceiro (L3) estágios de Cochliomyia hominivorax foram estudados em laboratório. Os perfis protéicos foram obtidos por eletroforese em géis de poliacrilamida (SDS-PAGE). Os PE/S de cada fase larval (L1, L2 e L3), tratados com inibidores de proteases, contendo 30µg, 40µg e 50µg de proteína, foram aplicados em géis de poliacrilamida a 10%. A atividade proteolítica dos PE/S na sua forma nativa, foi analisada em géis co-polimerizados com gelatina e por testes colorimétricos usando a azocaseína como substrato, com a caracterização das proteases feita por meio de inibidores sintéticos. Diferentes perfis protéicos foram observados para os instares larvais, com L1 apresentando o perfil mais complexo. Apesar disso, foram observadas várias bandas protéicas comuns a todos os estágios larvais. Os PE/S de todos os instares mostraram atividade proteolítica sobre a gelatina, evidenciada por zonas de proteólise, com predominância de massas moleculares aparentes mais altas em L1, enquanto que para L2 e L3 as zonas de proteólise puderam ser observadas também em regiões de menores massas. Os testes com inibidores de proteases usando a gelatina como substrato mostraram que os PE/S de L1, L2 e L3 eram compostos principalmente de serina-proteases. Adicionalmente, inibição foi observada nos PE/S de L2 tratada previamente com EDTA, um inibidor de metalo-proteases. Os ensaios com a zocaseína revelaram um aumento gradual da atividade proteolítica sobre este substrato com o progresso do desenvolvimento larval, com a mais forte inibição sendo observada após o tratamento com 3,4 dicloroisocumarina (DCI) para os PE/S de L1, L2 e L3. Estes resultados sugerem que as larvas de C. hominivorax produzem diferentes proteases, fato que pode estar relacionado a processos vitais para a sobrevivência do parasita, tais como a penetração nos tecidos dos hospedeiros e nutrição durante os estágios larvais.(AU)

A review on the Scorpaena plumieri fish venom and its bioactive compounds

The most poisonous fish species found along the Brazilian coast is the spotted scorpionfish Scorpaena plumieri. Though hardly ever life-threatening to humans, envenomation by S. plumieri can be quite hazardous, provoking extreme pain and imposing significant socioeconomic costs, as the victims may require days to weeks to recover from their injuries. In this review we will walk the reader through the biological features that distinguish this species as well as the current epidemiological knowledge related to the envenomation and its consequences. But above all, we will discuss the challenges involved in the biochemical characterization of the S. plumieri venom and its compounds, focusing then on the successful isolation and pharmacological analysis of some of the bioactive molecules responsible for the effects observed upon envenomation as well as on experimental models. Despite the achievement of considerable progress, much remains to be done, particularly in relation to the non-proteinaceous components of the venom. Therefore, further studies are necessary in order to provide a more complete picture of the venoms chemical composition and physiological effects. Given that fish venoms remain considerably less studied when compared to terrestrial venoms, the exploration of their full potential opens a myriad of possibilities for the development of new drug leads and tools for elucidating the complex physiological processes.

A review on the Scorpaena plumieri fish venom and its bioactive compounds

The most poisonous fish species found along the Brazilian coast is the spotted scorpionfish Scorpaena plumieri. Though hardly ever life-threatening to humans, envenomation by S. plumieri can be quite hazardous, provoking extreme pain and imposing significant socioeconomic costs, as the victims may require days to weeks to recover from their injuries. In this review we will walk the reader through the biological features that distinguish this species as well as the current epidemiological knowledge related to the envenomation and its consequences. But above all, we will discuss the challenges involved in the biochemical characterization of the S. plumieri venom and its compounds, focusing then on the successful isolation and pharmacological analysis of some of the bioactive molecules responsible for the effects observed upon envenomation as well as on experimental models. Despite the achievement of considerable progress, much remains to be done, particularly in relation to the non-proteinaceous components of the venom. Therefore, further studies are necessary in order to provide a more complete picture of the venom's chemical composition and physiological effects. Given that fish venoms remain considerably less studied when compared to terrestrial venoms, the exploration of their full potential opens a myriad of possibilities for the development of new drug leads and tools for elucidating the complex physiological processes.(AU)

Production and partial characterization of proteases from Mucor hiemalis URM3773 / Produção e caracterização parcial de proteases a partir de Mucor hiemalis URM3773

The current study evaluated the proteases production from 11 fungal species belonging to the genera Mucor, Rhizomucor and Absidia. The species were obtained from the Collection of Cultures URM at the Mycology Department-UFPE, Brazil. The best producing species was Mucor hiemalis URM 3773 (1.689 U mL-1). Plackett-Burman design methodology was employed to select the most effective parameter for protease production out of 11 medium components, including: concentration of filtrate soybean, glucose, incubation period, yeast extract, tryptone, pH, aeration, rotation, NH4Cl, MgSO4 and K2HPO4. Filtrated soybean concentration was the significant variable over the response variable, which was the specific protease activity. The crude enzyme extract showed optimal activity in pH 7.5 and at 50ºC. The enzyme was stable within a wide pH range from 5.8 to 8.0, in the phosphate buffer 0.1M and in stable temperature variation of 40-70ºC, for 180 minutes. The ions FeSO4, NaCl, MnCl2, MgCl2 and KCl stimulated the protease activity, whereas ZnCl2 ion inhibited the activity in 2.27%. Iodoacetic acid at 1mM was the proteases inhibitor that presented greater action.The results indicate that the studied enzyme have great potential for industrial application.

Foi avaliada a produção de proteases por 11 espécies fúngicas pertencentes aos gêneros Mucor, Rhizomucor e Absidia, obtidas da Coleção de Culturas URM do Departamento de Micologia- UFPE, Brasil. A melhor espécie produtora foi Mucor hiemalis URM3773 (1,689 U mL-1). A metodologia de planejamento Plackett-Burman foi empregada para selecionar o parâmetro mais efetivo para a produção de proteases através de 11 componentes do meio, incluindo: concentração do filtrado de soja, glicose, período de incubação, extrato de levedura, triptona, pH, aeração, rotação, NH4Cl, MgSO4 e K2HPO4. A variável significante sobre a variável- resposta, atividade proteásica específica, foi a concentração do filtrado de soja. O extrato enzimático bruto apresentou atividade ótima ao pH 7,5 a 50ºC. A enzima foi estável em uma ampla variação de pH de 5,8­8,0 em tampão fosfato 0,1M e termicamente estável a uma variação de 40-70°C, durante 180 minutos. Os íons FeSO4, NaCl, MnCl2, MgCl2 e KCl estimularam a atividade proteásica, enquanto que o íon ZnCl 2 inibiu 2,27% da atividade. O inibidor de proteases que teve maior ação foi o ácido iodoacético a 1mM. Os resultados obtidos indicam que a enzima estudada tem grande potencial de aplicação industrial.

Potencial do latex da fruta pão (Artocarpus altilis) como agente coagulante do leite

Breadfruit is an exotic tree in Brazil, very well acclimatized. Despite of being a laticifer plant, the knowledge about its latex is scarce. However, it is known that proteolytic enzymes represents over 50% of the latex composition in laticifers plants. The aim of this study was to evaluate the potential of breadfruit (Artocarpus altilis var. Apyrena) latex as a source of milk clotting proteases and partially characterize it. The proteolytic activity of the fraction of crude extract was assessed using azocasein and quantitation of total protein, the bicinchoninic acid (BCA). Milk-clotting activity was analyzed using skim milk at 12%. The enzyme activity was analyzed under different temperature conditions (35 to 80°C) and pH (5.8 to 10.7) presenting optimum activity in alkaline pH (8.5) and 50°C; being stable to the two variables at 120 minutes during the test. The clotting activity was directly proportional to the temperature at the better concentration of CaCl2 (10mmol L-1). The results indicate that enzyme is a possible replacement for calf rennet.

A fruta-pão é uma árvore exótica no Brasil, onde se aclimatou muito bem. Embora seja uma planta lactífera, o conhecimento sobre seu látex é escasso. No entanto, sabe-se que enzimas proteolíticas correspondem a mais de 50% da composição do látex em plantas lactíferas. O objetivo deste estudo foi avaliar o potencial do látex da fruta-pão (Artocarpus altilis var. Apyrena) como fonte de protease coagulante do leite e caracterizar parcialmente a enzima. A atividade proteolítica da fração de extrato bruto foi avaliada utilizando azocaseína e, para a quantificação das proteínas totais, o ácido bicinconínico (BCA). A atividade de coagulação do leite foi testada utilizando leite desnatado a 12%. A protease foi testada em diferentes condições de temperatura (35 a 80°C) e pH (5,8 a 10,7) e apresentou atividade ótima a 50°C e pH alcalino (8,5) sendo estável a estas variáveis durante 120 minutos. A atividade coagulante no leite foi diretamente proporcional à temperatura na melhor concentração de CaCl2 a 10µmol L-1. Os resultados indicam que a enzima analisada é uma possível alternativa à quimosina.

Characterization of Trypsin-Like Serine Protease from Lethocerus indicus Salivary Venom and its Cytotoxic Effect against Human Epidermoid Carcinoma Cell, A431.

Objective: Lethocerus indicus salivary venom characterization and evaluation of extracellular degradation activity and cytotoxic effect against native human collagen type 1 and epidermoid carcinoma cell, A431. Method: Salivary venom extract was collected from adult insects by injecting 2% pilocarpine of 50 μml. Enzyme presence was detected by the apiZYM assay. The proteolytic activity was tested by the photometric and zymogram methods using specific fluorescent substrates and inhibitors. The cytotoxic activity was determined by the MTT assay and Trypan blue exclusion method. Apoptosis induction was observed using AO/EB staining solution. Digestion of extracellular matrix protein was detected against native human type I collagen. Result: L. indicus salivary venom presents amylases, proteases, carbohydrases, phosphatases and lipases. Among them, protease enzyme showed highest composition. The highest rate of proteolytic activity observed at pH 8 in 35ºC (100 %). Serine proteases present predominantly in salivary venom. Cysteine and metalloproteases are also detected. The activation energy of salivary venom is 49.86 kJ. Use of serine inhibitor, PMSF inhibited 92.77% which indicated that the maximum activity was due to serine protease. Detection of trypsin-like protease was confirmed by using PMSF and TLCK with specific substrate, BApNA. It shows significant inhibitions, 82% and 78% respectively suggesting maximum influence in salivary venom. Degradation of the fibrillar native state collagen Type I into 8 smaller peptide bands showed it importance in medical application. IC50 concentration of venom that induces cytotoxicity in epidermoid carcinoma cells, A431was 2.3 μg/ml only. It gives prominent apoptotic features such as cytoplasmic membrane blebbing, nuclear contraction, nuclear fragmentation and contact inhibition. Conclusion: We suggest that further investigation of the venom will lead to identification of active compound in L. indicus salivary venom for its potential use in therapeutic application.

Effect of citric acid on the proteolytic activity of Zea mays L. / Efeito do ácido cítrico sobre a atividade proteolítica de Zea mays L.

Hybrids of Zea mays L. (Buffalo, Falcon, H360 y HV313) were treated with citric acid (2000 ppm). Grain yield, soluble protein and proteolytic activity were monitored when the crop reached physiological maturity. Citric acid was applied before the appearance of the flag leaf, and induced an increase in the grain yield from 4222 to 5780 kg/ha, in the soluble protein from 6.34 to 7.91 mg/mg and into the proteolytic activity from 14.3 to 65.7 µU mg prot-1. This is an increase of 2 to 12 times in the Falcon, H360 and HV313 hybrids, while the Buffalo hybrid responded with less intensity to the treatment with citric acid. In the H360 hybrid treated with citric acid, an increase in the proteolytic activity of aspartyl serine proteases was observed. The results indicate that citric acid differentially induces proteolytic activity and vigor in the corn hybrids analyzed.

Zea mays L. híbridos (Buffalo, Falcão, H360 e HV313) foram tratados com ácido cítrico (2000 ppm). O rendimento de grãos, proteína solúvel e atividade proteolítica foram monitorados na fase de maturação fisiológica do cultivo. O ácido cítrico aplicado antes do aparecimento da folha bandeira induziu um aumento na produção de grãos 4222 a 5780 kg/ha, na proteína solúvel de 6.34 7.91 mg/mg de peso seco e atividade proteolítica de 14.3 to 65.7 µU mg prot-1, esto é, um incremento de 2-12 vezes nos híbridos Falcao, H360 e HV313, enquanto o híbrido Buffalo responderam com menor intensidade ao tratamento com ácido cítrico. No híbrido H360, tratadas com ácido cítrico, apresentou-se um aumento na atividade proteolítica de aspartil proteases e serin protease. Os resultados indicam que o ácido cítrico diferencialmente induz a atividade proteolítica e o vigor de híbridos de milho testados.

Maternal and fetal indicators of oxidative stress during Pregnancy-Induced Hypertension (PIH).

The present study demonstrates the possibility of increased lipid peroxidation and protein oxidation in both maternal and fetal erythrocytes as markers of oxygen radical activity during pregnancy induced hypertension (PIH). The erythrocyte malondialdehyde (MDA) levels were significantly elevated in mothers with PIH when compared to controls (p<0.001). The endogenous protein damage due to oxidative stress was significantly higher in mothers with PIH when compared to controls (p<0.01). Similarly the proteolytic activity in erythrocyte lysates against oxidatively damaged hemoglobin was significantly increased in mothers with PIH compared to controls (p<0.001). In babies born to mothers with PIH, erythrocyte MDA levels were significantly elevated in comparison those of normal newborns (p<0.01). Both the endogenous oxidative protein damage and erythrocyte proteolytic activity were significantly higher in newborns born to mothers with PIH than in newborns in the control group (p<0.01). The results of this study indicate that oxidative stress is induced both in mothers with PIH as well as their babies which is manifested as increased lipid peroxidation and protein oxidant damage.

Response surface analysis on the effect of temperature and pH on Growth and proteolytic activity of thermophilic Bacillus sp

Proteolytic activity and cell biomass of thermophilic Bacillus sp. strain were evaluated at various levels of initial pH and temperature by applying response surface methodology. The mineral medium containing yeast extract (0.01 percent) and starch (1 percent) was used throughout the experiment. The results of statistical analysis revealed the polynomial model with high coefficient of determination (R² = 0.8) for the biomass and total proteolytic activity of the strain studied. This model showed a satisfactory adjustment of the statistic model with the experimental data. The p values showed that the temperature and pH had significant effect on biomass and proteolytic activity (P<0.05) of strain tested. The highest proteolytic activity (2.333 U/ml/h) of the Bacillus sp. was predicted at 41º C and pH 4.8. The high biomass values were observed at broad range of temperature and pH.

Atividade proteolítica total e biomassa de uma linhagem de Bacillus sp. termofílico foram analisados em vários níveis de pH inicial e temperatura utilizando a metodologia de superfície de resposta. O meio mineral com extrato de levedura (0.01 por cento) e amido (1 por cento) foi utilizado no experimento. Os resultados de análise estatística da metodologia de resposta de superfície definiram um modelo polinomial para a biomassa e atividade proteolítica da linhagem de Bacillus sp. com alto coeficiente de determinação (R² = 0.8), mostrando um ajuste satisfatório do modelo estatístico obtido com os dados experimentais. Os valores de p mostraram que a temperatura e pH tiveram efeito significante em biomassa e atividade total (P <0.05) da linhagem testada. A atividade proteolítica mais alta (2.368 U/ml/h) da linhagem de Bacilus sp. foi prevista pelas curvas de superfície de resposta em temperatura a 41º C e pH igual a 4.8. Os valores de biomassa altos foram previstos para ampla faixa de temperatura e pH.

The proteolytic activity of Pseudomonas fluorescens 07A isolated from milk is not regulated by quorum sensing signals

The proteolytic activity of Pseudomonas fluorescens 07A was investigated, and was optimal on tryptone-calcium medium. N-acyl-homoserine lactones (AHLs) were not detected on supernatants of late-exponential and stationary-phase culture broths. Synthetic AHLs or bacterial cell extracts added to the medium did not influence growth or proteolytic activity suggesting that quorum sensing might not regulate protease production in this strain.

Inhibition of local effects of Indian Daboia/Vipera russelli venom by the methanolic extract of grape (Vitis vinifera L.) seeds.

Although anti-venom therapy is available for the treatment of fatal bite by snakes, it offers less or no protection against the local effects such as dermo- and myonecrosis, edema, hemorrhage and inflammation at the bitten region. The viper species are known for their violent local effects and such effects have been commonly treated with plant extracts without any scientific validation in rural India. In this investigation, the methanolic extract of grapes (Vitis vinifera L.) seed was studied against the Indian Daboia/Vipera russelli venom-induced local effects. The extract abolished the proteolytic and hyaluronidase activities and also efficiently neutralized the hemorrhage, edema-inducing and myonecrotic properties of the venom. In addition, the extract also inhibited partially the pro-coagulant activity of the venom and abolished the degradation of Aα and Bβ chains of human fibrinogen. Thus, the extract possesses potent anti-snake venom property, especially against the local effects of viper bites.

Study on the proteolytic activity of pepsin, papain and their mixture

The reasonable pepsin + papain mix was studied. The results showed that the proteolytic activity of the mix at 3 areas pH: 20.5, 41 and 70.5 and temperature 38O2 was compared with either agent alone in the same conditions. These mixes will be improved and applied to the treatment of malnutrition in children

Studies on plant gums. Proteases in neem (Azadirachta indica) gum.

Proteolytic activity was detected in neem (Azadirachta indica) exudate gum when tested with casein and albumin as substrates. The enzyme activity was separated into two fractions by chromatography on TEAE-cellulose after EDTA treatment. Both the enzyme fractions were fairly stable to high temperatures and wide range of pH conditions. The pH optima were found to be around 6·5. Phenylmethyl sulphonylfluoride inhibited the activity of both the fractions. EDTA, ß-mercaptoethanol, tosylamide phenylethylchloromethylketone, tosyllysine chloroimethylketone, p-chloromercuribenzoate and dithiobis-2-nitrobenzoie acid did not affect the activity of the two enzyme fractions. The two fractions had no hydrolytic action on a variety of synthetic substrates tested.