Properties of thermostable extracellular FOS-producing fructofuranosidase from <Emphasis Type="Italic">Cryptococcus</Emphasis> sp.

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, showing a substrate inhibitory model. The K _m, K _i and v _max values were shown to be 64 mM, 3 M and 159.6 μmol mL⁻¹ min⁻¹ for FA and 131 mM, 1.6 M and 377.8 μmol mL⁻¹ min⁻¹ for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 °C, while the best stability was achieved at pH 4.5 and temperatures below 60 °C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).     

Evaluation of Functional Properties in Protein Hydrolysates from Bluewing Searobin (Prionotus punctatus) Obtained with Different Microbial Enzymes

Enzymatic hydrolysis of proteins from low commercial value fish could be produced for uses like functional ingredients in a wide and always increasing zone of application in different food products. The objective of this work was to evaluate the functional properties and the amino acid profile of enzymatic hydrolysates from Bluewing searobin (Prionotus punctatus), using two microbial enzymes, Alcalase and Flavourzyme. The enzymatic hydrolysate obtained through the addition of the enzyme Alcalase reached the maximum solubility (42%) at pH 9, water holding capacity (WHC) of 2.4 g_water  g_protein ⁻¹, 4.5 g_oil g_protein ⁻¹ of oil holding capacity (OHC) and an emulsifying activity index (EAI) of 54 m² g_solids ⁻¹ at pH 3. On the other hand, the hydrolysate obtained from Flavourzyme attained 38% of solubility at pH 9, 3.7 g_water  g_protein ⁻¹ and 5.5 g_oil g_protein ⁻¹ for the holding capacities, and an EAI of 71 m² g_solids ⁻¹ at pH 11. The hydrolysate with Flavourzyme produced best results for WHC, OHC, and EAI because it had solubility lower than the hydrolysate of Alcalase. The hydrolysate produced by Alcalase had a higher amino acid content compared with Flavourzyme’s hydrolysate. However, both showed a good essential amino acid amounts. In general, these results indicate the potential utilization of the hydrolysate from Bluewing searobin in food formulations for the direct human consumption.     

Optimization and Modeling of Process Parameters for Lipase Production by Bacillus brevis

Statistical evaluation of fermentation conditions and nutritional factors by Plackett–Burman two-level factorial design followed by optimization of significant parameters using response surface methodology for lipase production by Bacillus brevis was performed in submerged batch fermentation. Temperature, glucose, and olive oil were found to be the significant factors affecting lipase production. Maximum lipase activity of 5.1 U ml⁻¹ and cell mass of 1.82 g l⁻¹ at 32 h were obtained at the optimized conditions of temperature, 33.7 °C; initial pH, 8; and speed of agitation, 100 rpm, with the medium components: olive oil, 13.73 ml l⁻¹; glucose, 13.98 g l⁻¹; peptone, 2 g l⁻¹; Tween 80, 5 ml l⁻¹; NaCl, 5 g l⁻¹; CH₃COONa, 5 g l⁻¹; KCl, 2 g l⁻¹; CaCl₂·2H₂O, 1 g l⁻¹; MnSO₄·H₂O, 0.5 g l⁻¹; FeSO₄·7H₂O, 0.1 g l⁻¹; and MgSO₄·7H₂O, 0.01 g l⁻¹. The lipase productivity and specific lipase activity were found to be 0.106 U (ml h)⁻¹ and 2.55 U mg⁻¹, respectively. Unstructured kinetic models and artificial neural network models were used to describe the lipase fermentation. The kinetic analysis of the lipase fermentation by B. brevis shows that lipase is a growth-associated product.     

Angiotensin-I converting enzyme inhibitory and antioxidant activities of protein hydrolysates from Phaseolus lunatus and Phaseolus vulgaris seeds

Phaseolus lunatus and Phaseolus vulgaris protein concentrates were hydrolyzed with the enzymes Alcalase^® and Flavourzyme^® at different reaction times, and the angiotensin-I converting enzyme (ACE-I) inhibitory activity, antioxidant properties and amino acid composition measured in the hydrolysates. With Alcalase^®, the highest degree of hydrolysis (DH) in P. lunatus was 37.94% at 45 min, and in P. vulgaris was 49.48% at 30 min. With Flavourzyme^®, the highest DH's were 22.03% and 26.05%, respectively, both at 90 min. ACE-I inhibitory activity in the Alcalase^® hydrolysates was IC₅₀ = 0.056 mg mL⁻¹ for P. lunatus at 90 min, and IC₅₀ = 0.061 mg mL⁻¹ for P. vulgaris at 60 min. In the Flavourzyme^® hydrolysates this activity was IC₅₀ = 0.0069 mg mL⁻¹ for P. lunatus at 90 min and IC₅₀ = 0.127 mg mL⁻¹ for P. vulgaris at 45 min. In SDS-PAGE, the hydrolysates exhibited low molecular weight bands. Antioxidant activity was 11.55 mmol L⁻¹ TEAC mg⁻¹ protein for P. lunatus with Flavourzyme^® at 90 min and 10.09 mmol L⁻¹ TEAC mg⁻¹ protein for P. vulgaris with Alcalase^® at 60 min. Amino acid composition exhibited high amino acid hydrophobic residues content.     

Acidic β-mannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application

The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035 U mg^–1 towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining > 60% of its maximum activity at pH 3.0–7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The K_m and V_max values were 5.6 and 4.8 mg mL^–1, and 2785 and 1608 μmol min^–1 mg^–1, respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96 mg g^–1 reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed.     

Advantages of a proteolytic extract by Aspergillus oryzae from fish flour over a commercial proteolytic preparation

Proteolytic extract (CPE) was produced by Aspergillus oryzae 2095 under solid state fermentation using a mix of fish flour with polyurethane foam (70:30, w/w) of particle size of 0.5 mm. The proteolytic activity from CPE was compared to a commercial protease (Flavourzyme 500 MG^®). The maximal activity for both extracts was found at pH 8 and 50 °C. The half-lives of CPE and Flavourzyme 500 MG^® were 52 and 25 min at 50 °C, respectively. Furthermore, the hydrolytic activity for both extracts was tested on muscle of giant sea bass (Epinephelus morio), CPE produced a higher degree of hydrolysis (DH) than Flavourzyme 500 MG^® (22.2 and 9.1%, respectively) under optimal experimental conditions for each extract.     

Purification and properties of a heat-stable enzyme of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Rm12 from raw milk

Pseudomonas fluorescens Rm12 is a kind of Psychrotrophic bacteria growing in cold raw milk. It produced an extracellular heat resistant protease with an estimated molecular weight of 45 kDa by size exclusion chromatography and SDS-PAGE under both reducing and non-reducing conditions. The enzyme, designated Ht13, was purified to electrophoretic homogeneity from the culture supernatant by sequentially using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography and size exclusion chromatography. The specific activity of the enzyme increased 115.5-folds. The optimum pH value and temperature of Ht13 were 7.5 and 40 °C, respectively. Based on its biochemical characteristics, Ht13 can be included in the group of metalloproteases, which was inhibited by 1, 10-phenanthroline and EDTA but not by pepstatin A, chymostatin, STI, E-64, BBI, PMSF and pAPMSF. Mn²⁺ has positive effect on activity and can increased the heat resistance capability, while Ca²⁺ had a negligible effect. For the hydrolysis of azocasein, the Km was 0.012 mg mL⁻¹. The enzyme showed typical heat-stable behavior. After treatment of 160 °C 20 s, the residual activity was 9%. The half-life of the enzyme at 160 °C in buffer with Mn²⁺ was approximately 12 s. Among several main milk proteins, Ht13 can cleave α_s-casein, β-casein and κ-casein. The sequence of 1st–16th amino acids of N-terminal was MSKVKDKAIVSAAQAS, which was same as those proteases excreted from some other P. fluorescens. However, their molecular weights, the activation ion and amino acid composition were different, suggesting Ht13 from P. fluorescens Rm12 is a novel protease.     

Enzymatic hydrolysis of hard‐to‐cook bean (Phaseolus vulgaris L.) protein concentrates and its effects on biological and functional properties

Inadequate postharvest handling and storage under high temperature and relative humidity conditions produce the hard‐to‐cook (HTC) defect in beans. However, these can be raw material to produce hydrolysates with functional activities. Angiotensin I‐converting enzyme (ACE) inhibitory and antioxidant capacities were determined for extensively hydrolysed proteins of HTC bean produced with sequential systems Alcalase‐Flavourzyme (AF) and pepsin–pancreatin (Pep‐Pan) at 90 min ACE inhibition expressed as IC₅₀ values were 4.5 and 6.5 mg protein per mL with AF and Pep‐Pan, respectively. Antioxidant activity as Trolox equivalent antioxidant capacity (TEAC) was 8.1 mm  mg^?1 sample with AF and 6.4 mm  mg^?1 sample with Pep‐Pan. The peptides released from the protein during hydrolysis were responsible for the observed ACE inhibition and antioxidant activities. Nitrogen solubility, emulsifying capacity, emulsion stability, foaming capacity and foam stability were measured for limited hydrolysis produced with Flavourzyme and pancreatin at 15 min. The hydrolysates exhibited better functional properties than the protein concentrate.     

Fatty acid composition of several wild microalgae and cyanobacteria, with a focus on eicosapentaenoic, docosahexaenoic and α-linolenic acids for eventual dietary uses

A total of 13 species of microalgae and 14 strains of cyanobacteria, collected directly in the Portuguese coast and lagoons, were characterized for their fatty acid contents, focusing on two with a market potential — i.e. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA); and another already with alternative (yet somehow more expensive) natural sources — i.e. α-linolenic (ALA) acid. The purpose of this work was their eventual inclusion as additives in food or feed. ALA was the most abundant PUFA in Nannochloropsis sp. (0.616 ± 0.081 mg_FA.L_culture^{− 1}.d^{− 1}), and EPA in Phaeodactylum tricornutum (0.148 ± 0.013 mg_FA.L_culture^{− 1}.d^{− 1}); Pavlova lutheri was particularly rich in EPA (0.290 ± 0.005 mg_FA.L_culture^{− 1}.d^{− 1}) and DHA (0.140 ± 0.037 mg_FA.L_culture^{− 1}.d^{− 1}). Despite several previous reports on similar topics and encompassing some of our microalgal species, the wild nature of our strains accounts for the novelty of this work — in addition to the characterization of a few wild cyanobacteria. Eustigmatophyceae class was the best producer of ALA, while Prymnesiophyceae was the best for EPA and ALA. Nodularia harveyana exhibited the highest ALA level (0.611 ± 0.022 mg_FA.L_culture^{− 1}.d^{− 1}) and Gloeothece sp. was highest in EPA (0.030 ± 0.004 mg_FA.L_culture^{− 1}.d^{− 1}).     

Partial Purification and Characterization of Extracellular Fructofuranosidase with Transfructosylating Activity from <Emphasis Type="Italic">Candida</Emphasis> sp.

The present work was carried out with the aim to investigate some properties of an extracellular fructofuranosidase enzyme, with high transfructosylating activity, from Candida sp. LEB-I3 (Laboratory of Bioprocess Engineering, Unicamp, Brazil). The enzyme was produced through fermentation, and after cell separation from the fermented medium, the enzyme was concentrated by ethanol precipitation and than purified by anion exchange chromatography. The enzyme exhibited both fructofuranosidase (FA) and fructosyltransferase (FTA) activities on a low and high sucrose concentration. With sucrose as the substrate, the data fitted the Michaellis–Menten model for FA, showing rather a substrate inhibitory shape for fructosyltransferase activity. The K _m and v _max values were shown to be 13.4 g L⁻¹ and 21.0 μmol mL⁻¹ min⁻¹ and 25.5 g L⁻¹ and 52.5 μmol mL⁻¹ min⁻¹ for FA and FTA activities, respectively. FTA presented an inhibitory factor K _i of 729.8 g L⁻¹. The optimum conditions for FA activity were found to be pH 3.25–3.5 and temperatures around 69 °C, while for FTA, the optimum condition were 65 °C (±2 °C) and pH 4.00 (±0.25). Both activities were very stable at temperatures below 60 °C, while for FA, the best stability occurred at pH 5.0 and for FTA at pH  4.5–5.0. Despite the strong fructofuranosidase activity, causing hydrolysis of the fructooligosaccharides (FOS), the high transfructosilating activity allows a high FOS production from sucrose (44%).     

Immobilization and characterization of naringinase for the hydrolysis of naringin

The degree of hydrolysis of naringin was investigated at various temperatures (40, 50, 60 °C), enzyme concentrations (0.01–0.30 mg ml⁻¹), and pH values (2.5–5.5) for naringinase enzyme. Naringinase was immobilized on celite by simple adsorption. Naringin content was determined by HPLC method. The degree of hydrolysis of naringin showed a linear increase up to an enzyme concentration of 0.2 mg ml⁻¹ that corresponds to 82% hydrolysis. The optimum values of pH for the hydrolysis of naringin were 4.0 for free and 3.5 for immobilized enzymes. Maximum enzyme activities were found to be 70 and 60 °C for free and immobilized enzymes, respectively. The values of K _m,app and V _max,app calculated were 1.22 mM and 0.45 μmol min⁻¹ mg enzyme⁻¹ for free and 2.16 mM and 0.3 μmol min⁻¹ mg enzyme⁻¹ for immobilized enzyme, respectively. The mathematical modelling was applied to the experimental data for hydrolysis of naringin as a function of time at 30, 40 and 50 °C. The increase in temperature from 30 to 50 °C increased the rate constant 3.09 times for free enzyme. However, the rate constants found for immobilized enzyme applications did not increase in a similar trend as a function of temperature. The retained activity of celite-adsorbed naringinase was found to be 83% at their optimum conditions. The retained activity of immobilized enzyme was followed up to the fifth run and was found to be almost unchanged after the third use at optimum reaction conditions (pH 3.5, 60 °C).     

Effect of pH and Temperature on the Activity of Enzymatic Extracts from Pineapple Peel

This study was carried out to characterize a crude extract from pineapple peel after precipitation by three methods with the aim of obtaining an enzymatic extract from agro-industrial waste. The characterization of these extracts involved the determination of both protein content and specific protease activities. The effects of pH and temperature on specific protease activity and on the stability of the extracts were also evaluated. The optimal values of specific activity for the crude extract (CE) were pH 6.0 (5.76 U mg⁻¹ protein) and 7.0 (5.71 U mg⁻¹ protein) and a temperature of 70 °C (16 U mg⁻¹ protein). The average values for the relative specific activity were 17.4% (pH 3.0 to 9.0) and 42.7% (at 30, 50, and 70 °C). The ethanolic extract had the highest specific activity (10.7 U mg⁻¹ protein) in comparison to the best results obtained for the isoelectric precipitation (7.7 U mg⁻¹ protein) and the ammonium sulfate precipitation (4.7 U mg⁻¹ protein). Moreover, the ethanolic extract was more stable than the CE, retaining 60.9% and 53.7% of the initial specific activity during the evaluation of the stability at different pH and temperature values, respectively. The optimal values of pH and temperature were almost the same for the crude and the ethanolic extracts. In addition, the ethanolic extract was more stable than the CE in the experimental conditions tested in this work.     

Production and Application of Xylanase from Penicillium sp. Utilizing Coffee By-products

The lignocellulosic coffee by-products such as coffee pulp, coffee cherry husk, silver skin, and spent coffee were evaluated for their efficacy as a sole carbon sources for the production of xylanase in solid-state fermentation using Penicillium sp. CFR 303. Among the residues, coffee cherry husk was observed to produce maximum xylanase activity of 9,475 U/g. The process parameters such as moisture (50%), pH (5.0), temperature (30 °C), particle size (1.5 mm), inoculum size (20%), fermentation time (5 days), carbon source (xylose), and nitrogen source (peptone) were optimized and the enzyme activity was in the range of 19,560–20,388 U/g. The enzyme production was further improved to 23,494 U/g with steam as a pre-treatment. The extracellular xylanase from the fungal source was purified to homogeneity from culture supernatant by ammonium sulfate fractionation, DE32-cellulose with a recovery yield of 25.5%. It appeared as a single band on SDS-PAGE gel with a molecular mass of approximately 27 kDa. It had optimum parameters of 50 °C temperature, pH 5.0, K _m 5.6 mg/mL, and V _max 925 μmol mg⁻¹ min⁻¹ with brichwood xylan as a substrate. The crude enzyme hydrolysed lignocellulosic substrate as well as industrial pulp. Production of xylanase utilizing coffee by-products constitutes a renewable resource and is reported for the first time.     

Electrochemical sensor based on Prussian blue nanorods and gold nanochains for the determination of H<Subscript>2</Subscript>O<Subscript>2</Subscript>

In this paper, a novel electrochemical sensor for the detection of H₂O₂ was proposed based on gold nanochains and prussian blue nanorods (PB@MWCNTs), which were synthesized with multiwall carbon nanotubes (MWCNTs) as a template. With the introduction of MWCNTs and the gold nanochains, the proposed system shows synergy among the Prussian blue (PB), MWCNTs, and the gold nanochains, which amplifies the H₂O₂ sensitivity greatly. A linear range from 1.75 × 10⁻⁶ to 1.14 × 10⁻³ M with a detection limit of 0.5 × 10⁻⁶ M (S/N = 3) and a high sensitivity 300 μA mM⁻¹ cm⁻² for H₂O₂ detection is obtained. Moreover, the sensor exhibits good repeatability and stability.     

Properties of <Emphasis Type="Italic">Aspergillus subolivaceus</Emphasis> free and immobilized dextranase

Aspergillus subolivaceus dextranase is immobilized on several carriers by entrapment and covalent binding with cross-linking. Dextranase immobilized on BSA with a cross-linking agent shows the highest activity and considerable immobilization yield (66.7%). The optimum pH of the immobilized enzyme is shifted to pH 6.0 as compared with the free enzyme (pH 5.5). The optimum temperature of the reaction is resulted at 60 °C for both free and immobilized enzyme. Thermal and pH stability are significantly improved by the immobilization process. The calculated K _m of the immobilized dextranase (14.24 mg mL⁻¹) is higher than that of the free dextranase (11.47 mg mL⁻¹), while V _max of the immobilized enzyme (2.80 U μg protein⁻¹) is lower than that of the free dextranase (11.75 U μg protein⁻¹). The immobilized enzyme was able to retain 76% of the initial catalytic activity after 5.0 cycles.     

Analytical performance and characterization of antibody immobilized magnetoelastic biosensors

This article presents the results of an investigation into the enhancement of sensitivity and thermal stability of polyclonal antibody immobilized magnetoelastic biosensors. The Langmuir–Blodgett (LB) monolayer technique was employed for antibody (specific to Salmonella sp.) immobilization on rectangular shaped strip magnetoelastic sensors. Biosensor performance was investigated by exposing to graded concentrations (5 × 10¹–5 × 10⁸ cfu mL⁻¹) of Salmonella typhimurium solutions in a flow through mode. Bacterial binding to the antibody on the sensor surfaces changed the resonance parameters, and these changes were quantified by the sensor’s resonance frequency shift. An increase in the sensitivity from 159 Hz decade⁻¹ for a 2 mm sensor to 246 Hz decade⁻¹ for a 1 mm sensor was observed during the dose–response measurements. The stability of the biosensor was also investigated by storing the biosensor at 25, 45 and 65 °C. The binding activity of the stored biosensor was estimated by measuring the changes in resonance frequency after exposure to the bacterial solutions (10⁹ cfu mL⁻¹). Binding activity was also confirmed by counting bound S. typhimurium cells on the sensor surface using Scanning Electron Microscopy (SEM) micrographs. The results show that at each temperature, the binding activity of the biosensor gradually decreased over the testing period. Degradation of biosensor accelerated at higher storage temperatures. The activation energy of biosensor system degradation was determined to be 7.7 kcal mol⁻¹.     

In vivo immunomodulatory activity of polysaccharides derived from Chlorella pyrenoidosa

The effects of polysaccharides from Chlorella pyrenoidosa on immunomodulatory activity were investigated in this paper. Phagocytosis of peritoneal macrophages was detected through ingesting chicken red blood cells (CRBC) test to evaluate nonspecific immune response. The cellular immune response was measured using the delayed-type hypersensitivity response to sheep red blood cells (SRBC) in terms of antibody titration level. Oral administration of C. pyrenoidosa polysaccharides, 1 g and 2 g kg⁻¹ body weight significantly enhanced phagocytic rate (23.00 and 26.25%, respectively) and phagocytic index (0.30 and 0.36, respectively) compared with saline-treated control (P<0.05). The value of ear thickness rate was greatly elevated from 10.09% in saline-treated control to 16.22 and 19.48%, respectively in group treated with 1 g and 2 g kg⁻¹ body weight polysaccharides (P<0.05). There was a significant correlation between the dosage of C. pyrenoidosa polysaccharides and antibody titre (r=0.99, P=0.019) and the antibody titre was significantly increased by 11.80, 21.90 as well as 39.80 in 2 g kg⁻¹ bw compared to 10.30 in the control. Dietary supplementation with C. pyrenoidosa polysaccharides did not affect the growth of the mice during the experiment. These results indicated that immunomodulatory activities in mice were enhanced by administration of polysaccharides derived from C. pyrenoidosa.     

Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage

The potential antioxidant activities of three phlorotannins (phloroglucinol, eckol and dieckol) purified from Ecklonia cava collected in Jeju Island were investigated to evaluate their potential value as the natural products for foods or cosmetic application. In this study, antioxidant activities were measured by electron spin resonance spectrometry (ESR) technique for scavenging effects of free radicals such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), alkyl, hydroxyl (HO^•) and superoxide anion radical (O₂ ^•−) and by comet assay for protecting effects against H₂O₂-mediated DNA damage. The results show that all the phlorotannins have the potential DPPH, alkyl, hydroxyl and superoxide radical scavenging activities. Especially, eckol samples scavenged around 93% of DPPH at 0.25, 0.5, 1 mg/mL of concentrations and were higher than the other phlorotannins, such as phloroglucinol and dieckol samples. Also, protecting effects of the phlorotannins against H₂O₂-mediated DNA damage increased with increased concentrations of the samples in the L5178 mouse T-cell lymphoma cell lines (L5178Y-R). In conclusion, these results suggest that the three phlorotannins purified from E. cava have the potential inhibitory effect on H₂O₂-mediated DNA damage and harmful free radicals and can be used as antioxidants in cosmetic, foods and drug industry.     

Detection and Quantification of Enterobacter sakazakii by Real-time 5′-Nuclease Polymerase Chain Reaction Targeting the palE Gene

Enterobacter sakazakii is an emerging food-borne pathogen causing invasive infection with high mortality rates in neonates and infants. The aim of this study was to develop, optimize, and evaluate real-time 5′-nuclease polymerase chain reaction (PCR) for the specific detection and quantification of E. sakazakii. Original primers and TaqMan probe targeting a sequence of E. sakazakii palE gene were designed. The developed real-time PCR system was highly specific for E. sakazakii with 100% inclusivity determined using 54 E. sakazakii strains and 100% exclusivity determined using 99 other strains. Detection limits of 4 × 10² and 4 × 10¹ CFU ml⁻¹ were determined with 100% and 90% probability, respectively. The response of the 5′-nuclease PCR system was linear (correlation coefficient ≥ 0.997) in the range of 10¹ to 10⁸ CFU ml⁻¹. Five methods of DNA sample preparation were compared. The methods of DNA preparation using the InstaGene Matrix and the simple lysis by boiling with the Triton X-100 were the most sensitive with calibration lines applicable for quantification. The developed real-time PCR targeted to the palE gene provides an alternative possibility for the detection and quantification of E. sakazakii after the suitable sample preparation.     

Purification and identification of proteolytic enzymes from Aspergillus oryzae capable of producing the antihypertensive peptide Ile-Pro-Pro

Two proteolytic enzymes capable of releasing the angiotensin I-converting enzyme (ACE) inhibitor Ile-Pro-Pro from casein were identified by purification of an Aspergillus oryzae extract by three-step column chromatography. First, proteins capable of producing Ile-Pro-Pro from β-casein were eluted using a DEAE-sepharose FF column with a linear sodium chloride gradient. An endopeptidase capable of releasing Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro from Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln and an aminopeptidase producing Ile-Pro-Pro from Gln-Asn-Ile-Pro-Pro were separated from the resultant fraction using a hydroxyapatite column. Each active enzyme was then loaded onto a Develosil 300Diol gel filtration column for high performance liquid chromatography (HPLC) and purified to homogeneity.The endopeptidase had a molecular mass of approximately 46,000 Da and exhibited an N-terminal amino acid sequence identical to that of neutral protease I (NP I) of A. oryzae. Meanwhile, the aminopeptidase had a molecular mass of 36,000 Da and an N-terminal amino acid sequence similar to that of Leucine aminopeptidase (LAP), as reported in Aspergillus sojae and A. oryzae. The eluted endopeptidase and aminopeptidase were thus identified as NP I and LAP, respectively.Analysis of peptide production using synthetic proteins containing an Ile-Pro-Pro sequence showed that NP I processed the C-terminal end and LAP processed the N terminus to produce Ile-Pro-Pro. While Ile-Pro-Pro was successfully produced from casein by the addition of these two purified enzymes, it was not generated with the addition of only a single enzyme. Based on our experimental findings, we suggest that NP I and LAP are key proteolytic enzymes in the release of Ile-Pro-Pro from casein in A. oryzae.