Characterization of amylase and protease produced by Aspergillus awamori in a single bioreactor

The aim of this study was to characterize the glucoamylase and acid protease produced in a single bioreactor by Aspergillus awamori: nakazawa MTCC 6652. Both the enzymes were found stable in wide range of pH (3–9) and temperature (25–70 °C). Optimum activities of amylase and protease were obtained at pH 4 and 5, respectively, whereas 70 and 55 °C had been found as most suitable temperature for highest activities of amylase and protease, respectively. Half life of glucoamylase was 210, 120, 60 and 35 min at 50, 60, 70 and 80 °C, respectively, which was 150, 120, 65 and 15 min at 40, 50, 60 and 70 °C, respectively, for acid protease. K_m and V_max of glucoamylase and protease were 9.8 mg/ml, 56.2 mg/ml/min and 1.08 mg/ml, 8.8 mg/ml/min, respectively. In low amount (1 mM) almost all metal ions except Mn, such as Ca²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, Zn²⁺ and Hg²⁺ enhanced glucoamylase activity whereas protease activity was inhibited by all the ions except Zn²⁺. At low concentration, i.e., (0.03% w/v) Triton X-100 and SLS increased the activity of glucoamylase, while in higher concentration it inhibited activities of both the enzymes. β-mercaptoethanol at 0.25% (v/v) enhanced the amylase and protease activity by 1.6 and 3.0 fold, respectively, whereas the presence of 0.5% (v/v) β-mercaptoethanol inhibited the activities of both the enzymes drastically. At 0.5 M concentration of urea, glucoamylase activity increased but drastic inhibition took place at 5 M urea. In case of protease, 0.5 M of urea enhanced its activity and 1 M urea inhibited it completely. Thus, glucoamylase and protease produced by A. awamori nakazawa confirm their suitability for diverse applications in industries.     

Identification of fish species in smoked fish products by electrophoresis and isoelectric focusing

Summary Different species of fresh fish and smoked fish products were analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and isoelectric focusing. It was possible to differentiate and identify species of fish in smoked samples and draw some conclusions about the type of smoking process employed.

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Artbestimmung in geräucherten Fischprodukten durch Elektrophorese und isoelektrischer Focussierung

Zusammenfassung Proben von verschiedenen Arten von Frischfisch und von geräucherten Fischprodukten wurden mit Hilfe von SDS-Polyacrylamid-Gelektrophorese und isoelektrischer Focussierung analysiert. Es war möglich in den geräucherten Proben zwischen verschiedenen Fischarten zu unterscheiden und die Fischart zu bestimmen. Ebenso waren Rückschlüsse auf den angewendeten Räucherungsprozeß möglich.

     

Detection and partial characterisation of subtilisin inhibitors in legume seeds by isoelectric focusing

Seed extracts of ten legume species were subjected to isoelectric focusing in acrylamide gels and inhibitors of subtilisin, three other bacterial proteases, trypsin and chymotrypsin were detected by a negative-staining technique based on the chromogenic substrate, acetyl-D, L-phenylalanine-2-naphthylester. In addition to complex patterns of trypsin and chymotrypsin inhibitor zones, most legume seeds examined exhibited one major and one minor inhibitor of subtilisin, and the other bacterial proteases. However, in cowpea and black gram only the major, and in lentil and soya bean only two minor subtilisin inhibitor zones were detected. Isoelectric points of the subtilisin inhibitors range from 4.4 to 5.9. The inhibitor patterns obtained by isoelectric focusing of extracts mixed with bacterial protease confirmed that inhibitors of the bacterial proteases represent a new type of inhibitor different from the well characterised trypsin and chymotrypsin inhibitors.     

Identification of commercial prawn and shrimp species of food interest by native isoelectric focusing

Native isoelectric focusing (IEF) of water-soluble sarcoplasmic proteins was applied to the identification of 14 shrimp species of food interest belonging to the order Decapoda. These species have different commercial values, but due to their phenotypic similarities and carapace removal in their industrial processing, incorrect food labelling and deliberate or inadvertent adulteration can occur. Each of the 14 tested species showed species-specific protein band profiles and intra-specific polymorphism was low, not preventing the correct identification of the species. Therefore, IEF of water-soluble sarcoplasmic proteins allowed the differentiation of the 14 species considered. In addition, sarcoplasmic calcium-binding proteins (SCPs) were identified by tandem mass spectrometry (MS/MS) as the major species-specific proteins in these species, opening the way to further studies focusing on their potential use as specific biomarkers.     

Production of a Lactobacillus plantarum starter with linamarase and amylase activities for cassava fermentation

Lactobacillus plantarum strain A6 isolated from cassava, cultured on cellobiose MRS medium showed a growth rate of 0.41 h^?1, a biomass yield of 0.22 g g^?1, and produced simultaneously an intracellular linamarase (76.4 U g^?1 of biomass) and an extracellular amylase (36 U ml^?1). The synthesis of both enzymes was repressed by glucose. The use of such a strain as a cassava fermentation starter for gari production had the following influences: a change from a hetero-fermentative pattern observed in natural fermentation to a homofermentation, a lower final pH, a faster pH decline rate and a greater production of lactic acid (50 g kg^?1 DM). However, this starter did not appear to play a significant role in cassava detoxification, since it was observed that the level of endogenous linamarase released during the grating of the roots was sufficient to permit the complete and rapid breakdown of linamarin.     

Identification of the genetic variants of κ-casein in milk by isoelectric focusing electrophoresis

The genetic variants of κ-casein (κ-CN) A and B were analysed in milk from Holstein–Friesian (HF) and Jersey cows by means of isoelectric focusing (IEF) electrophoresis. Milk samples were obtained in triplicate from pure-breed HF and Jersey cows (three of each) to estimate the protein content, casein and purify κ-CN. The protein and casein contents in the milk from both breeds were statistically different ( P <  0.05). The κ-CN A migrates first with an isoelectric point (pI) of 7.2–7.3 and then B with a pI of 7.5–7.7. Differences in the expression proportion of both variants were detected.     

Identification of freshwater fish commercially labelled “perch” by isoelectric focusing and two-dimensional electrophoresis

Isoelectric focusing (IEF) and two-dimensional electrophoresis (2-DE) were used to distinguish four freshwater fish species which are sold under the generic label of “perch”: Perca fluviatilis (European perch), Lates niloticus (Nile perch), Stizostedion lucioperca (European pikeperch) and Morone chrysops x saxatilis (sunshine bass). These species have different commercial values but are easily “interchangeable” because they are sold already filleted, in view of the numerous bones of the whole fish. IEF of the water-soluble proteins extracted from fish muscle resolved in species-specific patterns. Intra-species polymorphism was low, and did not concern the bands identified as characteristic of the species. As well, 2-DE maps showed numerous species-specific protein spots. Interestingly, while none of the IEF bands was common to all four species, several major 2-DE spots were similar. Therefore, IEF of water soluble sarcoplasmic proteins is sufficient to unambiguously discriminate among the four species considered. Analysis by 2-DE, which has a higher resolution power but it is more expensive and time consuming, may be applied to obtain further knowledge of the proteome of poorly characterized species.     

高效半制备液相色谱法从橘皮中分离制备黄酮类化合物

采用高效半制备液相色谱法从橘皮中分离制备黄酮类化合物.橘皮提取液经过过滤、浓缩、D101大孔树脂吸附、甲醇洗脱后,在室温、283nm波长、1.0mL的进样量和以流速为15mL/min的MeOH:H2O(0.5%HAc)=19:31做洗脱液条件下,用YEG-C18色谱柱分离得到8种化合物.通过紫外分光光度法、标准品液相色谱对照法、质谱特征离子峰法初步确定其中5种化合物为新橙皮苷、橙皮苷、柚皮苷、异柚皮苷、新圣草苷,其它3种物质有待进一步鉴定.橙皮苷、柚皮苷、异柚皮苷、新橙皮苷的色谱分析纯度达到或超过99.0%.     

A comparison of DNA-hybridization, immunodiffusion, countercurrent immunoelectrophoresis and isoelectric focusing for detecting the admixture of pork to beef

Using DNA-hybridization at least 0·5% raw pork admixtured to beef could be detected using total genomic pig DNA as well as a cloned pig-specific DNA fragment as a DNA probe. Although signal intensity increased with increasing amounts of pig-DNA, a precise quantitation of pork in samples was not possible. Compared to this the sensitivity for detecting raw pork in beef found by Countercurrent Immunoelectrophoresis was 0·4%; by Immunodiffusion, 1·1%, and by Isoelectric Focusing, 5·0%.     

Production of phytase in a low phosphate medium by a novel yeast Candida krusei

A yeast strain producing high levels of phytase was isolated from soil and identified as Candida krusei. The phytase was located on the yeast cell wall and was a glucanase-extractable protein. The phytase production was controlled by the phosphate concentration in the medium used. The maximum production of phytase occurred in a medium containing 0.5 mg of phosphorus per 100 ml, and most of the cells were ellipsoid-shaped and did not exhibit budding. Increasing the concentration of phosphorus in the medium to more than 5 mg of phosphorus per 100 ml caused inhibition of phytase production and 90% of the cells exhibited budding. On the other hand, transferring cells grown in the high-phosphate medium into a phosphate-free one derepressed the phytase production. For example, transferring cells grown in 2 mg of phosphorus per 100 ml into the phosphate-free medium, enhanced the total phytase activity up to 5.5-fold that in the medium containing 0.5 mg of phosphorus per 100 ml. The phytase showed two optimum pHs of 2.5 and 5.5, an optimum temperature of 40 degrees C and the K(m) value for Na-phytate was 0.03 mM. Using in vitro experiments that simulated the conditions of the digestive tract, 50-80% phosphorus was liberated from different plant samples (wheat bran, rice bran and feeds) by the strain.     

Profiling of serine protease activities in human stratum corneum and detection of a stratum corneum tryptase-like enzyme

Seasonal variation in stratum corneum (SC) biophysical and biological characteristics has been described previously. In particular, the winter season has been shown to affect more severely the properties of facial skin compared with forearm skin. Moreover, when compromised, such as in dry skin conditions, facial SC has been shown to contain increased inflammatory cytokines and proteases. Nevertheless, there have been no comparative studies of the activities and depth activity of several proteases in the SC on different body sites and at different times of the year. In this study, we examined the distribution of key serine protease activities (kallikrein 5, kallikrein 7, urokinase, plasmin and a tryptase-like enzyme) in different layers of the SC on the cheek and the forearm by analysis of consecutive tape strippings of healthy Caucasian subjects during winter and summer. The protein content of the tape strippings was quantified by absorption measurements with a recently developed and novel infrared densitometer SquameScan 850A while the SC enzyme activities were determined using fluorogenic peptide substrates. Transepidermal water loss (TEWL), skin pH and skin hydration were higher on the cheek than on the forearm. In the same way, the activity of the inflammatory-related proteases plasmin, urokinase and tryptase was approximately five to eight times and the activity of the desquamatory-related proteases kallikrein 5 and kallikrein 7 approximately two to four times higher on the cheek than on the forearm. There were no gender-related differences in these enzyme activities except for the increased kallikrein 7 in the forearm skin of the female subjects in winter. Reduced kallikrein 5 was associated with increased SC cohesion, as judged by increased protein removal, in forearm skin in the winter months of the year although the skin was clinically normal. It can be concluded that (i) protected skin areas show lower TEWL, skin pH and skin hydration and less protease activities than skin areas that are exposed to the environment, possibly indicating subclinical inflammation on these body sites, (ii) in normal healthy forearm skin, the outer SC exhibits greater serine protease activity than its deeper layers, (iii) compared with the forearm, urokinase- and plasmin-like activities are elevated on SC strippings from the cheek, confirming activation of the plasminogen cascade, and (iv) tryptase-like activity in the SC is also elevated in samples from the cheek, possibly indicating involvement of mast cells in these barrier-compromised body sites or the synthesis of a novel tryptase-like enzyme by keratinocytes. Although elevation of the activities of urokinase, plasmin, kallikrein 5, kallikrein 7 and now a tryptase-like enzyme was observed on SC derived from skin of clinically normal cheeks, we anticipate even higher activities in skin conditions where the epidermal barrier is further impaired.     

Production of a certified reference material for the acrylamidecontent in toasted bread

The need for a certified matrix reference material (CRM) of acrylamide in a food type matrix was emphasized by the competent authorities as a tool to improve comparability, ensuring accuracy and traceability of analytical results. The institute for reference materials and measurements (IRMM) responded to the international request by producing a certified reference material, ERM-BD273, containing endogenous acrylamide in a toasted bread matrix. This work describes the production of the CRM, according to 2 and 3 [ISO Guide 34 (2000). General requirements for the competence of reference materials producers; ISO Guide 35 (2006). Reference materials – General and statistical principles for certification], which comprises the material processing, homogeneity and stability assessment, material characterisation and the acrylamide mass fraction value assignment in toasted bread. Heterogeneity of the material between the vials processed was determined by an in-house validated gas chromatographic methodology involving acrylamide derivatisation and mass spectrometric detection and found to be below 2%. Potential degradation during storage was also investigated and a shelf-life based on this value was established. A collaborative study for material characterisation involved sixteen laboratories applying different analytical methodologies including gas chromatography or high resolution liquid chromatography and isotopic dilution mass spectrometry. The certified value for acrylamide in ERM-BD273, traceable to the international system of units (SI), is (425 ± 29) ng g⁻¹.     

Antioxidant and antibacterial activities of peptide fractions from flaxseed protein hydrolysed by protease from Bacillus altitudinis HK02

Flaxseed protein (FP) hydrolysates by crude protease of strain Bacillus altitudinis HK02 were further separated into five fractions by ultrafiltration membranes with a molecular weight cut‐off of 10, 5, 3 and 1 kDa for the analysis of antioxidant activities and antibacterial ability. The results demonstrated that the fraction of 1‐ to 3‐kDa peptides exhibited higher antioxidant activities on the free radical‐scavenging ability and the reducing power than those of Vit C, Vit E, BHA and other fractions. The fraction with a low molecular weight (<1 kDa) of peptides demonstrated the highest ferrous ion‐chelating ability and a higher inhibition of lipid peroxidation than BHA and other fractions. Moreover, it also exhibited the best growth inhibition of Pseudomonas aeruginosa and Escherichia coli. The results, including the concentration‐dependent effect of peptides fractions, demonstrated that it is feasible to derive functional ingredients of natural antioxidants along with antimicrobial activity from FPs hydrolysed by protease from B. altitudinis HK02.     

Determination of bromate in bread additives and flours by flow injection analysis

A new, simple, rapid and sensitive spectrophotometric flow injection analysis (FIA) method was developed for the determination of bromate based on its reaction with 3,5-dibromo-PADAP and thiocyanate in a strongly acidic medium. This produced an unstable violet product with a maximum absorption at 602 nm. The calibration curve was linear in the range of 2.00×10⁻⁶–2.10×10⁻⁵ mol/l and the detection limit was 8.00×10⁻⁷ mol/l. The sampling frequency was 90 h⁻¹. The method has been successfully applied to the determination of bromate in commercial bread additives and flours.     

Measurement of fat and moisture in air-dried bread by near infrared reflectance

Near infrared calibrations have been derived and used routinely for a year in the measurement of fat and moisture in air-dried bread. First and second derivative calibrations were obtained using a Pacific Scientific mark II scanning spectrophotometer on samples sent from all over South Africa to the Wheat Board for analysis. Prediction analysis performed on further bread samples gave standard errors of prediction (s.e.p.) of 0.12% fat and 0.13% moisture.     

A databank able to be used for identifying and authenticating commercial flatfish (Pleuronectiformes) products at the species level using isoelectric focusing of native muscle proteins

Summary A computerized databank of IEF protein patterns for use in identifying flatfish species (in total 17 species, including 15 commercial ones) is presented. The databank includes all species regulated by the Belgian Law (22 May 1996) on the use of official names for fishes and seafood products. It was found that interspecimen similarity of the IEF patterns, as processed by digitization, was always larger than interspecies similarity, which allows for unequivocal authentication of unknown samples, as long as the authentic pattern is available in the databank. The databank was used to authenticate 17 commercial fish fillets.