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.     

Species identification of cooked fish by urea isoelectric focusing and sodium dodecylsulfate polyacrylamide gel electrophoresis: a collaborative study

The suitability and reliability of urea IEF and SDS-PAGE for the identification of cooked fish flesh was tested by a collaborative study among nine laboratories. Urea IEF was performed with CleanGels as well as with ImmobilineGels, and ExcelGels were used for SDS-PAGE, enabling all three types of gels to be run in the same flat bed electrophoresis chamber. By strictly following optimised standard operation procedures (SOPs), five unknown cooked samples had to be identified with each technique using a set of 10 raw reference samples. With urea IEF, only one out of 35 identifications was incorrect, and with SDS-PAGE a similar result was obtained. It was concluded that methods, as now developed, are suitable for checking the species declaration of fishery products.     

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.     

Comparison of capillary electrophoresis and isoelectric focusing for analysis of casein/caseinate addition in processed cheeses

Capillary electrophoresis (CE) and immobilized pH gradient isoelectric focusing were applied for the determination of the addition of isoelectric casein, rennet casein and sodium caseinate to processed cheeses of known composition containing different dairy products. Both methods allowed the determination of these protein ingredients by analysing the intact κ-CN. The comparison of the quantitative determination by both techniques demonstrated that they presented similar recoveries for isoelectric casein, but for sodium caseinate, a considerably better recovery was obtained by using the isoelectric focusing method (85.6% versus 47.2%). However, CE showed higher precision. CE was applied for the determination of the addition of rennet casein by the use of a calibration curve calculated by plotting the peak area ratio ∑β-CN A/para-κ-CN in the electropherograms versus the content of rennet casein of known composition cheeses from the same manufacture batch. This approach gave recoveries of added rennet casein between 94% and 102%.     

Optimization of encapsulation of maltogenic amylase into a mixture of maltodextrin and beeswax and its application in gluten-free bread

In this work maltogenic amylase (MAase) was encapsulated into the mixture of maltodexrin and beeswax (BW) for retarding staling of gluten-free bread. The effects of maltogenic amylase (MAase) concentration (8.2, 45, and 82 mg/ml), maltodextrin with dextrose equivalent (DE) 4–7 (MD) (1, 2.5, and 4%) and, BW (1, 2.5, and 4%) on the encapsulation efficiency (EE) of encapsulated enzyme were optimized using RSM. The optimized formulation was MAase with 8.2 mg/ml, MD 4% and BW 1%, leading to the highest EE (79.35%), thus chosen for subsequent experiments. The prepared particles were 1,190.50 nm with PDI of 0.336 and zeta potential of −8.30 mV. Surface morphologies of produced particles were almost spherical with layered appearance. Batter with this formulation led to higher cross over point in frequency sweep than free enzyme-loaded batter. Lower weight loss, higher volume index, darker crust color, whiter crumb color, more aerated microstructure, less hardness in crumb, and higher sensorial acceptability on the first day and during storage period in the breads containing encapsulated MAase was observed.     

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%.     

复合蛋白酶水解棉粕制备多肽的工艺优化

为提高棉粕蛋白资源的利用率,考察了复合蛋白酶水解脱酚棉粕制备多肽的工艺。以多肽得率和水解度为评价指标,比较了不同蛋白酶对棉粕蛋白水解能力的差异,确定了以胰蛋白酶与米曲霉蛋白酶按照酶活比3∶1构建的复合蛋白酶作为水解棉粕蛋白的最佳蛋白酶。采用单因素试验及响应面试验对影响棉粕蛋白水解的因素底物蛋白质量浓度、加酶量、p H、酶解温度以及酶解时间进行了分析,并确定了棉粕蛋白水解的最佳工艺条件为:棉粕蛋白质量浓度25 g/L,加酶量5 000 U/g,p H 8. 91,酶解温度50～55℃,酶解时间5. 92 h。在最佳工艺条件下,水解棉粕蛋白的多肽得率可以达到71. 85%。多肽的氨基酸分析结果表明,酶水解制备的多肽能基本保持原棉粕蛋白的氨基酸组成,其氨基酸种类较齐全,8种必需氨基酸含量丰富,营养价值较高。     

Supplementation of alkaline phytase (Ds11) in whole-wheat bread reduces phytate content and improves mineral solubility

Abstract: In this study, alkaline phytase was added to whole‐wheat bread and the phytate content and mineral profiles were compared to commercially available acidic phytase. At neutral pH, some phytate (approximately 20%) was degraded by endogenous phytase in wheat flour, while 40% of phytate was hydrolyzed by alkaline phytase DS11 and a 35% reduction was observed with acidic phytase. Most of the enzymatic activity occurred during the proofing stage, and the rate of reaction depended on pH. DS11 phytase effectively degraded the phytate level within a 30 min treatment at pH 7; however, at least 60 min was needed with acidic phytase to achieve the same hydrolysis level. Mineral profiles were also dramatically affected by the phytate reduction. The biggest increase was observed in Fe²⁺ by the phytase treatment. The Fe²⁺ content increased 10‐fold at pH 7 and 8‐fold at pH 5 with alkaline phytase DS11. Alkaline phytase DS11 was shown to be effective at phytate reduction in whole‐wheat bread preparation. Additionally, phytate degradation enhanced the mineral availability of bread.     

Detection of mechanically recovered meat and head meat from cattle in ground beef mixtures by multivariate analysis of isoelectric focusing protein profiles

The present work investigates the possibility of constructing a multivariate calibration model for predicting the composition of ground beef with respect to different meat quality types, based on intensity profiles from isoelectric focusing of water-soluble proteins. Beef mixtures containing various amounts of mechanically recovered meat, head meat and production meat from beef, were analysed by isoelectric focusing in immobilised pH-gradients. The gels were photographed and the images transferred to a digital format. By simple image processing procedures, background colour was virtually eliminated and signal strength was improved to a considerable degree. Multivariate analysis of protein profiles from the gels gave models explaining 75 to 90% of variance in sample composition. Manually deboned meat was explained to the highest degree, and with a precision of 7%. Two different qualities of mechanically recovered meat could be detected even when treated as one category. The present approach needs further refinement, but seems applicable for detecting intentional substitution of high quality meat products with low-price raw materials. One advantage of the approach is that evaluation of samples is not dependent on specific knowledge on the individual components to be analysed, so that such analytical methods are relatively easy to implement in any standard laboratory.     

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%.     

中性蛋白酶酶解缢蛏制备多肽工艺条件优化

本实验利用中性蛋白酶对缢蛏进行酶解制备多肽,以多肽含量为指标,在单因素实验基础上采用正交实验对鲜缢蛏的酶解工艺进行优化。结果表明,中性蛋白酶最优酶解工艺条件为:p H7、酶添加量1200 U/g、料液比(m∶v)为1∶2.5、温度40℃、时间2 h,在此工艺条件下多肽含量为(79.2±0.7)mg/g。