Partial purification and characterisation of dipeptidyl peptidase II from porcine skeletal muscle

The purification and study of biochemical properties of dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from porcine skeletal muscle have been carried out in the present work. The purification included ammonium sulphate fractionation and two HPLC chromatographic separations using a Resource-Q anion exchange column. The enzyme was purified 1270 fold, with a 1.6% recovery and was completely separated from DPP IV activity. The pure enzyme displayed one main protein band with a Mr of 58 kDa on SDS-PAGE. Maximum activity was reached at pH 5.5 and 65°C. Those synthetic substrates containing Pro in N-penultimate position were the most efficiently hydrolysed, whereas in the case of peptides, DPP II efficiently hydrolysed both X-Pro- and X-Ala- peptides. The serine peptidase inhibitors PMSF and Pefabloc SC suppressed DPP II activity in a high degree, whereas 3, 4-DCI and cysteine peptidase inhibitors exerted little effect. Alkaline metal salts inhibited the enzyme activity according to the size of the cation, and among the assayed divalent cations, only Cu(2+), Fe(2+) and Hg(2+) showed significant inhibition of the activity. This is the first time that porcine muscle DPP II has been purified and its biochemical characteristics studied. So, these results contribute to improve the knowledge in relation with the proteolytic chain and the generation of flavour characteristics in meat products.     

Isolation and characterisation of Arcobacter butzleri from meat

A survey was conducted to determine the prevalence of Arcobacter in ground chicken, pork, beef and lamb meats. Meat samples were enriched in Arcobacter broth (AB) containing cefoperazone, amphotericin and teicoplanin (CAT) supplement. Samples were screened for the presence of Arcobacter spp. using a multiplex polymerase chain reaction (PCR) followed by isolation on blood and selective agar. Arcobacter butzleri was the only species of Arcobacter isolated from 35% of 88 samples of ground meats. A. butzleri was more frequently isolated from poultry (73%) than pork (29%), beef (22%) or lamb (15%) samples. No significant differences were found in the isolation rates and from the different regions sampled. Isolates were characterised by pulsed-field gel electrophoresis (PFGE) using SacII, EagI and SmaI restriction endonucleases. A number of isolates with indistinguishable PFGE fingerprints were found to be epidemiologically related, which may indicate cross-contamination of common types of Arcobacter from different meat species or between meat species. The public health significance of Arcobacter in ground meat needs to be determined.     

Isolation and characterisation of a wheat albumin

A major albumin from Cappelle-Desprez wheat flour has been isolated by carboxymethyl-cellulose chromatography in a sufficiently pure state for characterisation. The water-soluble protein is free from phenylalanine and histidine, and this enables the purity to be estimated as > 95 %. It appears to consist of a single chain with a mol. wt. of 26,000. The absorbance coefficient at 280 nm is 1.9 × 10³, the high value being due to 8 tryptophan and 8 tyrosine residues per molecule. There are 10 S. S bonds but no sulphydryl groups or sugar residues in the molecule. Sedimentation studies indicate that the molecules tend to aggregate in 0.2 M-NaCl. No protease or amylase activity was detectable.     

Isolation and characterisation of disulphide peptides from wheat flour

A method has been devised for the isolation and fractionation of disulphide peptides from flour. Oxidised glutathione is only a minor member of a family of disulphide peptides, the rest of which are basic and of molecular weight about 2000. The basic disulphide peptides could not be fractionated into individual components, but they contained some disulphide groups that were very reactive towards glutathione at pH 5.8, which is approximately the pH of dough.     

UHT杀菌乳中耐热菌的分离与鉴定

采用2种不同的细菌富集方式对5种不同品牌UHT（Ultra High Temperature）杀菌牛乳中残留的细菌进行了分离，得到18株细菌，并对它们进行了耐热性和细菌学鉴定试验。结果表明，18株细菌全部耐热，初步将其归为3个属。其中，片球菌属10株，均为戊糖片球菌，肠球菌属3株，1株类鸟肠球菌，2株粪肠球菌；微球菌属5株，1株嗜冷微球菌，4株变异微球菌。虽然这些耐热菌均为非致病菌，但是遇到适宜条件，这些耐热菌会生长繁殖，导致乳产品在贮藏和流通过程中发生变质。     

Isolation and characterisation of a coffee melanoidin fraction

BACKGROUND: Melanoidins contribute to the colour and flavour of coffee brews and are also reported to be physiologically active components. However, structural information about coffee melanoidins is rare and current models are supported by few data. RESULTS: Using hydrophobic interaction chromatography (HIC), melanoidin fractions were isolated from different high‐molecular‐weight fractions of coffee brews obtained by ultrafiltration. Generally, melanoidin fractions isolated by HIC showed three‐ to fourfold higher antioxidant activities than the remaining high‐molecular‐weight material in the ultrafiltration fractions. Melanoidin fractions showed an intense brown colour: aqueous solutions with a concentration as low as 40 µg mL^?1 were distinguishable from pure water. The melanoidin fractions contained less than 6% each of releasable carbohydrates and amino acids. The molecular masses of the melanoidins were estimated to be between 3 and 22 kDa, irrespective of the ultrafiltration fraction from which they were obtained. Two‐dimensional nuclear magnetic resonance studies revealed an enrichment of phenolic/aromatic/olefinic structural units and did not support the idea of intact ferulate or caffeate moieties integrated into the melanoidin polymer. CONCLUSION: These studies support melanoidin models describing them as high‐molecular‐weight compounds themselves as opposed to models describing them as low‐molecular‐weight chromophores bound to polysaccharides or proteins. Phenolic constituents are more likely integrated into melanoidins as condensed phenolics than as intact hydroxycinnamates from chlorogenic acids. Copyright © 2008 Society of Chemical Industry     

Isolation and characterisation of a water-soluble wheat-flour protein

The main component of the water-soluble proteins of wheat flour has been isolated in sufficient quantity for chemical and physical characterisation. This was achieved by a combination of ion-exchange chromatography on carboxymethyl cellulose and gel filtration through Sephadex G75. The protein isolated was judged to be 90% pure by zone electrophoresis. Sedimentation analysis yielded a single symmetrical peak with an S°_20,w value of 2.45. The molecular weight was found to be 19,300 by gel filtration, and the diffusion coefficient estimated by the same method was 10.47 × 10^?7. A molecular weight of 16,000 was calculated from sedimentation equilibrium analysis in a dissociating medium. The ultra-violet spectrum of the isolated albumin exhibited a maximum at 278 nm and from this an E^1% ₂₇₈ value of 13.1 was calculated. No free sulphydryl groups could be detected. Amino acid analysis showed that this protein has a composition similar to those of other soluble wheat-flour preparations. A molecular weight of 16,300 was calculated from the amino acid analysis. End-group analysis of the protein showed that serine is the N-terminal amino acid. The C-terminal amino acid was found to be resistant to release by both carboxypeptidase A and carboxypeptidase B.     

Isolation and characterisation of a kallikrein-like enzyme from Agkistrodon halys pallas snake venom

BACKGROUND: Viper snake venoms contain a great variety of toxic proteins. These components mediate their toxicity by either stimulating or inhibiting the haemostatic system of human victims or experimental animals, resulting in common clinical complications of blood clotting or uncontrolled haemorrhage. Therefore it is deemed important to isolate the active component(s) from snake venom with kallikrein‐like activity. RESULTS: A kallikrein‐like proteinase of Agkistrodon halys pallas snake venom, designated AHP‐Ka, was purified by anion exchange chromatography and affinity chromatography. Physicochemical studies showed that the purified enzyme was a 34 kDa monomeric glycoprotein, the molecular weight of which decreased to 26 kDa after deglycosylation with peptide N‐glycosidase F (PNGase F). Sequence studies on the NH₂‐terminal region of the protein indicated that AHP‐Ka shared a high degree of sequence homology with other serine proteinases from snake venoms. AHP‐Ka showed high catalytic activity and kallikrein‐like activity on substrates such as arginine esterase BAEE and chromogenic H‐D‐Pro‐Phe‐Arg‐pNA·2HCl (S‐2302) and was inhibited by protease inhibitor phenylmethylsulfonyl fluoride (PMSF). CONCLUSION: The results showed that AHP‐Ka isolated from A. halys pallas snake venom and purified by anion exchange chromatography and affinity chromatography is in fact a kallikrein‐like enzyme. Copyright © 2011 Society of Chemical Industry     

Isolation and characterisation of the isoflavones from sprouted chickpea seeds

Seven isoflavones were isolated from sprouted chickpea seeds by chromatography on silica gel column, polyamide column, sephadex LH-20 column and preparing thin-layer chromatography (TLC), respectively. The structures were characterised with one- and two-dimensional NMR in combination with mass and IR spectrometry. The obtained isoflavones were biochanin A (5,7-dihydroxyflavone-4′-methoxyflavone, (1); calycosin (7,3′-dihydroxy-4′-methoxyisoflavone, (2); formononetin (7-hydroxy-4′-methoxyisoflavone, (3); genistein (5,7,4′-trihydroxyisoflavone, (4); trifolirhizin (maackiain-3-O-β-d-glucopyranoside, (5); ononin (7-O-β-d-glucosyl-7-hydroxy-4′-methoxyisoflavone, (6); sissotrin (7-O-β-d-glucosyl-5,7-dihydroxy-4′-methoxyisoflavone, (7).     

Isolation and characterisation of arabinogalactan-proteins from New Zealand kanuka honey

Fractionation of manuka, kanuka and clover honeys indicated the >10 kDa fraction contained small amounts of type II arabinogalactans (AGs), which are often present as arabinogalactan proteins (AGPs). AGPs were isolated from the >10 kDa fraction of kanuka honey using β-glucosyl Yariv reagent and their composition and structure analysed. Constituent sugar, glycosyl linkage and NMR spectroscopy analysis of the purified AGP fraction revealed a predominance of neutral sugars, mainly galactose and arabinose, linked in a highly-branched structure typical of type II AGs. The molecular weight of the major component of the purified AGPs was ∼110 kDa, as determined by size-exclusion chromatography-multi-angle laser light scattering (SEC-MALLS). The Yariv supernatant fraction contained less total sugar, especially galactose, and more protein than purified AGPs. Linkage analysis indicated this fraction also contained an AG-type polymer in addition to various other polysaccharides and SEC-MALLS indicated the molecular weight of the major component was ∼32 kDa.     

Isolation and characterisation of four trypsin-chymotrypsin inhibitors from lentil seeds

Twenty-three proteinase inhibitors were isolated from Syrian local small lentils (Lens culinaris) by ammonium sulphate fractionation of the acidic extract followed by affinity chromatography on anhydrotrypsin-Sepharose. They all inhibited human and bovine trypsin and chymotrypsin. Three inhibitors (LCI-11·7, -3·3 and -4·6) were separated and purified to homogeneity by anion exchange chromatography and preparative isoelectric focusing (IEF) with immobilised pH gradients; a fourth (LCI-2·2) required additional reversed-phase high-pressure liquid chromatography. The four inhibitors were similar in their amino acid composition, with high cystine and aspartic acid/asparagine content, and lack of free sulphydryl groups, methionine and tryptophan. The calculated minimum number of amino acid residues per molecule, the calculated molecular masses confirmed by gel liquid chromatography, gel-permeation high-pressure liquid chromatography and sodium-dodecylsulphate polyacrylamide gel electrophoresis, and the isoelectric points determined by IEF (immobilised pH gradients and carrier ampholytes) were 84, 77, 68 and 60 residues per molecule, 9200, 8500, 7200 and 6750, and 5·26, 5·88, 6·80 and 7·80 for LCI-1·7, -2·2, -3·3 and -4·6, respectively. All four inhibitors inhibited human trypsin less than bovine trypsin, and human chymotrypsin more than the bovine enzyme. All these properties are in accordance with the classification of the four lentil inhibitors as members of the Bowman-Birk proteinase inhibitor family. © 1998 Society of Chemical Industry.     

Manothermosonication of heat-resistant lipase and protease from Pseudomonas fluorescens: effect of pH and sonication parameters

The effect of different parameters (pH, ultrasonic amplitude and pressure) on the resistance to heat and manothermosonication (MTS) treatments of heat resistant lipase and protease produced by Pseudomonas fluorescens B52 and NCDO 2085, respectively, were studied. Lipase B52 thermoresistance decreases with an increase of pH. However, inactivation by MTS seems to be pH independent. There were only slight increases in the MTS efficiency when increasing pressure at UHT temperatures and the effect of amplitude was different depending on treatment temperature. Protease NCDO 2085, which was very resistant to MTS at 30 degrees C. was very sensitive to MTS at 76 degrees C. Increases in applied pressure had no effect on MTS efficiency at 140 degrees C and its inactivation by MTS was almost temperature independent between 76-109 degrees C. Data obtained are compared with previous published data and inactivation mechanisms are discussed.     

Isolation and characterisation of lytic bacteriophages against Pseudomonas spp., a novel biological intervention for preventing spoilage of raw milk

Thirteen novel lytic bacteriophages against 13 Pseudomonas strains were isolated from local sewage and initially identified by morphology using a transmission electron microscope. PP1 and PP5 were identified as Pedoviridae and Cystoviridae, respectively; while the other 11 phages were identified as Leviviridae. Most phages showed high infectivity at either 4 °C or 25 °C, and the optimum pH range for phage infectivity was pH 5–7. A strong antimicrobial effect of the phage cocktail was evidenced by a 2-log reduction in Pseudomonas cell number of UHT milk inoculated with Pseudomonas, and a 1-log reduction in the psychrotrophic bacteria and total bacteria counts of raw milk at 4 °C over 5 d. A similar result was obtained at 25 °C over 8 h. Results indicated that the 13 phages with different morphological and physiological characteristics may have a potential application as biological preservative agent in raw milk.     

The Extracellular Protease AprX from Pseudomonas and its Spoilage Potential for UHT Milk: A Review

The negative effects of proteases produced by psychrotrophic bacteria on dairy products, especially ultra‐high‐temperature (UHT) milk, are drawing increasing attention worldwide. These proteases are especially problematic, because it is difficult to control psychrotrophic bacteria during cold storage and to inactivate their heat‐resistant proteases during dairy processing. The predominant psychrotrophic species with spoilage potential in raw milk, Pseudomonas, can produce a thermostable extracellular protease, AprX. A comprehensive understanding of AprX on the aspects of its biological properties, regulation, proteolytic potential, and its impact on UHT milk can contribute to finding effective approaches to minimize, detect, and inactivate AprX. AprX also deserves attention as a representative of all extracellular metalloproteases produced by psychrotrophic bacteria in milk. The progress of current research on AprX is summarized in this review, including a view on the gap in current understanding of this enzyme. Reducing the production and activity of AprX has considerable potential for alleviating the problems that arise from the instability of UHT milk during shelf‐life.     

Isolation and characterisation of a novel antibacterial peptide from bovine αS1-casein

Bovine casein was hydrolysed with a range of proteolytic enzymes including pepsin, trypsin, α-chymotrypsin and β-chymotrypsin, and assessed for antibacterial activity. The pepsin digest of bovine casein, which showed antibacterial activity, was fractionated using reverse phase high performance liquid chromatography and the antibacterial peptides isolated were characterised using electrospray ionisation mass spectrometry. Two antibacterial peptides were identified, a novel peptide (Cp1) which corresponded to residues 99–109 of bovine α_S1-casein and a previously reported peptide (Cp2) which corresponded to residues 183–207 of bovine α_S2-casein. The minimum inhibitory concentration (MIC) of Cp1 and Cp2 were determined against a range of bacterial cultures. Cp1 exhibited an MIC of 125 μg mL⁻¹ against all Gram-positive bacteria tested, and MIC ranging between 125 and >1000 μg mL⁻¹ against the Gram-negative bacteria tested. Cp2 was generally far more potent against the Gram-positive bacteria, exhibiting an MIC of 21 μg mL⁻¹, compared to MICs ranging from 332 to >664 μg mL⁻¹ against most of the Gram-negative bacteria tested.     

进口肉类中恶臭假单胞菌的分离与鉴定

目的选择具有较高选择性的培养基,分离与鉴定进口肉类中恶臭假单胞菌。方法参照GB4789.4-2010《食品安全国家标准食品微生物学沙门氏菌检验》、SN/T2099-2008《进出口食品中绿脓杆菌检测方法》,根据VITEK2显示的结果做生化鉴定。结果在选择性较高的沙门氏菌显色培养基上生长的菌落形态与沙门氏菌极为相似,VITEK2显示结果为恶臭假单胞菌,后经生化鉴定,结果与VITEK2一致,为恶臭假单胞菌。结论沙门氏菌显色培养基难以区分假单胞菌,但是亚硫酸铋琼脂(bismuth agar,BS)却可以明显区分,所以在日常检测过程中建议不要省去BS平板,在配制BS平板时需严格按照说明进行,以保证实验效果。     

Isolation and characterisation of invertase inhibitor from sweet potato storage roots

BACKGROUND: Plant invertases play important roles in sucrose metabolism. Cell wall invertase has been reported to participate in phloem loading and unloading. Soluble invertases are involved in hexose level regulation in mature tissues and in utilisation of stored sucrose within vacuoles. Invertase inhibitory proteins have been described as one of the possible components for invertase activity regulation in some plant species. RESULTS: In this work an invertase inhibitor (ITI) coding sequence was cloned by differential display from sweet potato (SP) storage roots. SPITI codes for a protein of 192 amino acids with a predicted molecular mass of 20 624 Da containing a 20‐amino‐acid signal peptide and four cysteines. Computer analysis of the deduced amino acid sequences of the conserved domain revealed that the protein belonged to the plant invertase/pectin methylesterase inhibitor. Both the corresponding mRNA and protein levels were found to be highest in storage roots, followed by veins. Recombinant SPITI protein from the storage root cDNA clone overproduced in Escherichia coli (M15) was purified by affinity chromatography. This protein effectively inhibited the invertase activity in a dose‐dependent manner. The results presented in the Lineweaver‐Burk plots indicated that the invertase inhibitor displayed a mode of competitive inhibition towards the invertase tested, with a K_i of 3.82 × 10^?6 mol L^?1. CONCLUSION: These results suggested that SPITI is a novel member of the ITI family in plants. SPITI genes of sweet potato storage roots display differential gene expression patterns, which may be associated with sucrose metabolism to cope with particular developmental requirements. Copyright © 2008 Society of Chemical Industry     

Isolation and chemical characterisation of a polysaccharide from green tea (Camellia sinensis L.)

BACKGROUND: To contribute towards understanding the relationship of structure and bioactivity, a protein‐bound acidic polysaccharide named TPC3‐1 was isolated and purified from low‐grade green tea (Camellia sinensis L.). The homogeneity and weight average molecular weight of TPC3‐1 was determined by agarose gel electrophoresis and high‐performance gel permeation chromatography. The monosaccharide and amino acid composition of TPC3‐1 were analysed by gas chromatography and an amino acid analyser. The molecular structure of TPC3‐1 was characterised by Fourier transform infrared spectroscopy, ¹³C nuclear magnetic resonance spectroscopy and atomic force microscopy. RESULTS: Based on the data obtained, the average peak molecular weight of TPC3‐1 was about 120 kDa. TPC3‐1 was composed of L ‐arabinose, D ‐ribose, D ‐xylose, D ‐glucose and D ‐galactose with a molar ratio of 4.9:2.2:3.1:1.8:1.0. Fifteen amino acids were identified as components of the polymer. The TPC3‐1 molecule was found to have an anomeric carbon sign of both α and β configurations and high‐branched chains. The network structure of TPC3‐1 was observed. CONCLUSION: The tea polysaccharide TPC3‐1 was an acid protein‐bound polysaccharide with an image of network structure. The results presented here will facilitate further study of the relationship between the chemical structure and biological role of tea polysaccharide. Copyright © 2008 Society of Chemical Industry     

Thermal inactivation of a heat-resistant lipase produced by the psychotrophic bacterium Pseudomonas fluorescens.

Lipase from Pseudomonas fluorescens was studied for thermostability at temperatures ranging from 100 C to 160 C. The heat treatments were in two media, and heating times necessary to inactivate 90% of the enzyme at constant temperature were extremely long even at high temperatures, e.g. 3.6 min at 140 C in nutrient broth and 2.0 min at 170 C in skim milk. The increments of temperature to reduce these heating times 90% were 37.0 C in nutrient broth and 38.9 C in skim milk. The lipase was inactivated only partly after 20 h at 20 C in 8 M urea, 6 M guanidine hydrochloride, and 1.0% sodium dodecyl sulfate. Four percent 2-mercaptoethanol showed no effect.     

禽肉中铜绿假单胞菌的分离及其耐药性

采用GB 4789.4—2010《食品安全国家标准食品微生物学检验沙门氏菌检验》中的选择性前增菌方法从鲜鹅肉、鸭肉样本中检测到2株疑似沙门氏菌的菌株,但经ATB生化鉴定和基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization-time of flight-mass spectrometry,MALDI-TOF-MS)鉴定为铜绿假单胞菌。比对2株铜绿假单胞菌和沙门氏菌的生化图谱,其中鸟氨酸脱羧酶、精氨酸双水解酶和L-阿拉伯糖的实验结果同为阳性,其余生化反应结果差异较大;2株铜绿假单胞菌的质谱标识峰足迹与肠炎沙门氏菌CICC21482有明显不同。微生物耐药实验结果表明:大部分抗生素对铜绿假单胞菌无抑制作用,只有喹诺酮类抗生素环丙沙星、氨基糖苷类抗生素庆大霉素和链霉素对其有一定的抑制作用。