焦磷酸测序法分析中国荷斯坦牛、娟姗牛及 水牛的乳蛋白基因多态性

目的 本研究旨在分析和比较中国荷斯坦牛、娟姗牛和水牛的乳蛋白基因多态性。方法 根据奶牛多态位点设计引物, 采用焦磷酸测序法分析乳蛋白基因多态性, 并采用高效液相色谱法进行验证。结果 荷斯坦牛和娟姗牛的乳蛋白(β-casein, κ-casein和β-lactoglobulin)均存在基因多态性, 而水牛仅在κ-casein上存在多态性且多态位点与另两种奶牛不同。结论 中国荷斯坦牛、娟姗牛和水牛的乳蛋白基因均存在多态性, 焦磷酸测序法能高通量、快速测定乳蛋白基因多态性。     

Growth and development of water buffalo and Friesian crossbred cattle, with special reference to the 'entire' and 'boneless' cuts

Twelve buffaloes, nine Friesian × Baladi and nine Friesian × (Friesian × Baladi) bulls were slaughtered over the live weight ranges 161–560 kg for buffaloes and 176–448 kg for cattle. Right sides of all carcasses were jointed and dissected and the increase in the weight of ‘entire’ and ‘boneless’ cuts and cut groups (i.e. pistol; BLRC) relative to the ‘entire’ and ‘boneless’ side weights, respectively, were examined using covariance analyses.

Increasing distoproximal and dorsoventral growth gradients were found in both species. Most noticeably, the sticking was early developing in buffaloes and late developing in cattle, whereas the shortloin developed approximately at an average rate in buffaloes and at a lower rate in cattle. Statistically significant but relatively slight differences were recorded between buffaloes and cattle in the adjusted means of the ‘entire’ and ‘boneless’ hind shank, sirloin (favouring buffaloes) and brisket (favouring cattle). Buffaloes were superior to cattle in weight of pistol. At an equal side weight of 73 kg buffaloes had significantly higher weight of pistol (maximum difference = 1·4 kg). At a 115 kg side weight, the maximum difference in ‘entire’ and ‘boneless’ pistol reached 3·58 and 5·04 kg, respectively.     

Hempseed protein derived antioxidative peptides: Purification,identification and protection from hydrogen peroxide-induced apoptosis in PC12 cells

Hempseeds have not been utilised to any significant extent by the industrial food markets. A novel antioxidant peptide derived from hempseed by-product was investigated. Hempseed protein isolate was prepared by alkaline extraction and subsequent isoelectric precipitation. Its enzymatic hydrolysate, obtained by Alcalase® treatment, was subjected to DA201-C macroporous absorption resin, with simultaneous desalting and concentrating of hydrophobic fragments with improved free radical-scavenging activities. The active fraction was further separated by gel filtration and reversed-phase high performance liquid chromatography to obtain two purified peptides; the peptides were characterised as NHAV and HVRETALV, by MALDI-TOF/TOF MS/MS. The protective effects of the purified peptides on hydrogen peroxide-induced cell apoptosis were investigated in rat pheochromocytoma line PC12 cells. The peptides, at a concentration of 10 μg/ml, possessed protective effects (P < 0.05) against cell death and oxidative apoptosis. The findings are significant for the discovery and development of natural antioxidants from hempseed by-products.     

Physicochemical, molecular and immunological characterization of camel calf rennet: a comparison with buffalo rennet

Camel calf rennet (CCR) and buffalo calf rennet (BCR) were prepared from dried abomasa to study their physicochemical properties and electrophoretic behaviour and to carry out an immunological characterization of the rennet proteins. CCR was more thermostable than BCR. The milk clotting activity of both rennets increased as pH decreased. The optimum temperatures for CCR and BCR were 50 and 45 degrees C respectively. CCR was more sensitive to increased CaCl2 in milk than BCR. Addition of NaCl to milk in the range 0-100 g/l resulted in a marked decrease in the clotting activity of both rennets. When the rennets were treated with acetone, the activity of BCR was completely destroyed, but that of CCR was unaffected. The proteolytic activity of CCR was higher than that of BCR and pepsin towards both camel and cows' milk caseins at pH 6.0. SDS-PAGE electrophoretic patterns of CCR and BCR proteins gave two major bands with molecular masses estimated as 52 and 39 kDa for CCR and 50 and 35 kDa for BCR. Immunodiffusion and immunoelectrophoresis using anti-CCR serum demonstrated immunological cross reactivity between CCR and BCR.     

Short communication: Phenotypic characterization of total antioxidant activity of buffalo,goat, and sheep milk

Free radicals are reactive and unstable waste molecules produced by cells, responsible of damages and alteration on DNA, proteins, and fat. The daily intake of antioxidant compounds, acting against free radicals and their detrimental effects, is essential for human health. Milk contains several compounds with antioxidant activity, and the sum of their reducing potential blocking free radicals development is defined as total antioxidant activity (TAA). This novel trait has been described in literature both in individual and bulk cow milk, but there are no reports from other dairy species. Therefore, the present study aimed to investigate phenotypic variation of TAA in individual samples of buffalo (n = 105), goat (n = 112), and sheep (n = 198) milk. Total antioxidant activity was measured through a reference spectrophotometric method, and expressed as millimoles per liter of Trolox equivalents (TE). The greatest TAA was observed in sheep milk, averaging 7.78 mmol/L of TE and showing also the broadest phenotypic variation expressed as coefficient of variation (13.98%). Significantly lower TAA values were observed for buffalo (7.35 mmol/L of TE) and goat (6.80 mmol/L of TE) milk, with coefficients of variation of 8.18 and 8.47%, respectively. Total antioxidant activity exhibited weak correlations with milk yield and chemical composition. Phenotypic values of TAA presented in this study will be used to assess the ability of mid-infrared spectroscopy to predict this new trait and thus to collect data at the population level.     

Characterization of heme-coordinating histidyl residues of an engineered six-coordinated myoglobin mutant based on the reactivity with diethylpyrocarbonate, mass spectrometry, and electron paramagnetic resonance spectroscopy

A genetically engineered porcine myoglobin triple mutant (H64V/V68H/H93A) (VHA-Mb) contains 6 non-axial His residues (His24, His36, His48, His81, His82, and His119) besides two candidate axial His residues (His68 and His97). Although previous resonance Raman study on the ferric VHA-Mb were not conclusive for its coordination structure, present EPR parameters of the ferric VHA-Mb were consistent with bis-imidazole coordination of His68/His97. We further investigated the reactivity of these possible His ligands with diethylpyrocarbonate (DEPC) to clarify the coordination structure and their protonation states in ferric form. We found that the non-axial His residues were easily modified with a low concentration of DEPC based on UV spectral changes and MALDI-TOF-MS analyses. On the other hand, the two candidate axial His ligands were protected from the modification due to a limited steric exposure of their imidazoles to solvent, the Fe(3+)-N(epsilon2) coordination bond, and the protonation of N(delta1) by forming a hydrogen bond with their immediate surroundings. However, once N-carbethoxylation occurred at N(epsilon2) of His97, resulting in a disruption of the heme Fe(3+)-N(epsilon2) coordination bond, it facilitated the second N-carbethoxylation to take place at N(delta1) of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. These phenomena were consistent with the bis-His68/His97 coordination. Further, these were not observed at all for cytochrome b(561), a transmembrane di-heme containing protein responsible for the ascorbate-specific transmembrane electron transfer, where only a specific N(delta1)-carbethoxylation of axial His occurred at a low concentration of DEPC, leading to an inhibition of the electron acceptance from ascorbate without a release of the heme. These distinct results might be related to a specific physiological mechanism being operative at the cytosolic heme center of cytochrome b(561).     

Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing,purification, and characterization

An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.     

Short communication: Identification and technological characterization of yeast strains isolated from samples of water buffalo Mozzarella cheese

Sixty yeast cultures were isolated from samples of water buffalo Mozzarella, a popular “pasta filata” cheese, originating on 16 farms located in the provinces of Salerno, Caserta, and Frosinone (Italy). Strains were identified by means of 5.8S internal transcribed spacer rDNA PCR-RFLP combined with 26S rRNA gene partial sequencing and characterized for their ability to exert biochemical properties of technological interest. The recorded dominance of fermenting yeasts such as the lactose-fermenting Kluyveromyces marxianus (38.3% of the total isolates) and the galactose-fermenting Saccharomyces cerevisiae (21.6% of the total isolates) suggests that these yeasts contribute to the organoleptic definition of the water buffalo Mozzarella. The speciographic analysis revealed the presence of 7 other species rarely or never reported in a dairy environment belonging to the genera Pichia and Candida, whose role in Mozzarella cheese organoleptic properties need to be further investigated.     

Gluco-oligosaccharides as potential prebiotics: Synthesis,purification, structural characterization,and evaluation of prebiotic effect

Prebiotics have long been used to modulate the gut microbiota and improve host health. Most established prebiotics are nondigestible carbohydrates, especially short-chain oligosaccharides. Recently, gluco-oligosaccharides (GlcOS) with 2–10 glucose residues and one or more O-glycosidic linkage(s) have been found to exert prebiotic potentials (not fully established prebiotics) because of their selective fermentation by beneficial gut bacteria. However, the prebiotic effects (non-digestibility, selective fermentability, and potential health effects) of GlcOS are highly variable due to their complex structure originating from different synthesis processes. The relationship between GlcOS structure and their potential prebiotic effects has not been fully understood. To date, a comprehensive summary of the knowledge of GlcOS is still missing. Therefore, this review provides an overview of GlcOS as potential prebiotics, covering their synthesis, purification, structural characterization, and prebiotic effect evaluation. First, GlcOS with different structures are introduced. Then, the enzymatic and chemical processes for GlcOS synthesis are critically reviewed, including reaction mechanisms, substrates, catalysts, the structures of resultant GlcOS, and the synthetic performance (yield and selectivity). Industrial separation techniques for GlcOS purification and structural characterization methods are discussed in detail. Finally, in vitro and in vivo studies to evaluate the non-digestibility, selective fermentability, and associated health effects of different GlcOS are extensively reviewed with a special focus on the GlcOS structure–function relationship.     

6-phosphofructokinase from Pichia pastoris: purification,kinetic and molecular characterization of the enzyme

6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.     

Hot and cold water infusion aroma profiles of Hibiscus sabdariffa: fresh compared with dried

Abstract: Calyxes from the Roselle plant (Hibiscus sabdariffa L.) were used to prepare cold (22 °C for 4 h) and hot (98 °C for 16 min) infusions/teas from both fresh and dried forms. Aroma volatiles were extracted using static headspace SPME and analyzed using GC‐MS and GC‐O with 2 different columns (DB‐5 and DB‐Wax). Totals of 28, 25, 17, and 16 volatiles were identified using GC‐MS in the dried hot extract (DHE), dried cold extract (DCE), fresh hot extract (FHE), and fresh cold extract (FCE) samples, respectively. In terms of total GC‐MS peak areas DHE ? DCE > FHE ? FCE. Nonanal, decanal, octanal, and 1‐octen‐3‐ol were among the major volatiles in all 4 beverage types. Thirteen volatiles were common to all 4 teas. Furfural and 5‐methyl furfural were detected only in dried hibiscus beverages whereas linalool and 2‐ethyl‐1‐hexanol were detected only in beverages from fresh hibiscus. In terms of aroma active volatiles, 17, 16, 13, and 10 aroma active volatiles were detected for DHE, DCE, FHE, and FCE samples, respectively. The most intense aroma volatiles were 1‐octen‐3‐one and nonanal with a group of 4 aldehydes and 3 ketones common to all samples. Dried samples contained dramatically higher levels of lipid oxidation products such as hexanal, nonanal, and decanal. In fresh hibiscus extracts, linalool (floral, citrus) and octanal (lemon, citrus) were among the highest intensity aroma compounds but linalool was not detected in any of the dried hibiscus extracts. Practical Application: Hibiscus teas/infusions are one of the highest volume specialty botanical products in international commerce. The beverage is consumed for both sensory pleasure and health attributes and is prepared a number of ways throughout the world. Although color and taste attributes have been examined, little information is known about its aroma volatiles and no other study has compared extractions from both fresh and dried as well as extraction temperature differences. This is also, apparently, the first study to identify the aroma active volatiles in hibiscus beverages using GC‐olfactometry. Manufacturers and consumers will now have a better understanding of why hibiscus teas prepared in different ways from either fresh or dried forms have a different flavor quality and intensity.     

Expression in Escherichia coli of a recombinant adenosine kinase from Saccharomyces cerevisiae: purification, kinetics and substrate analyses

The Saccharomyces cerevisiae ADO1 gene is known to encode a homologue of eukaryotic adenosine kinases. This gene was expressed in Escherichia coli as a recombinant protein fused to a polyhistidine tag by using the rhamnose-inducible bacterial promoter rhaB. The recombinant protein was purified to apparent homogeneity and its ability to phosphorylate different substrates was evaluated. Adenosine (Km 3 microM) is its primary substrate. In addition, it also phosphorylates, albeit less efficiently, 3'-deoxyadenosine (cordycepin; Km 1.84 mM) and 3'-amino-3'-deoxyadenosine (Km 0.26 mM). Other kinetic properties of the recombinant enzyme have also been determined.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

Extraction of ginger rhizome: kinetic studies with dichloromethane, ethanol, 2-propanol and an acetone—water mixture

The kinetics of extraction of [6]-gingerol from ground Jamaican ginger rhizome have been determined at 30°C in dichloromethane, ethanol, isopropanol and an 80% (v/v) acetone + 20% (v/v) water mixture. The extractions all proceeded in three stages: an initial 'washing' stage, a fast stage and a subsequent much slower stage. The rate of extraction of hexahydrocurcumin in ethanol was found to follow a simpler pattern. From the first order plots, the diffusion coefficients of the extracted solubles within the ginger particles were calculated. They varied inversely with the 0.6 power of the solvent viscosity, which explained why the rates of [6]-gingerol extraction decreased in the sequence: acetone < acetone + water < dichloromethane < ethanol < isopropanol. These results show that solvents of low viscosity should be chosen to attain fast extraction rates. The diffusion coefficient of [6]-gingerol was also measured at 30°C in pure acetone, ethanol and isopropanol. The values in these bulk solvents were 13–20 times greater than the diffusion coefficients of [6]-gingerol within the ginger particles for the fast stage and over 900–1800 times greater than those for the slow stage. These hindrance factors quantify the effect of the ginger matrix environment on internal diffusion.     

Using water to cool cattle: behavioral and physiological changes associated with voluntary use of cow showers

Water is commonly used to cool cattle in summer either at milking or over the feed bunk, but little research has examined how dairy cows voluntarily use water separate from these locations. The objectives were to describe how and when dairy cattle voluntarily used an overhead water source separate from other resources, such as feed, and how use of this water affected behavioral and physiological indicators of heat stress. Half of the 24 nonlactating cattle tested had access to a “cow shower” composed of 2 shower heads activated by a pressure-sensitive floor. All animals were individually housed to prevent competition for access to the shower. Over 5 d in summer (air temperature = 25.3 ± 3.3°C, mean ± standard deviation), cattle spent 3.0 ± 2.1 h/24 h in the shower, but considerable variability existed between animals (individual daily values ranged from 0.0 to 8.2 h/24 h). A portion of this variation can be explained by weather; shower use increased by 0.3 h for every 1°C increase in ambient temperature. Cows preferentially used the shower during the daytime, with 89 ± 12% of the time spent in the shower between 1000 and 1900 h. Respiration rate and skin temperature did not differ between treatments [53 vs. 61 breaths/min and 35.0 vs. 35.4°C in shower and control cows, respectively; standard error of the difference (SED) = 5.6 breaths/min and 0.49°C]. In contrast, body temperature of cows provided with a shower was 0.2°C lower than control cows in the evening (i.e., 1800 to 2100 h; SED = 0.11°C). Cows with access to a shower spent half as much time near the water trough than control animals, and this pattern became more pronounced as the temperature-humidity index increased. In addition, cattle showed other behavioral changes to increasing heat load; they spent less time lying when heat load index increased, but the time spent lying, feeding, and standing without feeding did not differ between treatments. Cows had higher respiration rate, skin temperature, and body temperature as heat load index increased, regardless of treatment. These data suggest that cattle, when given the opportunity, will make considerable use of a shower to reduce heat load, but that individuals are highly variable in their use of this resource. The variability between cows indicates that the behavioral response to water is likely an important, but poorly understood, consideration in the design of sprinkler systems used for summer cooling.     

Dibenzothiophene desulfurizing enzymes from moderately thermophilic bacterium Bacillus subtilis WU-S2B: purification, characterization and overexpression

The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzymes, DBT monooxygenase (BdsC) and DBT sulfone monooxygenase (BdsA), were purified from the wild-type strain, and the last one, 2'-hydroxybiphenyl 2-sulfinic acid desulfinase (BdsB), was purified from the recombinant Escherichia coli overexpressing the gene, bdsB, with chaperonin genes, groEL/ES. The genes of BdsC and BdsA were also overexpressed. The molecular weights of BdsC and BdsA were determined to be 200 and 174 kDa, respectively, by gel filtration chromatography, suggesting that both enzymes had four identical subunits. BdsB had a monomeric structure of 40 kDa. The three enzymes were characterized and compared with the corresponding enzymes (DszC, DszA, and DszB) of mesophilic desulfurization bacteria. The specific activities of BdsC, BdsA, and BdsB were 84.2, 855, and 280 units/mg, respectively, and the latter two activities were higher than those of DszA and DszB. The heat stability and optimum temperature of BdsC, BdsA, and BdsB were higher than those of DszC, DszA, and DszB. Other enzymatic properties were investigated in detail.