Synergistic degradation of arabinoxylan with alpha-L-arabinofuranosidase, xylanase and beta-xylosidase from soy sauce koji mold, Aspergillus oryzae, in high salt condition

This study addresses induction and some properties of alpha-L-arabinofuranosidase from a soy sauce koji mold, Aspergillus oryzae HL15, cultured on solid and liquid media. Alpha-L-arabinofuranosidase was induced by soybean polysaccharide and secreted into media on solid cultivation; the enzyme was associated with mycelium as a cell-wall-bound form in liquid cultivation. A major alpha-L-arabinofuranosidase, which was purified homogeneously on SDS-PAGE, showed highest activity in the presence of 10% of NaCl; also, somewhat higher activity was observed even in 25% NaCl than in the absence of NaCl. Arabinoxylan was synergistically degraded by xylanase, beta-xylosidase, and alpha-L-arabinofuranosidase from A. oryzae HL15 in the condition of imitative pH 5.0 and 15% NaCl concentration of the soy sauce moromi mash. These results suggest that arabinose and xylose, which are closely related to melanoidin formation, can be released by synergistic action of these enzymes in soy sauce moromi mash.     

固相萃取-高效液相色谱法测定饼干中的维生素B12

目的建立固相萃取-高效液相色谱法测定饼干中维生素B12的分析方法。方法样品中的维生素B12用0.1mol/L乙酸钠溶液(pH=4.5)提取,二乙烯苯和N-乙烯基吡咯烷酮共聚物(N-vinylpyrrolidonecopolymer)HLB固相萃取柱净化, 10%的乙腈水溶液定容后用高效液相色谱仪测定。结果维生素B12含量在0.5～10 mg/L线性关系良好,线性相关系数r=0.9999,检出限为0.1 mg/kg。在精密度实验中,相对标准偏差为0.74%～3.75%,在0.3、0.8、1.7mg/kg的添加水平下,加标回收率为93.9%～99.4%。结论该方法准确、高效,适合饼干中维生素B12的测定。     

Purification,characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39.3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56.5% homology with bovine milk lysozyme and 30.4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.     

固相萃取-高效液相色谱法同时测定食品中16种禁限用色素

目的建立一种高效、准确、同时测定食品中16种禁限用色素(柠檬黄、新红、苋菜红、胭脂红、日落黄、酸性品红6B、诱惑红、酸性红13、丽春红2R、丽春红SX、酸性橙I、赤藓红、酸性黄11、酸性蓝113、罗丹明B、分散红1)的方法。方法待测样品先后经0.5%氨水和乙醇+氨水+水提取、经OasisHLB固相萃取柱净化后,以Eclipse XDB C18(150 mm×4.6 mm, 5?m)为分离柱,甲醇和10 mmol/L乙酸铵为流动相进行梯度洗脱,流速为0.8 mL/min,检测波长为470、520、550和490 nm的条件下用高效液相色谱法进行测定,外标法定量。结果 16种禁限用色素在此次方法下有较好的线性关系(r≥0.9997),检出限(S/N=3)为33.0～144.0μg/L。结论本研究建立的固相萃取-高效液相色谱法可以同时测定食品中16种禁限用色素,且方法高效、准确。     

Calcium binding of peptides derived from enzymatic hydrolysates of whey protein concentrate

This study was carried out to investigate the peptides derived from enzymatic hydrolysates of whey protein concentrate. The physiological activity of peptides in whey protein may be used in food additives to promote the absorption of calcium and prevent bone disorders. The whey protein was hydrolysed by trypsin, and the separation of peptides, the properties of hydrolysates and the analysis of the ability to inhibit the formation of calcium phosphates were then investigated. Calcium-binding peptides were produced by tryptic hydrolysis of whey protein concentrate and further purified by precipitation and chromatography on DEAE-cellulose. The hydrolysates were loaded onto an ion-exchange column, followed by stepwise elution with 0, 0.25, 0.5, and 0.75 m NaCl in equilibration buffer to separate the peptides. Trypsin hydrolysates were shown to peak with 0.25 m NaCl and 0.5 m NaCl. The results of SDS-PAGE analysis showed that the peptides with a small molecular weight of about 1.4 to 3.4 kDa were present in the fraction resulting from 0.25 m and 0.5 m NaCl stepwise elution by ion-exchange chromatography of tryptic hydrolysates. The results of this study show that the whey protein hydrolysates produced by the action of trypsin have the ability to inhibit the formation of calcium phosphates.     

肌酐水解酶菌株S-09发酵条件优化及酶的分离纯化

该实验以海洋来源的微小杆菌（Exiguobacterium sp.）S-09为出发菌株,对其发酵培养基和产酶条件进行优化。并将粗酶液经硫酸铵盐析、透析、超滤离心和Sephadex G-100凝胶过滤层析进行分离纯化。结果表明,其最佳发酵培养基为0.5%的肌酐作为碳源、0.3%胰蛋白胨和0.2%玉米浆作为复合氮源,诱导物肌酸含量为0.3%;其最佳发酵条件为作用pH值7.5、温度25 ℃、装液量50 mL/250 mL、接种量3%。Fe2+和Mn2+能显著促进产酶。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶电泳结果显示,纯化后的肌酐水解酶分子质量为24.3 ku。     

液相色谱法测定肉制品中万古霉素的残留量

采用液相色谱法测定肉制品中万古霉素的残留量。样品经过0.1%甲酸水-乙腈(体积比为7∶3)提取,二氯甲烷去脂,固相萃取柱净化,利用液相色谱仪,采用外标法定量检测。结果显示,万古霉素液相色谱检测方法的线性范围是8.5μg/mL^170μg/mL,线性相关系数大于0.99。方法检出限为0.5 mg/kg,定量限为8 mg/kg。添加回收率在75%~95%,精密度为5%~8%。该方法适用于肉制品中万古霉素残留量的测定。     

Antimicrobial synergism of partially-denatured lysozyme with glycine: effect of sucrose and sodium chloride

Previously we reported that heat-treatment of lysozyme at 80°C for 20 min in pH 6.0 (HL80/6) greatly promoted its antimicrobial action to Gram-negative bacteria without detrimental effects on its inherent action to Gram-positive ones. In this study the effects of sucrose and NaCl on the promoted antimicrobial activity of HL80/6 and synergy with glycine were investigated. This potent antimicrobial lysozyme (HL80/6) retained 54% of the native enzymatic activity. The enhanced bactericidal action of HL80/6 against Escherichia coli K12 was gradually decreased with increasing sucrose and NaCl concentration in the medium, where complete suppression was observed at 2% sucrose and 0.1% NaCl. On the other hand, 1% NaCl concentration was required to suppress the antimicrobial activity against Staphylococcus aureus of either native lysozyme (NLz) or HL80/6, while sucrose up to 8% had no detectable inhibitory effect on this strain. However, similar doses of sucrose or NaCl had no effect on the enzymatic activity of both NLz and HL80/6, indicating that the antimicrobial action of HL80/6 is independent of its muramidase activity. HL80/6 lysozyme, but not NLz, exhibited good synergy with glycine against Gram-negative bacteria even in the presence of the inhibitory doses of sucrose or NaCl, suggesting a possible application in food industry as a food preservative. Thus, the results introduce an interesting finding that partial denaturation of lysozyme can induce its antimicrobial specificity to include the food-borne Gram-negative pathogens and heralded fascinating opportunities for application of HL80/6 with glycine in formulated food systems.     

高效液相色谱-串联质谱法测定蜂蜡中痕量氯霉素残留

目的建立正己烷预处理-固相萃取净化-高效液相色谱-串联质谱法检测蜂蜡中痕量氯霉素的分析方法。方法试样采用正己烷预溶解,经水提取后进行亲水亲脂平衡(hydrophilic-lipophilic-balance, HLB)固相萃取小柱净化,用Accucore XL C18柱分离,多反应监测(multiple reaction monitoring, MRM)模式串联质谱进行测定,内标法定量。结果氯霉素在0.3~10 ng/mL范围内与峰面积具有较好的线性关系,相关系数大于0.998。该方法检出限(S/N>3)为0.05μg/kg,方法定量限(S/N>10)为0.3μg/kg,在0.3、1.0和2.0μg/kg 3个添加水平下的回收率为80.2%~105.2%,相对标准偏差均小于8%(n=6)。结论本方法快速、灵敏、准确,适用于日常大批量蜂蜡样品中痕量氯霉素残留量的分析。     

枯草芽孢杆菌内切木聚糖酶的纯化与性质研究

建立了一种快速、简便分离纯化木聚糖酶的方法。采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合 ,从枯草芽孢杆菌 (Bacillussubtilis)固态培养基发酵产物中分离得到了 2种内切木聚糖酶 ,分别定义为xylⅠ和xylⅡ ,它们水解桦木木聚糖的主要产物有木二糖、木三糖和聚合度更高的木聚寡糖 ,没有木糖。SDS PAGE显示内切木聚糖酶xylⅡ为单肽链结构 ,分子质量为 98 8ku。内切木聚糖酶xylⅡ的酶反应最适温度为 5 0℃ ,酶反应的最适 pH为 7 0。Mn2 + 对xylⅡ酶反应具有促进作用 ,将酶活提高了 2 7倍 ,而Fe3+ 对该酶反应起完全抑制作用。     

多功能免疫亲和柱净化-超高效液相色谱串联质谱法快速检测咖啡豆中的11种真菌毒素

建立了同时测定咖啡豆中11种真菌毒素的超高效液相色谱-串联质谱分析方法,实验考察了色谱柱选择、提取溶剂组成、固相萃取柱选择、流动相比例对分析结果的影响。以咖啡豆为研究对象,样品均质后,经乙腈-水-甲酸（85+14+1,V/V）振荡提取,离心,过Oasis PRiME HLB柱,取净化液10 mL用磷酸缓冲液稀释至50 mL,经多功能免疫亲和柱净化,氮吹、50%乙腈复溶,以0.1%甲酸-2 mmol/L乙酸铵溶液-乙腈为流动相,用Waters BEH C₁₈柱分离,超高效液相色谱-质谱仪分析,内标法定量。结果表明,11种真菌毒素在各自线性范围内线性关系良好,相关系数均大于0.998,检出限为0.008~0.544 μg/kg。在低、中、高3个添加水平下的平均回收率为80.2%~114%,相对标准偏差为1.4%~7.9%。本方法步骤简便、快速高效,适用于咖啡豆中11种真菌毒素的同时测定分析。     

燕麦加工中苦味物质的分离与初步鉴定

目的通过极性分离与感官评价相结合的方法,采用高效液相色谱-四极杆飞行时间质谱(高分辨率液相色谱质谱)联用方法对燕麦加工中的苦味物质进行初步鉴定。方法燕麦样品经过正己烷、75%甲醇萃取,经固相萃取柱净化,经半制备液相色谱富集,利用高分辨率液相色谱质谱技术鉴定。结果 5类苦味物质的可能分子式有C_(27)H_(16)N_2O_4、C_(23)H_(42)N_3O_(14)、C_(24)H_(30)N_(2O)、C_(19)H_(30)N_(4O)、C_(45)H_(64)O_4、C_(22)H_(32)N_2O、C_(17)H_(37)N_(12)O、C_(35)H_(33)N_4O_(14)以及C_(25)H_(38)N_4O_(13)。结论本研究初步鉴定出5类苦味物质的可能分子式,为后续结构鉴定奠定基础。     

一种胰蛋白酶亲和材料的制备及应用

基于抑制剂与酶的特异亲和作用,将具有良好稳定性的荞麦胰蛋白酶抑制剂(Buckwheat trypsin inhibitor,BTI)固定化,制备成一种胰蛋白酶的亲和吸附剂,实现胰蛋白酶的高效纯化。通过原核表达、Ni2+-NTA亲和层析和Superdex G-25凝胶过滤技术得到电泳纯的BTI,以溴化氰活化琼脂糖凝胶(CNBr-Sepharose CL-4B)作为亲和层析载体,制备亲和吸附剂BTI-Sepharose,检测其对胰蛋白酶的特异性吸附作用,并进一步研究BTI-Sepharose对胰蛋白酶吸附及解吸附的条件。制备的BTI-Sepharose对胰蛋白酶具有特异吸附性,可用于胰蛋白酶的亲和纯化。缓冲液p H对BTI-Sepharose与胰蛋白酶的吸附及解吸附均有重要影响,二者吸附作用的最适pH为7.2,在pH为3.5时两者能够完全分离,且不影响胰蛋白酶的生物活性。试验制备的BTI-Sepharose可实现一步层析制备高纯度胰蛋白酶,为胰蛋白酶的高效纯化及新型胰蛋白酶的研究开发提供理论依据。     

Aspergillus ficuum菊粉酶的分离纯化与鉴定

Aspergillus ficuum菊粉酶系经硫酸铵分级沉淀、透析脱盐、DEAE-Cellulose离子交换色谱、Sepharose CL-6B凝胶过滤色谱和制备电泳技术分离纯化,获得五个电泳纯酶:三个外切菊粉酶组分Exo-I、Exo-II、Exo-III和两个内切菊粉酶组分Endo-I、Endo-II。SDS-PAGE分析测得五种酶的分子量分别为:Exo-I:69961Da,Exo-II:39973Da,Exo-III:45561Da,Endo-I:33574Da,Endo-II:30769Da。     

基于宰后蛋白质磷酸化研究L-组氨酸和L-赖氨酸对鸡胸肉加工特性的影响

为研究低盐（1% NaCl）条件下添加L-组氨酸（L-histidine，L-His）及L-赖氨酸（L-lysine，L-Lys）调节宰后鸡胸肉肌原纤维蛋白磷酸化的变化规律及其对鸡胸肉加工特性（蒸煮损失、质构、蛋白溶解度、流变特性）的影响，提取腌制后鸡胸肉肌原纤维蛋白，进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，再通过荧光染色和液相色谱-串联质谱鉴定。结果表明：L-His和L-Lys的添加不影响肌原纤维蛋白的整体磷酸化水平，但是可以改变单个蛋白质的磷酸化水平；相比于1% NaCl处理组，1% NaCl＋0.06% L-His/L-Lys处理组硬度和蛋白溶解度均有显著提高，蛋白流变特性改善，蒸煮损失无显著变化；相关性分析得出，多个蛋白质磷酸化水平与肉的蒸煮损失及蛋白溶解度等加工特性呈显著正相关或负相关，且单个蛋白质的磷酸化水平还会受到其他蛋白质磷酸化水平的影响；将11 条磷酸化水平差异显著的条带进行质谱鉴定后，发现大多是与肌肉收缩或糖酵解有关的酶，表明L-His、L-Lys的添加可能通过对肌原纤维蛋白磷酸化水平产生正面效应影响宰后僵直成熟过程，进而影响肉品品质。     

固相萃取-气相色谱法同时检测草莓中13种 农药残留

目的 建立一种固相萃取-毛细管柱气相色谱方法,可以同时检测草莓中13种农药残留量。方法 草莓样品匀浆后,经乙腈提取,NH₂固相萃取柱净化,采用HP-5毛细管气相色谱柱进行分离,GC-ECD 进行定性及定量分析。结果 13 种农药残留的色谱图分离效果良好,线性相关系数均大于0.996,方法检出限在0.01 mg.kg_-1~0.5 mg.kg_-1之间。添加回收试验表明,该方法平均回收率在70.5~114.5 % 之间,相对标准偏差在2.17~6.85 %之间。结论 该方法简单、快速、灵敏、净化效果好、回收率高,适合草莓中多种农药残留的检测和安全监控。通过对50份草莓样品进行检测,检出百菌清、乙草胺、毒死蜱、粉唑醇、醚菌酯、烯酰吗啉6种农药。     

海洋细菌Pseudoalteromonas sp.HL9的筛选鉴定及产磷脂酶B性质

从黄海海泥中筛选产磷脂酶菌株,其中酶活力最高的HL9鉴定为假交替单胞菌属(Pseudoalteromonas).该菌株最佳生长培养基组成为米糠与酵母粉,最佳生长pH 8.0、NaC1质量浓度30 g/L、培养温度25℃.优化后产酶最高发酵条件:发酵培养基配方为10 g/L麸皮和5 g/L鱼粉蛋白胨,NaC120 g/L...     

指状青霉DSM62840生物转化柠檬烯生产α-松油醇过程中关键酶的分离纯化及质谱鉴定

以指状青霉DSM62840为研究对象,通过高压法破碎细胞,采用超高速离心、硫酸铵沉淀、羟基磷灰石层析和凝胶过滤层析等方法对指状青霉DSM62840生物转化柠檬烯生产a-松油醇过程中的关键酶进行分离纯化.经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测,结果显示在分子质量约为50 kDa处有一条明显的蛋白条带,并通过液相色谱-串...     

Purification and characterization of intracellular and extracellular, thermostable and alkali-tolerant alcohol oxidases produced by a thermophilic fungus, Thermoascus aurantiacus NBRC 31693

Intracellular and extracellular alcohol oxidases (AO int and AO ext) were purified from the liquid and solid cultures of a thermophilic fungus, Thermoascus aurantiacus NBRC 31693, as electrophoretically and isoelectrophoretically homogeneous proteins, respectively. Both enzymes contained a flavin adenine dinucleotide (FAD) cofactor and were stained with Schiff's reagent. The molecular weight of AO int was estimated to be about 320 kDa and its subunit was 75 kDa. The molecular weight of AO ext was about 560 kDa, and it was composed of two types of subunits (75 kDa and 59 kDa). The pIs of AO int and AO ext were 5.88 and 6.08, respectively. AO int and AO ext were stable up to 60 degrees C and 55 degrees C, respectively. The enzymes were stable over a wide range of pH from 6 to 11. AO int oxidized short straight-chain alcohols (K(m) for methanol, 13.5 mM and K(m) for ethanol, 15.8 mM). On the other hand, AO ext could oxidize secondary alcohols and aromatic alcohols (veratryl alcohol and benzyl alcohol) in addition to straight-chain alcohols (K(m) for methanol, 0.5 mM and K(m) for ethanol, 10.2 mM).     

Saio法提取大豆7S球蛋白的纯化及鉴定

研究利用Sephadex G-200凝胶柱层析法和纤维素DE-52离子交换柱层析法分离纯化经过Saio法提取的大豆7S球蛋白样品,采用SDS-聚丙烯酰胺凝胶电泳和高效毛细管电泳进一步鉴定Saio法提取的大豆7S球蛋白样品的组分及含量。结果表明,Saio法提取的大豆7S球蛋白纯度约为81%。