特殊医学用途婴儿配方食品中乳铁蛋白的检测

建立特殊医学用途婴儿配方食品中乳铁蛋白的高效液相色谱（high performance liquid chromatography,HPLC）检测方法。试样中的乳铁蛋白经磷酸盐缓冲液提取,肝素亲和柱净化后,用反相-HPLC分离,以0.1%三氟乙酸和乙腈作为流动相进行梯度洗脱,HPLC仪紫外检测器检测,外标法定量。结果表明：在乳铁蛋白质量浓度50～500 μg/mL范围内,检测结果呈现良好线性关系;该方法的检出限为5 mg/100 g,定量限为15 mg/100 g,平均回收率为93.9%;同一质量浓度乳铁     

高效液相色谱法测定婴儿配方奶粉中乳铁蛋白含量.

本研究建立了一种用高效液相色谱测定婴儿配方奶粉中乳铁蛋白的方法。色谱条件:色谱柱为Hi Trap Heparin肝素琼脂糖亲和柱(0.7×2.5cm,1m L),流动相A为50mmol/L的Na2HPO4溶液(p H7.00),流动相B为50mmol/L的Na2HPO4+1.0mol/L的Na Cl溶液(p H7.00),检测波长为230nm。结果表明:乳铁蛋白的检出限为3.0mg/100g,回收率在86%~99%之间,相对标准偏差(RSD)为3.4%。用此方法检测婴儿配方奶粉中乳铁蛋白,操作简单,结果准确可靠,精密度高,重现性好。     

利用高效液相色谱仪测定猪乳中乳清蛋白含量的方法及其应用

为了建立快速准确检测猪乳中溶菌酶、乳铁蛋白、α-乳白蛋白、血清白蛋白和β-乳球蛋白高效液相色谱方法,采用0.1%三氟乙酸水溶液为流动相A,0.1%三氟乙酸乙腈溶液为流动相B进行程序洗脱,用Xbridge~(TM) Sneild RP C18色谱柱对α-乳白蛋白、β-乳球蛋白和溶菌酶进行分离检测,用Supelcosil C18色谱柱对乳铁蛋白和血清白蛋白进行分离检测。结果显示:5种乳清蛋白分离良好,在25~1 000 mg·L~(-1)范围内呈良好线性,标准曲线回归系数均大于0.99,RSD小于5%,回收率在91%~97%,溶菌酶、乳铁蛋白、α-乳白蛋白、血清白蛋白和β-乳球蛋白的检测限分别为0.2、4.0、0.6、0.3和1.0 mg·L~(-1);对猪乳中乳清蛋白进行测定显示,β-乳球蛋白、溶菌酶、乳铁蛋白和血清白蛋白含量在母猪分娩后16 d内逐渐降低,其含量均在母猪分娩后第1天最高,分别为17.2、0.224、2.0和15.6 g·L~(-1),在第16天含量最低,分别为5.6、0.092、0.5和3.6 g·L~(-1);α-乳白蛋白含量在母猪分娩后逐渐升高,在第7天时含量达到最高为3.3 g·L~(-1),随后降低,保持在3.1 g·L~(-1)。结果表明:建立的乳清蛋白高效液相色谱法,能够快速准确检测猪乳中5种乳清蛋白的含量。     

卫生部批准四种新型食品添加剂

卫生部发布今年第六号公告,批准了竹叶抗氧化物、喹啉黄、海萝胶、乳铁蛋白4个食品添加剂新品种, 30个扩大使用范围、使用量的品种,并公布了119种食品用香精的名单,新规定从即日起开始实施。在4个新品种中,竹叶抗氧化物可用于食用油脂、肉制品、水产品、膨化食品,最大使用量不超过0.5g/kg;喹啉黄作为着色剂,可用于预调酒,最大使用量为100mg/L;海萝胶为增稠剂,可用于胶基糖果,最大使用量为10g/kg;乳铁蛋白可用于婴儿配方奶粉、较大婴儿和幼儿配方奶粉,最大使用量为30-100mg/100g。     

高效液相色谱法测定乳粉中5种核苷酸的含量

建立乳粉中核苷酸高效液相色谱的测定方法.采用超纯水作为提取剂,0.5％的乙酸沉淀蛋白,SAX固相萃取小柱净化,C18色谱柱分离,流动相为0.1 mol/L的磷酸二氢钾溶液.每100 g乳粉中核苷酸添加量为0.3～50 mg时,回收率大于90％,定量限为0.3 mg/100 g.该方法具有结果准确、操作简便、重复性好等优点,可用于乳粉中核苷酸的检测.     

高效液相色谱法测定发酵乳中着色剂的含量

建立发酵乳中8 种着色剂（新红、苋菜红、诱惑红、胭脂红、柠檬黄、日落黄、靛蓝和亮蓝）的高效液相色谱测定方法。采用pH值为7.7～7.9的水为提取剂,聚酰胺固相萃取小柱净化,C18色谱柱（赛默飞RP-MS,4.6 mm×100 mm,2.6 μm）分离,采用乙酸铵溶液（0.02 mol/L）-甲醇作为流动相进行梯度洗脱。结果表明：每1 000 g发酵乳中8 种着色剂添加量为0.5～5.0 mg时,除靛蓝外的其他着色剂回收率均大于90%,靛蓝的回收率接近80%;方法定量限为0.5 mg/kg;发酵乳加标样品分     

高效液相色谱法测定调制乳中低聚果糖的含量

建立调制乳中低聚果糖高效液相色谱的测定方法.采用水作为提取剂,乙腈沉淀蛋白,氨基色谱柱分离,流动相为70％乙腈溶液,示差检测器检测.每100 g调制乳中低聚果糖添加量为20～500 mg时,回收率为80.2％～107.1％,定量检出限为20 mg/100g.该方法操作简便、检测准确,可用于调制乳中低聚果糖的检测.     

高效液相色谱法测定乳品与饲料中阿维菌素类药物残留

建立乳品与饲料中阿维菌素类药物(埃普利诺菌素、阿维菌素、多拉菌素和伊维菌素)残留液相色谱的测定方法。乙腈为提取剂,tC18固相萃取小柱净化,三氟乙酸酐和N-甲基咪唑柱前衍生,C18柱分离,甲醇-乙腈的混合溶剂与水梯度洗脱,液相色谱仪荧光检测器检测,外标法定量。每1 000 g乳粉(或每1 000 mL液态乳)中阿维菌素添加量为1.5 μg时,回收率大于95％。本方法液态乳、酸乳、乳粉的定量限为2μg/kg,饲料的定量限为10 μg/kg。该方法具有结果准确、操作简便、重复性好等优点,可用于乳品和饲料中阿维菌素类药物残留的检测。     

乳铁蛋白及其水解物对直投式乳酸菌发酵剂生长的影响

以直投式乳酸发酵剂为实验菌,研究乳铁蛋白及其水解产物对发酵剂各生长阶段的影响。实验条件为乳铁蛋白质量浓度范围0.5～2.5mg/mL,乳铁蛋白水解物的质量浓度范围0.05～0.3mg/mL,无氧条件下37℃培养。结果表明,37℃恒温培养,乳铁蛋白在0.5～2.0mg/mL范围内对乳酸菌生长有促进作用,最适添加质量浓度为1.0mg/mL,抑菌质量浓度为大于2.5mg/mL;乳铁蛋白水解物在0.05～0.25mg/mL范围内对乳酸菌生长有促进作用,最适添加质量浓度为0.1mg/mL,抑菌质量浓度为大于0.3mg/mL。     

牛乳铁蛋白多克隆抗体的制备、纯化及定量检测

本试验旨在制备高纯度和特异性的牛乳铁蛋白多克隆抗体,为鉴定并定量检测牛奶样品中及奶牛乳腺组织中合成的乳铁蛋白提供试验材料。选用4只健康新西兰大白兔,初次免疫乳铁蛋白4周后进行加强免疫,每2周加强免疫1次,待血清达到抗体效价后,对兔进行心脏采血并分离血清,利用饱和硫酸铵法和蛋白A树脂纯化抗体,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western-blot)法分别用于鉴定纯化后抗体的纯度和特异性。测定纯化后抗体效价,并绘制抗体抑制曲线。最后利用所得抗体对市售液态奶、奶牛乳腺组织匀浆液、奶粉样品中的乳铁蛋白进行定量检测。结果表明,本试验制备的兔抗牛乳铁蛋白多克隆抗体纯度较高、特异性较强,抗体浓度为11.02 mg/m L,效价达到1∶128 000;采用该抗体测定的乳腺组织样品中乳铁蛋白浓度为16.13μg/g,液态奶中接近于0μg/g,奶粉中为5.28μg/g。总之,本试验采用经过饱和硫酸铵法和蛋白A树脂2步纯化后得到了纯度较高、特异性较强的兔抗牛乳铁蛋白多克隆抗体,可以用于牛奶等产品的乳铁蛋白鉴定。     

Seminal plasma lactoferrin but not transferrin reflects gonadal function in dogs

Lactoferrin purified from canine seminal plasma by a three-step chromatography procedure had a molecular mass of 75.2 kDa and cross-reacted with antiserum to equine seminal plasma lactoferrin. Seminal plasma lactoferrin concentrations were determined by a competitive enzyme-linked immunosorbent assay (ELISA) by using rabbit anti-equine lactoferrin antibody and alkaline phosphatase-labeled goat anti-rabbit IgG antibody in 14 normal dogs and found to range from 12 to 197 micro g/ml, with a mean value of 77 +/- 59 micro g/ml (the mean +/- SD). Seminal plasma transferrin concentrations were determined by a sandwich ELISA with goat antibody to canine serum transferrin and alkaline phosphatase-conjugated goat anti-canine transferrin antibody and found to range from 0.32 to 12.6 micro g/m l, with a mean value of 2.44 +/- 3.25 micro g/m l. The lactoferrin concentration significantly correlated with the sperm concentration (r=0.7025, P<0.01), but there was no significant correlation between the seminal plasma transferrin concentration and sperm density. These results indicate that seminal plasma lactoferrin, but not transferrin, reflects gonadal function.     

紫花苜蓿基因转化的影响因素分析

通过农杆菌介导法对紫花苜蓿不同品种植株进行了抗旱基因转化的研究,得到了一套紫花苜蓿基因转化的优化体系。研究表明,100 mg/L的卡那霉素对苜蓿愈伤组织的生长有着显著的抑制效应。250 mg/L头孢霉素能够有效地抑制农杆菌菌株LBA4404的生长。紫花苜蓿供试材料被切后直接用OD600值为0.3~0.5的农杆菌LBA4404菌液侵染10~15 min;培养材料共培养3 d后在愈伤组织诱导培养基MS 2 mg/L 2,4-D 0.5 mg/LKT 30 g/L蔗糖 9 g/L琼脂 250 mg/L Cef上诱导出愈伤组织;在体细胞胚分化培养基MS 1.0 mg/L BA 0.3 mg/L NAA 30 g/L蔗糖 9 g/L琼脂 50 mg/L Kan 250 mg/L Cef上促进体细胞胚的分化;分化出的体细胞胚在再生培养基上(同分化培养基,Kan为80 mg/L)进一步发育成抗性转化苗;转化的无根小苗在生根培养基1/2 MS 1.0 mg/L IBA 1%蔗糖 0.8%琼脂 100 mg/L卡那霉素上可生长出根系。     

Expression of lactoferrin in the boar epididymis: effects of reduced estrogen

Lactoferrin is regulated by estrogen in the female reproductive tract and evidence in immature mice suggests that it may be estrogen regulated in males as well. The estrogen regulation of lactoferrin in the epididymis of the boar, a high estrogen-producing male, is unknown. This study was designed to test the hypothesis that lactoferrin expression in the boar epididymis is regulated by estrogen. Twenty-one littermate pairs of boars were treated with vehicle or Letrozole, an aromatase inhibitor, from 1 week of age until castration at 2 through 8 months. Epididymal tissue was collected at castration and fixed for immunolocalization of lactoferrin. Epididymal and testicular tissues were also collected from five mature boars (1-2.5 years) and fixed for immunocytochemistry (ICC). Lactoferrin was localized in the principal cell cytoplasm of the caput, corpus and cauda of developing boars but only in the corpus and cauda of mature boars. Basal cells were negative for lactoferrin. Sperm in the corpus and cauda was also positive for lactoferrin. The efferent ducts and testes were negative for lactoferrin. Intensity of lactoferrin immunostaining increased with age in the corpus and cauda regardless of treatment. Reduced endogenous estrogen in the epididymis during development did not affect the intensity of immunostaining between control and Letrozole-treated animals. Lactoferrin expression in the epididymis of the developing boar does not appear to be regulated by estrogen.     

草地早熟禾新格莱德胚性愈伤组织原生质体培养及植株再生的研究

以草地早熟禾品种——新格莱德成熟种子为供试材料,在含3.0mg/L2,4-二氯苯氧乙酸(2,4-D)、0.5mg/L6-苄氨基嘌呤(6-BA)的MB₅培养基中进行胚性愈伤组织诱导的培养。并从生长了7～9个月的胚性愈伤组织中分离出原生质体,将该原生质体置于KM₈P培养基(含3.0mg/L2,4-D、0.5mg/L6-BA、100mg/L水解酪蛋白、100mg/L水解乳蛋白、1%蔗糖、0.4mol/L甘露醇)中进行了液体浅层培养。结果表明,新格莱德原生质体在上述KM₈P培养基中培养3d后出现第1次细胞分裂,2～3周后形成小细胞团,此时添加低渗培养液2～3次,小细胞团持续分裂并形成愈伤组织。当愈伤组织块长至3～5mm时,转入固体培养基MS+3.0mg/L2,4-D+0.5mg/L6-BA和MS+0.5mg/L萘乙酸(NAA)+5.0mg/L6-BA上进行培养,使其细胞增殖和分化,且逐步形成完整的植株。     

Disposition kinetics of lactoferrin in milk after intramammary administration

Disposition kinetics of lactoferrin (Lf) purified from cheese whey was studied in the milk of Finnish Ayrshire cows after intramammary administration of 1 g of Lf into one udder quarter. Intramammary administration of 1 g of Lf increased Lf concentration in milk for several hours. Mean elimination half-life of Lf was 2.2 h and a mean maximum concentration of 6.3 g/L was reached between 1 and 4 h. After 8 h of administration, Lf concentrations in milk decreased to almost the same level as before the infusion. Forty-eight hours postinfusion, the mean Lf concentration was again higher than in the milk samples taken before the infusion of Lf, being on average 1.5 g/L. Lactoferrin caused some local tissue irritation in the udder quarter. Severity of the irritation reactions varied between cows. The udder quarters of primiparous cows reacted faster than those of multiparous cows, but irritation reactions decreased more rapidly in the older cows than in primiparous cows. The cows had no general signs such as fever or anorexia. The somatic cell count returned to baseline level 4 days after the administration.     

Purification and quantification of lactoferrin in equine seminal plasma

Lactoferrin with a molecular mass of 80 kDa was purified from equine seminal plasma by heparin-Agarose affinity chromatography and Sephacryl S-200 gel filtration. Purified lactoferrin was found to be highly homogeneous on the bases of its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of the monospecificity of rabbit antibodies to the purified protein in immunoblotting of seminal plasma proteins. A sandwich enzyme-linked immunosorbent assay was developed for quantifying lactoferrin in equine seminal plasma. Seminal plasma lactoferrin concentrations in 23 normal stallions ranged from 42 to 453 microg/ml, with a mean value of 157 +/- 118 microg/ml (S.D.).