乳铁蛋白和溶菌酶在婴儿配方奶粉中添加量的计算及应用

从婴儿配方奶粉模拟母乳含量和成分的角度出发，提出了在婴儿配方奶粉中添加乳铁蛋白和溶菌酶(两种功能性活性蛋白质)的依据。文章重点对乳铁蛋白和溶菌酶的添加量进行了精确计算，并与国外的同类产品的添加量进行了对比．最后对添加工序的关键点进行了分析。     

人乳铁蛋白在原核中的融合表达

设计了特异引物，通过PCR扩增人乳铁蛋白（HLF）基因，将扩增的DNA片段重组到原核表达载体pGEX-4T3中，重组体转化大肠杆菌BL21菌株，经IPTG诱导后，得到了高效融合表达。分析结果表明，获得了特异表达的乳铁蛋白。     

Genetic analysis of lactoferrin content in bovine milk

Bovine lactoferrin (LF) is mainly present in milk and shows important physiological and biological functions. The aim of this study was to estimate the heritability and correlation values of LF content in bovine milk with different economic traits as milk yield (MY), fat and protein percentages, and somatic cell score (SCS). Variance components of the studied traits were estimated by REML using a multiple-trait mixed model. The obtained heritability (0.22) for LF content predicted using mid-infrared spectrometry (pLF) suggested the possibility of animal selection based on the increase of LF content in milk. The phenotypic and genetic correlation values calculated between pLF and SCS were moderate (0.31 and 0.24, respectively). Furthermore, a preliminary study of bovine LF gene polymorphism effects was performed on the same production traits. By PCR, all exons of the LF gene were amplified and then sequenced. Three new polymorphisms were detected in exon 2, exon 11, and intron 8. We examined the effects of LF gene polymorphisms of exons 2, 4, 9, 11, and 15, and intron 8 on pLF, MY, fat and protein percentages, and SCS. The different observed effects did not reach a significant level probably because of the characteristics of the studied population. However, the results were promising, and LF may be a potential indicator of mastitis. Further studies are necessary to evaluate the effect of genetic selection based on LF content on the improvement of mastitis resistance.     

乳铁蛋白的功能特性及其在婴儿配方奶粉中的应用

介绍了母乳中乳铁蛋白的功能特性，及其应用在婴儿配方奶粉中对非母乳喂养婴儿营养的重要性，国外有关乳铁蛋白在婴幼儿配方奶粉中的应用情况。探讨了乳铁蛋白在婴儿配方奶粉中应用的研究过程，包括有关配方设计依据、使用的原料、生产工艺流程、检验方法、检验结果等。     

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value.     

牛乳中乳铁蛋白的分离纯化研究

牛乳经酸沉淀除酪蛋白后,通过超滤、离子交换法分离纯化得到乳铁蛋白,利用考马斯亮蓝、SDS-PAGE电泳定性定量检测。结果表明:每毫升牛乳能提取乳铁蛋白0.22mg,纯度为95%。     

Expression and purification of goat lactoferrin from Pichia pastoris expression system

ABSTRACT:  The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZαC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZαC into Pichia pastoris X-33 expression host, the GLF-pGAPZαC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using α-factor preprosequence. SDS-PAGE and PAS staining analysis indicated that the rGLF could be purified to electrophoretic homogeneity by heparin-Sepharose 6 Fast Flow affinity chromatography and glycosylated by the expression host. The yield of purified rGLF was approximately 2.0 mg/L of culture broth. The N-terminal sequence was identical to the native goat lactoferrin (nGLF). The iron-binding behavior, papain-inhibiting property, and thermal stability of the purified rGLF were comparable to nGLF. This is the 1st report of intact goat lactoferrin expression using the P. pastoris system.     

Genetic variability of lactoferrin content estimated by mid-infrared spectrometry in bovine milk

The effects of lactoferrin (LF) on the immune system have already been shown by many studies. Unfortunately, the current methods used to measure LF levels in milk do not permit the study of the genetic variability of lactoferrin or the performance of routine genetic evaluations. The first aim of this research was to derive a calibration equation permitting the prediction of LF in milk by mid-infrared spectrometry (MIR). The calibration with partial least squares on 69 samples showed a ratio of standard error of cross-validation to standard deviation equal to 1.98. Based on this value, the calibration equation was used to establish an LF indicator trait (predicted LF; pLF) on a large number of milk samples (n = 7,690). A subsequent study of its variability was conducted, which confirmed that stage of lactation and lactation number influence the overall pLF level. Small differences in mean pLF among 7 dairy breeds were also observed. The pLF content of Jersey milk was significantly higher than that in Holstein milk. Therefore, the choice of breed could change the expected LF level. Heritability estimated for pLF was 19.7%. The genetic and phenotypic correlations between somatic cell score and pLF were 0.04 and 0.26, respectively. As somatic cell score increases in presence of mastitis, this observation seems to indicate that pLF, or a function of observed pLF, compared with expected LF might have potential as an indicator of mastitis. The negative genetic correlation (−0.36) between milk yield and pLF could indicate an undesirable effect of selection for high milk production on the overall LF level.     

生牛乳中乳铁蛋白的蛋白质芯片检测平台的评价研究

目的对本研究前期建立的用于乳铁蛋白的蛋白质芯片检测平台进行评价。方法使用3份生牛乳样品,批内重复8次检测,计算批内精密度;另对3份生牛乳分别重复8个批次检测,计算批间精密度。对高浓度标准品稀释3个浓度后,进行稀释回收率试验;另对3个浓度基础液分别进行加标回收率试验;取10份生牛乳样品,分别用蛋白质芯片检测平台与商用酶联免疫吸附测定(ELISA)试剂盒进行检测并对结果进行方法学比较。结果用于生牛乳中乳铁蛋白的蛋白质芯片检测平台的批内精密度在9.79%~15.90%之间,批间精密度在11.63%~23.38%之间;稀释回收率在79.8%~129.7%之间,加标回收率在105.7%~133.9%之间;经比较,两种方法的相关系数r=0.825 2,差异有统计学意义(t=4.132,P0.05),配对比较t检验结果显示两种方法差异无统计学意义(t=1.282,P0.05)。结论本研究建立的用于乳铁蛋白的蛋白质芯片检测平台能够满足实验室检测需要,尚有操作偏倚需进一步优化。     

高效液相色谱法测定羊乳中的乳铁蛋白

为开发羊乳中乳铁蛋白的高效液相色谱测定方法及报告羊乳中乳铁蛋白的分布规律。本研究采集哈尔滨地区羊乳样品(n=24)和经过巴氏杀菌的羊乳样品(n=24),采用高效液相色谱测定其乳铁蛋白质量浓度。样品脱脂后,调整pH沉淀酪蛋白,采用C8色谱柱分离,二极管阵列检测器210nm检测,以水、乙腈、三氟乙酸为流动相进行线性梯度洗脱。方法回收率94.2%,线性范围50~300μg/mL,检出限12μg/mL,定量限40μg/mL。原料羊乳中乳铁蛋白的质量浓度的平均值122.1μg/mL,巴氏杀菌后羊乳中乳铁蛋白的平均值103.9μg/mL。巴氏杀菌对羊乳中乳铁蛋白浓度无影响。      

反相高效液相色谱法测定婴儿奶粉中乳铁蛋白的含量

建立了反相高效液相色谱法(RP-HPLC)测定婴儿奶粉中乳铁蛋白的含量,采用C4色谱柱洗脱,二极管阵列检测器(波长220nm)测定.流动相A为0.1％三氟乙酸水溶液,流动相B为乙腈-水-三氟乙酸(体积比90∶10∶0.1).线性范围为5mg/L～1000mg/L,相关系数为0.9999,定量下限为30mg/kg,加标回收率为86.0％～91.7％.该方法用于实际样品的测定,样品处理简单,结果令人满意.     

Antimicrobial activity of recombinant human lactoferrin from <Emphasis Type="Italic">Aspergillus awamori</Emphasis>, human milk lactoferrin and their hydrolysates

The antibacterial activity of human lactoferrin from milk (hLF), recombinant human lactoferrin from Aspergillus awamori (rhLF) and their hydrolysates obtained with pepsin was investigated against Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) were determined for all the bacteria and the proteins assayed. Taking into account the MICs found for both lactoferrins studied, we can say that they behave very similarly, except for L. monocytogenes for which rhLF was more active. We studied the effect that heat treatments exerted on the antibacterial activity of the two types of lactoferrin and the only heat treatment that had a negative effect on that activity was 85 °C for 10 min. The activity of hLF and rhLF in UHT milk and whey against E. coli O157:H7 and L. monocytogenes, was also assayed. Our results showed a reduction in the number of viable cells for both microorganisms when were incubated with rhLF or hLF, but this decrease was lower than in broth media.     

Production and processing of milk from transgenic goats expressing human lysozyme in the mammary gland

The potential for applying biotechnology to benefit animal agriculture and food production has long been speculated. The addition of human milk components with intrinsic antimicrobial activity and positive charge to livestock milk by genetic engineering has the potential to benefit animal health, as well as food safety and production. We generated one line of transgenic goats as a model for the dairy cow designed to express human lysozyme in the mammary gland. Here we report the characterization of the milk from 5 transgenic females of this line expressing human lysozyme in their milk at 270 μg/mL or 68% of the level found in human milk. Milk from transgenic animals had a lower somatic cell count, but the overall component composition of the milk and milk production were not different from controls. Milk from transgenic animals had a shorter rennet clotting time and increased curd strength. Milk of such nature may be of benefit to the producer by influencing udder health and milk processing.     

Effects of thermized donkey milk with lysozyme activity on altered gut barrier in mice exposed to water-avoidance stress

Nutrition plays a crucial role in human gut health through the improvement of gut barrier functionality. Donkey milk represents an interesting source of natural antimicrobial factors such as lysozyme. Recently, anti-inflammatory properties of donkey milk lysozyme activity were described in a mouse model of ileitis. The current increase of donkey milk consumption highlights the necessity to propose a healthy milk compliant with microbiological standards. This study aims to define a heat treatment of donkey milk, retaining its high lysozyme activity, and to evaluate its beneficial effects on a gut barrier impairment model due to chronic stress in mice. To perform this experiment, samples of raw donkey milk were collected in 15 distinct French farms. Microbiological analysis and lysozyme content and activity were evaluated for each sample. Then, several heat treatments were carried out to define a time and temperature combination that allowed for both a reduction in the number of total micro-organisms, increasing the shelf-life of the product, and preservation of lysozyme activity. The beneficial effect of heated donkey milk on the gut barrier of mice was evaluated and compared with raw donkey milk. We found that samples of raw donkey milk showed low total mesophilic microbial counts, and no pathogens were detected. Among the different heat-treatment procedures tested, a 2-min, 72°C combination was determined to be the most optimal time and temperature combination to preserve lysozyme activity and increase the shelf-life of donkey milk. Oral administration of this heat-treated donkey milk in mice counteracted chronic stress-induced intestinal damage, illustrated by gut hyper-permeability and low-grade inflammation, similar to raw donkey milk. We have demonstrated for the first time that oral intervention with donkey milk, optimally heat-treated to retain enzymatic lysozyme activity, improves intestinal barrier damage linked to psychological stress in mice.     

婴儿配方粉中溶菌酶活力的测定及其校正系数的研究

在婴儿配方粉中添加溶菌酶已成为高档婴儿粉的一种趋势，因此奶粉中溶菌酶活力成为判定其质量的重要指标。根据经典的分光光度法探索出测定奶粉中溶菌活力的方法，并以纯酶液人对照初步得出校正系数r=1.2；本试验测得市售婴儿粉中每克粉中溶菌酶活力为25960u。     

Abatement of the clostridial load in the teats of lactating cows with lysozyme derived from donkey milk

The use of a sterilized product for washing cows' udders before milking may be useful to reduce or prevent Clostridium tyrobutyricum contamination, the main cause of the late-blowing defect in hard and semi-hard cheeses. The aim of this research was to evaluate the antibacterial efficacy of an experimental formula containing 15% condensed donkey milk (lysozyme content 825 mg/L). The antimicrobial activity of condensed milk was first evaluated in vitro, using the disk diffusion method, on the following microorganisms: Bacillus megaterium, Bacillus mojavensis, Clavibacter michiganensis, and Clostridium tyrobutyricum. These results were compared with the effects of 2 antibiotics, ampicillin (100 mg/mL) and kanamycin (50 mg/ mL), and a commercial pre-dipping formula. The results showed that the inhibitory activity of lysozyme from donkey milk on all the considered microorganisms was higher than that of the commercial product and similar to that of the 2 antibiotics. Next, the formula with lysozyme was compared with a commercial pre-dipping formula on 48 lactating cows (24 cows in each group). Skin tests were performed on teats before and after pre-dipping. Results showed that the formula with condensed milk significantly reduced the clostridial load detected on the skin of cows' teats before cleaning (−55.61% vs. −27.99%) and in the bulk milk of the experimental group compared with the control group with commercial product (−52.53% vs. −32.42%).     

高效液相色谱法检测牛奶及其制品中的溶菌酶

目的建立一种测定牛奶及奶酪制品中低含量溶菌酶的高效液相色谱-荧光检测新方法。方法样品经p H 6.0的氯化钠溶液活化,再在低p H值条件下除蛋白后,采用反相色谱柱(PLRP-S 250 mm×4.6 mm,300?,5μm)用A水(0.1%的三氟乙酸(trifluoroacetic acid,TFA)),B乙腈(0.1%TFA)体系作为流动相进行梯度洗脱,使用荧光检测器在激发波长(λ_(ex))276 nm,发射波长(λ_(em))345 nm处检测。结果最优实验条件下,溶菌酶在2.0~30.0 mg/L浓度范围内线性良好,加标实验结果显示回收率和相对标准偏差(relative standard deviation,RSD)分别在92.3%~104.3%和0.81%~3.26%之间,对牛奶和奶酪制品的检出限分别为20和40 mg/kg。结论该方法操作简便,结果准确、可靠,可用于乳制品中溶菌酶含量测定。