HPLC Determination of Lactulose in Heat Treated Milk

Lactulose (4-O-β-d-galactopyranosyl-d-fructose), formed from lactose (4-O-β-d-galactopyranosyl-d-glucose) by the Lobry de Bruyn–Alberda van Ekenstein rearrangement during severe heat treatments, is considered a useful indicator of heat-induced modifications in milk. Its chromatographic determination in milk is particularly troublesome due to the concomitant presence of a lactose amount two orders of magnitude larger than the lactulose amount and to a similar retention time of the two compounds. In this work, four HPLC methods have been compared with the aim to develop a more accurate analytical procedure to determine lactulose in milk, together with lactose. The developed method is based on a Carrez precipitation followed by a HPLC separation on two in-series amino-based columns, using CH₃CN:H₂O 75:25 (v:v) as the mobile phase at 1 ml/min flow rate and a refractive index as the detector. The linearity test for the quantitation of lactulose has been carried out over the range 0.060–1.006 mg/ml, the limit of detection, is 0.013 mg/ml (256 ng injected) and the limit of quantitation is 0.028 mg/ml (556 ng injected). The proposed method, simple, cheap and time-saving, allows an accurate lactulose–lactose separation, with conventional HPLC equipment.     

Efficient Alkaline Isomerization of Lactose to Lactulose in the Presence of an Organogermanium Compound

Lactulose, a disaccharide widely used in pharmaceuticals and functional foods, is produced by lactose isomerization. Lactose and lactulose have an aldose–ketose relationship. Less than 25 % conversion of lactose into lactulose is achieved using the Lobry de Bruyn–Alberda van Ekenstein transformation with heating, whereas the conversion is increased to 80 % by the addition of an approximately equimolar concentration of the organogermanium compound 3-(trihydroxygermyl)propanoic acid (THGP) to the reaction mixture. To further understand this phenomenon, in this study, we analyzed the affinity between THGP and sugar isomers using ¹H nuclear magnetic resonance spectroscopy. For the dimethyl derivative of THGP with lactose and lactulose, the complex formation ratios at 0.1 M (1:1 mixing ratio) were 14 and 59 %, respectively, with complex formation constants of 1.8 and 43 M^–1, respectively. The complex formation capacity was approximately 24-fold higher for lactulose than for lactose. Moreover, THGP is considered to protect lactulose from alkaline degradation, resulting in high production yield of lactulose. Therefore, we concluded that high affinity for the isomerization product may promote isomerization and that promotion of sugar isomerization using an organogermanium compound is an effective method for converting lactose to lactulose.     

高效液相色谱法测定液态乳中乳果糖方法的优化

为了对液态乳中乳果糖含量进行简便准确地定量,改进高效液相色谱法测定乳果糖含量的方法。采用化学稳定的改性酰胺基色谱柱进行分离,并以乙腈-水流动相梯度洗脱40 min,结果表明,样品进样体积为3 μL时乳糖与乳果糖的分离度为1.5,满足色谱分离检测。优化后的方法经验证,乳果糖在50~2500 mg/kg范围内线性关系良好,（R²=0.9997）,乳果糖检出限和定量限分别为15和50 mg/kg,3个加标水平（n=6）乳果糖平均回收率在90.3%~105.6%之间,相对标准偏差为3.4%~3.9%。利用该法对市场中的27种实际乳样进行分析,结果均在合理范围之内。该方法经济、准确适用于液态乳中乳果糖含量的日常检测。     

乳糖异构生产乳果糖的条件优化

首先对乳糖异构生产乳果糖的异构剂进行筛选,选出最佳异构剂为亚磷酸氢二钠,然后对异构条件进行优化,得出乳糖异构生产乳果糖最佳条件为:异构剂加量为2.0%,反应时间为3 h,反应温度为110℃,乳糖浓度为70%,在上述最佳条件下得到异构混合液中乳果糖含量为56.84%。     

高效液相色谱法测定发酵乳中的乳糖、葡萄糖和半乳糖

利用高效液相色谱法测定发酵乳中乳糖、葡萄糖、半乳糖的含量。样品通过Carrez法处理沉淀蛋白,采用Aminex HPX 87H色谱柱, 5mmol/L H2SO4溶液为流动相,流速0.6mL/min。结果表明：利用该法进行色谱分析各组分达到较好的分离,各糖组分在0.5～12mg/mL范围内呈现良好线性关系,相关系数为0.9986～0.9999(n＝ 5),样品回收率为96.45%～101.78%;精密度实验表明,各组分峰面积RSD在0.62%～4.21%之间(n＝ 5);重现性实验表明,各组分含量测定值RSD为1.86%～3.44%。该法测定样品快速、准确、可靠,为发酵乳贮藏过程中关键糖含量的测定提供参考依据。     

应用高效液相色谱快速测定奶粉中的乳糖,蔗糖,葡萄糖

讨论了应用高效液相色谱（ＨＰＬＣ）对奶粉及调制奶粉中的乳糖,蔗糖,葡萄糖的一种快速测定方法。样品经简单的前处理后由Ｃａｒｂｏｈｙｄｒａｔｅ Ａｎａｌｙｓｉｓ柱分离,流动相为乙腈／水,示差折光检测,奶粉中乳糖,蔗糖,葡萄糖完全分离并定量测定。方法简便,快速,结果令人满意。     

Multivariate Classification of UHT Milk as to the Presence of Lactose Using Benchtop and Portable NIR Spectrometers

Lactose hydrolyzed milk was developed in the 1970s to serve individuals with lactose intolerance. This demand for lactose-free products by lactose-intolerant consumers has created a market segment for this food whose quality control has to be guaranteed. In order to assess milk samples for lactose content, this work proposes an analytical methodology to classify regular and lactose-free ultra high temperature (UHT) milks using multivariate classification methods and NIR spectra obtained in FT-NIR and ultra-compact NIR spectrometers, aiming at field analysis. For this, 71 samples were purchased; 41 were lactose-free UHT milk and 30 regular UHT milk. Diffuse transflectance spectra were obtained by FT-NIR (833 to 2500 nm, 4 cm^?1 of resolution and mean of 16 scans), and by ultra-compact NIR (908 to 1676 nm, with 12.5 nm of resolution and mean of 50 scans). The classification models were obtained by PLS-DA and LDA techniques with robust variables selection by SPA and GA, evaluating different spectral pre-processing (MSC, SNV, and derivatives). The three models developed (PLS-DA, GA-LDA, and SPA-LDA) with benchtop equipment spectra correctly classified all samples with sensitivity and specificity of 100%. For the portable equipment spectra, PLS-DA and GA-LDA models obtained sensitivity and specificity of 100%. The SPA-LDA model, however, presented sensitivity and specificity of 80 and 100%, respectively. These results indicate that methodologies using NIR equipment, especially the ultra-compact NIR, with multivariate classification techniques are feasible in discrimination between regular and lactose-free milk in the field, thus enabling a quick and precise analysis.     

液态乳中掺有复原乳的乳果糖检测方法

目的:比较乳果糖的3种紫外分光光度检测方法,优选出一较好的方法来检测液态乳中是否掺有复原乳。方法:以生乳、复原乳、掺有复原乳的巴氏杀菌乳和UHT灭菌乳为试样,分别采用Adhikari、蒽酮和Seliwanoff’s比色方法来检测生乳和复原乳的差别,选定一种较好的方法测定复原乳的掺入率。结果:用Seliwanoff’s比色法检测液态乳中乳果糖的含量,在生乳和巴氏杀菌乳中掺有15%的复原乳,UHT灭菌乳中掺有20%的复原乳。结论:乳果糖的Seliwanoff’s测定法可以快速检测液态乳中是否掺有复原乳。     

Determination of Lactose and Lactose Hydrolysis in Milk Using Cerium(IV)

Lactose and lactose hydrolysis in milk were determined using a novel method involving a measurement of the time for 0.39M Cerium(IV) [Ce(IV)] in 0.5M nitric acid to be reduced to Cerium(III) [Ce(III)] after an initial precipitation of milk protein and fat. The time for the absotbance at 445 nm to decrease to 0.4 could be directly correlated with standard curves to determine either the amount of lactose or the degree of lactose hydrolysis. This Ce(IV) oxidation method was rapid (15 min) and exhibited good agreement with infrared methods of determining lactose and enzymatic methods of determining percentage lactose hydrolysis. Recoveries of lactose found using the Ce(IV) oxidation method from a number of milk samples containing different amounts of fat were comparable to recoveries using the infrared method of lactose determination.     

离子色谱电化学测定牛奶中的乳糖和乳果糖

本方法成功地对牛奶中的乳糖和乳果糖进行了分离和测定。以CarboPacPA1色谱柱,5mmol/LNaOH+1mmol/LCH3COONa等梯度淋洗,脉冲安培检测,该方法具有良好的重现性、线性关系(r2>0.9989)和较低的检测限。乳糖和乳果糖的检测限分别是:0.012、0.02mg/L;样品测定的回收率为95%～102.8%。     

低乳糖牛奶褐变抑制剂的筛选

利用正交实验方法,以5-HMF的含量为指标,对低乳糖牛奶褐变抑制剂A、B、C、D进行优化配比的研究.直观分析、因素指标分析的结果表明:A、B、C和D四种褐变抑制剂,对低乳糖牛奶褐变抑制效应依次为:C＞B＞D＞A.四种褐变抑制剂的最优化组合为A0.01%、B 0.02%、C 0.02%、D 0.02%.该组合的褐变抑制剂使低乳糖牛奶产品中的5-HMF的含量降到0.63μg/mL,抑制率达到59.5%.     

羊乳中掺入牛乳的间接ELISA定量检测

制备抗β- 酪蛋白多克隆血清,免疫吸收封闭与羊乳反应的抗体,获免疫吸收抗体用于乳样的间接ELISA检测中,建立羊乳中掺入牛乳成分的定量免疫学方法。实验表明,该间接ELISA 法用于原乳检测时,掺入牛乳的百分含量与A450nm 在4%～50% 呈线性关系,该方法的最低检出量为4%。所建立的ELISA 方法变异系数＜ 5%,回收率在94%～105% 之间,符合方法学要求,可用于牛乳掺假的定量检测。对灭菌乳的检测表明,热处理不改变β- 酪蛋白与抗体反应的特性,方法还可用于经热加工的乳品检测中。     

HPLC-PAD外标法及UPLC-MS/MS内标法测定乳及乳制品中三聚氰胺的含量

分别建立高效液相色谱-二极管阵列法(HPLC-PAD)、超高压液相色谱-串联质谱法(UPLC-MS/MS)测定乳及乳制品中三聚氰胺含量的方法。利用HPLC-PAD外标法进行样品中三聚氰胺的初筛,利用UPLC-MS/MS法进行确证,以13C315N3-三聚氰胺为同位素内标定量。乳制品经乙腈、乙酸锌、盐酸溶液提取,质谱检测时,样品再经MCX固相萃取柱富集净化。HPLC-PAD法的定量限为4mg/kg,UPLC-MS/MS的定量限为0.05mg/kg;HPLC-PAD的添加回收率为95.7%～102.3%,UPLC-MS/MS的添加回收率为91.3%～95.7%(基质匹配曲线校正),两种方法的相对标准偏差(RSD)分别为1.98%～4.51%,1.60%～3.27%。     

马克斯克鲁维酵母对发酵乳中糖代谢的影响

采用高效液相色谱等方法,对乳酸菌单独发酵和向其中添加马克斯克鲁维酵母这两种发酵乳中乳糖代谢主要产物及关键酶活力的变化情况进行对比分析,以确定添加酵母菌对乳糖无氧代谢产生乳酸途径的影响。结果表明：添加酵母菌后乳糖降解速率明显加快（P＜0.05）,贮藏期间马克斯克鲁维酵母可以对积累的半乳糖进行利用;发酵过程中,由于酵母菌的添加使β-半乳糖苷酶及乳酸脱氢酶活力有显著提高（P＜0.05）,糖酵解途径关键限速酶--己糖激酶和丙酮酸激酶活力增加（P＜0.05）;含有酵母菌的发酵乳pH值下降（滴定酸度上升）较乳酸菌单菌发酵快（P＜0.05）,这与添加酵母菌后发酵乳中乳酸含量显著增加（P＜0.05）有关;丙酮酸含量变化不显著（P＞0.05）。该研究揭示了马克斯克鲁维酵母的添加对乳糖酵解具有一定促进作用。     

Fractionation of Nonfat Dry Milk Powder into Protein and Lactose Products

Five processes were used to fractionate low heat NDM into a number of products including crude casein, three types of lactalbumin, crude lactose, United States Pharmacopeia-grade lactose, and a partially deproteinated whey product. Regular lactalbumin and crude lactose were produced by process 1 with a total recovery of 91% of starting NDM solids including those obtained from the casein fraction. Process 2 led to the production of both high and low ash lactalbumin and crude lactose, but the total solids recovery was about 10% lower than process 1 due to extra material handling. Both Processes 3 and 4 allowed for the crystallization of crude lactose from condensed whey containing the full protein complement. Process 4 was similar to process 3 except provision was made to return the mother liquor from the United States pharmacopeia lactose preparation back to the starting casein whey; this increased the solids recovery from 91 to 96%. The fifth process was used to purify the crude lactose of processes 1 to 4 for the preparation of United States Pharmacopeia grade lactose.     

Membrane Fractionation Processes for Removing 90% to 95% of the Lactose and Sodium from Skim Milk and for Preparing Lactose and Sodium‐Reduced Skim Milk

ABSTRACT: Pilot‐scale microfiltration (MF), microfiltration‐diafiltration (MDF), ultrafiltration (UF), ultrafiltration‐diafiltration (UDF), and nanofilration (NF) membrane fractionation processes were designed and evaluated for removing 90% to 95% of the lactose and sodium from skim milk. The study was designed to evaluate several membrane fractionation schemes as a function of: (1) membrane types with and without diafiltration; (2) fractionation process temperatures ranging from 17 to 45 °C; (3) sources of commercial drinking water used as diafiltrant; and (4) final mass concentration ratios (MCR) ranging from about 2 to 5. MF and MDF membranes provided highest flux values, but were unsatisfactory because they failed to retain all of the whey proteins. UDF fractionation processes removed more than 90% to 95% of the lactose and sodium from skim milk. NF permeate prepared from UDF cumulative permeate contained sodium and other mineral concentrations that would make them unsuitable for use as a diafiltrant for UDF applications. A method was devised for preparing simulated milk permeate (SMP) formulated with calcium, magnesium, and potassium hydroxides, and phosphoric and citric acids for use as UDF diafiltrant or for preparing lactose and sodium reduced skim milk (L‐RSM). MF retentates with MCR values of 4.7 to 5.0 exhibited extremely poor frozen storage stabilities of less than 1 wk at ?20 °C, whereas MCR 1.77 to 2.95 MDF and UDF retentates and skim milk control exhibited frozen storage stabilities of more than 16 wk. L‐RSM exhibited a whiter appearance and a lower viscosity than skim milk, lacked natural milk flavor, and exhibited a metallic off‐flavor.     

离子色谱法测定奶粉中的葡萄糖、蔗糖和乳糖

建立高效阴离子交换色谱-脉冲安培检测法测定奶粉中葡萄糖、蔗糖和乳糖的方法。以METROSEP CARB 1(150mm×4.0mm)阴离子交换柱为分离柱,脉冲安培检测,37mmol/L NaOH溶液为淋洗液,以柠檬酸作为蛋白质沉淀剂。葡萄糖、蔗糖和乳糖的线性范围分别为1～40、1～50、1～50mg/L,相关系数分别0.9966、0.9968、0.9985,检出限分别为0.014、0.091、0.083mg/L,精密度为1.10%～4.96%,回收率为90.13%～104.83%。该方法分析时间短,前处理简单,适用于快速测定奶粉中的葡萄糖、蔗糖和乳糖。