乳蛋白中可利用赖氨酸及其封闭产物含量的测定

乳蛋白在热处理条件下,其中的氨基酸与还原糖发生美拉德反应,使得蛋白中可利用氨基酸残基发生锁闭从而降低蛋白的营养价值。本研究以可利用赖氨酸、封闭产物呋喃素与羧甲基赖氨酸(CML)含量作为美拉德反应的主要指标,建立并优化了乳蛋白样品中可利用赖氨酸、呋喃素和CML的检测方法并完成方法验证。结果表明所建方法均具有良好的线性、精密度与准确度,适用于乳蛋白样品中上述3种指标的检测。并采用该方法对6种不同热处理方式的乳制品,包括:巴氏杀菌全脂牛奶、巴氏杀菌脱脂牛奶、超高温瞬时灭菌(UHT)全脂牛奶、超高温瞬时灭菌(UHT)脱脂牛奶、低热脱脂乳粉(LH-SMP)、中热脱脂乳粉(MH-SMP)中可利用赖氨酸、呋喃素以及CML的含量进行测定。结果显示不同乳制品中的美拉德反应水平所受到热处理方式的影响:(1)液态奶中可利用赖氨酸含量高于乳粉而呋喃素与CML含量低于乳粉(P0.01);(2)LH-SMP中可利用赖氨酸含量高于MH-SMP(P0.05)而呋喃素含量低于MH-SMP(P0.01)。因此,在乳品加工过程中较低温度的热处理工艺有利于保留牛乳中的可利用赖氨酸并减少美拉德反应产物的生成,有助于更好的保留乳蛋白的营养价值。     

不同热处理方式对牛奶乳清蛋白的影响

目的：比较不同热处理方式对牛奶中乳清活性蛋白的影响。方法：以奶场牛乳、巴氏杀菌乳、超高温瞬时灭菌(UHT)乳和蒸汽侵入式直接杀菌(INF)乳4种不同热处理方式的牛奶作为研究对象，观察4种牛奶活性蛋白含量及热处理后牛奶中美拉德反应产物含量等的变化情况；采用SEM、FTIR、荧光光谱分析仪和Malvern纳米粒度仪分析微观结构。结果：4种热处理牛奶中，奶场牛乳的乳清蛋白含量最高，INF杀菌乳与巴氏杀菌乳中乳清蛋白含量和美拉德反应产物含量相当；UHT灭菌乳的粒径及其乳清蛋白极性环境均显著增加；4种牛奶乳清蛋白二级结构中无规则卷曲逐渐增加，乳清蛋白结构从有序向无序转化。结论：蒸汽侵入式直接杀菌对乳清活性蛋白的损伤程度小于超高温瞬时灭菌，与巴氏杀菌相当，且此方法处理的牛乳保存期高于巴氏杀菌乳。     

美拉德反应对鱼糜漂洗液回收蛋白酶解物结构的影响

文章以鱼糜漂洗液回收蛋白酶解物为原料,与葡萄糖反应制备美拉德反应产物,并采用红外光谱、荧光光谱、气质联用以及扫描电镜等分析技术对酶解物及美拉德反应产物的结构和风味成分进行了分析。研究结果发现:酶解物美拉德反应产物在激发波长347nm、发射波长425nm处有最大荧光强度;同时,红外光谱分析结果显示:酶解物美拉德反应产物在3700~3200cm~(-1)处吸收峰变宽,其结果符合美拉德反应产物的光谱特征,说明酶解物与葡萄糖之间发生了美拉德反应。扫描电镜分析中也显示酶解物美拉德反应前后表面结构有明显不同,这些都表明美拉德反应对蛋白酶解物有一定的影响。     

一种快速检测复原乳的方法

用表面荧光方法,以美拉德反应的产物糠氨酸为指标,检测生乳中添加复原乳的含量,可以检测到添加量5%以上的复原乳。该方法快速简便,不用任何复杂的前处理,5min可检验一个样品,能适应快速检测的需要。同时用高效液相色谱法加以对照,作为表面荧光方法检验的依据。     

美拉德反应产物丙烯酰胺含量与抗氧化活性的关系

探讨美拉德反应产物抗氧化活性与丙烯酰胺含量之间的关系。以自由基清除率和还原能力吸光值表示抗氧化活性,高效液相色谱(HPLC)检测丙烯酰胺含量,研究不同条件下美拉德反应产物抗氧化活性值与丙烯酰胺含量之间的相关性。结果显示:在10～15 min、120～180℃范围内,丙烯酰胺含量随温度变化趋势与美拉德反应产物自由基清除率的基本一致;美拉德反应产物还原能力在140～160℃范围内达到较大值,基本不随温度的变化而变化。结论:丙烯酰胺生成量和美拉德反应产物的抗氧化活性呈明显的正向关系。     

超声对小麦蛋白水解物美拉德反应进程的影响

该文以小麦蛋白水解物为原料,研究超声时间对小麦蛋白水解物美拉德反应进程的影响。测量美拉德反应褐变程度、中间产物变化、接枝度、荧光强度、紫外吸收光谱以及表面疏水性等指标。结果表明,超声时间40 min时,美拉德反应的褐变度、中间产物和接枝度最佳。同时,荧光光谱、紫外吸收光谱以及表面疏水性表明,超声时间导致多肽结构变得比较松散,超声时间为40 min时效果最佳,然而超声时间过长导致蛋白水解物聚集,对美拉德反应的进行造成不利的影响。综上所述,超声时间可影响美拉德反应的反应进程以及产物结构,进而影响产物的性能,如抗氧化作用、乳化作用。     

超声对小麦蛋白水解物美拉德反应产物抗氧化性影响的研究

以小麦蛋白水解物和半乳糖的混合物为参照,以他们的美拉德反应产物为研究对象,研究超声波水浴加热对其抗脂质氧化能力、还原能力、荧光强度以及ABTS+自由基清除活性的影响。研究结果表明,超声处理能够增加美拉德反应产物中的吡咯和噻吩等的含量,并产生杂环物质或焦糖化等物质,增加美拉德反应产物的抗脂质氧化能力、还原能力、ABTS+自由基清除活性均显著提高。但是,随着超声时间增加到60 min时荧光产物逐渐转变为了褐色素,使荧光强度呈现下降的趋势。     

烤烟自然醇化过程美拉德反应产物变化及与感官质量的关系

为探究醇化过程中美拉德反应产物含量的变化,合理指导烤烟醇化,利用气质联用仪对诸城地区烤烟醇化烟叶进行美拉德反应产物检测。试验结果表明,上部叶美拉德反应产物含量在醇化前18个月期间逐步提高,18个月时达到最大值50.13 μg/g,18个月之后趋于稳定;中部叶美拉德反应产物含量在醇化前30个月期间缓慢增加,30~36个月期间显著提升,醇化36个月时达到最大值52.44μg/g。醇化过程中3-羟基-2-丁酮、戊醇、2-正戊基呋喃等含量是上升的,其他物质含量变化不显著。上部叶美拉德反应产物总量与香气质、余味、杂气和刺激性、总分指标呈显著或极显著正相关关系;吲哚、2-甲基四氢呋喃-3-酮、壬醛、氧化沉香醇-1、糠醇等物质对感官质量有显著或极显著正向作用;2,6-壬二烯醛、2-正戊基呋喃对感官质量有显著或极显著负向作用;中部叶美拉德反应产物总量与香气量极显著正相关,与杂气和刺激性指标显著正相关;吲哚、糠醛、2-乙酰基呋喃等对感官质量有显著或极显著正向作用。因此,烟叶经过一定时间的醇化可以提高美拉德反应产物总量,并有利于烟叶感官质量的提升。     

反应时间对牛肉、牛骨美拉德反应产物风味品质的影响

以牛肉、牛骨等为研究对象,分析不同反应时间对美拉德反应产物风味品质的影响。结果表明:反应时间不同,美拉德反应产物中挥发性风味物质种类及相对含量均存在明显差异,反应时间为30、45、60、75、90 min条件下分别检测出42、40、43、43、41种挥发性风味物质。反应温度为105℃,反应时间为60 min时,美拉德反应产物中挥发性风味物质含量高达92.95%,具有特征风味的醛、酮类化合物的含量为47.38%。运用电子鼻分析可知,不同反应时间美拉德反应产物的主成分之间有显著性差异,利用主成分分析可以很好地区分不同反应时间下的美拉德反应产物。所得到的美拉德反应产物肉香味浓郁,回味悠长。     

紫薯蛋白-葡萄糖美拉德产物制备及其与花色苷相互作用研究

通过干法美拉德反应制备紫薯蛋白-葡萄糖美拉德产物,并分析其与花色苷相互作用。采用聚丙烯酰胺凝胶电泳(SDS-PAGE)测定产物的分子质量,运用傅里叶红外变换光谱(FIRT)、差示扫描量热(DSC)和荧光光谱等多种方法对产物进行结构表征。结果表明:美拉德反应主要产物分子量为25 kDa;相较于紫薯蛋白花色苷复合物,美拉德产物的红外吸收强度普遍较低,尤其体现于酰胺I带处的红外吸收,美拉德产物变性峰温度(76℃)较高,变性焓值(366.7 J/g)较低;美拉德产物的最大荧光发射波长为340 nm,花色苷猝灭美拉德产物荧光为动静态联合猝灭,静态猝灭为主导;花色苷与美拉德产物之间的相互作用力属于强作用力,包括范德华力和氢键。花色苷与紫薯蛋白-葡萄糖美拉德产物相互作用结合位点靠近色氨酸残基。     

Evaluation of Front-face Fluorescence for Assessing Thermal Processing of Milk

The use of front‐face fluorescence spectroscopy (FFFS) was assessed for its ability to monitor the development of Maillard browning in milk during thermal processing. Skim milk was processed using a Microthermics thermal processing system for a range of conditions from 70 °C to 140 °C from 3 to 30 s. Milk was analyzed using FFFS Hunter L*, a*, b*, hydroxymethylfurfural (HMF), tryptophan, and optical density. FFFS and HMF were found to be the most sensitive methods for distinguishing the heat treatment of milk. Activation energies of 126 and 190 kJ/ mol were found for HMF and FFFS, respectively. A strong correlation was found between these 2 methods. As a fast nonpreparatory method, FFFS is quite useful for evaluating the effect on the 1st stages of the Maillard reaction caused by the heat processing of milk. This work indicates that FFFS with no sample preparation has the potential to be of use as an online instrument for monitoring and control of thermal processing of milk; it can be applied as a process analytical technology (PAT) as is being done in the pharmaceutical industry with other methods.     

Short communication: Suitability of fluorescence spectroscopy for characterization of commercial milk of different composition and origin

Thirteen milk brands comprising 76 pasteurized and UHT milk samples of various compositions (whole, reduced fat, skimmed, low lactose, and high protein) were obtained from local supermarkets, and milk samples manufactured in various countries were discriminated using front-face fluorescence spectroscopy (FFFS) coupled with chemometric tools. The emission spectra of Maillard reaction products and riboflavin (MRP/RF; 400 to 600 nm) and tryptophan (300 to 400 nm) were recorded using FFFS, and the excitation wavelengths were set at 360 nm for MRP/RF and 290 nm for tryptophan. Principal component analysis (PCA) was applied to analyze the normalized spectra. The PCA of spectral information from MRP/RF discriminated the milk samples originating in different countries, and PCA of spectral information from tryptophan discriminated the samples according to composition. The fluorescence spectral data were compared with liquid chromatography-mass spectrometry results for the glycation extent of the milk samples, and a positive association (R² = 0.84) was found between the degree of glycation of α-lactalbumin and the MRP/RF spectral data. This study demonstrates the ability and sensitivity of FFFS to rapidly discriminate and classify commercial milk with various compositions and processing conditions.     

Application of front-face fluorescence spectroscopy as a tool for monitoring changes in milk protein concentrate powders during storage

This study investigated the feasibility of front-face fluorescence spectroscopy (FFFS) to predict the solubility index and relative dissolution index (RDI) of milk protein concentrate (MPC) powders during storage. Twenty MPC powders with varying protein contents from 4 different commercial manufacturers were used in this study. The MPC powders were stored at 2 temperatures (25 and 40°C) for 0, 1, 2, 4, 8, and 12 wk. The front-face fluorescence spectra of tryptophan and Maillard products were recorded and analyzed with chemometrics to predict solubility of MPC powders. The similarity maps showed clear discrimination of the MPC samples stored at 25 and 40°C. Partial least squares regression models were developed using the fluorescence spectra of tryptophan and Maillard products to predict the solubility index and RDI measurements of MPC powders, and the prediction models were validated using an independent test set. Coefficients of determination (R²) of 0.76, 0.84, and 0.68 were obtained between fluorescence spectra (tryptophan emission, Maillard emission, and Maillard excitation, respectively) and solubility index. The R² values for the RDI predictions were 0.58 and 0.60 for the data set of tryptophan emission and Maillard emission, respectively. The ratio of prediction error to standard deviation was >2 for Maillard emission fluorescence spectra and solubility index measurements, indicating good practical utility of the partial least squares regression prediction models. The results indicated that the solubility and dissolution behavior of MPC powders were related to their protein content and storage conditions that could be measured using FFFS. Hence, FFFS can be used as a rapid nondestructive analytical technique to predict the solubility and dissolution characteristics of MPC powders.     

Effect of green tea flavonoids on Maillard browning in UHT milk

The ability of green tea flavonoids to control Maillard browning was investigated. Epicatechin (EC) and epigallocatechin gallate (EGCG) were added at levels of 0.l and 1.0 mmol/l to a glucose/glycine model system as well as into milk that was then thermally processed in a Microthermics processing system. Samples were assessed with (1) front-face fluorescence spectroscopy for Maillard browning, (2) Hunter L*, a*, and b*; and (3) sensory analysis. In the model gluocose/glycine system, EC and EGCG reduced Maillard fluorescence at the 0.1 mmol/l level, while fluorescence was negligible with added flavonoids at 1.0 mmol/l. When these flavonoids were added to milk, they reduced the production of Maillard associated fluorescence with UHT processing. EC and EGCG also reduced the ΔE (Total color difference) during thermal processing. Throughout shelf-life testing, these compounds reduced Maillard associated fluorescence in milk. Milk samples processed with these extracts were monitored by sensory analysis during extended storage. The sensory evaluation found the green tea milk samples to be of similar liking to the control milk. These flavonoids may be of use to the food industry to control Maillard browning.     

Effect of pH on heat-induced interactions in high-protein milk dispersions and application of fluorescence spectroscopy in characterizing these changes

This study investigated casein-whey protein interactions in high-protein milk dispersions (5% protein wt/wt) during heating at 90°C for 1.5 to 7.5 min at 3 different pH of 6.5, 6.8, and 7.0, using both conventional methods (gel electrophoresis, physicochemical properties) and fluorescence spectroscopy. Conventional methods confirmed the presence of milk protein aggregates during heating, similar to skim milk. These methods were able to help in understanding the denaturation and aggregation of milk proteins as a function of heat treatment. However, the results from the conventional methods were greatly affected by batch-to-batch variations and, therefore, differentiation could be drawn only in nonheated samples and samples heated for a longer duration. The front-face fluorescence spectroscopy was found to be a useful tool that provided additional information to conventional methods and helped in understanding differences between nonheated, low-, and high-heated samples, along with the type of sample used (derived from liquid or powder milk protein concentrates). At all pH values, tryptophan maxima in nonheated samples derived from powdered milk protein concentrates presented a blue shift in comparison to samples derived from liquid milk protein concentrates, and tryptophan maxima in heated samples presented a red shift. With the heating of the sample, Maillard emission and excitation spectra also showed increases in the peak intensities from 408 to 432 and 260 to 290 nm, respectively. As the level of denaturation increased with heating, a marked differentiation can be seen in the principal component analysis plots of tryptophan, Maillard emission, and excitation spectra, indicating that the front-face fluorescence technique has a potential to monitor and classify samples according to milk protein interactions as a function of pH and heat exposure. Overall, it can be said that the pattern of protein-protein interactions in high-protein dispersions was similar to the observation reported in skim milk systems, and fluorescence spectroscopy with chemometrics can be used as a rapid, nondestructive, and complementary method to conventional methods for following heat-induced changes.     

Development and validation of a front-face fluorescence spectroscopy-based method to determine casein in raw milk

A front-face fluorescence spectroscopy (FFFS)-based method for measuring casein in raw milk was developed and validated. Calibration samples (n_cal = 20) with different casein contents (0.36–3.7%) were prepared by mixing ultrafiltered retentate (2 × concentrate) and permeate at different ratios. A principal component regression model was developed using the calibration fluorescence spectra, showing a useful prediction power based on a residual prediction deviation of 3.1. The calibration model was independently validated using 20 raw milk samples. The FFFS-based method showed a root mean square error of prediction of 0.15% and 6.7% relative prediction error. A larger sample size should be included in the future to further validate the method and potentially implement it for routine measurement of casein levels in raw milk.     

A rapid method to quantify casein in fluid milk by front-face fluorescence spectroscopy combined with chemometrics

Casein in fluid milk determines cheese yield and affects cheese quality. Traditional methods of measuring casein in milk involve lengthy sample preparations with labor-intensive nitrogen-based protein quantifications. The objective of this study was to quantify casein in fluid milk with different casein-to-crude-protein ratios using front-face fluorescence spectroscopy (FFFS) and chemometrics. We constructed calibration samples by mixing microfiltration and ultrafiltration retentate and permeate in different ratios to obtain different casein concentrations and casein-to-crude-protein ratios. We developed partial least squares regression and elastic net regression models for casein prediction in fluid milk using FFFS tryptophan emission spectra and reference casein contents. We used a set of 20 validation samples (including raw, skim, and ultrafiltered milk) to optimize and validate model performance. We externally tested another independent set of 20 test samples (including raw, skim, and ultrafiltered milk) by root mean square error of prediction (RMSEP), residual prediction deviation (RPD), and relative prediction error (RPE). The RMSEP for casein content quantification in raw, skim, and ultrafiltered milk ranged from 0.12 to 0.13%, and the RPD ranged from 3.2 to 3.4. The externally validated error of prediction was comparable to the existing rapid method and showed practical model performance for quality-control purposes. This FFFS-based method can be implemented as a routine quality-control tool in the dairy industry, providing rapid quantification of casein content in fluid milk intended for cheese manufacturing.     

The FAST index – A highly sensitive indicator of the heat impact on infant formula model

Infant formulas are highly sensitive to the Maillard reaction during manufacturing, while this reaction induces significant loss in protein nutritional value and safety. The indicators mostly used to monitor the reaction during heat treatment are furosine, carboxymethyllysine and hydroxymethylfurfural, but analysis of these molecules is time-consuming and expensive. The FAST method, based on simple fluorescence measurements on clear milk supernatant, is a good alternative for Maillard reaction monitoring in milk products.     

Application of fluorescence spectroscopy for monitoring changes in nonfat dry milk during storage

The objective of this study was to determine if fluorescence spectroscopy could be used to characterize the biochemical characteristics of nonfat dry milk (NDM) caused by manufacturing and storage conditions. Nine low-heat NDM samples were collected from 3 manufacturers and stored at 4 temperatures (4, 22, 35, and 50°C) for 8 wk. The spectra of Maillard products, tryptophan, and riboflavin were recorded and analyzed with principal components analysis. Colorimetric indices L*, a*, and b* were also determined. The before-storage NDM samples collected from each manufacturer had different fluorescent characteristics. Inconsistency was observed for the NDM samples collected from 1 manufacturer, whereas the samples from the other 2 manufacturers displayed consistent fluorescence characteristics. Biochemical reactions, such as Maillard reaction, modification of the tryptophan environment, and degradation of riboflavin occurred during the manufacturing process. For each of the data collections, discrimination of the NDM samples stored at 50°C from the samples stored at 4, 22, and 35°C was observed in the similarity maps. The factor loadings of the first 2 principal components for the fluorescence spectra of the samples before storage were similar to the principal components analysis results of the samples during storage. It appears that similar factors are responsible for the variation in the samples before storage and their changes during storage. Additionally, storage of the samples at 50°C accelerated these reactions. The results demonstrate that front-face fluorescence spectroscopy, coupled with multivariate statistical methods, can be utilized as an analytical technique to monitor variation in NDM samples from different manufacturers and changes during storage.     

Utilization of front-face fluorescence spectroscopy for analysis of process cheese functionality

The purpose of this study was to evaluate the feasibility of front-face fluorescence spectroscopy (FFFS) to predict the meltability of process cheese spreads or products. Twenty-seven commercial samples from 3 manufacturers were used in this study. Each sample was analyzed using dynamic stress rheometry, which was used to calculate the meltability index (temperature at tandelta = 1). Additionally, fluorescence spectra of tryptophan (excitation: 290 nm; emission: 305 to 400 nm) were collected on each sample at 20 degrees C using a front-face accessory. Fluorescence spectrum for each sample consisted of an average of 36 scans (6 scans performed on 6 replicates). The spectral data set consisted of normalized and mean-centered spectra from all the samples. Multivariate statistical analysis was used to correlate spectral data with cheese meltability index as measured by dynamic stress rheometry. A prediction model was developed using partial least square regression and was calibrated using a cross-validation method. A correlation coefficient of 0.93 was obtained between fluorescence spectra and cheese meltability. The regions 335 to 350 nm and 385 to 400 nm had the highest correlation to cheese meltability. A negative correlation between the peak height of tryptophan (335 to 350 nm) and cheese meltability index was observed. This correlation may be due to presence of tryptophan residues in a more hydrophobic environment in stronger emulsions as compared with a more polar environment in weak emulsions. These results indicate that the melt properties of process cheese spreads or products are related to molecular structure that can be measured using FFFS. Hence, FFFS can be used as an analysis technique to predict process cheese meltability.