Lactose oxidase: Enzymatic control of Pseudomonas to delay age gelation in UHT milk

Shelf-stable milk is consumed worldwide, and this market is expected to continue growing. One quality challenge for UHT milk is age gelation during shelf life, which is in part caused by bacterial heat-stable proteases (HSP) synthesized during the raw milk storage period before heat processing. Some Pseudomonas spp. are HSP producers, and their ability to grow well at refrigeration temperature make them important spoilage organisms for UHT processors to control. Previous studies have shown that lactose oxidase (LO), a natural and commercially available enzyme that produces hydrogen peroxide and lactobionic acid from lactose, can control bacterial growth in raw milk. In this research, we investigated the ability of LO to control HSP producer outgrowth, and thus delay age gelation in UHT milk. Six strains of Pseudomonas spp. were selected based on their ability to synthesize HSP and used as a cocktail to inoculate both raw and sterile (UHT) milk at a level of 1 × 10⁵ cfu/mL. Groups were treated with and without LO, stored for 4 d at 6°C, and monitored for cell count and pH. Additionally, a sample from each was tested for HSP activity via particle size analysis (average effective diameter at 90° angle and 658 nm wavelength) and visual inspection on each day of the storage period. The HSP activity results were contrasted using Tukey's HSD test, which showed that in UHT milk, a LO treatment (0.12 g/L) effectively prevented gelation as compared with the control. In raw milk, however, a concentration of 0.24 g/L of LO was needed to obtain a similar effect. This test was scaled up to 19-L pilot plant batches of raw milk where they were challenged with Pseudomonas cocktail, treated with LO for 3 d, and then UHT processed. Resulting UHT milk bottles were monitored for gelation. Significant differences in particle size between the LO-treated samples and the control were observed as early as 1 mo after processing, and gelation was not detected in the LO-treated samples through 6 mo of storage. These results demonstrated that LO can be used to delay age gelation in UHT milk induced by HSP-producing Pseudomonas spp., representing an opportunity to improve quality and reduce postproduction losses in the shelf-stable milk market sector.     

酶凝干酪素凝胶性研究进展及其在干酪中的应用

介绍酶凝干酪素凝胶形成机理和冷却机制研究进展,概述乳化盐、蛋白和脂肪、pH 值、温度、钙离子对凝胶形成的影响：乳化盐能促进蛋白质溶解形成凝胶,蛋白和脂肪含量可对凝胶的复合模量和黏弹性产生影响,适当的温度和pH 值条件能形成良好的凝胶结构,钙离子增加对凝胶形成不利,以及酶凝干酪素在加工干酪中的应用。     

Effect of pH on the gelation properties of skim milk gels made from plant coagulants and chymosin

The effect of three milk pH values, 6.0, 6.3 and 6.7, on gelation properties was monitored by small amplitude oscillatory rheology as well as a large deformation (yield) test for gels induced by the plant coagulants, Cynara cardunculus L. and Cynara humilis L., and chymosin. Gel microstructure was studied using confocal scanning laser microscopy. For each coagulant, a decrease in pH of milk resulted in a decrease in gelation time (tg), and an increase in the rate of increase in storage modulus (G'). At pH 6.0 and 6.3, plant coagulant-induced gels reached a maximum value in G' (G'max) followed by a decrease in G' values during the rest of the experiment, reflecting higher proteolytic activity of plant coagulants towards caseins as determined by SDS-PAGE. Gels produced at pH 6.0 and 6.3, exhibited a minimum in loss tangent (tan delta) followed by slight increase in tan delta during gel aging, that may have been associated with faster rearrangements of the gel network structure. In gels aged for approximately 6 h, the values for G', tan delta at low frequency (0.006 Hz) and yield stress were higher for chymosin than for plant-induced gels. For gels with the same pH value, no major differences were observed in microstructure between coagulants. Gels made at low pH values (6.3 and 6.0) appeared to have a denser or more interconnected structure than gels made at pH 6.7. Our results suggest that, at a low pH, the type of coagulant used in gelation is likely to have a considerably impact on gel/cheese structure.     

High-pressure structuring of milk protein concentrate: Effect of pH and calcium

In this study, we investigated the effect of pH and calcium on the structural properties of gels created by high-pressure processing (HPP, 600 MPa, 5°C, 3 min) of milk protein concentrate (MPC, 12.5% protein). The pH level of the MPC was varied between 6.6 and 5.1 by adding glucono-δ-lactone (GDL), and the calcium content was varied from 24 to 36 mg of Ca/g of protein by adding calcium chloride. The rheological properties and microstructure of the pressure-treated MPC were assessed. The pressurization treatments and analytical testing were conducted in triplicate. Data were analyzed statistically using one-way ANOVA with Tukey's honestly significant difference post hoc tests. A pressurization time of 3 min was sufficient to induce gel formation in MPC at pH 6.6, so it was used throughout the study. Adjusting either pH or calcium affected the structure of the HPP-created milk protein gels, likely by influencing electrostatic interactions and shifting the calcium–phosphate balance. Gels were formed after pressurization of MPC at pH above 5.3, and increasing the pH from 5.3 to 6.6 resulted in stronger gels with higher values of elastic moduli (G′). At neutral pH (6.6), adding calcium to MPC further increased G′. Scanning electron microscopy showed that reducing pH or adding calcium resulted in more porous, aggregated microstructures. These findings demonstrate the potential of HPP to create a variety of structures using MPC, facilitating a new pathway from dairy protein ingredients to novel, gel-based, high-protein foods, such as puddings or on-the-go protein bars.     

Effect of pH adjustment at heating on the rheological properties of acid skim milk gels with added potato starch

The rheological properties of acid skim milk gels, prepared from milk with added potato starch and pH adjusted (pH 6.5–7.1) prior to heat treatment and acidification, were investigated. The storage modulus, G′, of the final acid gels was increased by heating the milk at higher pH and further increased by adding starch. The effect of pH at heating and addition of starch appeared to be additive and independent of each other up to a starch addition level of 1%. Above this starch level, the pH at heating had a lesser effect. This may have been due to the increased viscosity of the aqueous phase as a result of starch gelatinization or to direct contributions of the starch to the gel network structure. Confocal microscopy showed that milk proteins developed fewer but broader protein clusters at higher pH than at lower pH. Starch addition resulted in an increased density of the protein network.     

乳酸脱氢酶测定试剂盒鉴定复原乳

利用乳酸脱氢酶测定试剂盒测定巴氏杀菌乳、超高温灭菌乳和复原乳中乳酸脱氢酶活性的差异以鉴定复原乳。结果显示：巴氏杀菌乳中乳酸脱氢酶的活性显著高于超高温灭菌乳和复原乳。该方法操作简单、快速准确, 值得推广。     

超声处理改善羊乳理化性质及其酶凝胶流变学特性

在超声频率20 kHz,超声功率800 W,超声时间15、20、25、30 min的条件下处理生羊乳,分析超声处理对羊乳粒径、Zeta-电位、表面疏水性和二级结构的影响,并通过流变学分析和扫描电子显微镜观察进一步研究添加凝乳酶对羊乳酶凝胶的流变学特性和微观结构的影响.结果表明,超声处理降低了羊乳样品的粒径,使蛋白质的二...     

Distinct composition of bovine milk from Jersey and Holstein-Friesian cows with good,poor, or noncoagulation properties as reflected in protein genetic variants and isoforms

The objective of this study was to examine variation in overall milk, protein, and mineral composition of bovine milk in relation to rennet-induced coagulation, with the aim of elucidating the underlying causes of milk with impaired coagulation abilities. On the basis of an initial screening of 892 milk samples from 42 herds with Danish Jersey and Holstein-Friesian cows, a subset of 102 samples was selected to represent milk with good, poor, or noncoagulating properties (i.e., samples that within each breed represented the most extremes in regard to coagulation properties). Milk with good coagulation characteristics was defined as milk forming a strong coagulum based on oscillatory rheology, as indicated by high values for maximum coagulum strength (G′_max) and curd firming rate (CFR) and a short rennet coagulation time. Poorly coagulating milk formed a weak coagulum, with a low G′_max and CFR and a long rennet coagulation time. Noncoagulating milk was defined as milk that failed to form a coagulum, having G′_max and CFR values of zero at measurements taken within 1 h after addition of rennet. For both breeds, a lower content of total protein, total casein (CN) and κ-CN, and lower levels of minerals (Ca, P, Mg) were identified in poorly coagulating and noncoagulating milk in comparison with milk with good coagulation properties. Liquid chromatography/electrospray ionization-mass spectrometry revealed the presence of a great variety of genetic variants of the major milk proteins, namely, α_S1-CN (variants B and C), α_S2-CN (A), β-CN (A¹, A², B, I, and F), κ-CN (A, B, and E), α-lactalbumin (B), and β-lactoglobulin (A, B, and C). In poorly coagulating and noncoagulating milk samples of both breeds, the predominant composite genotype of α_S1-, β-, and κ-CN was BB-A²A²-AA, which confirmed a genetic contribution to impaired milk coagulation. Interestingly, subtle variations in posttranslational modification of CN were observed between the coagulation classes in both breeds. Poorly coagulating and noncoagulating milk contained a lower fraction of the least phosphorylated α_S1-CN form, α_S1-CN 8P, relative to total α_S1-CN, along with a lower fraction of glycosylated κ-CN relative to total κ-CN. Thus, apparent variation was observed in the milk and protein composition, in the genetic makeup of the major milk proteins, and in the posttranslational modification level of CN between milk samples with either good or impaired coagulation ability, whereas the composition of poorly coagulating and noncoagulating milk was similar.     

An indirect ELISA system for the detection of heat-stable Pseudomonas endopeptidases (AprX) in milk

Heat-stable endopeptidases in raw milk, especially the alkaline metallopeptidase AprX secreted by Pseudomonas spp., are a well-known challenge for the dairy industry. They can withstand UHT treatment and may cause quality defects over the shelf life of milk products. Therefore, we established an indirect ELISA for the detection of Pseudomonas AprX in milk. We developed a 2-step sample treatment for milk contaminated with AprX to avoid the interference of milk proteins with the detection system. First, casein micelles were destabilized by the detraction of Ca²⁺ using trisodium citrate; then, AprX was concentrated 10-fold using hydrophobic interaction chromatography. The recovery of AprX in spiked milk samples after the 2-step treatment was 43 ± 0.1%. Specific antibodies for purified AprX from Pseudomonas lactis were produced to establish the ELISA. Western blot experiments showed that the binding affinity of these antibodies depended on the sequence homology of the AprX from P. lactis and several other Pseudomonas spp. The indirect ELISA, which was completed in 6 to 7 h, had a limit of detection of 21.0 ng mL^?1 and a limit of quantification of 25.7 ng mL^?1. Milk proteins or milk endogenous peptidases were not detected by the antibodies. The ELISA had high precision, with a CV between 0.2 and 0.8% measured on the same day (intraday) and 5.6 and 6.8% measured on 5 separate days (interday). Milk samples were spiked with different AprX activity levels [7.5–150 nkat Na-caseinate/o-phthalaldehyde (OPA) mL^?1] and evaluated by ELISA. The recovery of the ELISA was 92.3 ± 1.6 to 105 ± 4.7%. The lowest AprX activity quantifiable in the spiked milk samples was 500 pkat Na-caseinate/OPA mL^?1. The proof of concept to detect heat-stable Pseudomonas AprX in milk by ELISA was established.     

Protective influence of several packaging materials on light oxidation of milk

Light-induced degradation reactions in milk create a serious problem for the dairy industry because of the development of off-flavors, the decrease in nutritional quality, and the severity and speed by which these phenomena develop. Packaging materials are essential to avoid this particular deterioration of milk. Therefore, efforts are being made to design protective polyethylene terephthalate (PET) packages. In the present study, a number of PET bottles were compared for their ability to avoid photo-oxidation in UHT semi-skimmed milk. The milk was packed in 3 types of PET bottles: one transparent bottle provided with an active oxygen-binding inner layer, one bottle with perfect light barrier, and one transparent bottle provided with a UV-absorbing additive. During 2 storage experiments, running parallel to each other for 2 mo, chemical milk quality parameters such as fat oxidation, vitamin and protein degradation, oxygen consumption, and color change were monitored. A trained taste panel compared the sensory quality of the illuminated milk stored in these bottles, with milk perfectly protected against light and oxygen. In the first study, milk was continuously illuminated at room temperature. A comparison was made for milk under storage conditions that simulated those expected during display in retail and supermarkets. The results of the 2 shelf-life studies showed that an adequate light barrier was apparently sufficient to avoid the light-induced oxidation of milk during extended storage. Oxygen barriers, on the other hand, did not provide a significant protection, nor did bottles with UV filter. If wavelengths detrimental to riboflavin were not completely excluded by the packaging material, incoming light could still give rise to photo degradation of milk. Accordingly, riboflavin and vitamin A were gradually degraded, milk fat was photo-oxidized, oxygen dissolved in the milk was consumed, and the sensorial quality decreased significantly.     

Effect of polymorphisms in the leptin, leptin receptor, and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) genes and genetic polymorphism of milk proteins on cheese characteristics

Cheese production has increased worldwide during the last decade and is expected to increase within the coming decade as well. Despite this, the relations between cow genetics and cheese characteristics are not fully known. The aim of this study was to determine if polymorphisms in the leptin (LEP), leptin receptor (LEPR), and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) genes as well as genetic variants of β-casein (β-CN), κ-CN, and β-lactoglobulin (β-LG) affect technological properties important for cheese production and, hence, could act as genetic makers for cheese quality. Individual milk samples from the Swedish Red and the Swedish Holstein breeds were analyzed for sizes of CN micelles and fat globules as well as rennet-induced gel strength, gelation time, and yield stress. Model cheeses were produced to study yield, hardness, and pH of the cheeses. The A1457G, A252T, A59V, and C963T single nucleotide polymorphisms (SNP) were analyzed on the LEP gene, the T945M SNP on the LEPR gene, and the Nt984+8(A-G) SNP on the DGAT1 gene. In addition, genetic variants of β-CN, κ-CN, and β-LG were determined. The results indicate that technological properties were influenced by the LEPR_T945M polymorphism, which had an association with gel strength, yield stress, and cheese hardness (T > C). However, also LEP_A252T was shown to affect gel strength (T > A), whereas the LEP_A59V had an effect on fat globule size (T > C). For the milk protein genes, favorable effects were found for the A and B variants of β-LG and κ-CN, respectively, on gel strength, gelation time, and yield stress. In addition, the B variant of κ-CN was shown to be associated with smaller CN micelles than the A variant. Thus, the results demonstrate potential genetic markers for cheese characteristics. However, milk composition traits also affected the obtained results, thus making it necessary to thoroughly assess the different aspects regarding the influence of gene effects on cheese characteristics before directly selecting for certain alleles or genetic variants to improve the processing and quality of cheese.     

气相色谱-质谱法分析比较超高温灭菌乳和巴氏杀菌乳中的10种内酯类风味物质

目的 建立气相色谱-质谱法(gas chromatography-mass spectrometry, GC-MS)分析比较超高温灭菌乳(ultra-high temperature milk, UHT)和巴氏杀菌乳中10种内酯类风味物质的分析方法。方法 液体乳样品用乙腈提取、无水硫酸镁干燥、正己烷除脂后,经Stabile-WAX色谱柱分离,在单离子监测模式(single ion monitoring, SIM)下进行测定,采用基质匹配外标法进行定量。结果 10种风味物质在1~20 μg/L浓度范围内线性关系良好,线性相关系数均大于0.99,方法的检出限为0.2~4 μg/kg,定量限为0.7~13 μg/kg。各目标化合物在3个基质加标浓度（1、5、10 μg/L）下的平均回收率为85.94%-116.44%,相对标准偏差为1.06%~6.36%（n=6）。超高温灭菌乳样品中检出δ-癸内酯、γ-十二内酯、δ-十二内酯,巴氏杀菌乳样品中检出δ-癸内酯和δ-十二内酯,前者内酯类风味物质的种类和含量略高于后者。结论 建立的方法操作简单,适用于液体乳中内酯类风味物质的检测。     

超高压对食品凝胶特性影响的研究进展

蛋白质和多糖是食品中两大重要组成成分,凝胶性是它们共有的功能特性之一,超高压技术可改变它们的空间构象并进一步相互作用而形成凝胶。本文对超高压技术的概念、原理、特点进行概述,并将其对蛋白凝胶、多糖凝胶、蛋白-蛋白凝胶、多糖-多糖凝胶、蛋白-多糖凝胶的特性影响进行分析,旨在全面了解超高压技术对食品凝胶特性的影响。在此基础上提出该领域未来可能的研究方向,为扩大其应用范围提供理论依据。      

Gelation upon long storage of milk drinks with carrageenan

The carrageenan-induced stabilization and gelation of ultra-high-temperature-treated milk was studied during long storage. Severe heating (causing increased protein denaturation), lowering of the pH, or the use of κ-carrageenan (instead of ι-carrageenan) led to excessive gelation. It is suggested that the balance between carrageenan-carrageenan interactions and carrageenan-protein interactions determines the gel strength. If the interactions between carrageenan and proteins are decreased, more carrageenan is available for carrageenan-carrageenan interactions, leading to a stronger gel. This is the case if κ-carrageenan is used instead of ι-carrageenan because the former forms weaker interactions with proteins than the latter. Also, heating and pH influence the attachment of whey proteins to the casein micelle surface, hindering the attachment of carrageenan to the casein proteins. Upon storage, gel strength increased. Particle size and rheology measurements indicated that upon storage, tenuous carrageenan-protein aggregates are formed. The firming of the gel was probably related to slow structural arrangements of the gel and not related to slowly changing calcium equilibria or age gelation.     

原生态与人工添加二十二碳六烯酸羊乳制品贮藏期脂肪酸稳定性比较

通过饲喂奶山羊富含二十二碳六烯酸（docosahexaenoic acid,DHA）的微藻粉,获得原生态DHA羊乳（DHA含量为30 mg/100 g原料乳）,然后将其制作成超高温瞬时灭菌（ultra-high temperature instantaneous sterilization,UHT）乳及全脂乳粉,同时设立人工添加富含DHA微胶囊粉的UHT乳及全脂乳粉作为对照组,在常温（25 ℃）和高温（37 ℃）下进行为期28 d的贮藏实验,研究原生态与人工添加DHA羊乳制品贮藏期脂肪酸稳定性。结果表明,与人工添加组相比,贮藏期间原生态UHT乳及全脂乳粉的DHA含量下降速率明显减缓,在UHT乳中,人工添加组乳制品DHA含量降低率在37 ℃下最高达（40.92±3.52）%（贮藏第28天）,此时原生态组DHA降低率为（36.70±4.84）%。贮藏期间,原生态与人工添加DHA的UHT乳及全脂乳粉中多不饱和脂肪酸相对含量总体均下降,且与人工添加DHA的乳制品相比,原生态组中多不饱和脂肪酸相对含量更高,更易氧化生成碳链更短的脂肪酸。此外,随着贮藏期的延长,原生态DHA乳制品组中的油脂氧化指标过氧化值和酸价上升速率明显低于人工添加DHA乳制品组。综上,本实验可为制备富含DHA的天然奶制品提供理论参考。     

Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30 min (heating temperature). The particle size of the casein micelles was measured at pH 4.75-7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215 nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40 nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20 nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.     

Effect of temperature and pH on the properties of skim milk gels made from a tamarillo (Cyphomandra betacea) coagulant and rennet

Reconstituted skim milk was gelled with a crude protease extract from tamarillo [Cyphomandra betacea or Solanum betacea (syn.)] fruit and compared with gels prepared with calf rennet. The effects of temperature and pH on the gelation of skim milk were investigated by small deformation oscillatory rheology. The tamarillo extract-induced gels had a faster rate of increase in the elastic modulus (G′) at the early stage of gelation than rennet-induced milk gels. This was probably due to the broader proteolytic activity of tamarillo protease extracts as shown by sodium dodecyl sulfate–PAGE analysis. Confocal microscopy also showed that the milk gels resulting from the addition of tamarillo extracts had larger voids than rennet-induced milk gels. The proteolytic activity of tamarillo extracts was found to be optimal at pH 11. For both rennet and tamarillo extracts, the aggregation time was similar between pH 6.7 and 6.5, but the aggregation time of rennet-induced milk gels was lower than that of milk gels obtained by the addition of tamarillo extracts at pH lower than 6.5. An increase in temperature was found to have a significant effect on aggregation time, particularly at 20°C, where rennet did not coagulate milk in 3 h but the tamarillo extracts coagulated milk within 2 h. The results of this study suggest that extracts from tamarillo fruit could be used for milk gelation, particularly under lower temperature or high pH conditions.