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.     

Effect of beta-lactoglobulin A and B whey protein variants on the rennet-induced gelation of skim milk gels in a model reconstituted skim milk system

The effect of fortifying reconstituted skim milk with increasing levels of the β-lactoglobulin (β-LG) genetic variants A, B, and an A-B mixture on rennet-induced gelation was studied by small deformation-sensitive rheology. Free-zone capillary electrophoresis and high-sensitivity oscillatory rheology were used to elucidate the role of potential heterotypic associative interactions between whey proteins and casein in a mixed colloidal system, subjected to moderate heating (65°C for 30 min) prior to renneting, on the gelling properties of the system. Increasing levels of added whey protein, in the concentration range of 0.225 to 1.35% of added protein, led to a concomitant progressive increase in the equilibrium shear storage modulus, G′ (recorded after ∼10,800 s), in the order β-LG B > β-LG A and β-LG A-B, as the general expected consequence of the setup of denser casein gel networks. The preferential effect of β-LG B over β-LG A on the mechanical strength of the gels may be due to the formation of cross-links and aggregates involving whey proteins and rennet hydrolysis products or an increase in the size of the casein micelle caused by the grafting of β-LG B to its surface, or both. The results of free-zone capillary electrophoresis were consistent with the notion that β-LG B (and not β-LG A) binds to the casein micelle under an optimal stoichiometry of 1:0.045 (mg/mg), even in the absence of heat treatment. The liquid-like character of the gel networks formed, tan δ, was a parameter sensitive to the level of addition of β-LG A in particular. At low concentrations (up to 0.45%) of β-LG A, tan δ increased by almost twice as much, which was interpreted as a result of the increase in the loss modulus, G″, of the sol fraction because of the presence of unbound β-LG A. At greater incremental concentrations of β-LG (>0.45%), the formation of smaller whey protein aggregates confined to the sol fraction may have led to a progressive decrease in tan δ. The critical gel time, t_gel, was also affected by the concentration of added whey protein and described 3 zones of behavior, irrespective of the type of whey protein variant. The critical gel time was slightly shorter for β-LG B than for β-LG A at 0.45% of added whey protein, but this difference became larger at 0.67%. Even when only β-LG B was found to associate with casein prior to renneting, both β-LG A and β-LG B, either alone or mixed, had a profound influence on the mechanical strength and coagulation kinetics of the rennet-induced casein gels. This knowledge is expected to be useful to exert better control and optimize processing conditions during the manufacturing of cheese and cheese analogs.     

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。     

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.     

Electrophoretic characterization of protein interactions suggesting limited feasibility of accelerated shelf-life testing of ultra-high temperature milk

Accelerated shelf-life testing is applied to a variety of products to estimate keeping quality over a short period of time. The industry has not been successful in applying this approach to ultra-high temperature (UHT) milk because of chemical and physical changes in the milk proteins that take place during processing and storage. We investigated these protein changes, applying accelerated shelf-life principles to UHT milk samples with different fat levels and using native- and sodium dodecyl sulfate-PAGE. Samples of UHT skim and whole milk were stored at 20, 30, 40, and 50°C for 28 d. Irrespective of fat content, UHT treatment had a similar effect on the electrophoretic patterns of milk proteins. At the start of testing, proteins were bonded mainly through disulfide and noncovalent interactions. However, storage at and above 30°C enhanced protein aggregation via covalent interactions. The extent of aggregation appeared to be influenced by fat content; whole milk contained more fat than skim milk, implying aggregation via melted or oxidized fat, or both. Based on reduction in loss in absolute quantity of individual proteins, covalent crosslinking in whole milk was facilitated mainly by products of lipid oxidation and increased access to caseins for crosslinking reactions. Maillard and dehydroalanine products were the main contributors involved in protein changes in skim milk. Protein crosslinking appeared to follow a different pathway at higher temperatures (≥40°C) than at lower temperatures, making it very difficult to extrapolate these changes to protein interactions at lower temperatures.     

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。      

Importance of casein micelle size and milk composition for milk gelation

The economic output of the dairy industry is to a great extent dependent on the processing of milk into other milk-based products such as cheese. The yield and quality of cheese are dependent on both the composition and technological properties of milk. The objective of this study was to evaluate the importance and effects of casein (CN) micelle size and milk composition on milk gelation characteristics in order to evaluate the possibilities for enhancing gelation properties through breeding. Milk was collected on 4 sampling occasions at the farm level in winter and summer from dairy cows with high genetic merit, classified as elite dairy cows, of the Swedish Red and Swedish Holstein breeds. Comparisons were made with milk from a Swedish Red herd, a Swedish Holstein herd, and a Swedish dairy processor. Properties of CN micelles, such as their native and rennet-induced CN micelle size and their ζ-potential, were analyzed by photon correlation spectroscopy, and rennet-induced gelation characteristics, including gel strength, gelation time, and frequency sweeps, were determined. Milk parameters of the protein, lipid, and carbohydrate profiles as well as minerals were used to obtain correlations with native CN micelle size and gelation characteristics. Milk pH and protein, CN, and lactose contents were found to affect milk gelation. Smaller native CN micelles were shown to form stronger gels when poorly coagulating milk was excluded from the correlation analysis. In addition, milk pH correlated positively, whereas Mg and K correlated negatively with native CN micellar size. The milk from the elite dairy cows was shown to have good gelation characteristics. Furthermore, genetic progress in relation to CN micelle size was found for these cows as a correlated response to selection for the Swedish breeding objective if optimizing for milk gelation characteristics. The results indicate that selection for smaller native CN micelles and lower milk pH through breeding would enhance gelation properties and may thus improve the initial step in the processing of cheese.     

Association between the bovine milk metabolome and rennet-induced coagulation properties of milk

The milk metabolomes of 407 individual Swedish Red dairy cows were analyzed by nuclear magnetic resonance spectroscopy as part of the Danish-Swedish Milk Genomics Initiative. By relating these metabolite profiles to total milk protein concentration and rheological measurements of rennet-induced milk coagulation together using multivariate data analysis techniques, we were able to identify several different associations of the milk metabolome to technological properties of milk. Several novel correlations of milk metabolites to protein content and rennet-induced coagulation properties were demonstrated. Metabolites associated with the prediction of total protein content included choline, N-acetyl hexosamines, creatinine, glycerophosphocholine, glutamate, glucose 1-phosphate, galactose 1-phosphate, and orotate. In addition, levels of lactate, acetate, glutamate, creatinine, choline, carnitine, galactose 1-phosphate, and glycerophosphocholine were significantly different when comparing noncoagulating and well-coagulating milks. These findings suggest that the mentioned metabolites are associated with milk protein content and rennet-induced coagulation properties and may act as quality markers for cheese milk.     

乳清蛋白对直接超高温乳贮存稳定性的影响研究进展

直接超高温（direct ultra-high temperature,dUHT）牛乳是蒸汽直接接触牛乳快速加热,然后通过闪蒸迅速蒸发水分,具有热负荷能低、最大程度保留牛乳营养和风味等优点,有望成为继UHT乳的新一代高品质液态乳制品。目前我国dUHT乳产业尚处于起步阶段。虽然dUHT乳的营养全面,但其贮存过程中有时出现沉淀、老化凝胶等品质问题,影响了产品货架期。dUHT乳的贮存稳定性影响因素非常复杂,乳清蛋白的热不稳定性是影响dUHT乳贮存稳定性的主要原因之一。本文综述了dUHT乳生产基本原理,探讨了乳清蛋白分子间、乳清蛋白与酪蛋白和纤溶酶等物质之间的交联反应机制及其对dUHT乳贮存稳定性的影响,以期为dUHT乳的加工技术优化升级和我国dUHT乳的快速发展提供参考。     

超高压射流灭菌对牛奶理化特性的影响

利用超高压射流对撞式/碰撞式技术处理液态奶,考察其相对密度、滴定酸度、pH值、灰分碱度、浊度、颗粒粒径等理化性质。结果表明,超高压射流处理后,液态奶的灰分碱度及压力≥200 MPa时滴定酸度均符合灭菌乳国家标准(GB5408.2-1999);相对密度保持不变;浊度呈上升趋势;对撞方式均质效果明显优于碰撞方式。超高压射流处理牛奶的最佳工艺为200 MPa对撞方式处理。该研究为超高压射流杀菌奶的后续深入研究奠定了基础。     

Impaired rennetability of heated milk; study of enzymatic hydrolysis and gelation kinetics

Casein micelles in milk are stable colloidal particles with a stabilizing hairy brush of kappa-casein. During cheese production rennet cleaves kappa-casein into casein macropeptide and para-kappa-casein, thereby destabilizing the casein micelle and resulting in aggregation and gel formation of the micelles. Heat treatment of milk causes impaired clotting properties, which makes heated milk unsuitable for cheese production. In this paper we compared five different techniques, often described in the literature, for their suitability to quantify the enzymatic hydrolysis of kappa-casein. It was found that the technique is crucial for the yield of casein macropeptide and this yield then affects the calculated enzymatic inhibition caused by heat treatment, ranging from 5 to 30%. The technique, which we found to be the most reliable, demonstrates that heat-induced calcium phosphate precipitation does not affect the enzymatic cleavage, while whey protein denaturation causes a very slight reduction of enzyme activity. By using diffusing wave spectroscopy, a very sensitive technique to monitor gelation processes, we demonstrated that heat-induced calcium phosphate precipitation does not affect the clotting. Whey protein denaturation does not affect the start of flocculation but has a clear effect on the clotting process. This work adds to a better understanding of the processes causing the impaired clotting properties of heated milk.     

Addition of sodium caseinate to skim milk inhibits rennet-induced aggregation of casein micelles

The objective of this paper was to observe the rennet-induced aggregation behaviour of casein micelles in milk in the presence of additional sodium caseinate. Analysis of the centrifugal supernatants by size exclusion chromatography confirmed an increase in the soluble protein in the milk serum phase after addition of sodium caseinate. Although the total amount of κ-casein hydrolyzed over time was not affected, there was a significant effect of soluble casein on milk gelation, with a dose-dependent decrease of the gelation time as measured by rheology. Light scattering experiments also confirmed that the addition of soluble caseins inhibited the aggregation of casein micelles. Addition of 1 mM CaCl₂ prior to renneting increased the extent of rennet aggregation in samples containing additional sodium caseinate, but the inhibiting effect was still evident. The amount of soluble casein (as measured by chroma tography) significantly decreased after renneting, suggesting its association with the micellar fraction. Supporting experiments carried out with purified fractions of soluble caseins demonstrated that both α_s-casein and β-casein played a role as protective colloids (increasing steric repulsion) during renneting. It was concluded that the inhibiting effect observed during gelation was caused by the adsorption of soluble casein molecules on the surface of rennet-altered casein micelles.     

Effects of ultra-high pressure homogenization on the cheese-making properties of milk

The effects of single- or 2-stage ultra-high pressure homogenization (UHPH; 100 to 330 MPa) at an inlet temperature of 30°C on the cheese-making properties of bovine milk were investigated. Effects were compared with those from raw, heat-pasteurized (72°C for 15 s), and conventional homogenized-pasteurized (15 + 3 MPa, 72°C for 15 s) treatments. Rennet coagulation time, rate of curd firming, curd firmness, wet yield, and moisture content of curds were assessed. Results of particle size and distribution of milk, whey composition, and gel microstructure observed by confocal laser scanning microscopy were analyzed to understand the effect of UHPH. Single-stage UHPH at 200 and 300 MPa enhanced rennet coagulation properties. However, these properties were negatively affected by the use of the UHPH secondary stage. Increasing the pressure led to higher yields and moisture content of curds. The improvement in the cheese-making properties of milk by UHPH could be explained by changes to the protein-fat structures due to the combined effect of heat and homogenization.     

超高压诱变选育富锌啤酒酵母菌株的研究

以啤酒酵母为出发菌株,采用超高压诱变方法诱变选育高富集锌的酵母菌株。优化了超高压处理的工艺条件,并基于菌株对锌离子的耐受性和富集性,对菌株进行筛选。结果表明,培养10 h的啤酒酵母菌处于对数生长期,适宜于诱变;超高压处理压力250 MPa、保压时间20 min为诱变的最佳工艺条件;成功选育出一株高富集锌的突变菌株,其细胞锌含量达2.75 mg/g,是出发菌的6.55倍。     

乳中蛋白酶与UHT乳贮存中的胶凝现象

乳中的蛋白酶有2个主要的来源途径，乳中天然存在的蛋白酶和由某些微生物产生的蛋白酶，其中纤维蛋白溶酶和嗜冷菌产生的耐热性蛋白酶是存在于UHT乳中的主要蛋白酶，这些蛋白酶非常耐热，经UHT灭菌处理仍可存活。耐热性蛋白酶在UHT乳贮存中水解乳蛋白质从而导致了UHT乳的胶凝．简要介绍了纤维蛋白溶酶、嗜冷菌耐热性蛋白酶的性质、活性测定方法，论述了这些酶引起的UHT乳的胶凝性质、形成原因及影响因素，并提出了一些防止措施。     

Structure and shelf stability of milk protein gels created by pressure-assisted enzymatic gelation

In this work, pressure-assisted enzymatic gelation was applied to milk proteins, with the goal of enhancing the structure and stability of pressure-created milk protein gels. High-pressure processing (HPP) at 600 MPa, 3 min, and 5°C was applied to milk protein concentrate (MPC) samples of 12.5% protein concentration, both in the absence and in the presence of calf chymosin [up to 60 IMCU (international milk-clotting units)/kg of milk] or camel chymosin (up to 45 IMCU/kg of milk). Gel hardness, water-holding capacity, and degree of proteolysis were used to assess network strength and shelf stability. The processing trials and all measurements were conducted in triplicate. Statistical analyses of the data were performed by ANOVA, at a 95% confidence level. After HPP treatment, we observed significant structural changes for all samples. Pressurization of MPC, with or without chymosin addition, led to extensive protein aggregation and network formation. The strength of HPP-created milk protein gels without chymosin addition, as measured by the elastic modulus (G′), had a value of 2,242 Pa. The value of G′ increased with increasing chymosin concentration, reaching as high as 4,800 Pa for samples with 45 IMCU/kg of camel chymosin. During 4 wk of refrigerated storage, the HPP and chymosin MPC gels maintained higher gel hardness and better structural stability compared with HPP only (no chymosin) MPC gels. The water-holding capacity of the gels without chymosin remained at 100% during 28 d of refrigerated storage. The HPP and chymosin MPC gels had a lower water-holding capacity (91–94%) than the HPP-only counterparts, but their water-holding capacity did not decrease during storage. Overall, these findings demonstrate that controlled, fast structural modification of high-concentration protein systems can be obtained by HPP-assisted enzymatic treatment, and the created gels have a strong, stable network. This study provides insights into the possibility of using HPP for the development of milk-protein-based products with novel structures and textures and long refrigerated shelf life, along with the built-in safety imparted by the HPP treatment.     

Acid induced gelation behavior of skim milk concentrated by membrane filtration

This work reports a detailed study of the effect of ultrafiltration (UF) and diafiltration (DF) on the acid-induced gelation behavior of fresh milk retentates (2× and 4×). Concentrates were heated at 80°C for 15 min, and compared to unheated samples. The use of extensive DF caused a significantly greater amount of protein (both caseins and whey proteins) in the supernatant fraction, compared to UF retentates at the same concentration, both in unheated and heated samples. DF retentates showed higher pH of gelation, compared to the corresponding UF retentates. The development of tan δ is reported for the first time as a function of colloidal calcium release, and the protein gelation behavior discussed in light differences in composition of the soluble fraction. The results demonstrate how processing history can affect compositional changes and the gelation behavior of fresh milk retentates. Membrane filtration is a widespread unit operation in the dairy industry, employed either to prepare fresh concentrates for further processing, or ingredients with specific functional properties. This work describes in detail the effect of processing history during membrane filtration on the rheological properties of acid induced gels and will help in optimizing formulations and prepare the right ingredients for the right application. It will also be possible to determine new ways to define processing quality of the milk protein concentrates, as it relates to their ability to form texture in fermented dairy products.