转谷氨酰胺酶对乳蛋白质的改性作用

转谷氨酰胺酶是催化酰基转移反应的一种酶,可使蛋白质或多肽之间发生共价交联反应。本文介绍了转谷氨酰胺酶对牛乳蛋白质凝胶特性、乳化性、热稳定性、成膜性等特性的改性作用及其在乳品加工中的应用。     

Enzymatic cross-linking of ewe's milk proteins by transglutaminase

Enzymatic cross-linking of ewe's milk proteins in the presence of transglutaminase was studied and the extent of cross-linking was analysed by capillary gel electrophoresis. Up to now, no publications are available that study the relative susceptibility of individual ewe's milk proteins. Transglutaminase has been demonstrated to induce cross-linking of the ewe's milk proteins. Moreover, a heat treatment of the milk before the reaction with transglutaminase enhanced the susceptibility of the individual ewe's milk proteins towards the cross-linking reaction. The specificity of transglutaminase has been shown to vary with the type of ewe's milk proteins (α_s2-casein, α_s1-casein, α_s0-casein, κ-casein, β-casein A¹, β-casein A², α-lactalbumin and β-lactoglubulin). From our findings, the reactivity for ovine α-caseins was reduced with respect to that of ovine κ-casein and ovine β-caseins. An optimisation strategy based on desirability functions together with experimental design has been used to optimise the preheating conditions (temperature and time) of ovine milk that maximised the cross-linking reactions catalysed by transglutaminase.     

Inactivation of Cronobacter sakazakii by manothermosonication in buffer and milk

The inactivation of Cronobacter sakazakii by heat and ultrasound treatments under pressure at different temperatures [manosonication (MS) and manothermosonication (MTS)] was studied in citrate-phosphate pH 7.0 buffer and rehydrated powdered milk. The inactivation rate was an exponential function of the treatment time for MS/MTS treatments (35−68 °C; 200 kPa of pressure; 117 μm of amplitude of ultrasonic waves) in both media, and for thermal treatments alone when buffer was used as heating media. Survival curves of C. sakazakii during heating in milk had a concave downward profile. Up to 50 °C, the lethality of ultrasound under pressure treatments was independent of the treatment temperature in both media. At temperatures greater than 64 °C in buffer and 68 °C in milk, the inactivating effect of MTS was equivalent to that of the thermal treatments alone at the same temperature. Between 50 and 64 ºC for buffer and 50 and 68 °C for milk, the lethality of MTS was the result of a synergistic effect, where the total lethal effect was higher than the lethal effect of heat added to that of ultrasound under pressure at room temperature. The maximum synergism was found at 60 °C in buffer and at 56 °C in milk. A heat treatment of 12 min (60 °C) or 4 min of an ultrasound under pressure at room temperature treatment (35 °C; 200 kPa; 117 μm) would be necessary to guarantee the death of 99.99% of C. sakazakii cells suspended in milk. The same level of C. sakazakii inactivation can be achieved with 1.8 min of a MTS treatment (60 °C; 200 kPa; 117 μm). Damaged cells were detected after heat treatments and after ultrasound under pressure treatments at lethal but not at non-lethal temperatures.     

Adaptation of bovine milk towards mares’ milk composition by means of membrane technology for koumiss manufacture

This study was carried out to determine the physical, chemical, microbiological and organoleptic properties of koumiss made from bovine milk. The bovine milk was modified according to the composition of mares’ milk using ultrafiltration, microfiltration and nanofiltration. Koumiss made from modified bovine milk and mares’ milk were compared. In order to assess options for a consistent fermentation, a starter culture, instead of the prevailing spontaneous flora in traditional manufacture of koumiss, was used. Key compositional factors modified were the contents of dry matter, mineral, protein and lactose and the casein-to-whey protein ratio. Koumiss made from modified bovine milk and by starter cultures was found to be very similar to koumiss from mares’ milk in terms of pH, titratable acidity, alcohol, proteolytic activity, apparent viscosity, and microbiological composition, when assessed both in the freshly made product and that after 15 days storage at 4°C.     

Inactivation of bacterial pathogens in human milk by high-pressure processing

Low-temperature, long-time (LTLT) pasteurization assures the safety of banked human milk; however, heat can destroy important nutritional biomolecules. High-pressure processing (HPP) shows promise as an alternative for pasteurization of breast milk. The purpose of this study was to investigate the efficacy of HPP for inactivation of selected bacterial pathogens in human milk. Human milk was inoculated with one of five pathogens (10(8) to 10(9) CFU/ml), while 0.1% peptone solution solutions with the same levels of each organism were used as controls. The samples were subjected to 400 MPa at 21 to 31 degrees C for 0 to 50 min or to 62.5 degrees C for 0 to 30 min (capillary tube method) to simulate LTLT pasteurization. Tryptic soy agar and selective media were used for enumeration. Traditional thermal pasteurization resulted in inactivation (> 7 log) of all pathogens within 10 min. In human milk and in peptone solution, a 6-log reduction was achieved after 30 min of HPP for Staphylococcus aureus ATCC 6538. After 30 min, S. aureus ATCC 25923 was reduced by 8 log and 6 log in human milk and peptone solution, respectively. Treatments of 4 and 7 min resulted in an 8-log inactivation of Streptococcus agalactiae ATCC 12927 in human milk and peptone solution, respectively, while Listeria monocytogenes ATCC 19115 required 2 min for an 8-log inactivation in human milk. Escherichia coli ATCC 25922 was inactivated by 8 log after 10 min in peptone solution and by 6 log after 30 min in human milk. These data suggest that HPP may be a promising alternative for pasteurization of human milk. Further research should evaluate the efficacy of HPP in the inactivation of relevant viral pathogens.     

Inactivation of Listeria innocua in milk and orange juice by ultrahigh-pressure homogenization

The aim of this work was to evaluate the bactericidal efficacy of ultrahigh-pressure homogenization (UHPH) against Listeria innocua ATCC 33090 inoculated into milk and orange juice. We also intended to study the effect of inlet temperature on the lethality and production of sublethal injuries in this microorganism and its ability to survive, repair, and grow in refrigerated storage after UHPH treatment. Samples of ultrahigh-temperature whole milk and ultrahigh-temperature orange juice inoculated at a concentration of approximately 7.0 log (CFU per milliliter) were immediately pressurized at 300 MPa on the primary homogenizing valve and at 30 MPa on the secondary valve, with inlet temperatures of 6.0 +/- 1.0 degrees C and 20 +/- 1.0 degrees C. L. innocua viable counts and injured cells were measured 2 h after UHPH treatment and after 3, 6, and 9 days of storage at 4 degrees C for milk and after 3, 6, 9, 12, 15, 18, and 21 days of storage at 4 degrees C for orange juice. Both the inlet temperature and the food matrix influenced significantly (P < 0.05) the inactivation of L. innocua, which was higher in whole milk at the 20 degrees C inlet temperature. The UHPH treatment caused few or no sublethal injuries in L. innocua. During storage at 4 degrees C after treatments, counts increased by approximately 2 logarithmic units from day 0 to 9 in whole milk, whereas in orange juice counts diminished by approximately 2.5 logarithmic units from day 0 to 18.     

膜技术分离脱脂乳中酪蛋白胶束和乳清蛋白工艺的研究

对膜技术分离脱脂乳酪蛋白胶束和乳清蛋白工艺进行了研究,考察了不同筛分膜的分离效果,并研究了不同操作参数对渗透通量的影响.最终确定用切割分子量300kDa的陶瓷膜进行脱脂乳浓缩分离,酪蛋白胶束和乳清蛋白的分离效果较佳,透过液中乳清蛋白占真蛋白的百分比可达98.91%.确定了膜分离的最适操作压力为0.20MPa,最适操作温度为50℃.     

不同还原剂对大豆胰蛋白酶抑制剂的钝化研究

研究了二巯基苏糖醇(DTT)、亚硫酸钠(Na2SO3)和半胱氨酸(Cys)对大豆胰蛋白酶抑制剂活性的影响。实验在温度80℃,pH7.5条件下反应,并以BAPNA为底物,采用改进的方法测定DTT对大豆蛋白酶抑制剂的钝化作用,最后用SDS-PAGE方法和凝胶排阻色谱法研究其蛋白酶钝化敏感性。通过改进的BAPNA法得出还原剂钝化大豆胰蛋白酶抑制剂由大到小顺序依次为:DTT>Na2SO3>Cys,并通过SDS-PAGE进一步证实了比色法得出的结论。而凝胶排阻色谱法说明了还原剂对大豆胰蛋白酶抑制剂作用使得抑制剂中二硫键被打断,抑制剂结构发生改变,有新的物质生成。所以,得出大豆胰蛋白酶抑制剂的稳定性与二硫键的存在有关。     

Inactivation of Bacillus cereus spores in milk by mild pressure and heat treatments

The objective of this work was to study the germination and subsequent inactivation of Bacillus cereus spores in milk by mild hydrostatic pressure treatment. In an introductory experiment with strain LMG6910 treated at 40 degrees C for 30 min at 0, 100, 300 and 600 MPa, germination levels were 1.5 to 3 logs higher in milk than in 100 mM potassium phosphate buffer (pH 6.7). The effects of pressure and germination-inducing components present in the milk on spore germination were synergistic. More detailed experiments were conducted in milk at a range of pressures between 100 and 600 MPa at temperatures between 30 and 60 degrees C to identify treatments that allow a 6 log inactivation of B. cereus spores. The mildest treatment resulting in a 6 log germination was 30 min at 200 MPa/40 degrees C. Lower treatment pressures or temperatures resulted in considerably less germination, and higher pressures and temperatures further increased germination, but a small fraction of spores always remained ungerminated. Further, not all germinated spores were inactivated by the pressure treatment, even under the most severe conditions (600 MPa/60 degrees C). Two possible approaches to achieve a 6 log spore inactivation were identified, and validated in three additional B. cereus strains. The first is a single step treatment at 500 MPa/60 degrees C for 30 min, the second is a two-step treatment consisting of pressure treatment for 30 min at 200 MPa/45 degrees C to induce spore germination, followed by mild heat treatment at 60 degrees C for 10 min to kill the germinated spores. Reduction of the pressurization time to 15 min still allows a 5 log inactivation. These results illustrate the potential of high-pressure treatment to inactivate bacterial spores in minimally processed foods.     

Inactivation of Listeria innocua in skim milk by pulsed electric fields and nisin

Pulsed electric fields (PEF) is an emerging nonthermal processing technology used to inactivate microorganisms in liquid foods such as milk. PEF results in loss of cell membrane functionality that leads to inactivation of the microorganism. There are many processes that aid in the stability and safety of foods. The combination of different preservation factors, such as nisin and PEF, to control microorganisms should be explored. The objective of this research was to study the inactivation of Listeria innocua suspended in skim milk by PEF as well as the sensitization of PEF treated L. innocua to nisin. The selected electric field intensity was 30, 40 and 50 kV/cm and the number of pulses applied was 10.6, 21.3 and 32. The sensitization exhibited by PEF treated L. innocua to nisin was assessed for 10 or 100 IU nisin/ml. A progressive decrease in the population of L. innocua was observed for the selected field intensities, with the greatest reduction being 2 1/2 log cycles (U). The exposure of L. innocua to nisin after PEF had an additive effect on the inactivation of the microorganism as that exhibited by the PEF alone. As the electric field, number of pulses and nisin concentration increased, synergism was observed in the inactivation of L. innocua as a result of exposure to nisin after PEF. The reduction of L. innocua accomplished by exposure to 10 IU nisin/ml after 32 pulsed electric fields was 2, 2.7, and 3.4 U for an electric field intensity of 30, 40, and 50 kV/cm, respectively. Population of L. innocua subjected to 100 IU nisin/ml after PEF was 2.8-3.8 U for the selected electric field intensities and 32 pulses. The designed model for the inactivation of L. innocua as a result of the PEF followed by exposure to nisin proved to be accurate in the prediction of the inactivation of L. innocua in skim milk containing 1.2 or 37 IU nisin/ml. Inactivation of L. innocua in skim milk containing 37 IU nisin/ml resulted in a decrease in population of 3.7 U.     

预热处理对谷氨酰胺转氨酶催化羊乳热稳定性的影响

研究了羊乳经预热处理后谷氨酰胺转氨酶(transglutaminase,TGase)诱导催化羊乳热稳定性的影响。结果表明,随着预热处理温度的提高和处理时间的延长,羊乳热凝固时间(heat coagulation time,HCT)显著增加(P 0. 05);羊乳经60～70℃/60 min或80～90℃/30 min预热处理可使羊乳中天然TGase抑制剂完全灭活;预热处理可使羊乳中乳清蛋白变性,且随着处理温度的升高,乳清蛋白变性程度增大,这有利于TGase对乳蛋白的交联;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)进一步证实了羊乳中存在天然TGase抑制剂,预热处理可使羊乳中κ-酪蛋白在TGase作用下交联度提高,防止κ-酪蛋白从酪蛋白胶束中解离,进一步提高羊乳的热稳定性。     

Production of cheese from donkey milk as influenced by addition of transglutaminase

Donkey milk is characterized by low contents of total solids, fat, and caseins, especially κ-casein, which results in formation of a very weak gel upon renneting. The objective of this study was to evaluate the effect of fortification of donkey milk with microbial transglutaminase (MTGase) for cheesemaking in relation to different enzyme addition protocols (patterns, PAT). Four independent trials were performed using MTGase (5.0 U/g of milk protein) according to the following experimental patterns: control (no MTGase addition); MTGase addition (40°C) 15 min before starter inoculation (PAT1); addition of MTGase to milk simultaneously with starter culture (40°C) (PAT2); and MTGase addition simultaneously with rennet (42°C) in acidified milk (pH 6.3) (PAT3). Evolution of pH during acidification, cheesemaking parameters, and proximal composition and color of cheese at 24 h were recorded. The protein fractions of cheese and whey were investigated by urea-PAGE and sodium dodecyl sulfate-PAGE. Addition of MTGase had no significant effect on moisture, protein, fat, or cheese yield. The addition of MTGase with rennet (PAT3) improved curd firmness compared with the control. Among the different patterns of MTGase addition, PAT3 reduced gel formation time, time between rennet addition and cheese molding, and weight loss of cheese at 24 h. The PAT3 treatment also resulted in the lowest lightness and highest yellowness color values of the cheese. Sodium dodecyl sulfate-PAGE of cheeses revealed that MTGase modified the protein pattern in the high-molecular-weight zone (range 37–75 kDa) compared with the control. Of the MTGase protocols, PAT3 showed better casein retention in cheese, as confirmed by the lanes of α- and β-caseins in the electropherogram of the whey, which was subtler for this protocol. In conclusion, MTGase may be used in cheese production from donkey milk to improve curd firmness; MTGase should be added simultaneously with the rennet.     

超滤技术结合大孔吸附树脂纯化低聚果糖

菊粉酶解制备低聚果糖(FOS)过程中,酶解液中通常含有一定量的单糖、蔗糖和未酶解完全的菊粉;为了制备高纯度低聚果糖,酶解液先后经超滤(除去未完全酶解的菊粉)和大孔吸附树脂纯化(分离FOS、单糖和蔗糖)处理。因此,探讨超滤技术最佳工艺参数,比较4种树脂(CSR-1Na、CSR-2Na、CSR-3Na、CSR-1Ca)对FOS的纯化效果,选择最佳树脂并对其纯化工艺参数进行优化。结果表明,超滤技术的最佳操作工艺参数为:操作压差1.0 MPa,循环流量2L/min,pH6.0;该条件下,超滤的渗透通量为10.8L/(m2·h),FOS透过率为93.7%,纯度为63.84%。CSR-1Na型树脂对FOS的纯化效果较好;大孔树脂最佳纯化工艺参数为:操作温度60℃,体积流速1.5mL/min,操作pH 6.0~6.5;该条件下FOS(纯度大于95%)的回收率为83.26%。     

Physiological role of indigenous milk enzymes: An overview of an evolving picture

Over 60 indigenous enzymes have been identified so far in the milk of various mammalian species. The vast majority of research in this area has focused on their use as indicators of processing (mainly pasteurization), contribution to dairy product quality and investigating the factors that affect their level in milk. The aim of this article is to provide an overview of data accumulated during the last 5 years, mostly for bovine and human milk, which shows that milk indigenous enzymes play a key role in regulating lactogenesis, e.g., inducing active involution, and that they are essential components of antioxidation and the innate immune system of milk.     

Inactivation of trypsin inhibitor,lectin and urease in soybean by hydrothermal treatment

The effects of hydrothermal treating parameters on trypsin inhibitor (TI), lectin and urease activities in whole soybean seeds were investigated by Response Surface Analysis (RSA). Independent variables with equidistant levels examined in this study were the following: treating temperature (levels: 70, 85 and 100°C), treating time (levels: 10, 30 and 50 min), and soaking time (2, 4 and 6 h). The functions between treating parameters and responses values of TI, lectin and urease activities in treated soybean were calculated by multiple, non-linear regression analysis and analysis of variance. Mathematical models were developed in this study to predict TI, lectin and urease activities in soybean during hydrothermal processing and they have been found to be significant (P[%] = 0.1, 1.0, and 0.1, respectively). Differences between the effects of processing parameters on the inactivation of TI, lectin and urease in soybean were observed and they can be seen either from the mathematical models or the typical figures. The modeling of the effects should help in selection of optimal parameters of hydrothermal treatment for destruction of TI. lectin and urease in soybean. Based on the modeling, lectin and urease can be fully inactivated in soybean treated at proper conditions, while remaining TI activity can be expected in soybean treated at any conditions examined in this study.     

Physical properties of yoghurt manufactured with milk whey and transglutaminase

The effect of milk protein polymerization prior to the yoghurt fermentation process was evaluated by enzymatic reaction with microbial transglutaminase (Streptoverticillium mobaraense). Yoghurt samples were manufactured with 100% milk or by substituting milk with 20 or 30% of liquid milk whey, aimed at determining the use of natural milk whey in dairy products. Transglutaminase was added at a protein ratio of 0.5 U g⁻¹ to all samples and evaluated regarding rheological behavior, syneresis index and texture profile. The addition of enzyme transglutaminase contributed to syneresis prevention and increased the consistency index in yoghurt samples manufactured with milk whey. Yoghurt manufactured from 70% milk plus 20% milk whey, followed by enzymatic treatment, presented similar characteristics to traditionally manufactured yoghurt (C 100), with no alteration in the syneresis of the samples (p > 0.05) and presented texture parameters similar to the control yoghurt (C 100).     

不同杀菌方式对牛乳杀菌效果的比较

牛乳是天然营养品,微生物极易在其中繁殖,病源菌和其他有害菌也常存其中,这些微生物是造成牛乳腐败变质的主要原因。为了探讨不同杀菌方式对牛乳杀菌效果的优劣,对牛乳微波杀菌和热力杀菌进行了对比试验。通过试验表明:微波杀菌不同功率条件下,600W时杀菌效果最好,同时也表明微波杀菌比热力杀菌效果好。     

不补料酶膜耦合反应制备牛乳蛋白ACE抑制肽的研究

为了克服传统酶解技术的不足,提高酶解反应效率,采用不补料酶膜耦合反应制备牛乳蛋白ACE抑制肽。考察超滤膜对牛乳蛋白及酶的截留、膜通量及滤出液的ACE抑制率和IC₅₀的影响,确定出超滤膜最佳的截留分子量为5000 u;研究了反应时间、底物浓度、加酶量和循环泵转速对蛋白转化率的影响,确定最优酶解工艺条件:底物浓度7%（W/W）、加酶量2000 U/g、反应时间100 min、循环泵转速100 r/min,该条件下蛋白转化率、单位酶产肽量、ACE抑制率分别达到53.16%、39.59 g肽/g酶、76.81%,与对照组相比蛋白转化率、单位酶产肽量、ACE抑制率分别提高了16.99%、16.61%、18.55%;采用高效凝胶过滤色谱法测定超滤液中ACE抑制肽的分子量分布,发现样品中分子量≤2253 u组分质量分数为94.62%,超滤膜对截留分子量大小控制准确。因此不补料酶膜耦合反应可为酶法制备牛乳蛋白ACE抑制肽提供一种更为高效的方法。