Polyphenol oxidase activity in grass and its effect on plant‐mediated lipolysis and proteolysis of Dactylis glomerata (cocksfoot) in a simulated rumen environment

Little is known about the level or activity of polyphenol oxidase (PPO) in grasses and its potential impact on proteolysis and lipolysis. Six grass species were initially screened for PPO activity (740.6, 291.9, 213.6, 119.0, 16.3 and 6.5 U g^?1 fresh weight (FW) for cocksfoot, hybrid ryegrass, Italian ryegrass, perennial ryegrass, timothy and tall fescue respectively). Cocksfoot, which expressed the highest activity, was then used to determine the effect of PPO on plant‐mediated proteolysis and lipolysis in a simulated rumen environment. Sourced cocksfoot was macerated and incubated in an antibiotic‐containing anaerobic medium with or without ascorbate to deactivate PPO in the dark at 39 °C over five time points. At each time point (0, 1, 2, 6 and 24 h), six replicate samples were destructively harvested; three of the replicates were used for lipid analysis and the other three for protein, free amino acid and bound phenol determination. Characterisation of the herbage showed PPO activities of 649.6 and 0 U g^?1 FW, which were reflected in the extent of phenol (derived from quinones) binding to protein after 24 h of incubation, namely 65.1 and 29.6 mg bound phenol g^?1 protein (P < 0.001) for cocksfoot and cocksfoot + ascorbate respectively. Proteolysis, measured as free amino acids released into the incubation buffer, was significantly reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.03 and 0.07 mmol L^?1 g^?1 FW for cocksfoot and cocksfoot + ascorbate respectively. Lipolysis, measured as the proportional decline in the membrane lipid polar fraction, was likewise reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.43 and 0.65 for cocksfoot and cocksfoot + ascorbate respectively. Changes that occurred in protein and the lipid fractions (polar fraction, monoacylglycerol + diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce protein and lipid losses in silo and potentially in the rumen. Copyright © 2006 Society of Chemical Industry     

Lipolysis in red clover with different polyphenol oxidase activities in the presence and absence of rumen fluid

This experiment aims to determine whether polyphenol oxidase (PPO) can reduce the extent of lipolysis and the consequent polyunsaturated fatty acid loss through microbial biohydrogenation in red clover when incubated in the presence of rumen fluid. PPO is involved in the browning reaction of red clover leaves when cut or crushed and exposed to air. It starts the browning process by oxidizing endogenous phenols to quinones, which contain electrophilic sites. These sites react with nucleophilic sites of other compounds such as proteins and have been shown to reduce proteolysis and lipolysis in silo. Two lines of red clover (cv. Milvus), a genotypic mutant with reduced PPO activity (L) and the wild type (H) with a high level of PPO activity, were cut 3 cm above soil level, crushed and cut into 1 cm strips before being loaded into incubation bottles. These were then incubated in anaerobic buffer at 39 °C in either the absence (?) or the presence (+) of rumen microorganisms. The incubations were then compared over a 24 h time course in terms of lipolytic activity. Characterization of the tissues showed PPO activities of 25.3 and 5.13 U g^?1 fresh weight for H and L, respectively. Lipolysis, measured as the proportional decline in the membrane lipid, was reduced (P < 0.001) with increasing PPO activity in both the presence (+) and absence (?) of rumen microorganisms. However, values were significantly higher (P < 0.001) in the presence of rumen microorganisms, with values after the 24 h incubation of 0.28, 0.42, 0.72 and 0.82 for H?, L?, H+ and L+, respectively. Biohydrogenation of C18:2 and C18:3 polyunsaturated fatty acids were significantly lower in the H+ treatment than the L+ treatment, with mean values after 24 h incubation of 53% and 57% (P < 0.05) for C18:2 and 65% and 74% (P < 0.01) for C18:3, respectively. Changes that occurred in the lipid fractions (membrane lipid, diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce polyunsaturated fatty acid loses in the rumen. Copyright © 2007 Society of Chemical Industry     

Ruminal micro‐organisms do not adapt to increase utilization of poly‐phenol oxidase protected red clover protein and glycerol‐based lipid

BACKGROUND: The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants with subsequent increases in the efficiency of N utilization and the level of beneficial polyunsaturated fatty acids in their products (meat and milk). It has also been reported that red clover feeding alters the rumen microbial population compared to grass feeding. This study investigated whether the observed shifts in the microbial population of the rumen when ruminants are fed red clover silage (RC) as opposed to grass silage (G) represented an adaptation by the micro‐organisms to increase the utilization of PPO‐protected protein and glycerol‐based lipid. RESULTS: The experiment consisted of two periods where ruminally fistulated dairy cows were offered either RC or G for 2 weeks, followed by collection of rumen fluid, which was then used in in vitro incubations to investigate lipolysis and proteolysis over time in plant material derived from red clover plants with either wild type PPO expression (PPO+) or PPO expression reduced to undetectable levels by gene silencing (PPO?). Proteolysis and lipolysis (P < 0.05) were lower after 24 h of incubation in the PPO+ treatment than the PPO? treatment irrespective of rumen fluid. Biohydrogenation of C18 polyunsaturated fatty acids was also lower on the PPO+ treatment than the PPO? treatment, with no effect of rumen fluid. CONCLUSION: These results suggest that microbial changes to red clover feeding did not result in an increased ability of the micro‐organisms in the present study to utilize either PPO‐protected protein or glycerol‐based lipid. Copyright © 2008 Society of Chemical Industry     

Immunogold labelling to localize polyphenol oxidase (PPO) during wilting of red clover leaf tissue and the effect of removing cellular matrices on PPO protection of glycerol‐based lipid in the rumen

BACKGROUND: The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants. PPO catalyses the conversion of phenols to quinones, which can react with nucleophilic cellular constituents (e.g. proteins) forming protein–phenol complexes that may reduce protein solubility, bioavailability to rumen microbes and deactivate plant enzymes. In this study, we localized PPO in red clover leaf tissue by immunogold labelling and investigated whether red clover lipid was protected in the absence of PPO‐induced protein–phenol complexes and plant enzymes (lipases). RESULTS: PPO protein was detected to a greater extent (P < 0.001) within the chloroplasts of mesophyll cells in stressed (cut/crushed and wilted for 1 h) than freshly cut leaves for both palisade (61.6 and 25.6 Au label per chloroplast, respectively) and spongy mesophyll cells (94.5 and 40.6 Au label per chloroplast, respectively). Hydrolysis of lipid and C18 polyunsaturated fatty acid biohydrogenation during in vitro batch culture was lower (P < 0.05) for wild‐type red clover than for red clover with PPO expression reduced to undetectable levels but only when cellular matrices containing protein–phenol complexes were present. CONCLUSION: Damaging of the leaves resulted in over a doubling of PPO detected within mesophyll cells, potentially as a consequence of conversion of the enzyme from latent to active form. PPO reduction of microbial lipolysis was apparent in macerated red clover tissue but not in the absence of the proteinaceous cellular matrix, suggesting that the PPO mechanism for reducing lipolysis may be primarily through the entrapment of lipid within protein–phenol complexes. Copyright © 2009 Society of Chemical Industry     

Sensory threshold of off-flavors caused by proteolysis and lipolysis in milk

The objective of this study was to determine the sensory threshold of off-flavor caused by lipolysis in 2% fat milk and to establish the relationship between increased proteolytic activity in milk and the detection of bitter off-flavor. Homogenized raw milk was held at room temperature for 100 min to allow the native milk lipase to release free fatty acids from the triglycerides. Low and high lipolysis pasteurized milk containing 2% fat were blended together in varying amounts to create a series of six milks with increasing free fatty acid (FFA) concentration for sensory evaluation. Sensory threshold for lipolysis in 2% fat milk was determined by ascending forced-choice procedure, with a series of triangle tests in four sessions with 25 panelists in each session. The group best estimated threshold was the geometric mean of the individual thresholds within each of four panel sessions. The geometric mean best estimated detection thresholds for off-flavors caused by lipolysis in 2% fat milk carried out by native milk lipases were 0.320, 0.322, 0.351, and 0.316 meq of FFA/kg milk for panels 1 to 4, respectively. One third of the panelists detected an off-flavor at or below 0.250 meq of FFA/kg milk. To establish the relationship between proteolysis and detection of off-flavor in pasteurized skim milk, 2800 ppm of CO2 were added to pasteurized skim milk, and it was stored for 27 d at 6 degrees C. Another portion of the same milk was frozen on d 1 at -40 degrees C for use as a low proteolysis portion of the same milk. Decrease in casein as a percentage of true protein (CN/TP) was used as an index of proteolysis. After 27 d at 6 degrees C the milk had a decrease in CN/TP of 4.76% and a standard plate count of 430 cfu/ml. The novel approach of storing milk at 6 degrees C for 27 d with added CO2 blocked microbial growth but allowed proteolytic degradation by milk enzymes to proceed. Before sensory analysis, CO2 was removed by vacuum from the high proteolysis milk and the low proteolysis milk was given the same heat and vacuum. Two triangle tests were performed to determine whether panelists could detect off-flavors caused by proteolysis in milk. The threshold detection of off-flavor in skim milk produced by the action of native milk proteases was less than a decrease of CN/TP of 4.76%, but this value is probably near the threshold.     

Effects of mixing red clover with alfalfa at different ratios on dynamics of proteolysis and protease activities during ensiling

This study was conducted to study the effects of ensiled alfalfa (Medicago sativa) and red clover (Trifolium pratense) at different ratios on dynamics of fermentation parameters, N distribution, protein fractions, and protease activities during ensiling. Alfalfa and red clover were harvested and wilted to 35 and 25% dry matter, respectively, chopped to 1 cm, mixed, weighed into 1.0-L buckets at a density of 700 g/L, and ensiled for 1, 3, 7, 15, and 30 d at 30°C. The treatments were mixing ratio of alfalfa to red clover at 100:0, 70:30, 50:50, 30:70, and 0:100 (R0, R30, R50, R70, and R100, respectively; fresh weight). For each ensiling duration, 3 replicates of each treatment were prepared. With increasing proportion of red clover in silage, total N content and proportions of nonprotein N, peptide N, free amino acid N, and NH₃-N decreased linearly, and PC (indigestible true protein, acid detergent insoluble N) proportion increased linearly after ensiling. Moreover, the final pH was lower in R50 and R100 than R0 (4.29, 4.20 vs. 4.48, respectively) on d 30. Also, lactic acid concentration on d 30 was higher in R50, R70, and R100 silage compared with R0 (7.77, 7.66, and 8.76 vs. 6.34, % of dry matter, respectively). The proportion of NH₃-N in R50 was lower than in R0 but closer to R100 after ensiling. During ensiling, proteases including carboxypeptidase, aminopeptidase, and acid proteinase activities decreased as red clover proportion increased. However, no differences were detected in aminopeptidase and acid proteinase activities among R50, R70, and R100 during ensiling. Overall, 50:50 was the optimal mixing ratio of alfalfa with red clover, showing good fermentation quality with lower pH and higher lactic acid concentration, reduced protease activities and proteolysis compared with pure alfalfa silage, and also more total N content than pure red clover silage.     

Amino acid composition and anti‐polyphenol oxidase of peptide fractions from sericin hydrolysate

Sericin hydrolysate (SH), prepared from enzymatic hydrolysis of sericin, was investigated for its antipolyphenol oxidase (PPO) properties. SH decreased PPO activity from both purified mushroom PPO and extracts from apple and eggplant, and retarded browning in fresh‐cut apple and eggplant. SH was a competitive inhibitor using catechol as a substrate. SH exhibited copper ion‐chelating power and reducing power abilities. Fractionation of SH using size exclusion chromatography resulted in four fractions, designated as F1, F2, F3 and F4, with PPO inhibition of 35.75%, 3.89%, 24.52% and 14.75%, respectively. Ser and Asp were major amino acids found in F1. Amino acid sequences in F1, as investigated by LC‐MS/MS using de novo sequencing, contained a high ratio of amino acids with chelating ability. Moreover, amino acids with reducing power ability and with antityrosinase ability were also identified in the sequences.     

百合中多酚氧化酶的部分性质

通过丙酮酚法从百合中提取出多酚氧化酶(PPO)的粗酶液。以儿茶酚为底物时它有两个最适pH,分别为4.0、7.0,在pH5.0-6.5之间酶活力可以在4℃保持至少10h。PPO的最适温度为40℃,从40℃开始酶出现热失活,热失活速度符合一级反应动力学。PPO除对L-酪氨酸没有活力外,对儿茶酚、儿茶素、没食子酸均有活力,其中对儿茶素具有最好的底物特异性。亚硫酸钠对PPO的抑制作用最强,高浓度的抗坏血酸、半胱氨酸、硫脲也有很好的抑制作用,氯化钠、氯化钙、柠檬酸的抑制作用较差。     

多酚氧化酶的提取及分离纯化研究进展

多酚氧化酶(PPO)是自然界中分布极广的一种金属蛋白酶,能催化邻-苯二酚氧化成邻-苯二醌,也是许多果蔬等农产品酶促褐变的主要原因。本文综述了国内外多酚氧化酶的提取及分离纯化技术的研究进展,包括传统提取方法(如缓冲液匀浆法、丙酮提取法、丙酮粉提取法等)和新型提取方法(超声波辅助提取法和超高压提取法等),传统经典分离纯化方法(如溶剂法、沉淀法)和新型分离纯化方法(如凝胶色谱分离法和离子交换色谱分离法等),旨在为多酚氧化酶的研究和应用,特别是为抑制农产品酶促褐变提供参考。     

A comparative study of inactivation of peach polyphenol oxidase and carrot polyphenol oxidase induced by high‐pressure carbon dioxide

The inactivation of polyphenol oxidase (PPO) in peach juice and PPO in carrot juice was investigated by high‐pressure carbon dioxide (HPCD), and their inactivation kinetics was analysed and compared. The temperature was 35–55 °C, the pressure was 5–15 MPa under HPCD condition. Results showed that HPCD enhanced the inactivation effect of the temperature on the two PPOs. The inactivation kinetics of peach PPO was well fitted to a first‐order kinetic model, of carrot PPO to a fraction‐conversion model as a function of temperatures or pressures. Susceptibility of the rate constant k of peach PPO was not altered and of carrot PPO was lessened to the temperature, but the susceptibility of the rate constant k of peach PPO and carrot PPO to the pressure was not changed when the pressure was >8 or 12 MPa, indicating the presence of a threshold pressure.     

发酵剂用量对硬质蒙古干酪蛋白质、脂类水解和微观结构的影响

该文通过添加不同发酵剂用量制作了3种硬质蒙古干酪,并对硬质蒙古干酪的理化性质、蛋白质和脂肪水解程度、微观结构等进行了研究。结果表明,随着发酵剂用量增多,除D-乳酸含量外,其他理化指标均随发酵剂用量的增大而减小;粗蛋白质和粗脂肪的含量减少;pH 4.6-可溶性氮（SN）含量和12%三氯乙酸（TCA）-SN含量均增加,表明干酪中蛋白质和脂肪的水解程度增大;游离氨基酸和游离脂肪酸的含量增加,表明干酪的风味物质增多。随着发酵剂用量的增多,干酪酸化速度加快,质地相对松散。最终确定发酵剂使用量为1.0 g/L,在此条件下,脂肪和蛋白质的水解程度适中,游离氨基酸种类丰富,能够满足干酪风味化合物生成的需求。     

Properties of polyphenol oxidase in mango (Mangifera indica) kernel

Polyphenol oxidase (PPO) activity of filtered extract of ground mango kernel suspension (400 g litre⁻¹) was studied spectrophotometrically at 420 nm using catechol as substrate. The enzyme was most active at pH 6·0 and 25°C. Activity was reduced by 50% at pH values of 5·0 and 7·1, and also at temperatures of 14°C and 30°C. The calculated activation energy and the Michaelis constant (K_m) were 21·4 kcal mol⁻¹ °C⁻¹ and 24·6 mM , respectively. The V_max value was 2·14 units g⁻¹ mango kernel. The time to heat inactivate PPO decreased rapidly to < 10 min with increasing temperature of ⩾ 70°C at 50% activity. © 1998 SCI.     

Effect of CO2 addition to raw milk on proteolysis and lipolysis at 4 degrees C

Fresh raw milks, with low (3.1 x 10(4) cell/ml) and high (1.1 x 10(6) cells/ml) somatic cell count (SCC), were standardized to 3.25% fat, and from each a preserved (with 0.02% potassium dichromate) and an unpreserved portion were prepared. Subsamples of each portion were carbonated to contain 0 (control, pH 6.9) and 1500 (pH 6.2) ppm added CO2, and HCl acidified to pH 6.2 Milk pH was measured at 4 degrees C. For the preserved low- and high-SCC milks, two additional carbonation levels, 500 (pH 6.5) and 1000 (pH 6.3) ppm, were prepared. Milks were stored at 4 degrees C and analyzed on d 0, 7, 14, and 21 for microbial count, proteolysis, and lipolysis. The addition of 1500 ppm CO2, but not HCl, effectively delayed microbial growth at 4 degrees C. In general, in both the low- and high-SCC unpreserved milks, there was more proteolysis and lipolysis in control and HCl acidified milks than in milk with 1500 ppm added CO2. Higher levels of proteolysis and lipolysis in the unpreserved milks without added CO2 were related to higher bacteria counts in those milks. In preserved low- and high-SCC milks, microbial growth was inhibited, and proteolysis and lipolysis were caused by endogenous milk enzymes (e.g., plasmin and lipoprotein lipase). Compared with control, both milk with 1500 ppm added CO2 and milk with HCl acidification had less proteolysis. The effect of carbonation or acidification with HCl on proteolysis in preserved milks was more pronounced in the high SCC milk, probably due to its high endogenous protease activity. Plasmin is an alkaline protease and the reduction in milk pH by added CO2 or HCl explained the reduction in proteolysis. No effect of carbonation or acidification of milk on lipolysis was observed in the preserved low- and high-SCC milks. The CO2 addition to raw milk decreased proteolysis via at least two mechanisms: the reduction of microbial proteases due to a reduced microbial growth and the possible reduction of endogenous protease activity due to a lower milk pH. The effect of CO2 on lipolysis was mostly due to a reduced microbial growth. High-quality raw milk (i.e., low initial bacteria count and low SCC) with 1500 ppm added CO2 can be stored at 4 degrees C for 14 d with minimal proteolysis and lipolysis and with standard plate count < 3 x 10(5) cfu/ml.     

Effect of somatic cell count on proteolysis and lipolysis in pasteurized fluid milk during shelf-life storage

The general goal of this research was to provide fluid milk processors with data to enable them to estimate the economic benefits they might derive from longer fluid milk shelf-life or new marketing opportunities due to a reduction in raw milk SCC. The study objectives were: 1) to measure the time in days for pasteurized homogenized 2% milk to achieve a level of lipolysis and proteolysis caused by native milk enzymes present in milks of different somatic cell count (SCC) at 0.5 and 6 degrees C that would be sufficient to produce an off-flavor, 2) to determine whether milk fat content (i.e., 1, 2, and 3.25%) influences the level of proteolysis or lipolysis caused by native milk enzymes at 6 degrees C, and 3) to determine the time in days for milks containing 2% fat with different SCC to undergo sufficient lipolysis or proteolysis to produce an off-flavor due to the combination of the action of native milk enzymes and microbial growth at 0.5 and 6 degrees C. In experiment 1, pasteurized, homogenized milks, containing 2% fat were prepared from raw milk containing four different SCC levels from < 100,000 to > 1,000,000 cells/ml. Each of the four milks was stored at 0.5 and 6 degrees C for 61 d. In experiment 2, pasteurized, homogenized milks containing 1, 2, and 3.25% fat were prepared starting from two raw milks containing two different SCC levels, one < 100,000 and the other > 1,000,000 cells/ml. In experiment 3, pasteurized, homogenized 2% fat milks were prepared starting from raw milks containing two different SCC levels, one < 100,000 and the other > 1,000,000 cells/ml. For experiments 1 and 2, all milks were preserved with potassium dichromate to prevent microbial growth but to allow the activity of native milk proteases and lipases during storage. For experiment 3, one set of milk was preserved with potassium dichromate to prevent microbial growth but to allow the activity of native milk proteases and lipases, and a second set of milk was unpreserved during storage at 0.5 and 6 degrees C for 29 d. Based on previous work, an off-flavor due to proteolysis was detected by 50% of panelists when the decrease in casein as a percentage of true protein (CN/TP) was > 4.76%. Our data indicated (assuming 50% of consumers would detect an off-flavor when CN/TP decreases 5%) that pasteurized milk containing 2% fat would develop an off-flavor at a time long after 61 and at 54 d for the low SCC milk, and at about 54 and 19 d for the high SCC milk, at 0.5 and 6 degrees C, respectively. Previous research reported that 34% of panelists could detect an off-flavor in milk containing 2% fat due to lipolysis at a (free fatty acid) FFA concentration of 0.25 meq/kg of milk. Based on these results, it was estimated in the present study that 34% of panelists would detect an off-flavor in a 2% fat pasteurized milk with low SCC at a time long after 61 and just after 61 d at 0.5 and 6 degrees C, respectively, while for milk with high SCC, an off-flavor would be detected by 34% of panelists at slightly longer than 61 and 35 d at 0.5 and 6 degrees C, respectively. The combination of low SCC milk and low storage temperature when coupled with processing technology to produce very low initial bacteria count in fluid milk could produce fluid milk that will maintain flavor quality for more than 61 d of storage at temperatures < 6 degrees C.     

虾体多酚氧化酶特性及其抑制技术研究进展

多酚氧化酶是虾体中普遍存在的一种含铜金属蛋白,它是虾体发生黑变的主要原因,也是虾保鲜品质控制中亟待解决的技术关键。研究多酚氧化酶的酶学特性,抑制酶促褐变,对于提高虾的食用价值和商品价值至关重要。本文综合介绍了多酚氧化酶的虾体分布、活性特征、分离纯化、分析测定、黑变机理及其抑制方法。     

降解水解单宁的内孢霉多酚氧化酶的性能及动力学研究

通过对单宁降解能力较强的内孢霉降解性能的研究发现 :内胞霉不仅具有单宁酶活性 ,而且具有多酚氧化酶(PPO)活性。内孢霉PPO作用的最适温度和pH分别是 3 0℃和 5 .0 ,且在温度为 2 0～ 5 0℃以及 pH 4～ 6的范围内 ,表现出较高的稳定性 ;Mn2 、Mg2 对PPO的活性有明显的促进作用 ,Fe3 对PPO活性有明显的抑制作用 ,其余金属离子的影响较小。内胞霉PPO对间苯三酚不表现出活力 ,对邻苯二酚及邻苯三酚具有氧化作用 ,说明水解类单宁的降解不仅与单宁酶有关 ,还与多酚氧化酶有关     

不同体细胞数原料乳对UHT贮存期间蛋白和脂肪水解的影响

不同体细胞数(21.4×104mL-1,75.8×104mL-1,118.1×104mL-1和216.2×104mL-1)原料乳生产的4组UHT乳在37℃贮存84d,对其贮存期间的蛋白水解及脂肪水解进行研究。结果表明,4组UHT乳贮存期间的蛋白水解速率无显著性差异(P>0.05),原料乳体细胞数并未对蛋白水解造成影响;4组UHT乳贮存期间的脂肪水解速率具有显著性差异(P<0.005),原料乳体细胞数与脂肪水解速率间存在极明显的正相关(R=0.9886,P<0.05)。