4-HR与抗坏血酸对中华管鞭虾多酚氧化酶的抑制动力学模拟分析

研究无硫复合抑制剂主要作用成分4-己基间苯二酚（4-hexylresorcinol,4-HR）、抗坏血酸对中华管鞭虾多酚氧化酶（polyphenoloxidase,PPO）活性的抑制作用,并从抑制剂化学结构及动力学模型出发分析推断其抑制机理。结果表明,4-HR、抗坏血酸均能抑制PPO活力,且对酶促反应有延滞作用,两者半抑制浓度分别为0.072、2.02?mmol/L,对中华管鞭虾PPO的抑制作用都表现为竞争性抑制;化学动力学模拟分析显示4-HR、抗坏血酸均可与PPO双铜活性中心结合,其中4-HR可生成一种高稳定的多酚氧化酶络合物,持续有效地消耗PPO。4-HR、抗坏血酸均能有效抑制中华管鞭虾PPO酶促反应,其中4-HR抑制效果更显著。本实验为无硫复合抑制剂在海水虾捕捞与加工过程中的应用推广提供理论依据。     

鲜切牛蒡丝护色和制备工艺优化

通过对牛蒡中多酚氧化酶活性的分析,确定抑制酶活性的条件,研究鲜切牛蒡丝的最佳制备工艺。结果表明,牛蒡中多酚氧化酶的适宜温度30～40℃、适宜pH6.5～7.5、较有效的抑制剂是VC。通过正交试验确定鲜切牛蒡丝的最佳制备工艺条件为将牛蒡切片浸泡在0.5% VC、1.0% 柠檬酸和0.5% CaCl2 组成的混合护色液中30min,在80℃的混合护色液中热烫90s,冷却后切丝,于( 4 ± 1 ) ℃条件下冷藏14d 不变色,且无任何不良风味。     

Inhibition of browning on the surface of peach slices by short-term exposure to nitric oxide and ascorbic acid

The effect of 0.2% ascorbic acid (AA), 5 μM nitric oxide (NO), and the simultaneous use of 0.2% AA and 5 μM NO solutions on inhibiting surface browning of fresh-cut peach slices stored at 10 °C and RH 95% was investigated. The browning index, relative leakage rate, microstructure, total phenol content, and activity of the phenol metabolism-associated enzymes phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) were evaluated. The results indicate that treatment with 0.2% AA, 5 μM NO and simultaneous use of 0.2% AA and 5 μM NO resulted in higher total phenol content, inhibition of PPO and POD activity, reduced membrane permeability and protection of cell microstructure to maintain compartmentation between enzymes and their substrates. In addition, NO increased PAL activity. The causes of inhibition in the browning of peach slices by NO are discussed.     

茶叶多酚氧化酶三相分离纯化及酶学性质研究

为研究一种快速高效的茶叶多酚氧化酶(polyphenol oxidase,PPO)制备方法,本试验采用三相分离法(three phase partitioning,TPP)获取茶叶PPO,并研究其酶学特性.结果表明,通过两次三相分离纯化后可获得酶比活力210.46 U/mg、纯化15.76倍、回收率8.04％的茶叶PP...     

Inhibition of Mushroom Polyphenoloxidase by Ascorbic Acid Derivatives

Ascorbic acid (AA), dehydroascorbic acid (dehydroAA), isoascorbic acid (isoAA), ascorbic acid-2-phosphate (AA-2- PO₄), and ascorbic acid-2-sulfate (AA-2-SO₄) were tested as inhibitors of mushroom polyphenoloxidase (PPO). Kinetic analysis indicated that AA and isoAA were more effective than dehydroAA. The half times (t_1/2) that decreased 50% of PPO activity for AA, isoAA and dehydroAA were 2.5, 3.1, and 1.9 hr, respectively, and the concentrations that inhibited half of PPO activity were as follows: ascorbic acid, 0.04 mM, isoAA, 0.25 mM; and dehydroAA, 7.5 mM. Electron spin resonance studies demonstrated that the Cu²⁺ of PPO was reduced to Cu⁺ by AA. AA-2-PO₄ and AA-2-SO₄ were not inhibitors for PPO. However, the digestion of AA-2-PO₄ with acid phosphatase yielded AA to inhibit PPO activity. AA-2-SO₄ was found to be a poor substrate for sulfatase.     

POLYPHENOLOXIDASE ACTIVITY OF MINIMALLY PROCESSED 'JONAGORED' APPLES (MALUS DOMESTICA)

The influence of three chemical dips using ascorbic acid (AA), citric acid (CA) and calcium chloride (CC) on the polyphenoloxidase (PPO) activity and on the total phenolic content of minimally processed (MP) apple (Malus domestica, cv. Jonagored) during cold storage was evaluated and a potential relationship with enzymatic browning was investigated. An ascorbic acid dip (42.6 mM) of 5 min duration was the most efficient chemical treatment in reducing the PPO activity of apple cubes. A 92% inhibition was achieved after 7 days of storage at 4C. All treatments were advantageous in comparison to the control in reducing color changes. Color changes, determined by absorbance at 420 nm (soluble pigments) and lightness (L) (insoluble pigments) of apple cubes treated with ascorbic acid were correlated with total phenolic content. No correlation was observed between PPO activity and tristimulus color parameters, browning index or total phenolic content of AA‐treated apple cubes.     

The Effect of Antibrowning Agents on Inhibition of Potato Browning,Volatile Organic Compound Profile,and Microbial Inhibition

Abstract: Burbank and Norkotah potato slices were dipped into 3% sodium acid sulfate (SAS), citric acid (CA), sodium erythorbate (SE), malic acid (MA), sodium acid pyrophosphate (SAPP), or a combination of SAS‐CA‐SE. Browning by polyphenol oxidase (PPO) obtained from potato extract with 0.04 to 0.016 g/mL of antibrowning solutions at pH 2.0 to 6.9 were measured by UV‐Vis spectroscopy. The color of slices dipped in antibrowning solutions at pHs 2 to 7 and stored at 4 °C for 15 d was measured every 5 d by colorimeter. Headspace analysis of volatiles in raw and cooked potato samples was performed by selected ion flow tube mass spectrometer (SIFT‐MS) and soft independent modelling by class analogy (SIMCA) analysis of the calculated odor activity values (OAV) determined interclass distances. Microbial growth was measured at 15 d. At unadjusted pHs (1.1 to 7.1), the PPO browning of the control and samples with SAPP was not significantly different, SAS, CA, and MA produced some inhibition and SE and SAS‐CA‐SE prevented browning. At pH 5 to 7, only SE and SAS‐CA‐SE were effective browning inhibitors. Based on the color of potato slices, SE was the most effective at pH 2 to 7, but SAS was most effective at unadjusted pH. Cooking increased volatile levels in the treated potatoes and decreased differences between volatile profiles. Differences between cooked samples may not be noticeable by the consumer because volatiles with high discriminating powers have low OAVs. SAS, CA, and SAS‐CA‐SE treatments inhibited microbial growth but SAPP, control, and SE did not, most likely due to pH. Practical Application: Antibrowning agents inhibit polyphenol oxidase, increasing shelf life and consumer acceptability of processed raw potato products by preserving the color. Their effectiveness was shown to be mainly due to a pH effect, except SE, which was not pH dependent. MA, CA, and SAS‐CA‐SE are better acidulants for inhibition of color change as well as growth of spoilage bacteria, yeast, and mold than SAPP, the industry standard.     

Effect of Green Tea Extract in Combination with Ascorbic Acid on the Retardation of Melanosis and Quality Changes of Pacific White Shrimp During Iced Storage

Melanosis and quality changes of Pacific white shrimp (Litopenaeus vannamei) treated with 0.1% green tea extract (GTE) in combination with ascorbic acid (AA) at different levels (0%, 0.005%, and 0.01%) were monitored during iced storage of 12?days. Based on in vitro study, 0.1% GTE inhibited polyphenoloxidase (PPO) from cephalothorax of Pacific white shrimp by 60.2%. Nevertheless, 0.1% GTE in combination with 0.01% AA exhibited the greater PPO inhibitory activity (93.0%) (P?<?0.05). When shrimp treated with 0.1% GTE in combination with AA (0.005 or 0.01%; GTE + AA), the increase in psychrophilic bacteria and spoilage microorganisms including H₂S- producing bacteria and enterobacteriaceae were retarded to a higher extent, in comparison with the control and those treated with 1.25% sodium metabisulfite (SMS; P?<?0.05). The coincidental lowered rates of increase in pH, total volatile base content and thiobarbituric acid reactive substances were obtained in the shrimp treated with GTE + AA (P?<?0.05). Additionally, shrimp treated with GTE + AA had the lower melanosis score but higher score for color, odor, taste, flavor, and overall likeness, compared with the control and those treated with SMS (P?<?0.05). Generally, AA at levels of 0.005% and 0.01% showed a similar synergist effect with GTE on both melanosis inhibition as well as retardation of quality loss of shrimp.     

绿原酸复配水杨酸对采后芦笋冷藏保鲜的影响

以新鲜绿芦笋为试材,研究0.3 mmol/L绿原酸(CA)和0.3 mmol/L绿原酸复配0.1 mmol/L水杨酸(CA+SA)处理,以腐烂率、呼吸强度、叶绿素、纤维素、V_C、总酚、超氧阴离子(O₂^-·)、多酚氧化酶(PPO)、过氧化物酶(POD)、过氧化氢酶(CAT)和总超氧化物歧化酶(T-SOD)活性为指标进行测定,探究其对冷藏(4±2℃)保鲜效果的影响。结果表明:冷藏中CA和CA+SA处理能显著降低芦笋的呼吸峰(P<0.05),比对照(CK)分别降低了7.21%和6.22%,减缓叶绿素和V_C的降解速度,提升总酚、CAT、POD和T-SOD酶活性,抑制腐烂率、超氧阴离子(O₂^-·)和PPO酶活性,使过氧化氢酶(CAT)活性高峰提前4 d,CA+SA处理效果优于CA处理。CA+SA处理显著抑制了贮藏8 d后芦笋纤维素的增加(P<0.05),其CAT酶活性峰值是CK的1.92倍,而CA处理对芦笋纤维素和CAT峰值没有影响。绿原酸复配水杨酸能较...     

八角莲多酚氧化酶的酶学性质

以邻苯二酚为底物,研究八角莲多酚氧化酶（polyphenol oxidase,PPO）粗提液的酶学特性。结果表明,底物邻苯二酚最适浓度为1.0 mol/L,八角莲PPO的最适pH值为7.0,最适温度为30 ℃,90 ℃高温处理10 min,八角莲PPO酶活力仅剩11.92%。PPO催化的酶促褐变反应符合米氏动力学方程,相应动力学参数Km和vmax分别为0.230 7 mol/L和769.23 U/min。抗坏血酸、甘氨酸、乙二胺四乙酸、柠檬酸对酶活性具有抑制作用,十二烷基硫酸钠表现出激活作用,亚硫酸氢钠对酶活性的抑制作用强于硫代硫酸钠,Al3+和Cu2+对酶活性具有一定的激活作用,Ca2+对酶活性有一定的抑制作用,Mg2+、Fe2+和Fe3+对酶活性影响不显著。     

Effect of pre-soaking treatments on the nutritional profile and browning index of sweet potato and yam flours

The use of sweet potato and yams for product development is hindered by the discoloration from enzymatic and non-enzymatic browning. The effect of pre-soaking treatments on the nutritional changes and browning index (BI) of sweet potato and yam (greater yam and white yam) flours was investigated. Ascorbic acid (AsA), citric acid (CA), acetic acid (AA) and sodium metabisulfite (SMS) were used for soaking at three concentrations (0.25%, 0.50% and 1.00%) and two durations (1 h and 2 h). AsA and SMS removed more starch during soaking of sweet potato, while CA and AA removed more starch from greater yam and CA removed more starch from white yam. Highest removal of reducing sugars was observed in sweet potato in AA, while CA removed maximum reducing sugars from both the yams. Phenol and total free amino acid (TFA) levels were more in SMS treatment than the control sweet potato flour, and least in AA (2 h). Whilst AsA raised the phenols and TFA in greater yam, it removed phenols to the maximum in white yam. Maximum BI was in AsA treatment for the three crops, and least for flour from CA (0.25%) treated yams and AA (1.00%) treated sweet potato.Industrial RelevanceBrowning resulting from enzymatic and non-enzymatic reactions is a major drawback in the processing of sweet potato and yams for product development. The study showed that this could be successfully tackled through soaking the slices in low cost chemicals like acetic acid or citric acid and sodium metabisulfite at low concentrations. The treated slices yielded flour with very low browning indices compared to the respective native flours, which could enhance the potential of such flours in the development of food products.     

Principal component and hierarchical cluster analysis to select hurdle technologies for minimal processed radishes

The application of principal component and hierarchical cluster analysis as a mathematical tool to select hurdle technologies (physical and chemical) to inhibit polyphenol oxidase (PPO) activity and color stability of minimally sliced radishes was presented. Results of PPO activity and color were reported initially (day zero) and after 4 d of refrigerated storage. Based on the mentioned tools and from the thirteen mono-hurdles technologies that were initially selected, eight hurdles were chosen as the best and combined in pairs. From those hurdles, the following double-hurdles were selected as those capable of retaining the product color: Ascorbic Acid (AA) + Acetic Acid (ACA-05), AA + NaCl-1, ACA-05+NaCl-1, AA + Heat Treatment (TT-501), ACA-05 + Ultrasound (US), Citric Acid (CA) + AA, CA + ACA-05, NaCl-1+US. When comparing the best mono-hurdles (AA, TT-501, NaCl-1) to the best double hurdles, the following hurdles: AA, TT-501, CA + ACA-05, NaCl-1+US, CA + AA and NaCl-1 were chosen as those capable of maintaining the typical color of radish slices with acceptable sensory parameters and enzyme inhibition after 4 d of storage.     

双酶水解提取牛蒡菊糖的研究

采用双酶水解法提取牛蒡菊糖。首先从8 种酶中选取3 种对牛蒡菊糖提取率最高的酶,分别为木瓜蛋白酶、植物蛋白酶和酸性蛋白酶,对牛蒡菊糖的提取率分别为8.83%、8.67% 和8.21%。然后对每一种酶采用单因素试验方法研究pH 值、固液比、加酶量、温度以及时间对牛蒡菊糖提取率的影响;再通过3 种酶之间的相互组合试验,选出一组最佳组合为：木瓜蛋白酶+ 植物蛋白酶,其提取率为11.43%。最后采用单因素和正交试验设计方法,研究加酶量、固液比、温度、时间以及pH 值对牛蒡菊糖提取率的影响,得到双酶水解提取牛蒡菊糖的最佳条件组合为：在木瓜蛋白酶加酶量10%、温度50℃、pH7、时间4h、固液比1:15(m/V)的条件下进行酶解,4h 后加入植物蛋白酶,加酶量20%、温度45℃、pH8,时间4h、固液比1:15(m/V),提取液经乙醇沉淀、真空浓缩,得到粗菊糖,菊糖提取率为13.41%,产品中菊糖含量为67.86%,蛋白质含量为1.32%。     

紫外线照射与柠檬酸处理对鲜切苹果的保鲜作用

目的研究柠檬酸、紫外线照射、柠檬酸和紫外线照射联合处理对鲜切苹果生理生化的影响。方法以富士苹果为实验材料,分成4组,分别是0.5%柠檬酸(CA)溶液中浸泡5 min、紫外照射(UV)5 min/面、0.5%柠檬酸溶液中浸泡5 min+紫外照射(CA+UV)5 min/面、不处理的对照组(KB)。分别于0、1、3、5、7、10、13、15 d测定鲜切苹果的色差、多酚氧化酶(PPO)、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)活性。结果富士苹果经过切分后,褐变严重。PPO、POD活性升高,PAL活性变化曲折,总体呈上升趋势。柠檬酸、紫外、柠檬酸和紫外联合处理的3三种处理方式都可以抑制鲜切苹果的褐变,降低PPO、POD、PAL的活性。在4种处理方法比较中,柠檬酸和紫外联合处理的效果比较好。结论柠檬酸和紫外联合处理是鲜切苹果的理想的保鲜方法。     

Purification of Polyphenoloxidase from the Purple‐fleshed Potato (Solanum tuberosum Jasim) and its Secondary Structure

ABSTRACT: Polyphenoloxidase (PPO) was purified from purple‐fleshed potatoes (Solanum tuberosum Jasim) using membrane concentration, ammonium sulfate fractionation, Resource Q ion exchange chromatography, and Sephacryl S‐200 HR gel permeation chromatography. PPO was purified 78‐fold from a crude extract. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results showed that the purified enzyme has a major subunit molecular weight of 40 kDa. To elucidate the secondary structure of the purified PPO, circular dichroism (CD) was performed. The CD spectrum of the purified enzyme showed that PPO contains 35% α‐helix, 30% β‐turn, and 35% random coil structure.     

超声-咖啡酸联合处理对海鲈鱼冷藏期间品质变化的影响

为研究超声-咖啡酸联合处理对冷藏海鲈鱼品质变化影响,将新鲜鲈鱼片随机分为5 组,分别为20 kHz、600 W超声处理10 min（US组）,2.0 g/L咖啡酸浸渍10 min（CA组）,超声联合咖啡酸处理10 min（20 kHz、600 W超声处理5 min后,咖啡酸浸渍5 min）（US＋CA组）,无菌水（空白,CK组）与体积分数1%乙酸（对照,AA组）分别浸渍10 min。将5 组样品处理后沥干,装于聚乙烯保鲜袋中,置于4 ℃冷藏。每2 d测定样品的微生物指标（菌落总数）、理化指标（pH值、总挥发性盐基氮（total volatile base nitrogen,TVB-N）含量、硫代巴比妥酸（thiobarbituric acid,TBA）值、质构特性、持水力）等,结合内源荧光强度与感官评价,综合评价超声-咖啡酸联合处理对冷藏鲈鱼的保鲜效果。结果表明：US与CA处理能明显抑制样品贮藏期间菌落总数的增长,在第10天,CA组与US组样品的菌落总数比CK组分别低18.49%与15.53%,其pH值与TVB-N含量上升速度明显缓于CK组样品。贮藏第10天时,CK组样品的TVB-N含量已超过腐败限值,达（33.88±0.56）mg/100 g,而此时US＋CA组样品的TVB-N含量为（16.71±0.41）mg/100 g,明显低于CK组。US处理使鱼肉的保水性明显改善,CA处理对抑制鱼肉的脂肪氧化有较好效果,其还能使样品的持水力得到较高提升;其中以US＋CA联合处理组样品总体品质最佳。与CK组相比,US＋CA处理对鱼肉品质保持效果较好,联合处理可使海鲈鱼的冷藏货架期至少延长4 d。     

Apple polyphenoloxidase inactivation during heating in the presence of ascorbic acid and chlorogenic acid

It was recently reported that during osmotic dehydration of ascorbic acid (AA)-treated apple cubes, losses in AA and phenolics could partly arise from enzymatic oxidation, provided polyphenoloxidase (PPO) was still active under the processing conditions. To determine the impact of dehydration temperatures on PPO action, as well as chemical and enzymatic oxidation reactions, apple PPO inactivation alone or with AA (1 mM) and/or chlorogenic acid (CG, 3 mM), as well as AA and CG levels evolution, during heating of the model solutions at 45 and 60 °C were investigated. At pH 3.8, PPO was still functional, keeping 61 and 4% residual activity after 2 h of heating at 45 and 60 °C, respectively. The combined treatment of heating and AA was more effective in reducing PPO activity, while incubation at 60 °C with AA and CG minimised the losses of PPO activity. CG remained stable during heating, even in the presence of AA which, in turn, was more affected by heating. Thus, during heating, provided PPO remained active with enough available O₂ in the model systems, CG oxidation and coupled oxidoreduction with AA could readily develop.     

Dual effect of β-cyclodextrin (β-CD) on the inhibition of apple polyphenol oxidase by 4-hexylresorcinol (HR) and methyl jasmonate (MJ)

Inhibition of red delicious apple polyphenol oxidase (PPO) by three non-related antibrowning agents has been studied at different substrate concentrations. The main apple polyphenol, cholorogenic acid (CA), has been used as a substrate. The three agents (β-cyclodextrin [β-CD], 4-hexylresorcinol [HR] and methyl jasmonate [MJ]) showed an inhibitory effect at every substrate concentration tested. From the inhibition constant (K_i) values, it was concluded that the inhibitors’ strength decreased in the order HR > β-CD > MJ. A combination of β-CD-HR had a synergic effect, which was not observed for the combination of β-CD-MJ. A competitive-type inhibition was obtained for HR with a K_i = 0.26 mM. β-CD and MJ behaved as mixed-type inhibitors, although more than one inhibitory mechanism is discussed for both agents.     

栓皮栎橡子果仁多酚抗氧化与抑菌活性研究

采用溶剂萃取秦巴山区栓皮栎橡子果仁,大孔树脂分离纯化、紫外光谱分析、红外光谱分析、高效液相色谱(High performance liquid chromatography,HPLC)分析栓皮栎橡子果仁多酚的成分组成,DPPH自由基清除率、ABTS+自由基清除率等、抑菌圈直径研究了其抗氧化活性及抑菌特性。结果表明:栓皮栎橡子果仁粗多酚得率为20.5%,其多酚含量为345 mg/g没食子酸当量,经D4020大孔树脂纯化后多酚得率为34%,多酚含量提高至702 mg/g没食子酸当量;紫外光谱分析、红外光谱分析表明其结构中均具有酚羟基、羰基和芳烃C=C骨架等结构;HPLC分析纯化的橡子果仁多酚至少含有28种物质,其中包括咖啡酸、原儿茶酸、鞣花酸、阿魏酸、槲皮素和山奈酚6种多酚物质,含量分别为24.9、14.6、3.2、2.2、0.5、0.2 mg/g;橡子果仁纯多酚及对照V_C清除DPPH自由基的IC₅₀值分别为2.50、3.20 μg/mL,清除ABTS+自由基IC₅₀值分别为28.67、28.82 μg/mL,ABTS+自由基清除能力与V_C相当,对Fe3+还原能力与V_C差异较小,但对亚铁离子螯合能力远低于EDTA-2Na,抗脂质过氧化能力与V_C相当,说明栓皮栎橡子仁多酚具有较强抗氧化能力;栓皮栎橡子仁粗多酚、纯多酚均对阪崎杆菌、沙门氏菌、金黄色葡萄球菌及大肠杆菌具有较强抑制作用,对李斯特菌的抑制作用较弱。研究结果表明栓皮栎橡子果仁多酚含量丰富,其多酚具有较强抗氧化性和广谱抑菌活性,是开发保健品和天然抗菌剂的良好材料。     

Partial purification and characterisation of banana [Musa (AAA Group) 'Gros Michel'] polyphenol oxidase

Polyphenol oxidase (PPO) from pulp of banana [Musa (AAA Group) 'Gros Michel'] was extracted and precipitated with 80% saturated ammonium sulphate followed by conventional column chromatography on Sephacryl S-200 HR and fast protein liquid chromatography on Mono Q column. The lyophilised PPO obtained from Sephacryl S-200 HR column was used for characterisation and inhibition studies. The partially purified PPO obtained from the Mono Q column exhibited at least three isoenzymes. The banana PPO had optimum pH for activity at 7 and it was stable around the same pH. Only 48% of initial enzyme activity was lost after heating at 70 °C for 30 min. The enzyme was completely inhibited by 2 m m sodium metabisulphite, 2 m m l -cysteine, 4 m m ascorbic acid, and 100 m m 4-hexylresorcinol. The K _m and V _max of banana PPO for dopamine were 2.08 m m and 0.124 m m  min⁻¹ respectively.