鲜切荸荠/莲藕特异性变色机理及其控制研究
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摘要
鲜切果蔬(Fresh-cut fruits and vegetables)是指任何果蔬经鲜切加工后,虽然形状发生改变,但仍然保持新鲜状态的产品。变色是直接限制鲜切果蔬产品流通和货架期的重要因素之一。荸荠(Eleocharis tuberosa(Roxb.)Roem.et. Schult)和莲藕(Nelumbo nucifern guertn)适宜鲜切加工,但鲜切荸荠在贮藏中会发生黄化,鲜切莲藕片则表现为两个表面变色不同的现象。本研究以鲜切荸荠和莲藕为试材,深入研究了它们在贮藏过程中发生的特异性变色现象,探讨了变色机理,并在此基础上研究了鲜切莲藕变色的方向性调控。
本研究结果表明,鲜切荸荠表面黄化可能并非是酶促褐变的结果,因为除了贮藏初期,在鲜切荸荠贮藏过程中一直没有检测到常见的与褐变相关的酚类物质;两种酶促褐变的关键酶——多酚氧化酶(Polyphenol oxidase,PPO)和过氧化物酶(Peroxidase,POD)在整个贮藏过程中不仅活性很低,而且变化平缓;采用抗坏血酸处理并不能有效延缓鲜切荸荠黄化的发生;进一步研究发现鲜切荸荠表面黄化物质的甲醇提取液在200~400 nm范围内呈现三个明显的吸收峰,经化学鉴定推测是黄酮类物质或黄酮类和蒽醌类物质的混合物;此外,对鲜切荸荠的黄化表面组织进行培养分离得到五种微生物,通过形态学研究初步证实其中一种为地霉属(Geotrichum)真菌,其余四种分别属于盐水球菌属(Salinococcus)、链球菌属(Streptococcus)、黄杆菌属(Flavobacterium)和葡萄球菌属(Staphylococcus)细菌。其中地霉属真菌、链球菌属和葡萄球菌属细菌可导致鲜切荸荠严重黄化。
鲜切莲藕片变色具有明显的方向性,具体表现为朝向藕梢的一面较朝向藕头的一面先变色,且变色严重,这一现象是由莲藕组织内部因素决定的。深入研究鲜切莲藕片的两个不同方向表面相关的生理发现,在两个表面中不但与褐变密切相关的因素——PPO、POD、PAL酶活性及膜脂过氧化程度存在着明显的不同,而且水分含量也有显著差异。进一步研究结果表明,水分不均衡是导致鲜切莲藕变色方向性的关键。贮藏过程中,朝向藕头面组织中的水分含量一直明显高于朝向藕梢面的组织。而莲藕组织水分减少量与PPO、POD酶活性之间呈高度正相关。苯丙氨酸解氨酶(Phenylalanine ammonialyase,PAL)活性随着莲藕组织中水分的大量丧失,同样表现出上升趋势。水势和膜透性的差异可能是产生水分不平衡的原因所在。
通过硫酸铵分步盐析和DEAE-Spharose离子交换柱、Phenyl Sepharose 6 Fast Flow疏水柱层析,从鲜切莲藕组织中分离纯化到PPO,分子量约为65.9~66.1。该酶最适反应温度为20℃,在40℃下较稳定;最适pH值约为6.0,pH 5.6~ pH 6.5之间最稳定,超过pH 6.5,稳定性下降;抗坏血酸、异抗坏血酸、L-半胱氨酸和亚硫酸钠几乎都可以完全抑制莲藕组织中PPO的活性,而EDTA-Na、柠檬酸、氯化钠和氯化钙对PPO均只表现出轻微的抑制作用;FeSO4、FeCl3、和CuSO4对莲藕组织中的PPO则表现出一定的激活作用。
通过硫酸铵分步盐析、Sephadex G-75柱层析和DEAE-Sepharose离子交换柱,对鲜切莲藕组织中POD进行了部分分离纯化。该酶最适反应温度为30℃,热稳定性相对较差,随着温度的升高,POD活性随着时间的延长呈加速下降趋势;最适pH为6.0,在pH 4.4、pH 6.5和pH 9.0都比较稳定;抗坏血酸、L-半胱氨酸和亚硫酸钠对其有较强的抑制作用,Fe2+和Fe3+也表现出一定的抑制作用,并且Fe2+的抑制作用强于Fe3+,而CaCl2、NaCl和柠檬酸只表现出微弱的抑制作用; Cu2+对其表现出明显的激活作用。
抗坏血酸虽然可以延缓鲜切莲藕组织褐变,但并不能改变变色的方向性,而且会产生紫边等现象,0.4 %的抗坏血酸护色效果最好;壳聚糖处理不仅可以减缓鲜切莲藕组织褐变的发生,并较为有效地解决了鲜切莲藕变色的方向性问题。采用壳聚糖涂膜的鲜切莲藕组织两个不同方向表面水分差异明显减少,并与壳聚糖浓度呈反比,壳聚糖涂膜对PAL、PPO和POD酶活性产生不同程度的抑制作用,并与壳聚糖浓度呈正比。其中1.5 %浓度的壳聚糖处理对PPO酶活性产生了强烈的抑制作用,使其整个贮藏过程中一直维持在非常低的水平;壳聚糖对POD酶活性也表现出一定的抑制作用,但抑制程度较PPO低。壳聚糖涂膜处理还抑制了膜脂过氧化作用。但采用壳聚糖处理会导致鲜切莲藕组织膜透性增大,而且低浓度的壳聚糖会诱发鲜切莲藕组织腐烂的发生。
单独采用壳聚糖溶剂食用乙酸处理研究表明,其增加了组织膜透性,减小了鲜切莲藕两个不同方向表面水分的差异,从而消除了由其所引发的褐变相关酶活性的差异,当食用乙酸浓度达到1.8 %时,鲜切莲藕变色的方向性现象已基本消除。同时食用乙酸还明显降低了PAL酶活性,减少了酶促褐变底物酚类物质的生成;并对PPO酶也产生了强烈的抑制作用,对POD酶也表现出一定的抑制能力,阻止了膜脂的过氧化作用。因此食用乙酸在控制鲜切莲藕变色的方向性中起关键作用,但其浓度过高会导致组织色泽偏红,并且食用乙酸由于改变组织pH值可能是导致低浓度壳聚糖处理诱发腐烂的原因所在。
Fresh-cut fruits and vegetables are generally refered to the products that remain in a fresh state after they are processed, but the discoloration always takes place, being thus regarded as one of the important factors to the shelf life of fresh-cut fruits and vegetables. Both chufa (Eleocharis tuberosa(Roxb.)Roem.et. Schult) and lotus root (Nelumbo nucifern guertn) serve as appropriate fresh-cut materials; However, certain discolorations have been reported during processing and storing. Yellowing is often observed in fresh-cut chufa during storage, and for fresh-cut lotus root, variance in browning on the opposite sections has also frequently been noticed. The present study aims to investigate yellowing mechanism of fresh-cut chufa and browning orientation of fresh-cut lotus root during storage. Certain regulations for browning orientation of fresh-cut lotus root are also discussed.
It may not be some specific enzymatic activities that result in the yellowing of fresh-cut chufa; certain considerations are discussed. reasons as follows: (1) None of the phenols interrelated with browning are found in fresh-cut chufa during storage expect at the initial stage; (2) As the key enzymes for the enzymatic browning reaction, both PPO and POD remain very low in their activities and exhibit little change during storage; (3) The yellowing of fresh-cut chufa could not be prevented by employing ascorbic acid, which has been reported as most widely used for browning control.
Further researches have shown that there are three distinct absorption peaks between 200-400 nm in the methanol extract of the yellowed parts from the fresh-cut chufa. Exact chemical identification suggests that the yellowed matter may be flavone or mixture of flavone and anthraquinone. Meanwhile, five microbes of one fungus and four bacteria separated from the yellowed tissue of the fresh-cut chufa have been cultured. Morphologic identification shows that the only fungus falls into Geotrichum, while other four bacteria belong respectively to Salinococcus, Streptococcus, Flavobacterium and Staphylococcus. The microbes in Geotrichum, Streptococcus and Staphylococcus have been proved to play a leading role in causing fresh-cut chufa yellow seriously.
Many experiments have substantiated that fresh-cut lotus root represents an obvious browning orientation in that the end-ward sections became brown earlier and more seriously than the head-ward ones. This phenomenon is regarded as induced by the lotus root itself other than the external factors. Furthermore, distinct difference was observed in the water contents of the opposite sections as well as that found in other factors including PAL, PPO, POD activities and the level of membrane lipid peroxidation. It indicated that the water imbalance of the opposite sections of fresh-cut lotus root acted as a key factor in causing browning orientation. A high positive correlation existed between the water content and the activities of browning-related enzymes such as PPO and POD. The activities of these enzymes rose as the tissues lost moisture. As for PAL activity, the trend was similar. Accordingly, the water imbalance of the opposite sections of fresh-cut lotus root might result from the diverse water potential and membrane permeability.
Purified PPO was obtained from the fresh-cut lotus root by ammonium sulfate fractionation, DEAE-Sepharose and Phenyl Sepharose 6 Fast Flow columns chromatography. The enzyme was found to be homogenous by SDS-PAGE. The molecular weight of the fresh-cut lotus root PPO as estimated by SDS-PAGE ranged from 65.9 kD to 66.1 kD according to Sephadex G-100 gel fractionation. The purified enzyme had an optimal pH of 6.0 for activity. It remained rather stable when kept in pH 5.6~ pH 6.5 citrate/phosphate or phosphate buffer and at 4℃. Its optimum reaction temperature was 20℃and stable below 40℃. However, the activity decreased dramatically when temperature rose above 60℃. Ascorbic acid, Erythorbic acid, L-cysteine and sodium sulfite could almost completely inhibit PPO activity, while EDTA-Na, citrate acid, NaCl and CaCl2 showed slight inhibition. FeSO4, FeCl3 and CuSO4 could remarkably activate the fresh-cut lotus root PPO activity, especially at a high concentration.
Partly purified POD was obtained from the fresh-cut lotus root by ammonium sulfate fractionation, Sephadex G-75 and DEAE-Sepharose columns chromatography. The purified enzyme had an optimal pH of 6.0 for activity. It appeared very stable at pH 4.4, 6.5 and 9.0 respectively when kept at 4℃. The optimum reaction temperature was 30℃, and the thermal stability of it was low. The activity decreased rapidly with the rising temperature. Ascorbic acid, L-cysteine and sodium sulfite almost completely inhibited the enzyme activity, Fe2+ and Fe3+ also appeared some inhibition to POD, whereas citrate acid, NaCl and CaCl2 had only slight inhibition. CuSO4 had a remarkable activating effect on the fresh-cut lotus root POD activity.
Ascorbic acid could not be expected to reverse the browning orientation of the fresh-cut lotus root, although it was certainly effective in delaying the browning phenomenon. Even worse, the tissue showed purple stains on the epidermis after treated with ascorbic acid. It had been proved that 0.4 % AA treatment could most efficiently inhibit the browning process. However, chitosan coating treatment was shown to be effective not only to postpone the browning but also to regulate the orientation of browning with an optimal concentration of 1.5 %. It indicated that such a chitosan coating had meliorated the water imbalance of the opposite sections of the fresh-cut lotus root. Simultaneously, PAL, PPO and POD activities had been inhibited; thereinto PPO activity was nearly entirely inhibited by 1.5 % chitosan, whereas POD activity was merely partly inhibited. Negative correlations existed between the enzyme activity and the chitosan concentration. LOX activity also decreased, leading the decrease in the superoxide anion free radicles and MDA. However, the membrane permeability of the fresh-cut lotus root would increase and the tissue decay could be induced by chitosan coating treatment.
The reason why the membrane permeability of fresh-cut lotus root increased and the tissue decay was induced by chitosan coating treatment might rest with the action of edible acetic acid as the chitosan solution was composed of both edible acetic acid and chitosan. This edible acetic acid changed the pH value, which served as a key factor for microbe growth. In the meantime, the difference of water content between the opposite sections was reduced due to the increasing of membrane permeability, which was induced by the acetic acid. As a result, the diversity of the enzyme activity that induced water imbalance was also eliminated. The browning orientation was almost completely removed by 1.8 % acetic acid concentration. Similarly, PAL, PPO and POD activities were all inhibited but with diverse degrees. The membrane lipid peroxidation was also prevented. Consequently, the edible acetic acid in the chitosan coating was the real crux for regulating the browning orientation of the fresh-cut lotus root. However, acetic acid in high concentration can lead a redder chroma in tissue color, which deserves further attention.
本研究结果表明,鲜切荸荠表面黄化可能并非是酶促褐变的结果,因为除了贮藏初期,在鲜切荸荠贮藏过程中一直没有检测到常见的与褐变相关的酚类物质;两种酶促褐变的关键酶——多酚氧化酶(Polyphenol oxidase,PPO)和过氧化物酶(Peroxidase,POD)在整个贮藏过程中不仅活性很低,而且变化平缓;采用抗坏血酸处理并不能有效延缓鲜切荸荠黄化的发生;进一步研究发现鲜切荸荠表面黄化物质的甲醇提取液在200~400 nm范围内呈现三个明显的吸收峰,经化学鉴定推测是黄酮类物质或黄酮类和蒽醌类物质的混合物;此外,对鲜切荸荠的黄化表面组织进行培养分离得到五种微生物,通过形态学研究初步证实其中一种为地霉属(Geotrichum)真菌,其余四种分别属于盐水球菌属(Salinococcus)、链球菌属(Streptococcus)、黄杆菌属(Flavobacterium)和葡萄球菌属(Staphylococcus)细菌。其中地霉属真菌、链球菌属和葡萄球菌属细菌可导致鲜切荸荠严重黄化。
鲜切莲藕片变色具有明显的方向性,具体表现为朝向藕梢的一面较朝向藕头的一面先变色,且变色严重,这一现象是由莲藕组织内部因素决定的。深入研究鲜切莲藕片的两个不同方向表面相关的生理发现,在两个表面中不但与褐变密切相关的因素——PPO、POD、PAL酶活性及膜脂过氧化程度存在着明显的不同,而且水分含量也有显著差异。进一步研究结果表明,水分不均衡是导致鲜切莲藕变色方向性的关键。贮藏过程中,朝向藕头面组织中的水分含量一直明显高于朝向藕梢面的组织。而莲藕组织水分减少量与PPO、POD酶活性之间呈高度正相关。苯丙氨酸解氨酶(Phenylalanine ammonialyase,PAL)活性随着莲藕组织中水分的大量丧失,同样表现出上升趋势。水势和膜透性的差异可能是产生水分不平衡的原因所在。
通过硫酸铵分步盐析和DEAE-Spharose离子交换柱、Phenyl Sepharose 6 Fast Flow疏水柱层析,从鲜切莲藕组织中分离纯化到PPO,分子量约为65.9~66.1。该酶最适反应温度为20℃,在40℃下较稳定;最适pH值约为6.0,pH 5.6~ pH 6.5之间最稳定,超过pH 6.5,稳定性下降;抗坏血酸、异抗坏血酸、L-半胱氨酸和亚硫酸钠几乎都可以完全抑制莲藕组织中PPO的活性,而EDTA-Na、柠檬酸、氯化钠和氯化钙对PPO均只表现出轻微的抑制作用;FeSO4、FeCl3、和CuSO4对莲藕组织中的PPO则表现出一定的激活作用。
通过硫酸铵分步盐析、Sephadex G-75柱层析和DEAE-Sepharose离子交换柱,对鲜切莲藕组织中POD进行了部分分离纯化。该酶最适反应温度为30℃,热稳定性相对较差,随着温度的升高,POD活性随着时间的延长呈加速下降趋势;最适pH为6.0,在pH 4.4、pH 6.5和pH 9.0都比较稳定;抗坏血酸、L-半胱氨酸和亚硫酸钠对其有较强的抑制作用,Fe2+和Fe3+也表现出一定的抑制作用,并且Fe2+的抑制作用强于Fe3+,而CaCl2、NaCl和柠檬酸只表现出微弱的抑制作用; Cu2+对其表现出明显的激活作用。
抗坏血酸虽然可以延缓鲜切莲藕组织褐变,但并不能改变变色的方向性,而且会产生紫边等现象,0.4 %的抗坏血酸护色效果最好;壳聚糖处理不仅可以减缓鲜切莲藕组织褐变的发生,并较为有效地解决了鲜切莲藕变色的方向性问题。采用壳聚糖涂膜的鲜切莲藕组织两个不同方向表面水分差异明显减少,并与壳聚糖浓度呈反比,壳聚糖涂膜对PAL、PPO和POD酶活性产生不同程度的抑制作用,并与壳聚糖浓度呈正比。其中1.5 %浓度的壳聚糖处理对PPO酶活性产生了强烈的抑制作用,使其整个贮藏过程中一直维持在非常低的水平;壳聚糖对POD酶活性也表现出一定的抑制作用,但抑制程度较PPO低。壳聚糖涂膜处理还抑制了膜脂过氧化作用。但采用壳聚糖处理会导致鲜切莲藕组织膜透性增大,而且低浓度的壳聚糖会诱发鲜切莲藕组织腐烂的发生。
单独采用壳聚糖溶剂食用乙酸处理研究表明,其增加了组织膜透性,减小了鲜切莲藕两个不同方向表面水分的差异,从而消除了由其所引发的褐变相关酶活性的差异,当食用乙酸浓度达到1.8 %时,鲜切莲藕变色的方向性现象已基本消除。同时食用乙酸还明显降低了PAL酶活性,减少了酶促褐变底物酚类物质的生成;并对PPO酶也产生了强烈的抑制作用,对POD酶也表现出一定的抑制能力,阻止了膜脂的过氧化作用。因此食用乙酸在控制鲜切莲藕变色的方向性中起关键作用,但其浓度过高会导致组织色泽偏红,并且食用乙酸由于改变组织pH值可能是导致低浓度壳聚糖处理诱发腐烂的原因所在。
Fresh-cut fruits and vegetables are generally refered to the products that remain in a fresh state after they are processed, but the discoloration always takes place, being thus regarded as one of the important factors to the shelf life of fresh-cut fruits and vegetables. Both chufa (Eleocharis tuberosa(Roxb.)Roem.et. Schult) and lotus root (Nelumbo nucifern guertn) serve as appropriate fresh-cut materials; However, certain discolorations have been reported during processing and storing. Yellowing is often observed in fresh-cut chufa during storage, and for fresh-cut lotus root, variance in browning on the opposite sections has also frequently been noticed. The present study aims to investigate yellowing mechanism of fresh-cut chufa and browning orientation of fresh-cut lotus root during storage. Certain regulations for browning orientation of fresh-cut lotus root are also discussed.
It may not be some specific enzymatic activities that result in the yellowing of fresh-cut chufa; certain considerations are discussed. reasons as follows: (1) None of the phenols interrelated with browning are found in fresh-cut chufa during storage expect at the initial stage; (2) As the key enzymes for the enzymatic browning reaction, both PPO and POD remain very low in their activities and exhibit little change during storage; (3) The yellowing of fresh-cut chufa could not be prevented by employing ascorbic acid, which has been reported as most widely used for browning control.
Further researches have shown that there are three distinct absorption peaks between 200-400 nm in the methanol extract of the yellowed parts from the fresh-cut chufa. Exact chemical identification suggests that the yellowed matter may be flavone or mixture of flavone and anthraquinone. Meanwhile, five microbes of one fungus and four bacteria separated from the yellowed tissue of the fresh-cut chufa have been cultured. Morphologic identification shows that the only fungus falls into Geotrichum, while other four bacteria belong respectively to Salinococcus, Streptococcus, Flavobacterium and Staphylococcus. The microbes in Geotrichum, Streptococcus and Staphylococcus have been proved to play a leading role in causing fresh-cut chufa yellow seriously.
Many experiments have substantiated that fresh-cut lotus root represents an obvious browning orientation in that the end-ward sections became brown earlier and more seriously than the head-ward ones. This phenomenon is regarded as induced by the lotus root itself other than the external factors. Furthermore, distinct difference was observed in the water contents of the opposite sections as well as that found in other factors including PAL, PPO, POD activities and the level of membrane lipid peroxidation. It indicated that the water imbalance of the opposite sections of fresh-cut lotus root acted as a key factor in causing browning orientation. A high positive correlation existed between the water content and the activities of browning-related enzymes such as PPO and POD. The activities of these enzymes rose as the tissues lost moisture. As for PAL activity, the trend was similar. Accordingly, the water imbalance of the opposite sections of fresh-cut lotus root might result from the diverse water potential and membrane permeability.
Purified PPO was obtained from the fresh-cut lotus root by ammonium sulfate fractionation, DEAE-Sepharose and Phenyl Sepharose 6 Fast Flow columns chromatography. The enzyme was found to be homogenous by SDS-PAGE. The molecular weight of the fresh-cut lotus root PPO as estimated by SDS-PAGE ranged from 65.9 kD to 66.1 kD according to Sephadex G-100 gel fractionation. The purified enzyme had an optimal pH of 6.0 for activity. It remained rather stable when kept in pH 5.6~ pH 6.5 citrate/phosphate or phosphate buffer and at 4℃. Its optimum reaction temperature was 20℃and stable below 40℃. However, the activity decreased dramatically when temperature rose above 60℃. Ascorbic acid, Erythorbic acid, L-cysteine and sodium sulfite could almost completely inhibit PPO activity, while EDTA-Na, citrate acid, NaCl and CaCl2 showed slight inhibition. FeSO4, FeCl3 and CuSO4 could remarkably activate the fresh-cut lotus root PPO activity, especially at a high concentration.
Partly purified POD was obtained from the fresh-cut lotus root by ammonium sulfate fractionation, Sephadex G-75 and DEAE-Sepharose columns chromatography. The purified enzyme had an optimal pH of 6.0 for activity. It appeared very stable at pH 4.4, 6.5 and 9.0 respectively when kept at 4℃. The optimum reaction temperature was 30℃, and the thermal stability of it was low. The activity decreased rapidly with the rising temperature. Ascorbic acid, L-cysteine and sodium sulfite almost completely inhibited the enzyme activity, Fe2+ and Fe3+ also appeared some inhibition to POD, whereas citrate acid, NaCl and CaCl2 had only slight inhibition. CuSO4 had a remarkable activating effect on the fresh-cut lotus root POD activity.
Ascorbic acid could not be expected to reverse the browning orientation of the fresh-cut lotus root, although it was certainly effective in delaying the browning phenomenon. Even worse, the tissue showed purple stains on the epidermis after treated with ascorbic acid. It had been proved that 0.4 % AA treatment could most efficiently inhibit the browning process. However, chitosan coating treatment was shown to be effective not only to postpone the browning but also to regulate the orientation of browning with an optimal concentration of 1.5 %. It indicated that such a chitosan coating had meliorated the water imbalance of the opposite sections of the fresh-cut lotus root. Simultaneously, PAL, PPO and POD activities had been inhibited; thereinto PPO activity was nearly entirely inhibited by 1.5 % chitosan, whereas POD activity was merely partly inhibited. Negative correlations existed between the enzyme activity and the chitosan concentration. LOX activity also decreased, leading the decrease in the superoxide anion free radicles and MDA. However, the membrane permeability of the fresh-cut lotus root would increase and the tissue decay could be induced by chitosan coating treatment.
The reason why the membrane permeability of fresh-cut lotus root increased and the tissue decay was induced by chitosan coating treatment might rest with the action of edible acetic acid as the chitosan solution was composed of both edible acetic acid and chitosan. This edible acetic acid changed the pH value, which served as a key factor for microbe growth. In the meantime, the difference of water content between the opposite sections was reduced due to the increasing of membrane permeability, which was induced by the acetic acid. As a result, the diversity of the enzyme activity that induced water imbalance was also eliminated. The browning orientation was almost completely removed by 1.8 % acetic acid concentration. Similarly, PAL, PPO and POD activities were all inhibited but with diverse degrees. The membrane lipid peroxidation was also prevented. Consequently, the edible acetic acid in the chitosan coating was the real crux for regulating the browning orientation of the fresh-cut lotus root. However, acetic acid in high concentration can lead a redder chroma in tissue color, which deserves further attention.
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