双孢蘑菇采后褐变的相关生理生化变化及其保鲜技术研究
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摘要
双孢蘑菇由于味道鲜美、营养丰富,深受人们喜爱。但是采后的双孢蘑菇水分含量高,组织非常细嫩,菌盖表面没有明显的保护结构,常温下贮藏1-2天后,菌盖及菌褶就会出现褐变、破膜、开伞、萎缩等现象,失去商品价值,尤其是褐变较其他食用菌更加严重。为了探求双孢蘑菇的褐变机理,延缓其采后褐变的发生,本文通过测定双孢蘑菇子实体采后部分可能促发酶促褐变或非酶促褐变的生理生化变化,推测分析其与褐变的相关性,进一步通过HPLC分析比较双孢蘑菇不同部位褐变前后的多酚提取物种类,推断其发生褐变反应的主要褐变底物,并研究其褐变相关酶POD的性质,为揭示双孢蘑菇褐变机理提供了一些理论依据。最终选择使用稳定态二氧化氯处理和N-取代羧甲基壳聚糖涂膜处理两种方法保鲜双孢蘑菇,分别评价其控制褐变的有效性及对双孢蘑菇采后贮藏品质的影响,试图为解决双孢蘑菇采后褐变问题,延长其货架期提供理论依据和实践指导。主要的研究结论如下：
1.与酶促褐变有关的主要生化指标——酚类底物、PPO(多酚氧化酶)、POD(过氧化物酶)酶活性变化与褐变密切相关,PPO和POD共同作用影响褐变；与非酶促褐变有关的主要生化指标——还原糖和Vc含量的动态变化情况也与褐变的发生存在一定的关系,其中Vc含量与色度L值变化显著相关,说明双孢蘑菇中可能有非酶促褐变存在；与褐变有关的主要生理指标——细胞膜透性与色度L值呈显著正相关,可能在一定程度上促进了褐变的发生。
2.双孢蘑菇发生褐变反应的主要褐变底物可能是愈创木酚和L-酪氨酸,且外源愈创木酚酶促褐变产物的最大吸收峰与双孢蘑菇自然褐变产物最大吸收峰接近。
3.双孢蘑菇POD的最适pH为5.5,最适温度为40℃,在此条件下,测得POD的Km=0.091mol/L, Vmax=454.55mol.L-1.min-1,拟合直线为y=0.0002x+0.0022,相关系数R2=0.9829,表明双孢蘑菇POD对愈创木酚有很好的亲和力。
4.稳定态二氧化氯处理保鲜双孢蘑菇的最佳使用浓度为150mg／L,最佳处理时间为10min。稳定态二氧化氯处理保鲜双孢蘑菇能够在贮藏期间保持蘑菇的白度,降低呼吸强度,抑制PPO活性,推迟POD活性高峰的出现,降低可溶性蛋白含量的损失,低温下保持SOD(超氧化物歧化酶)活性在较高水平,但对抑制开伞、萎焉作用不大。处理后在7℃～15℃的室温条件下,能够获得5天左右的货架期,若结合低温贮藏能够获得9天左右的货架期。
5.N-取代羧甲基壳聚糖涂膜处理双孢蘑菇能够有效降低细胞膜透性的变化幅度,降低细胞膜脂过氧化程度,减少水分损耗,延缓失重、失鲜现象的出现。涂膜处理能够在一定程度上延长了双孢蘑菇的贮藏保鲜期,常温17℃贮藏空白组在4天左右已几乎丧失食用价值,而涂膜组可将保鲜期延长2-3天,如果能结合低温4℃冷藏可继续将保鲜期延长5-6天,达到10-11天的货架期。
Consumption of Agaricus bisporus has been increasing substantially due to their delicacy, flavor and nutritional value. Agaricus bisporus have a short shelf life of 1-2 days during the ambient storage because that they are rich in wate and have no cuticle to protect them from physical or microbial attack or water loss. Especially, browning is a most important role in the decrease in appearance quality. However, there are so many researches on the browning of vegetable and fruit, based on the browning information is lack for the mushroom, Agaricus bisporus is selected as experiment material in this study to investigate the changes of physiological and biochemical characteristics associated with enzymatic browning and non-enzymatic browning during storage; and analyse some polyphenolic compounds before or after browning of Agaricus bisporus by HPLC technology to infer the main browning substrates; and study some properties of peroxidase of Agaricus bisporus to offer some theoretical bases for the browning mechanism; and study the effects of chlorine dioxide (ClO2) treatment or N,O-carboxymethyl chitosan coating on the storage quality of Agaricus bisporus. The conclusions of this thesis as follows:
1. The major biochemical indices associated with enzymatic browning of phenol content, PPO, POD activity were all related to the change of L-value. In the course of enzumatic browning, the key enzyme was PPO and POD.The major biochemical indices associated with non-enzymatic browning of reduced sugar and Vc content were also related to the change of L-value, and Vc content was significantly related. The major physiological indices associated with enzymatic browning of membrane permeability was significantly related to the change of L-value, it might promote the browning.
2. The main browning substrates of Agaricus bisporus might be guaiacol and L-tyrosine, and the maximum absorption peak of the enzymatic browning products of exogenous guaiaco was similar with natural browning.
3 The most suitable reaction pH of POD from Agaricus bisporus was 5.5, and the suitable temperature was 40℃.Under these conditions, Km=0.091mol/L Vmax=454.55mol.L-1.min-1 and the fitting straight line was y=0.0002x+0.0022, R2=0.9829.Therefore, POD from Agaricus bisporus had a high affinity with guaiaco.
4. The most suitable concentration of ClO2 solution is 150mg/L with the most suitable time is 10min. Agaricus bisporus were treated with CIO2 solution could maintain the whiteness, reduce browning degree, reduce the respiration rate and PPO activity, delay the activity peak of POD, reduce the loss degree of soluble protein and keep SOD activity at a high level at refrigerated storage.But it had little effect on cap opening and wilting. The one was treated at ambient(7℃-15℃) storage, had a shelf life about 5 days. The one was treated at refrigerated storage could maintain a good whiteness in first 5 days, and have a shelf life about 9 days.
5. Chemical modified chitosan coating was seen to delay browning changes and dehydration, decrease PPO activities and reduce membrane lipid peroxidation, especially during ambient storage, with extending the shelf life to 5-6 days. Incorporation of low temperature storage (refrigerated storage), the shelf life could reach 10 days.
1.与酶促褐变有关的主要生化指标——酚类底物、PPO(多酚氧化酶)、POD(过氧化物酶)酶活性变化与褐变密切相关,PPO和POD共同作用影响褐变；与非酶促褐变有关的主要生化指标——还原糖和Vc含量的动态变化情况也与褐变的发生存在一定的关系,其中Vc含量与色度L值变化显著相关,说明双孢蘑菇中可能有非酶促褐变存在；与褐变有关的主要生理指标——细胞膜透性与色度L值呈显著正相关,可能在一定程度上促进了褐变的发生。
2.双孢蘑菇发生褐变反应的主要褐变底物可能是愈创木酚和L-酪氨酸,且外源愈创木酚酶促褐变产物的最大吸收峰与双孢蘑菇自然褐变产物最大吸收峰接近。
3.双孢蘑菇POD的最适pH为5.5,最适温度为40℃,在此条件下,测得POD的Km=0.091mol/L, Vmax=454.55mol.L-1.min-1,拟合直线为y=0.0002x+0.0022,相关系数R2=0.9829,表明双孢蘑菇POD对愈创木酚有很好的亲和力。
4.稳定态二氧化氯处理保鲜双孢蘑菇的最佳使用浓度为150mg／L,最佳处理时间为10min。稳定态二氧化氯处理保鲜双孢蘑菇能够在贮藏期间保持蘑菇的白度,降低呼吸强度,抑制PPO活性,推迟POD活性高峰的出现,降低可溶性蛋白含量的损失,低温下保持SOD(超氧化物歧化酶)活性在较高水平,但对抑制开伞、萎焉作用不大。处理后在7℃～15℃的室温条件下,能够获得5天左右的货架期,若结合低温贮藏能够获得9天左右的货架期。
5.N-取代羧甲基壳聚糖涂膜处理双孢蘑菇能够有效降低细胞膜透性的变化幅度,降低细胞膜脂过氧化程度,减少水分损耗,延缓失重、失鲜现象的出现。涂膜处理能够在一定程度上延长了双孢蘑菇的贮藏保鲜期,常温17℃贮藏空白组在4天左右已几乎丧失食用价值,而涂膜组可将保鲜期延长2-3天,如果能结合低温4℃冷藏可继续将保鲜期延长5-6天,达到10-11天的货架期。
Consumption of Agaricus bisporus has been increasing substantially due to their delicacy, flavor and nutritional value. Agaricus bisporus have a short shelf life of 1-2 days during the ambient storage because that they are rich in wate and have no cuticle to protect them from physical or microbial attack or water loss. Especially, browning is a most important role in the decrease in appearance quality. However, there are so many researches on the browning of vegetable and fruit, based on the browning information is lack for the mushroom, Agaricus bisporus is selected as experiment material in this study to investigate the changes of physiological and biochemical characteristics associated with enzymatic browning and non-enzymatic browning during storage; and analyse some polyphenolic compounds before or after browning of Agaricus bisporus by HPLC technology to infer the main browning substrates; and study some properties of peroxidase of Agaricus bisporus to offer some theoretical bases for the browning mechanism; and study the effects of chlorine dioxide (ClO2) treatment or N,O-carboxymethyl chitosan coating on the storage quality of Agaricus bisporus. The conclusions of this thesis as follows:
1. The major biochemical indices associated with enzymatic browning of phenol content, PPO, POD activity were all related to the change of L-value. In the course of enzumatic browning, the key enzyme was PPO and POD.The major biochemical indices associated with non-enzymatic browning of reduced sugar and Vc content were also related to the change of L-value, and Vc content was significantly related. The major physiological indices associated with enzymatic browning of membrane permeability was significantly related to the change of L-value, it might promote the browning.
2. The main browning substrates of Agaricus bisporus might be guaiacol and L-tyrosine, and the maximum absorption peak of the enzymatic browning products of exogenous guaiaco was similar with natural browning.
3 The most suitable reaction pH of POD from Agaricus bisporus was 5.5, and the suitable temperature was 40℃.Under these conditions, Km=0.091mol/L Vmax=454.55mol.L-1.min-1 and the fitting straight line was y=0.0002x+0.0022, R2=0.9829.Therefore, POD from Agaricus bisporus had a high affinity with guaiaco.
4. The most suitable concentration of ClO2 solution is 150mg/L with the most suitable time is 10min. Agaricus bisporus were treated with CIO2 solution could maintain the whiteness, reduce browning degree, reduce the respiration rate and PPO activity, delay the activity peak of POD, reduce the loss degree of soluble protein and keep SOD activity at a high level at refrigerated storage.But it had little effect on cap opening and wilting. The one was treated at ambient(7℃-15℃) storage, had a shelf life about 5 days. The one was treated at refrigerated storage could maintain a good whiteness in first 5 days, and have a shelf life about 9 days.
5. Chemical modified chitosan coating was seen to delay browning changes and dehydration, decrease PPO activities and reduce membrane lipid peroxidation, especially during ambient storage, with extending the shelf life to 5-6 days. Incorporation of low temperature storage (refrigerated storage), the shelf life could reach 10 days.
引文
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