超高压处理对猕猴桃果汁杀菌钝酶效果和品质的影响
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摘要
选用热敏性果汁——猕猴桃果汁为超高压处理对象,以温度、压力和时间为超高压处理参数,对猕猴桃果汁的超高压处理杀菌钝酶效果和品质变化进行研究。
以菌落总数、霉菌酵母菌为指标,研究微生物在超高压处理后的变化规律,发现菌落数随着压力的升高呈明显下降趋势,30℃、400 MPa压力下猕猴桃果汁即可达到商业无菌(GB19297-2003),说明超高压对细菌的致死效果显著。当协同处理温度为50℃时,经处理的猕猴桃果汁样品在4℃贮藏30 d后,仍然满足商业无菌要求。
设计正交实验优化猕猴桃中过氧化物酶的提取工艺条件,最终提取工艺条件为缓冲液pH值7.5、缓冲液浓度0.05 mol/L、NaCl浓度1.0 mol/L、PVPP添加量2%。
研究不同温度不同保压时间下压力对猕猴桃中过氧化物酶活力影响趋势,发现压力对过氧化物酶活力有显著影响,较明显的失活过程主要发生在400 MPa以上的较高压力处理阶段。钝化酶的效果随温度升高而明显增加。随着时间延长,酶活的降低就越明显。但在15 min以后,下降趋势渐缓。部分条件超高压处理后出现过氧化物酶活力反弹现象。真实的食品体系中复杂的食品成分对于过氧化物酶的酶活具有一定的保护作用。
采用响应面分析和支持向量回归分析方法建立了猕猴桃过氧化物酶的超高压钝化动力学模型。经由响应面回归分析,采用Box-Behnken设计方法,对压力、温度和时间三个因素分别以X1、X2、X3表示,并以+1、0、-1分别代表自变量的高、中、低水平,得到相对残余酶活At/A0(Y)标准回归方程为:Y=0.90-0.15A-0.17B-0.056C-0.086A2 -0.075B2-0.023C2-0.022AB-0.025AC-0.027BC。采用支持向量回归分析方法,使用matlab 7.0软件,以压力、时间、温度作X轴归一化处理,以相对残留酶活为Y轴,得到X-Y关系图。对响应面法和支持向量回归分析方法进行比较,发现,由于两种方法拟合极限状态方程都存在偏差,故两种方法不可能得到精确解。但是结果误差较小,并且响应面法的计算效率较高。但支持向量回归预测值更符合实际测试值,且其泛化适用能力要远比响应面法好。在对超高压处理钝化过氧化物酶进行预测时,可以将两者结合进行分析。
猕猴桃过氧化物酶的最适pH范围在6.0-8.5之间,这一范围之外,在偏酸和偏碱的两侧均迅速下降。可能是由于猕猴桃中存在多种同工酶,且各个同工酶的最适pH值不同,从而产生了较宽的最适pH范围。Native-PAGE电泳发现未处理过氧化物酶样品中存在两条同工酶酶带,在较低压力处理后活性上升,并且出现新的同工酶;在较高压力处理后,新酶带消失,活性有所抑制。由SDS-PAGE电泳分析可知,不同压力大小处理后的过氧化物酶的SDS-PAGE电泳谱图没有发生显著的变化,说明超高压对酶蛋白分子的亚基不能产生明显的影响。采用DEAE Sepharose Fast Flow离子交换色谱分离,得到酸性过氧化物酶(记为组分1)和碱性过氧化物酶(记为组分2)。并进一步通过Superdex 75凝胶过滤色谱分离,得到过氧化物酶同工酶POD I和POD II(纯度分别为99.93%和93.37%)。过氧化物酶同工酶I和II的最适pH值分别为6.0和7.5。超高压处理后酶活力随压力变化曲线也不相同,说明两者的高压稳定性不同,从而为两者结合表现出的过氧化物酶活力在高压下的不规律变化提供了新的依据。并且,CD谱也显示了其二级结构构象单元含量在压力处理后发生了不同的变化。
超高压处理能够较好的保存果汁原有的感官性质,能够保持猕猴桃果汁中的天然营养成分(还原糖、Vc、氨基酸等)。温度和压力协同处理后,果汁的褐变减轻。且随着贮藏时间的延长,各样品色泽ΔE值之间的差距逐渐缩小。鲜榨猕猴桃果汁的主要挥发性风味物质分别是2-己烯醛(69.35%)和己醛(11.23%),鲜榨猕猴桃果汁的挥发性组分中最主要的是醛类化合物(85.139%)。超高压处理后,猕猴桃果汁中醛类化合物含量有所提高,说明高压中温协同处理对猕猴桃风味有加强作用。
高压和热协同处理不仅改变了果汁中粒子的形态,同时影响了网状结构的形成,使得大分子之间、颗粒之间、大分子和颗粒之间的相互作用发生了变化。猕猴桃果汁体系符合Herschel-Bulkley方程,表现出非牛顿流体特性。未经超高压处理的猕猴桃果汁,属于假塑性流体。在经超高压处理后,逐渐显示出触变性,在高于400 MPa压力处理后产生了触变环。动态流变特性研究表明,压力处理对于猕猴桃果汁体系有明显的增加黏性并降低弹性的作用。同时,处理温度也会影响高压处理对猕猴桃果汁流变特性作用的效果。猕猴桃果汁中的大分子在经过压力温度协同处理后,同时存在聚合和解聚两种现象。升高温度可以加速以上两个过程。
The effects of high pressure and heat treatments on sterilization, enzyme inactivation and quality of kiwifruit juice were investigated. Pressure levels ranging from 200 to 600 MPa and temperatures varying from 10 to 50℃were applied for up to 30 min.
Results show that total number of bacteria declined with pressure and temperature increased and arrived commercial asepsis under 400 MPa at 30℃. After 30-day storage period at 4℃, kiwifruit juice was still safe while 50℃was used as treated temperature combined high pressure treatment.
Results revealed that, at each temperature, an increase in pressure level results in a decrease of enzyme activity. Pressures higher than 400 MPa could be combined with mild heat (≤50℃) to accelerate enzyme inactivation. Prolongation of the exposure time had no great effect after the first 15 min. Regarding temperature, results showed that POD activity decreased when the temperature increased.
Compared with control, POD activity at 10℃showed a slight increase (P>0.05) under treatment at 200 MPa from 10 to 20 min, whereas at 400 and 600 MPa, the activity decreased with regard to that observed at 200 MPa. POD activity at 30℃was higher after treatment at 200 MPa for 15 min and the activity decreased following longer exposure time intervals. The slope of POD in kiwifruit juice at 30℃was slightly decreased compared with that in a model system. Real food systems show a protective effect of food ingredients at pressures applied for POD inactivation. Effect of three main factors (pressure, temperature and time) in high pressure treatment was investigated, and all experiments were designed with RSM and SVM.
Polyacrylamide gel electrophoresis (Native PAGE) and activity detection were carried out on the partially purified enzyme before and after treatment. At the beginning, two staining bands were observed on the untreated enzyme. This suggested that there may be two isoenzymes. After 15 min of treatment at 200 MPa at 30℃, a new band was observed. All bonds disappeared as pressure increased at 600 MPa. Purified POD isoenyme I and II changed differently after high pressure. From this observation, there may be several isoenzymes that have different resistance to high pressures.
There were no significant difference on pH, Brix and conductivity after treatment, while little difference on content of Vc, amino acid and color. And the effect of pressure is less than temperature. The main aromatic compounds of kiwifruit juice was 2-Hexenal(69.35) and Hexanal(11.23%). And the main component was aldehyde compounds(85.139%). The high pressure treatment enhanced the flavors of kiwifruit juice since the aldehyde contents increased after treatment.
The effect of high pressure treatment on rheological properties of kiwifruit juice was investigated. The results show that kiwifruit juice samples behaved like pesudoplastic non-Newtonian fluids and were coincident with Herschel-Bulkley model under all conditions. Kiwifruit juice displayed thixotropy when pressure up to 400 MPa. The value of G″increased while G′decreased after high pressure treatment, which indicated the enhancement of viscosity and reduce of flexibility. And the difference affected the rheological properties of kiwifruit juice after high pressure treatment. The particle size distribution of kiwifruit juice was changed after high pressure treatment.
以菌落总数、霉菌酵母菌为指标,研究微生物在超高压处理后的变化规律,发现菌落数随着压力的升高呈明显下降趋势,30℃、400 MPa压力下猕猴桃果汁即可达到商业无菌(GB19297-2003),说明超高压对细菌的致死效果显著。当协同处理温度为50℃时,经处理的猕猴桃果汁样品在4℃贮藏30 d后,仍然满足商业无菌要求。
设计正交实验优化猕猴桃中过氧化物酶的提取工艺条件,最终提取工艺条件为缓冲液pH值7.5、缓冲液浓度0.05 mol/L、NaCl浓度1.0 mol/L、PVPP添加量2%。
研究不同温度不同保压时间下压力对猕猴桃中过氧化物酶活力影响趋势,发现压力对过氧化物酶活力有显著影响,较明显的失活过程主要发生在400 MPa以上的较高压力处理阶段。钝化酶的效果随温度升高而明显增加。随着时间延长,酶活的降低就越明显。但在15 min以后,下降趋势渐缓。部分条件超高压处理后出现过氧化物酶活力反弹现象。真实的食品体系中复杂的食品成分对于过氧化物酶的酶活具有一定的保护作用。
采用响应面分析和支持向量回归分析方法建立了猕猴桃过氧化物酶的超高压钝化动力学模型。经由响应面回归分析,采用Box-Behnken设计方法,对压力、温度和时间三个因素分别以X1、X2、X3表示,并以+1、0、-1分别代表自变量的高、中、低水平,得到相对残余酶活At/A0(Y)标准回归方程为:Y=0.90-0.15A-0.17B-0.056C-0.086A2 -0.075B2-0.023C2-0.022AB-0.025AC-0.027BC。采用支持向量回归分析方法,使用matlab 7.0软件,以压力、时间、温度作X轴归一化处理,以相对残留酶活为Y轴,得到X-Y关系图。对响应面法和支持向量回归分析方法进行比较,发现,由于两种方法拟合极限状态方程都存在偏差,故两种方法不可能得到精确解。但是结果误差较小,并且响应面法的计算效率较高。但支持向量回归预测值更符合实际测试值,且其泛化适用能力要远比响应面法好。在对超高压处理钝化过氧化物酶进行预测时,可以将两者结合进行分析。
猕猴桃过氧化物酶的最适pH范围在6.0-8.5之间,这一范围之外,在偏酸和偏碱的两侧均迅速下降。可能是由于猕猴桃中存在多种同工酶,且各个同工酶的最适pH值不同,从而产生了较宽的最适pH范围。Native-PAGE电泳发现未处理过氧化物酶样品中存在两条同工酶酶带,在较低压力处理后活性上升,并且出现新的同工酶;在较高压力处理后,新酶带消失,活性有所抑制。由SDS-PAGE电泳分析可知,不同压力大小处理后的过氧化物酶的SDS-PAGE电泳谱图没有发生显著的变化,说明超高压对酶蛋白分子的亚基不能产生明显的影响。采用DEAE Sepharose Fast Flow离子交换色谱分离,得到酸性过氧化物酶(记为组分1)和碱性过氧化物酶(记为组分2)。并进一步通过Superdex 75凝胶过滤色谱分离,得到过氧化物酶同工酶POD I和POD II(纯度分别为99.93%和93.37%)。过氧化物酶同工酶I和II的最适pH值分别为6.0和7.5。超高压处理后酶活力随压力变化曲线也不相同,说明两者的高压稳定性不同,从而为两者结合表现出的过氧化物酶活力在高压下的不规律变化提供了新的依据。并且,CD谱也显示了其二级结构构象单元含量在压力处理后发生了不同的变化。
超高压处理能够较好的保存果汁原有的感官性质,能够保持猕猴桃果汁中的天然营养成分(还原糖、Vc、氨基酸等)。温度和压力协同处理后,果汁的褐变减轻。且随着贮藏时间的延长,各样品色泽ΔE值之间的差距逐渐缩小。鲜榨猕猴桃果汁的主要挥发性风味物质分别是2-己烯醛(69.35%)和己醛(11.23%),鲜榨猕猴桃果汁的挥发性组分中最主要的是醛类化合物(85.139%)。超高压处理后,猕猴桃果汁中醛类化合物含量有所提高,说明高压中温协同处理对猕猴桃风味有加强作用。
高压和热协同处理不仅改变了果汁中粒子的形态,同时影响了网状结构的形成,使得大分子之间、颗粒之间、大分子和颗粒之间的相互作用发生了变化。猕猴桃果汁体系符合Herschel-Bulkley方程,表现出非牛顿流体特性。未经超高压处理的猕猴桃果汁,属于假塑性流体。在经超高压处理后,逐渐显示出触变性,在高于400 MPa压力处理后产生了触变环。动态流变特性研究表明,压力处理对于猕猴桃果汁体系有明显的增加黏性并降低弹性的作用。同时,处理温度也会影响高压处理对猕猴桃果汁流变特性作用的效果。猕猴桃果汁中的大分子在经过压力温度协同处理后,同时存在聚合和解聚两种现象。升高温度可以加速以上两个过程。
The effects of high pressure and heat treatments on sterilization, enzyme inactivation and quality of kiwifruit juice were investigated. Pressure levels ranging from 200 to 600 MPa and temperatures varying from 10 to 50℃were applied for up to 30 min.
Results show that total number of bacteria declined with pressure and temperature increased and arrived commercial asepsis under 400 MPa at 30℃. After 30-day storage period at 4℃, kiwifruit juice was still safe while 50℃was used as treated temperature combined high pressure treatment.
Results revealed that, at each temperature, an increase in pressure level results in a decrease of enzyme activity. Pressures higher than 400 MPa could be combined with mild heat (≤50℃) to accelerate enzyme inactivation. Prolongation of the exposure time had no great effect after the first 15 min. Regarding temperature, results showed that POD activity decreased when the temperature increased.
Compared with control, POD activity at 10℃showed a slight increase (P>0.05) under treatment at 200 MPa from 10 to 20 min, whereas at 400 and 600 MPa, the activity decreased with regard to that observed at 200 MPa. POD activity at 30℃was higher after treatment at 200 MPa for 15 min and the activity decreased following longer exposure time intervals. The slope of POD in kiwifruit juice at 30℃was slightly decreased compared with that in a model system. Real food systems show a protective effect of food ingredients at pressures applied for POD inactivation. Effect of three main factors (pressure, temperature and time) in high pressure treatment was investigated, and all experiments were designed with RSM and SVM.
Polyacrylamide gel electrophoresis (Native PAGE) and activity detection were carried out on the partially purified enzyme before and after treatment. At the beginning, two staining bands were observed on the untreated enzyme. This suggested that there may be two isoenzymes. After 15 min of treatment at 200 MPa at 30℃, a new band was observed. All bonds disappeared as pressure increased at 600 MPa. Purified POD isoenyme I and II changed differently after high pressure. From this observation, there may be several isoenzymes that have different resistance to high pressures.
There were no significant difference on pH, Brix and conductivity after treatment, while little difference on content of Vc, amino acid and color. And the effect of pressure is less than temperature. The main aromatic compounds of kiwifruit juice was 2-Hexenal(69.35) and Hexanal(11.23%). And the main component was aldehyde compounds(85.139%). The high pressure treatment enhanced the flavors of kiwifruit juice since the aldehyde contents increased after treatment.
The effect of high pressure treatment on rheological properties of kiwifruit juice was investigated. The results show that kiwifruit juice samples behaved like pesudoplastic non-Newtonian fluids and were coincident with Herschel-Bulkley model under all conditions. Kiwifruit juice displayed thixotropy when pressure up to 400 MPa. The value of G″increased while G′decreased after high pressure treatment, which indicated the enhancement of viscosity and reduce of flexibility. And the difference affected the rheological properties of kiwifruit juice after high pressure treatment. The particle size distribution of kiwifruit juice was changed after high pressure treatment.
引文
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