摘要
本文主要研究了以菠萝废弃物为原料,采用硫酸铵盐析法结合超滤浓缩法的技术从菠萝皮中提取菠萝蛋白酶。
首先,在硫酸铵盐析分离提取菠萝皮浸提液中菠萝蛋白酶的过程中,实验研究结果表明,硫酸铵液体加入法要优于固体加入法；随着菠萝皮浸提液的pH值的增大,菠萝蛋白酶的酶活回收率先增大后减小,pH值为7时,得到的菠萝蛋白酶酶活回收率最大,为83.6%；随着硫酸铵浓度的增大,菠萝蛋白酶的酶活回收率先增大后减小,当硫酸铵的饱和度达到40%的时侯,菠萝蛋白酶的酶活回收率最大,为83.6%；在选定的实验温度范围内,随着温度的升高,菠萝蛋白酶的酶活回收率先增大后减小,20℃时菠萝蛋白酶的酶活回收率最大,为86.9%；随着盐析时间的增加,菠萝蛋白酶的酶活回收率先增大后减小,当盐析时间为60min时菠萝蛋白酶的酶活回收率最大,为89.2%。
其次,在超滤法浓缩提取菠萝皮浸提液中的菠萝蛋白酶的过程中,实验研究结果表明,选择截留分子量为20KD的超滤膜对菠萝皮浸提液进行超滤,收集其截留液,酶活截留率可达94%,或者选择截留分子量为100KD的超滤膜,酶活截留率为10.3%,可收集透过液；随着离心分离转速的增加,菠萝蛋白酶的酶活回收率逐渐增大,当离心分离转速当达到8000rpm转速时,菠萝蛋白酶的酶活截留率不再发生变化,为89.3%；使用20KD的超滤膜时,随着操作压力的增大,超滤膜的渗透通量先增大后减小,当操作压力为0.35MPa时,渗透通量达到最大值为5.57L·m-2·h-1；使用100KD的超滤膜时,随着操作压力的增大,超滤膜的渗透通量先增大后减小,当操作压力为0.25MPa左右时,渗透通量达到最大值为7.69L·m-2·h-1；无论是未经过处理,还是经过处理的超滤膜,随着使用次数的增加,其本身的渗透通量都逐渐减小,同时,经超滤菠萝皮浸提液得到的蛋白酶活截留率也逐渐减小,但是,经过纯水低压湍流清洗和酸碱溶液浸泡处理过的超滤膜,其渗透通量和得到的菠萝蛋白酶活截留率都明显大于未经处理过的超滤膜。
第三,在盐析-超滤菠萝皮浸提液的过程中,实验研究结果表明,L-半胱氨酸、苯甲酸钠、EDTA和维生素C等对菠萝蛋白酶活性的影响,它们都能提高菠萝蛋白酶的活性,其中, L-半胱氨酸对菠萝蛋白酶活性提高最大其浓度达到0.6mg/mL时,酶活回收率最大,为112.5%；EDTA和苯甲的浓度达到0.5mg/mL时,菠萝蛋白酶的酶活回收率就趋于稳定状态；维生素C虽然对菠萝蛋白酶酶活的提高不是很大,但从经济角度来讲,维生素C较其他三种便宜得多；将盐析得到的湿菠萝蛋白酶沉淀溶解后,先选用100KD的超滤膜在0.25MPa的压力下进行超滤,收集透过液,然后再选用20KD的超滤膜在0.35MPa的压力下进行超滤浓缩,收集截留液,进行冷冻干燥得到精酶,其酶活力为3944.0U·g-1,总酶活回收率为88.6%,总提取率为0.228%。
This paper mainly studied the technique of bromelain extracting from pineapple waste by ammonium sulfate precipitation combined with ultrafiltration enrichment.
First, the study showed that ammonium sulfate liquid addition was superior to solid in the process of bromelain extracting from pineapple peel extraction solution by ammonium sulfate precipitation; activity recovery of bromelain climbed up first and then declined with the pH value increased, and it was maximum for83.6%when the pH of the pineapple peel extraction solution was7; the activity recovery of bromelain increased first and then decreased with the ammonium sulfate concentration increased, and it was maximum for83.6%when the saturation of the ammonium sulfate was40%; in the range of experiment temperature, the activity recovery of bromelain increased first and then decreased with the temperature rose, and it was maximum for86.9%when the temperature of the pineapple peel extraction solution was20℃; the activity recovery of bromelain lifted first and then descended with precipitation time prolonged, and it was maximum for89.2%when the time of precipitation was60min.
Second, the paper studied concentrating bromelain by ultrafiltration method. The result indicated that activity interception rate of bromelain was94%when using20KD ultrafiltration membrane, the ultrafiltration retentate should be collected, the activity interception rate was10.3%when using100ultrafiltration membrane, so that the ultrafiltration permeate should be colle the activity recovery rate of bromelain was gradually increased with centri speed increased, and it was89.3%kept the stable when the speed was up to8000rpm; To the20KD membrane, permeation flux was increased gradually and then decreased with operating pressure increased, and it reached5.57L·m-2-h-1when operating pressure was0.35MPa; To the100KD membrane, permeation flux was increased gradually and then decreased with operating pressure increased, and it was7.69L·m-2·h-1when operating pressure was0.25MPa; the ultrafiltration membrane's permeation flux was gradually decreased with the using time increase whether it was processed or not, meanwhile, the activity interception rate of bromelain was gradually decreased; the permeation flux and the activity interception rate of bromelain were recovered to some degree when the ultrafiltration membrane was washed by pure water in low-pressure turbulence and then soaked in acid-base solution.
Third, the study indicated that addition of L-cysteine, sodium benzoate, EDTA or vitamin C could improve the activity recovery of bromelain in the extraction process by coupling ammonium sulfate precipitation with ultrafiltration method. Among the additives above, L-cysteine had the greatest impact, the activity recovery of bromelain was maximum for112.5%when the concentration of L-cysteine was0.6mg/mL; the activity of bromelain was stabilized when the concentration of EDTA and sodium benzoate were0.5mg/mL; although vitamin C addition resulted in a low effect on bromelain activity recovery, considering it was much cheaper than the others, it should be used in bromelain extraction; the bromelain activity was3944.0U·g-1with88.6%activity recovery in the ultrafiltration operation as:dissolving the bromelain precipitation, ultrafiltrating and collecting the permeate by using100KD ultrafiltration membrane under0.25MPa, ultrafiltrating and collecting the ultrafiltration retentate by using20KD ultrafiltration membrane under0.35MPa; the collection was freeze-dried; finally, the total extraction rate of bromelain was0.228%.
引文
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