耐热植酸酶产生菌株的筛选及其酶应用特性的研究
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摘要
植酸(phytic acid,myo-inositol—hexakisphosphate)是谷物和豆类作物籽实中磷的重要贮藏形式。由于植酸磷不能被单胃动物利用,因此饲料中大部分的植酸磷被动物排出体外,造成严重的环境污染。此外,植酸作为一种抗营养因子在单胃动物消化道中能与多种金属离子如钙、锌、铜、铁等及蛋白质鳌合,形成不溶复合物,降低单胃动物对这些营养元素的吸收利用率。植酸酶(phytase)是催化植酸及植酸盐水解成肌醇和磷酸(或磷酸盐)的一类酶的总称,可通过催化、水解反应将磷盐从植酸中释放出来,提高磷利用率、解除植酸抗营养作用及减少磷污染。
目前,热稳定性是在植酸酶的推广应用中较为突出的问题。饲料生产中的制粒过程需要经过高温加热,一般在73℃～95℃,植酸酶在这样的温度下很容易失活,通常活性降为原来的30%,甚至更低。所以,进一步筛选耐热性更好的植酸酶以及探索提高植酸酶的热稳定性的方法仍是研究的重点。
本研究从自然筛选着手,以开发耐热植酸酶为目标,进行了以下几方面的研究:
(1)耐热植酸酶产生菌的分离筛选。
(2)耐热植酸酶产生菌的初步鉴定。
(3)耐热植酸酶液体发酵条件的优化。
(4)耐热植酸酶粗酶酶学性质的研究
(5)耐热植酸酶在小麦粉及全麦面包中的应用。
本论文主要研究结果和结论如下:
(1)菌株的筛选
本研究从云南腾冲热泉水样中,初步筛选得菌株TB-1,pH5.5、60℃条件下测定植酸酶酶活,结果表明菌株TB-1所产酶表现出耐热性,酶活达0.10U/ml。
(2)菌株的鉴定
菌株TB-1在LB琼脂平板上菌落为黄白色,边缘波状,呈不规则形,表面具有皱褶,不透明。在液体LB培养基中生长具有菌膜。光学显微镜和扫描电子显微镜观察结果表明菌株TB-1菌体为杆状,具一中生芽孢。以菌株TB-1基因组为模板,进行16S rDNA PCR扩增,PCR扩增所产生的DNA片段为单一条带,片段大小约为1.5kb左右。由宝生物工程(大连)有限公司完成PCR产物测序。将16S rDNA序列在GeneBank中进行相似性搜索,与Bacillus subtilis(DQ993674,AM237342.1,DQ401073.1)相似性最高,为100%。因此,菌株TB-1初步鉴定为枯草芽孢杆菌(Bacillus subtilis)。
(3)液体发酵条件的优化
通过单因素液体发酵条件的优化实验和液体培养基发酵条件的正交实验获得了实验室条件下的优化发酵条件:麸皮4.0 g,牛肉浸膏1.5 g,吐温80 400ppm,pH 6.5,蒸馏水100 ml;37℃,200rpm/min条件下,培养5d,植酸酶的酶活由优化前的0.1U/ml提高到0.15U/ml。
在优化液体发酵条件下对枯草芽孢杆菌TB-1进行5批次的发酵培养,结果表明耐热植酸酶产量与正交结果相近,平均值达0.147U/ml。
(4)粗酶酶学性质的研究
本研究所筛选到的枯草芽孢杆菌TB-1所产植酸酶,最适作用温度为60℃,最适作用pH值为5.5。将该酶在70℃分别保温60min,120min和180min,残余酶活性分别为70%,68%和63%;将该植酸酶在80℃分别保温60min,120min和180min,残余酶活性分别为43%,23%和15%;将该酶在90℃分别保温60min和120min,酶活损失明显,残余酶活性分别为21%,10%;说明该酶在70℃具有良好耐温性。此酶被P~(5+)、I~-、Co~(2+)和Cu~(2+)明显抑制。
(5)耐热植酸酶在小麦粉和全麦面包中的应用
采用枯草芽孢杆菌TB-1发酵后所得到的粗酶液对小麦粉和全麦面包进行处理,37℃分别处理10、20、30、40、50、60min,无机磷、蛋白质和单糖释放量随时间的延长均不断增加。小麦粉处理液中无机磷含量由1.6μmol/ml增加到11.72μmol/ml;单糖含量由0.037mmol/ml增加到0.133mmol/ml;蛋白质含量由4.10μg/ml增加到5.72μg/ml。全麦面包处理液中无机磷含量由1.98μmol/ml增加到14.2μmol/ml;单糖含量由0.10mmol/ml增加到0.92mmol/ml;蛋白质含量由4.76μg/ml增加到5.72μmol/ml。实验结果说明应用该植酸酶处理小麦粮食及全麦食品,不仅能提高无机磷的释放量,还能减少植酸对碳水化合物及蛋白质的鳌合,提高粮食的营养价值。因此该酶非常适合于食品工艺,可以作为一种新型酶制剂应用到麦面食品生产当中。
Phytic acid(myo-inositol-hexakisphosphate) is a major storage form of phosphorus in cereals and legumes,representing 18-88%of total phosphorus content.Phytase hydrolyzes phytic acid to various lower myo-inositol derivatives and inorganic phosphate.Phytate phosphorus is not metabolized by monogastric animals due to the lack of these enzymes in gastrointestinal tract and therefore contributes to the phosphorus pollution in area of intensive livestock production.
Phytic acid also acts as an antinutritional factor in monogastrics because of its chelation of various minerals and proteins and thus reduces the availability of the nutritional factors.
Many animal feeds are sold in the form of pellets and the pelleting process involves exposure to high temperature during pelleting and drying. Therefore,it is crucial to achieve a phytase with theromstability for animal pellets feed.
So the aim of the study is to screen a thermostable phytase-producing bacterium.
This thesis is consisted of 5 parts:
(1) Isolating and screening of the thermostable phytase-producing bacterium.
(2) Studying on the identification of TB-1,included morphological, physiological,biochemical characteristics and 16S rDNA sequence analyses.
(3) Studying on the fermentation condition of TB-1.
(4) Studying on enzymatic properties of thermostable phytase produced by TB-1.
(5) The application of thermostable phytase on wheat flour and wholewheat bread.
The detailed results and conclusions were as follows:
(1) Phytase-producing bacteria were screened and isolated from the water of hot spring of Tengchong Yunnan Province on screening agar media.TB-1 showed the thermostable phytase activity.
(2) TB-1 was identified as Bacillus subttilis on the basis of morphological, physiological,physiochemical characteristics and 16S rDNA sequence analyses.
(3) The fermentation medium conditions were optimized for phytase production with respect to TB-1 by the methods of single factor assay and orthogonal test.Under the optimum culture condition,TB-1 could produce 0.15U/ml phytase in liquid state fermentation.
(4) The optimum pH and temperature were pH5.5 and 60℃respectively.After 60min,120min,180min-storage at 70℃,the remaining activity were 70%, 68%and 63%respectively.After 60min,120min,180min-storage at 80℃, the remaining activity were 43%,23%and 15%respectively.After 60min, 120min-storage at 90℃,the remaining activity were 21%and 10% respectively.The crude phytase was inhibited by Cu~(2+),Co~(2+),I~- and Zn~(2+).
(5) The application of thermostable phytase on wheat flour and wholewheat bread indicated that when phytase produced by TB-1 added,the presence of Pi,protein and monosaccharide was enhanced.
According to above,the functional condition of thermostable phytase produced by TB-1 is suit for using in food industry.
目前,热稳定性是在植酸酶的推广应用中较为突出的问题。饲料生产中的制粒过程需要经过高温加热,一般在73℃～95℃,植酸酶在这样的温度下很容易失活,通常活性降为原来的30%,甚至更低。所以,进一步筛选耐热性更好的植酸酶以及探索提高植酸酶的热稳定性的方法仍是研究的重点。
本研究从自然筛选着手,以开发耐热植酸酶为目标,进行了以下几方面的研究:
(1)耐热植酸酶产生菌的分离筛选。
(2)耐热植酸酶产生菌的初步鉴定。
(3)耐热植酸酶液体发酵条件的优化。
(4)耐热植酸酶粗酶酶学性质的研究
(5)耐热植酸酶在小麦粉及全麦面包中的应用。
本论文主要研究结果和结论如下:
(1)菌株的筛选
本研究从云南腾冲热泉水样中,初步筛选得菌株TB-1,pH5.5、60℃条件下测定植酸酶酶活,结果表明菌株TB-1所产酶表现出耐热性,酶活达0.10U/ml。
(2)菌株的鉴定
菌株TB-1在LB琼脂平板上菌落为黄白色,边缘波状,呈不规则形,表面具有皱褶,不透明。在液体LB培养基中生长具有菌膜。光学显微镜和扫描电子显微镜观察结果表明菌株TB-1菌体为杆状,具一中生芽孢。以菌株TB-1基因组为模板,进行16S rDNA PCR扩增,PCR扩增所产生的DNA片段为单一条带,片段大小约为1.5kb左右。由宝生物工程(大连)有限公司完成PCR产物测序。将16S rDNA序列在GeneBank中进行相似性搜索,与Bacillus subtilis(DQ993674,AM237342.1,DQ401073.1)相似性最高,为100%。因此,菌株TB-1初步鉴定为枯草芽孢杆菌(Bacillus subtilis)。
(3)液体发酵条件的优化
通过单因素液体发酵条件的优化实验和液体培养基发酵条件的正交实验获得了实验室条件下的优化发酵条件:麸皮4.0 g,牛肉浸膏1.5 g,吐温80 400ppm,pH 6.5,蒸馏水100 ml;37℃,200rpm/min条件下,培养5d,植酸酶的酶活由优化前的0.1U/ml提高到0.15U/ml。
在优化液体发酵条件下对枯草芽孢杆菌TB-1进行5批次的发酵培养,结果表明耐热植酸酶产量与正交结果相近,平均值达0.147U/ml。
(4)粗酶酶学性质的研究
本研究所筛选到的枯草芽孢杆菌TB-1所产植酸酶,最适作用温度为60℃,最适作用pH值为5.5。将该酶在70℃分别保温60min,120min和180min,残余酶活性分别为70%,68%和63%;将该植酸酶在80℃分别保温60min,120min和180min,残余酶活性分别为43%,23%和15%;将该酶在90℃分别保温60min和120min,酶活损失明显,残余酶活性分别为21%,10%;说明该酶在70℃具有良好耐温性。此酶被P~(5+)、I~-、Co~(2+)和Cu~(2+)明显抑制。
(5)耐热植酸酶在小麦粉和全麦面包中的应用
采用枯草芽孢杆菌TB-1发酵后所得到的粗酶液对小麦粉和全麦面包进行处理,37℃分别处理10、20、30、40、50、60min,无机磷、蛋白质和单糖释放量随时间的延长均不断增加。小麦粉处理液中无机磷含量由1.6μmol/ml增加到11.72μmol/ml;单糖含量由0.037mmol/ml增加到0.133mmol/ml;蛋白质含量由4.10μg/ml增加到5.72μg/ml。全麦面包处理液中无机磷含量由1.98μmol/ml增加到14.2μmol/ml;单糖含量由0.10mmol/ml增加到0.92mmol/ml;蛋白质含量由4.76μg/ml增加到5.72μmol/ml。实验结果说明应用该植酸酶处理小麦粮食及全麦食品,不仅能提高无机磷的释放量,还能减少植酸对碳水化合物及蛋白质的鳌合,提高粮食的营养价值。因此该酶非常适合于食品工艺,可以作为一种新型酶制剂应用到麦面食品生产当中。
Phytic acid(myo-inositol-hexakisphosphate) is a major storage form of phosphorus in cereals and legumes,representing 18-88%of total phosphorus content.Phytase hydrolyzes phytic acid to various lower myo-inositol derivatives and inorganic phosphate.Phytate phosphorus is not metabolized by monogastric animals due to the lack of these enzymes in gastrointestinal tract and therefore contributes to the phosphorus pollution in area of intensive livestock production.
Phytic acid also acts as an antinutritional factor in monogastrics because of its chelation of various minerals and proteins and thus reduces the availability of the nutritional factors.
Many animal feeds are sold in the form of pellets and the pelleting process involves exposure to high temperature during pelleting and drying. Therefore,it is crucial to achieve a phytase with theromstability for animal pellets feed.
So the aim of the study is to screen a thermostable phytase-producing bacterium.
This thesis is consisted of 5 parts:
(1) Isolating and screening of the thermostable phytase-producing bacterium.
(2) Studying on the identification of TB-1,included morphological, physiological,biochemical characteristics and 16S rDNA sequence analyses.
(3) Studying on the fermentation condition of TB-1.
(4) Studying on enzymatic properties of thermostable phytase produced by TB-1.
(5) The application of thermostable phytase on wheat flour and wholewheat bread.
The detailed results and conclusions were as follows:
(1) Phytase-producing bacteria were screened and isolated from the water of hot spring of Tengchong Yunnan Province on screening agar media.TB-1 showed the thermostable phytase activity.
(2) TB-1 was identified as Bacillus subttilis on the basis of morphological, physiological,physiochemical characteristics and 16S rDNA sequence analyses.
(3) The fermentation medium conditions were optimized for phytase production with respect to TB-1 by the methods of single factor assay and orthogonal test.Under the optimum culture condition,TB-1 could produce 0.15U/ml phytase in liquid state fermentation.
(4) The optimum pH and temperature were pH5.5 and 60℃respectively.After 60min,120min,180min-storage at 70℃,the remaining activity were 70%, 68%and 63%respectively.After 60min,120min,180min-storage at 80℃, the remaining activity were 43%,23%and 15%respectively.After 60min, 120min-storage at 90℃,the remaining activity were 21%and 10% respectively.The crude phytase was inhibited by Cu~(2+),Co~(2+),I~- and Zn~(2+).
(5) The application of thermostable phytase on wheat flour and wholewheat bread indicated that when phytase produced by TB-1 added,the presence of Pi,protein and monosaccharide was enhanced.
According to above,the functional condition of thermostable phytase produced by TB-1 is suit for using in food industry.
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