灵芝孢子低聚糖的制备及其改性研究
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摘要
本文以开平健之源保健食品有限公司提供的破壁去油后的灵芝孢子粉为原料,经羧甲基化改性获得羧甲基灵芝孢子粉,再经酶降解获得羧甲基灵芝孢子低聚糖。
1、对破壁去油后的灵芝孢子粉进行羧甲基化改性,以取代度为指标优化改性条件,得出最佳条件为:乙醇浓度80%、碱化时间2h、NaOH浓度1.5mol/L、氯乙酸浓度0.625mol/L、醚化温度45℃、醚化时间20h。此条件下,羧甲基取代度达到0.78;接着研究了影响改性样品溶解率的因素,得出较佳溶解条件为:pH值5.15、取代度0.78(最高DS)、溶解温度50℃、溶解时间3d;分别用水煮法、冻融-水煮法、超声波-水煮法提取破壁去油后的灵芝孢子粉中总糖,测得总糖提取率分别为:3.12%、3.68%、4.36%;DS为0.78的羧甲基灵芝孢子粉(以下简称改性样品)充分溶解后,测得总糖提取率为24.6%,是水煮法提取率的7倍以上;
2、苯酚硫酸法测定了取代度为0.721的改性样品溶液以及高速离心后上清液中总糖含量,实验得出改性样品中总糖含量占样品干重的26.7%,其中可溶性糖含量占样品干重的23.8%,即可溶性糖占改性样品中总糖的80.9%;改性样品通过纤维素酶降解,获得羧甲基灵芝孢子低聚糖。为获得聚合度在6～10的低聚糖,确定了纤维素酶降解改性样品的适合条件为:pH 4.4,酶解温度50℃,酶用量200U/g底物,酶解时间2h。在此条件下,所得到的羧甲基灵芝孢子低聚糖的平均聚合度为7.7;
3、分别以1%的葡萄糖和不同浓度羧甲基灵芝孢子低聚糖作为碳源,培养青春双歧杆菌。通过测定培养液的pH值、620nm波长下的吸光值以及菌体质量浓度,考察青春双歧杆菌的生长繁殖情况。实验得出,羧甲基灵芝孢子低聚糖对双歧杆菌的增殖效果明显,且糖浓度越高,增殖倍数越大。糖浓度从0.5%～3%,菌体增长倍数依次为:2.2、2.9、3.3、3.6。但在同一糖浓度水平上,葡萄糖在双歧杆菌体外增殖方面要优于羧甲基灵芝孢子低聚糖;
4、对羧甲基灵芝孢子粉和羧甲基灵芝孢子低聚糖的工业化生产作了粗略设计,主要设计内容有:工艺流程、关键工艺参数、车间组成及其平面布局,为其工业化生产提供一定的依据。
In this paper, SDGLS (sporoderm-broken and defatted Ganoderma lucidum spores) provided by Kaiping healthwise Co., LTD was taken as the raw material. Carboxy methyl SDGLS was prepared by carboxy methylated reaction. And carboxy methyl oligosaccharides were made by the enzyme degradation of Carboxy methyl SDGLS.
1 The raw material was carboxy methylated modified. Taking the DS (degree of substitution) as the standard, the reaction conditions were optimized. The results indicated the optimal conditions: the SDGLS was 1g, the concentration of thanol was 80%, the dose of sodium hydroxide was 1.20g, the dose of chloroactic acid was 1.18g, alkalization was 2h, reaction temperature was 45℃, and etherification time was 20h. In these conditions, the DS of carboxy methyl reached to 0.78. Then we studied the factors which would influence the solubility of the modified samples. And the results indicated the optimum solution conditions: pH5.15, DS 0.78(the maximum value of DS), temperature was 50℃and solution time was 3d. Three kinds of methods were used to extract the saccharides. There were water boiling method, freeze thaw and water boiling method, ultrasonic and water boiling method. And the extraction yields of these methods were: 3.12%, 3.68% and 4.36%.The sample, whose DS was 0.78, was dissolved in these conditions and its extraction yield of total saccharides was 24.6%, which was 7 times more than water boiling method’s.
2 The total saccharides contents of the sample solution and its liquid supernatant were determined using the phenol-sulfuric method. The results showed that the total saccharides contents were 26.7% and 23.8%. The oligosaccharides with carboxymethyl group were prepared by cellulose enzyme degradation. In order to get the oligosaccharides with a certain range (6～10) of polymerization degree, the conditions of enzyme degradation were studied. The appropriate conditions were that pH was 4.4, temperature was 50℃, concentration of the enzyme was 200U/g of substrate, and time was 2h. In these conditions, the oligosaccharides with an average polymerization degree of 7.7 were abtained.
3 The Bifidobacterium was proliferation in vitro taking glucose with the concentration of 1% and homemade oligosaccharides with different concentrations as the carbon source. And we studied its proliferation situation by determining the pH, the absorbance at 620nm and the bacteria concentration of the culture solution. The results showed that the effect of homemade oligosaccharides on proliferation of Bifidobacterium was obvious and the effect would become better and better with the concentration of oligosaccharides increasing. From 0.5% to 3%, its growth times would be 2.2, 2.9, 3.3, 3.6. And when their concentrations were the same, the effect of glucose would be better than homemade oligosaccharides.
4 A preliminary industrial design was made for Carboxy methyl SDGLS and carboxy methyl oligosaccharides. The main jobs were: process, key parameters, workshop design. And this design provided a certain basis for their industrial production.
1、对破壁去油后的灵芝孢子粉进行羧甲基化改性,以取代度为指标优化改性条件,得出最佳条件为:乙醇浓度80%、碱化时间2h、NaOH浓度1.5mol/L、氯乙酸浓度0.625mol/L、醚化温度45℃、醚化时间20h。此条件下,羧甲基取代度达到0.78;接着研究了影响改性样品溶解率的因素,得出较佳溶解条件为:pH值5.15、取代度0.78(最高DS)、溶解温度50℃、溶解时间3d;分别用水煮法、冻融-水煮法、超声波-水煮法提取破壁去油后的灵芝孢子粉中总糖,测得总糖提取率分别为:3.12%、3.68%、4.36%;DS为0.78的羧甲基灵芝孢子粉(以下简称改性样品)充分溶解后,测得总糖提取率为24.6%,是水煮法提取率的7倍以上;
2、苯酚硫酸法测定了取代度为0.721的改性样品溶液以及高速离心后上清液中总糖含量,实验得出改性样品中总糖含量占样品干重的26.7%,其中可溶性糖含量占样品干重的23.8%,即可溶性糖占改性样品中总糖的80.9%;改性样品通过纤维素酶降解,获得羧甲基灵芝孢子低聚糖。为获得聚合度在6～10的低聚糖,确定了纤维素酶降解改性样品的适合条件为:pH 4.4,酶解温度50℃,酶用量200U/g底物,酶解时间2h。在此条件下,所得到的羧甲基灵芝孢子低聚糖的平均聚合度为7.7;
3、分别以1%的葡萄糖和不同浓度羧甲基灵芝孢子低聚糖作为碳源,培养青春双歧杆菌。通过测定培养液的pH值、620nm波长下的吸光值以及菌体质量浓度,考察青春双歧杆菌的生长繁殖情况。实验得出,羧甲基灵芝孢子低聚糖对双歧杆菌的增殖效果明显,且糖浓度越高,增殖倍数越大。糖浓度从0.5%～3%,菌体增长倍数依次为:2.2、2.9、3.3、3.6。但在同一糖浓度水平上,葡萄糖在双歧杆菌体外增殖方面要优于羧甲基灵芝孢子低聚糖;
4、对羧甲基灵芝孢子粉和羧甲基灵芝孢子低聚糖的工业化生产作了粗略设计,主要设计内容有:工艺流程、关键工艺参数、车间组成及其平面布局,为其工业化生产提供一定的依据。
In this paper, SDGLS (sporoderm-broken and defatted Ganoderma lucidum spores) provided by Kaiping healthwise Co., LTD was taken as the raw material. Carboxy methyl SDGLS was prepared by carboxy methylated reaction. And carboxy methyl oligosaccharides were made by the enzyme degradation of Carboxy methyl SDGLS.
1 The raw material was carboxy methylated modified. Taking the DS (degree of substitution) as the standard, the reaction conditions were optimized. The results indicated the optimal conditions: the SDGLS was 1g, the concentration of thanol was 80%, the dose of sodium hydroxide was 1.20g, the dose of chloroactic acid was 1.18g, alkalization was 2h, reaction temperature was 45℃, and etherification time was 20h. In these conditions, the DS of carboxy methyl reached to 0.78. Then we studied the factors which would influence the solubility of the modified samples. And the results indicated the optimum solution conditions: pH5.15, DS 0.78(the maximum value of DS), temperature was 50℃and solution time was 3d. Three kinds of methods were used to extract the saccharides. There were water boiling method, freeze thaw and water boiling method, ultrasonic and water boiling method. And the extraction yields of these methods were: 3.12%, 3.68% and 4.36%.The sample, whose DS was 0.78, was dissolved in these conditions and its extraction yield of total saccharides was 24.6%, which was 7 times more than water boiling method’s.
2 The total saccharides contents of the sample solution and its liquid supernatant were determined using the phenol-sulfuric method. The results showed that the total saccharides contents were 26.7% and 23.8%. The oligosaccharides with carboxymethyl group were prepared by cellulose enzyme degradation. In order to get the oligosaccharides with a certain range (6～10) of polymerization degree, the conditions of enzyme degradation were studied. The appropriate conditions were that pH was 4.4, temperature was 50℃, concentration of the enzyme was 200U/g of substrate, and time was 2h. In these conditions, the oligosaccharides with an average polymerization degree of 7.7 were abtained.
3 The Bifidobacterium was proliferation in vitro taking glucose with the concentration of 1% and homemade oligosaccharides with different concentrations as the carbon source. And we studied its proliferation situation by determining the pH, the absorbance at 620nm and the bacteria concentration of the culture solution. The results showed that the effect of homemade oligosaccharides on proliferation of Bifidobacterium was obvious and the effect would become better and better with the concentration of oligosaccharides increasing. From 0.5% to 3%, its growth times would be 2.2, 2.9, 3.3, 3.6. And when their concentrations were the same, the effect of glucose would be better than homemade oligosaccharides.
4 A preliminary industrial design was made for Carboxy methyl SDGLS and carboxy methyl oligosaccharides. The main jobs were: process, key parameters, workshop design. And this design provided a certain basis for their industrial production.
引文
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