大豆糖蜜中功能性低聚糖的纯化分离
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摘要
本文以大豆糖蜜为原料,通过高效液相对其中的糖分进行检测,分别采用了发酵降解,大孔吸附树脂脱色,离子交换树脂脱盐等方法对大豆糖蜜中的功能性糖分进行纯化分离。
糖蜜中总固形物含量为66.58%,总糖占48.61 %,蛋白占8.14%,灰分5.54%,其余还包括脂类、异黄酮、皂苷等。其中蔗糖、棉子糖、水苏糖含量分别是317.53mg·mL~(-1),43.66mg·mL~(-1),175.42mg·mL~(-1)。
通过对酵母菌、乳酸菌、红曲及它们的混合菌进行筛选,确定了酵母7号菌才能满足去除大豆糖蜜中蔗糖,同时保留棉子糖,水苏糖的要求。可以使功能性低聚糖占总糖的比例由原来的40.24%提高到88.57%。
经过单因素实验优化,确定酵母7号菌发酵大豆糖蜜的最佳条件为:pH 5.0,接种量2%,稀释8倍,28℃,180rpm·min~(-1),发酵时间12h。大豆糖蜜发酵液中各糖分的保留率为:蔗糖:5.76%,棉子糖:99.61%,水苏糖:95.72%。
用20L的发酵罐进行中试发酵,在实验室的最优条件下,对中试发酵中的pH是否恒定、溶氧量两个因素进行研究,确定了最优条件是:发酵期间不需要控制pH值和溶氧量。此条件下,蔗糖的保留率达到19.69 %,棉子糖和水苏糖的保留率分别为98.07%、99.96%。
确定了发酵液的酸沉蛋白的最佳条件:4%HCL、pH为3.0、搅拌时间为10min,6000rpm·min~(-1)离心10min,正交实验的最佳结果显示蛋白质清除率可达到55.50%,总糖的保留率为99.70%。总糖占总固含物百分比由酸沉前的53.30%提高至58.65%。
利用大孔树脂对酸沉后的发酵液进行脱色,选用三种不同的大孔树脂,进行静态吸附比较,确定AB-8为最佳脱色树脂,其脱色最佳条件为:pH 3,吸附时间2h,脱色率达到93.96%,总糖保留率达到96.87%;动态实验优化,最终确定最佳条件为流速在1.5BV/h,流出体积4.5BV,脱色率为92%,总糖保留率为98.30%,总糖占总固含物的75.40%。此时蔗糖为2.23mg·mL~(-1)、棉子糖为5.31mg·mL~(-1)、水苏糖为20.51mg·mL~(-1)、蛋白含量为0.14mg·mL~(-1)。
选用强酸性阳离子交换树脂001×7联用强碱性阴离子交换树脂201×7或弱碱性阴离子交换树脂D301对大孔树脂脱色后的粗糖浆进行离子交换,确定了001×7和D301组合对脱色糖浆的离子交换效果较好。操作条件为:先阳后阴的方式连接,室温、流速1.2BV/h,001×7型树脂的交换能力约为9BV,D301型树脂的交换能力为6BV左右。最终得到的糖液的所有的含氮物质被去除,糖的保留率为89.74%;总糖占总固含物94.00%,此时蔗糖为2.05 mg·mL~(-1),棉子糖4.88 mg·mL~(-1),水苏糖18.87mg·mL~(-1)。功能性成分占总固形物的86.51%。
将纯化的低聚糖液体经过冷冻干燥,001×7和D301组合的回收液得到了白色透明的粉末,味微甜的大豆低聚糖,总糖含量0.94g·g~(-1);001×7和201×7组合回收的冻干样品成浅黄色,总糖含量201×7为0.83 g·g~(-1)。
This paper was studied on using fermentation, macroporous resins and ion exchange resins to extract soybean oligosaccharides detected by HPLC from soy molasses.
There are 66.58% soluble solid, 8.14% protein, 5.54% ash content and 48.61% total sugar which contain sucrose 317.53mg mL~(-1), raffinose 43.66mg mL~(-1) and stachyose 175.42mg mL~(-1).
Screening of yeasts, lactobacillus, Monascus rubber and mixed culture fermentation of them, 7 yeast is selected which can decompose sucrose as well as retain raffinose and stachyose, raise functionality of oligosaccharides from 40.24 % to 88.57% in total sugar.
The results of odd fator tests 7 yeast showed that the optimum fermentation conditions for soybean molasses was: the inoculation concentration was 2%, the temperature was 28℃, the initial pH was 5.0, the soybean oligosaccharide dilute 8 times,after 12 h the remainder rate of residule sucrose 5.76%, raffinose 99.61%, stachyose 95.72% in the fermentation broth of soybean molasses.
Evaluating whether fixed pH and dissolved oxygen content to ferment by fermenter of volum 20L, we defined the optimization condition is not necessary to control pH and oxygen content in the course of fermentation, at this time, the remainder rate of residule sucrose 19.69%, raffinose 98.07%, stachyose 99.96%.
The optimum conditions of Acid-Precipitatio protein was: 4% HCL, pH 3.0, stir 10min, centrifuge 10min. The result of perpendicular is deposit protein rate 55.50%, total sugar remainder rate 99.70%, functionality of oligosaccharides raise from 53.30% to 58.65 in soluble solid.
We selected three macroporous resins to decoloratio the fermentation solution after Acid-Precipitatio and definited AB-8 is best for decoloratio. The optimization condition is taht the flow rate is 1.5BV/h, the volum of decoloration is 4.5BV, decoloratio rate is 92%, total sugar remainder rate is 99.70%. The total sugar occupied 75.40% of soluble solid. The content of sucrose is 2.23mg mL~(-1), raffinose is 5.31mg mL~(-1) and stachyose is 20.51mg mL~(-1), the protein is 0.14mg mL~(-1).
001×7 cation exchange resin combined 201×7 anion exchange resin or D301 anion exchange resin to ion exchange the crude soybean oligosaccharides and the result is 001×7 combining D301 is better. The optimum conditions is that the ion exchange resins combined according to the cation antecedently, ordinary temperature, flow rate is 1.2BV/h and under this condition 001×7 exchange capacity is about 9BV, D301 is about 6BV. The result of ion exchange is that the solution of soybean oligosaccharides has no nitrogen-containing material. The total sugar is 89.74% times of soluble solid. The content of sucrose is 2.05mg mL~(-1), raffinose is 4.88mg mL~(-1) and stachyose is 18.87mg mL~(-1), and functionality of oligosaccharides occupied 86.51% of total soluble solid.
The freeze-dried solution of soybean oligosaccharides obtained from 001×7 combining D301 is white, taste somewhat sweet, the total sugar content is 0.94 g·g~(-1) and from 001×7 combining 201×7 is light yellow, and the total sugar content is 0.83 g.g~(-1).
糖蜜中总固形物含量为66.58%,总糖占48.61 %,蛋白占8.14%,灰分5.54%,其余还包括脂类、异黄酮、皂苷等。其中蔗糖、棉子糖、水苏糖含量分别是317.53mg·mL~(-1),43.66mg·mL~(-1),175.42mg·mL~(-1)。
通过对酵母菌、乳酸菌、红曲及它们的混合菌进行筛选,确定了酵母7号菌才能满足去除大豆糖蜜中蔗糖,同时保留棉子糖,水苏糖的要求。可以使功能性低聚糖占总糖的比例由原来的40.24%提高到88.57%。
经过单因素实验优化,确定酵母7号菌发酵大豆糖蜜的最佳条件为:pH 5.0,接种量2%,稀释8倍,28℃,180rpm·min~(-1),发酵时间12h。大豆糖蜜发酵液中各糖分的保留率为:蔗糖:5.76%,棉子糖:99.61%,水苏糖:95.72%。
用20L的发酵罐进行中试发酵,在实验室的最优条件下,对中试发酵中的pH是否恒定、溶氧量两个因素进行研究,确定了最优条件是:发酵期间不需要控制pH值和溶氧量。此条件下,蔗糖的保留率达到19.69 %,棉子糖和水苏糖的保留率分别为98.07%、99.96%。
确定了发酵液的酸沉蛋白的最佳条件:4%HCL、pH为3.0、搅拌时间为10min,6000rpm·min~(-1)离心10min,正交实验的最佳结果显示蛋白质清除率可达到55.50%,总糖的保留率为99.70%。总糖占总固含物百分比由酸沉前的53.30%提高至58.65%。
利用大孔树脂对酸沉后的发酵液进行脱色,选用三种不同的大孔树脂,进行静态吸附比较,确定AB-8为最佳脱色树脂,其脱色最佳条件为:pH 3,吸附时间2h,脱色率达到93.96%,总糖保留率达到96.87%;动态实验优化,最终确定最佳条件为流速在1.5BV/h,流出体积4.5BV,脱色率为92%,总糖保留率为98.30%,总糖占总固含物的75.40%。此时蔗糖为2.23mg·mL~(-1)、棉子糖为5.31mg·mL~(-1)、水苏糖为20.51mg·mL~(-1)、蛋白含量为0.14mg·mL~(-1)。
选用强酸性阳离子交换树脂001×7联用强碱性阴离子交换树脂201×7或弱碱性阴离子交换树脂D301对大孔树脂脱色后的粗糖浆进行离子交换,确定了001×7和D301组合对脱色糖浆的离子交换效果较好。操作条件为:先阳后阴的方式连接,室温、流速1.2BV/h,001×7型树脂的交换能力约为9BV,D301型树脂的交换能力为6BV左右。最终得到的糖液的所有的含氮物质被去除,糖的保留率为89.74%;总糖占总固含物94.00%,此时蔗糖为2.05 mg·mL~(-1),棉子糖4.88 mg·mL~(-1),水苏糖18.87mg·mL~(-1)。功能性成分占总固形物的86.51%。
将纯化的低聚糖液体经过冷冻干燥,001×7和D301组合的回收液得到了白色透明的粉末,味微甜的大豆低聚糖,总糖含量0.94g·g~(-1);001×7和201×7组合回收的冻干样品成浅黄色,总糖含量201×7为0.83 g·g~(-1)。
This paper was studied on using fermentation, macroporous resins and ion exchange resins to extract soybean oligosaccharides detected by HPLC from soy molasses.
There are 66.58% soluble solid, 8.14% protein, 5.54% ash content and 48.61% total sugar which contain sucrose 317.53mg mL~(-1), raffinose 43.66mg mL~(-1) and stachyose 175.42mg mL~(-1).
Screening of yeasts, lactobacillus, Monascus rubber and mixed culture fermentation of them, 7 yeast is selected which can decompose sucrose as well as retain raffinose and stachyose, raise functionality of oligosaccharides from 40.24 % to 88.57% in total sugar.
The results of odd fator tests 7 yeast showed that the optimum fermentation conditions for soybean molasses was: the inoculation concentration was 2%, the temperature was 28℃, the initial pH was 5.0, the soybean oligosaccharide dilute 8 times,after 12 h the remainder rate of residule sucrose 5.76%, raffinose 99.61%, stachyose 95.72% in the fermentation broth of soybean molasses.
Evaluating whether fixed pH and dissolved oxygen content to ferment by fermenter of volum 20L, we defined the optimization condition is not necessary to control pH and oxygen content in the course of fermentation, at this time, the remainder rate of residule sucrose 19.69%, raffinose 98.07%, stachyose 99.96%.
The optimum conditions of Acid-Precipitatio protein was: 4% HCL, pH 3.0, stir 10min, centrifuge 10min. The result of perpendicular is deposit protein rate 55.50%, total sugar remainder rate 99.70%, functionality of oligosaccharides raise from 53.30% to 58.65 in soluble solid.
We selected three macroporous resins to decoloratio the fermentation solution after Acid-Precipitatio and definited AB-8 is best for decoloratio. The optimization condition is taht the flow rate is 1.5BV/h, the volum of decoloration is 4.5BV, decoloratio rate is 92%, total sugar remainder rate is 99.70%. The total sugar occupied 75.40% of soluble solid. The content of sucrose is 2.23mg mL~(-1), raffinose is 5.31mg mL~(-1) and stachyose is 20.51mg mL~(-1), the protein is 0.14mg mL~(-1).
001×7 cation exchange resin combined 201×7 anion exchange resin or D301 anion exchange resin to ion exchange the crude soybean oligosaccharides and the result is 001×7 combining D301 is better. The optimum conditions is that the ion exchange resins combined according to the cation antecedently, ordinary temperature, flow rate is 1.2BV/h and under this condition 001×7 exchange capacity is about 9BV, D301 is about 6BV. The result of ion exchange is that the solution of soybean oligosaccharides has no nitrogen-containing material. The total sugar is 89.74% times of soluble solid. The content of sucrose is 2.05mg mL~(-1), raffinose is 4.88mg mL~(-1) and stachyose is 18.87mg mL~(-1), and functionality of oligosaccharides occupied 86.51% of total soluble solid.
The freeze-dried solution of soybean oligosaccharides obtained from 001×7 combining D301 is white, taste somewhat sweet, the total sugar content is 0.94 g·g~(-1) and from 001×7 combining 201×7 is light yellow, and the total sugar content is 0.83 g.g~(-1).
引文
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