蚕、桑中1-脱氧野尻霉素含量的测定及提取工艺条件优化
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摘要
为研究不同桑树品种桑叶和不同品种家蚕体内1-脱氧野尻霉素(DNJ)含量变化规律、筛选对食下桑叶中DNJ有较强富集能力的家蚕品种,本课题以10个桑树品种和18个家蚕品种为材料,采用反相高效液相色谱-紫外检测法(RP-HPLC-UV)测定供试桑叶和家蚕品种5龄第3天幼虫制备全蚕粉中的DNJ含量,并采用星点设计-效应面法优化乙醇提取桑叶和全蚕粉中DNJ的工艺条件,期望为进一步开发蚕桑资源用于功能性食品及药物研究提供理论依据。主要结果如下:
1. RP-HPLC-UV测定DNJ方法:以9-芴甲基氯甲酸甲酯(FMOC-C1)为衍生化剂,在pH=8.5的硼酸钾缓冲液中进行衍生化反应,生成具有紫外吸收的DNJ-FMOC络合物,色谱条件为:流动相为V(乙腈):V(0.1%乙酸)=40:60的混合液,流速1.0mL/min,检测波长254nm。
2. RP-HPLC-UV法测定10个桑树品种春季与秋季桑叶中DNJ含量,结果表明:陕桑305秋季桑叶中DNJ含量最高,为0.3795%,与其它品种桑叶中DNJ含量差异达极显著水平(P<0.01),说明不同桑树品种桑叶中DNJ含量存在明显差异;测定同一品种不同采集期桑叶中DNJ含量,结果表明:707、藤桑、蒲选一号、陕桑305、陕桑306、陕桑402等6个桑树品种秋季桑叶中DNJ含量极显著高于春季桑叶中DNJ含量(P<0.01);0206、吴堡桑、陕桑403等3个桑树品种秋季桑叶中DNJ含量极显著低于春季桑叶中DNJ含量(P<0.01),说明同一品种不同采集期桑叶中DNJ含量存在明显差异;测定不同染色体组数(二倍体、三倍体、四倍体)桑树品种桑叶中DNJ含量,结果表明:秋季桑叶三倍体中DNJ含量最高,平均质量分数为0.3439%,极显著高于二倍体、四倍体中DNJ含量(P<0.01),说明不同染色体组数桑叶中DNJ含量存在明显差异。
3. RP-HPLC-UV法测定不同家蚕品种5龄第3天制备的全蚕粉中DNJ含量,结果表明:18个供试家蚕品种中,四眠中系家蚕品种797对桑叶中DNJ的富集能力最强,其全蚕粉中的DNJ质量分数达到0.4345%,与其它家蚕品种全蚕粉中DNJ含量的差异均达极显著水平(P<0.01),说明不同家蚕品种对食下桑叶中DNJ的富集能力存在明显差异;测定不同地理系统(日系、中系、欧系)家蚕品种中DNJ含量,结果表明:日系品种与欧系品种对桑叶中DNJ的富集能力有显著差异(前者DNJ质量分数平均为0.3007%,后者为0.2469%),中系品种的DNJ质量分数平均为0.2880%,与日系品种与欧系品种之间无显著差异,说明不同地理系统家蚕品种对食下桑叶中DNJ的富集能力存在明显差异;不同眠性(三眠与四眠)家蚕品种间对桑叶中DNJ的富集能力差异不明显;选择6个家蚕品种分别取食不同桑树品种的桑叶,分析全蚕粉中的DNJ和桑叶中DNJ含量的相关性,其相关系数为0.8260,全蚕粉中DNJ含量最高的家蚕品种与桑品种组合为家蚕乌2与陕桑403,其次为家蚕武功土种与甜桑、家蚕798与陕桑403。
4.采用星点设计-效应面法,以乙醇浓度、提取温度、提取时间和液料比为考察因素,DNJ提取得率为效应值,优化桑叶中DNJ的提取工艺,初步建立了乙醇提取桑叶中DNJ的二次多项式数学模型,确定了最优提取工艺条件。结果表明:乙醇提取桑叶中DNJ最优工艺条件为:乙醇浓度70%,温度70℃,提取时间4h,液料比32.5mL/g。在此优化工艺条件下,DNJ提取得率的预测值为0.247%,实测值为0.263%,偏差率-6.478%。结果说明所建立的数学模型预测性好。
5.采用星点设计-效应面法,以乙醇浓度、提取温度、提取时间和液料比为考察因素,DNJ提取得率为效应值,优化全蚕粉中DNJ的提取工艺,初步建立了乙醇提取全蚕粉中DNJ的二次多项式数学模型,确定了最优提取工艺条件。结果表明:乙醇提取全蚕粉中DNJ最优工艺条件为:乙醇浓度80%,温度75℃,提取时间4h,液料比32.5mL/g。在此优化工艺条件下,DNJ提取得率的预测值为0.339%,实测值为0.327%,偏差率为3.540%。结果说明所建立的数学模型预测性好。
In order to study the variation of 1-Deoxynojirimycin (DNJ)content in different mulberry varieties leaves and silkworm varieties, to screen silkworm varieties with better enrichment ability from the forage mulberry leaves, 10 mulberry varieties and 18 silkworm varieties were used as the materials for determination DNJ contents in whole body powder of day 3 silkworm larvae of the 5th instar by reverse-phase high-performance liquid chromatography-ultraviolet spectrometry(RP-HPLC-UV), optimization of extraction process using central composite design-response surface methodology(CCD-RSM), so as to exploring sericultural resources are used to functionnal fooding and medicine study for providing theoretical basis. The main results are as follows:
1. The method of RP-HPLC-UV. In potassium borate buffer (pH=8.5), FMOC-Cl was added to derive DNJ. The DNJ-FMOC was produced which can absorb ultraviolet radiation and then was tested with ultraviolet radiation inspector. The results showed that the peak area of DNJ-FMOC was correspond with the concentration of DNJ. All of the precision test, recurring test, stability test and the recovery test showed that the method was credible and stable, and it could be used as an effective method in determining the DNJ content in mulberry leaves, silkworm and other related health products.
2.The DNJ content in 10 mulberry varieties leaves in Spring and Autumn were determined by RP-HPLC-UV. The rusults showed that The DNJ content in Shansang305 mulberry leaves in Autumn was the highest, DNJ mass fraction was 0.3795%, had extremely significant difference with other mulberry varieties leaves(P<0.01), it indicated that the DNJ content had remarkable difference in different mulberry varieties. Determinating DNJ content in the same mulberry variety in different collection seasons, the rusults showed that the mulberry leaves of 707, Tengsang, Puxuanyihao, Shansang305, Shansang306, Shansang402 in Autumn had extremely significant difference with DNJ content in spring(P<0.01), it indicated that the DNJ content had remarkable difference in the same mulberry variety in different collection seasons. Determinating DNJ content in different genome number of mulberry varieties(diploid, triploid and tetraploid) the rusults showed that the DNJ content in triploid mulberry in Autumn was the highest, DNJ mass fraction was 0.3439%, had extremely significant difference with diploid and tetraploid(P<0.01), it indicated that the DNJ content had remarkable difference in different genome number of mulberry varieties.
3. The DNJ contents in whole body powder of day 3 silkworm larvae of the 5th instar in 18 silkworm varieties were determined by RP-HPLC-UV. The rusults showed that silkworm variety 797 of Chinese strain had the best enrichment result to DNJ from mulberry leaves, DNJ mass fraction in the whole body powder reached 0.4345%, had extremely significant difference with other silkworm varieties (P<0.01), it indicated that different silkworm varieties had remarkable different enrichment ability to DNJ from forage mulberry leaves. Determinating DNJ content in silkworm varieties different geographical strains(Japanese strains, Chinese strains and European strains), The rusults showed that Silkworm varieties of Japanese and European strains had significantly different enrichment ability to DNJ from forage mulberry leaves (DNJ mass fraction of the former was 0.3007% and that of the latter was 0.2469%). The average DNJ mass fraction in silkworm varieties of Chinese strain was 0.2880%, having no significant difference with those of Japanese and European strains. Silkworm varieties of different moltinisms (trimolter or tetramoulter) had no remarkable difference in their enrichment ability to DNJ from forage mulberry leaves. The correlation between DNJ mass fraction in the whole body powder and DNJ content in mulberry leaves were analyzed by feeding larvae of 6 silkworm varieties with mulberry leaves of different varieties. The result showed that the correlation coefficient was 0.8260. The combination with the highest DNJ content in the whole body powder was silkworm variety Wu2 and mulberry variety Shansang403, followed by silkworm variety Wugongtuzhong and mulberry variety Tiansang, silkworm variety 798 and mulberry variety Shansang403.
4. In order to optimize the DNJ extraction process from mulberry leaves, central composite design-response surface methodology was used to optimise DNJ extraction yield. Four independent variables, extraction concentration, temperature, time and liquid-solid ratio were investigated to obtain the optimum extraction yield. Established preliminarily the model of quadratic polynomial and definited the optimum conditions. The result showed that optimum conditions of DNJ extraction from mulberry leaves were: 70% ethanol, 70°C, 4h for extraction time and 32.5 at liquid-solid ratio. Under the optimized condition the predict DNJ extraction yield was 0.247%, the actual DNJ extraction yield was 0.263%, bias was -6.478%. It indicated that the model was predictive.
5. In order to optimize the DNJ extraction process from silkworm powder, central composite design-response surface methodology was used to optimise DNJ extraction yield. Four independent variables, extraction concentration, temperature, time and liquid-solid ratio were investigated to obtain the optimum extraction yield. Established preliminarily the model of quadratic polynomial, definited the optimum conditions. The result showed that optimum conditions of DNJ extraction from silkworm powder were: 80% ethanol, 75°C, 4h for extraction time and 32.5 at liquid-solid ratio. Under the optimized condition the predict DNJ extraction yield was 0.339%, the actual DNJ extraction yield was 0.327%, bias was 3.540%. It indicated that the model was predictive.
1. RP-HPLC-UV测定DNJ方法:以9-芴甲基氯甲酸甲酯(FMOC-C1)为衍生化剂,在pH=8.5的硼酸钾缓冲液中进行衍生化反应,生成具有紫外吸收的DNJ-FMOC络合物,色谱条件为:流动相为V(乙腈):V(0.1%乙酸)=40:60的混合液,流速1.0mL/min,检测波长254nm。
2. RP-HPLC-UV法测定10个桑树品种春季与秋季桑叶中DNJ含量,结果表明:陕桑305秋季桑叶中DNJ含量最高,为0.3795%,与其它品种桑叶中DNJ含量差异达极显著水平(P<0.01),说明不同桑树品种桑叶中DNJ含量存在明显差异;测定同一品种不同采集期桑叶中DNJ含量,结果表明:707、藤桑、蒲选一号、陕桑305、陕桑306、陕桑402等6个桑树品种秋季桑叶中DNJ含量极显著高于春季桑叶中DNJ含量(P<0.01);0206、吴堡桑、陕桑403等3个桑树品种秋季桑叶中DNJ含量极显著低于春季桑叶中DNJ含量(P<0.01),说明同一品种不同采集期桑叶中DNJ含量存在明显差异;测定不同染色体组数(二倍体、三倍体、四倍体)桑树品种桑叶中DNJ含量,结果表明:秋季桑叶三倍体中DNJ含量最高,平均质量分数为0.3439%,极显著高于二倍体、四倍体中DNJ含量(P<0.01),说明不同染色体组数桑叶中DNJ含量存在明显差异。
3. RP-HPLC-UV法测定不同家蚕品种5龄第3天制备的全蚕粉中DNJ含量,结果表明:18个供试家蚕品种中,四眠中系家蚕品种797对桑叶中DNJ的富集能力最强,其全蚕粉中的DNJ质量分数达到0.4345%,与其它家蚕品种全蚕粉中DNJ含量的差异均达极显著水平(P<0.01),说明不同家蚕品种对食下桑叶中DNJ的富集能力存在明显差异;测定不同地理系统(日系、中系、欧系)家蚕品种中DNJ含量,结果表明:日系品种与欧系品种对桑叶中DNJ的富集能力有显著差异(前者DNJ质量分数平均为0.3007%,后者为0.2469%),中系品种的DNJ质量分数平均为0.2880%,与日系品种与欧系品种之间无显著差异,说明不同地理系统家蚕品种对食下桑叶中DNJ的富集能力存在明显差异;不同眠性(三眠与四眠)家蚕品种间对桑叶中DNJ的富集能力差异不明显;选择6个家蚕品种分别取食不同桑树品种的桑叶,分析全蚕粉中的DNJ和桑叶中DNJ含量的相关性,其相关系数为0.8260,全蚕粉中DNJ含量最高的家蚕品种与桑品种组合为家蚕乌2与陕桑403,其次为家蚕武功土种与甜桑、家蚕798与陕桑403。
4.采用星点设计-效应面法,以乙醇浓度、提取温度、提取时间和液料比为考察因素,DNJ提取得率为效应值,优化桑叶中DNJ的提取工艺,初步建立了乙醇提取桑叶中DNJ的二次多项式数学模型,确定了最优提取工艺条件。结果表明:乙醇提取桑叶中DNJ最优工艺条件为:乙醇浓度70%,温度70℃,提取时间4h,液料比32.5mL/g。在此优化工艺条件下,DNJ提取得率的预测值为0.247%,实测值为0.263%,偏差率-6.478%。结果说明所建立的数学模型预测性好。
5.采用星点设计-效应面法,以乙醇浓度、提取温度、提取时间和液料比为考察因素,DNJ提取得率为效应值,优化全蚕粉中DNJ的提取工艺,初步建立了乙醇提取全蚕粉中DNJ的二次多项式数学模型,确定了最优提取工艺条件。结果表明:乙醇提取全蚕粉中DNJ最优工艺条件为:乙醇浓度80%,温度75℃,提取时间4h,液料比32.5mL/g。在此优化工艺条件下,DNJ提取得率的预测值为0.339%,实测值为0.327%,偏差率为3.540%。结果说明所建立的数学模型预测性好。
In order to study the variation of 1-Deoxynojirimycin (DNJ)content in different mulberry varieties leaves and silkworm varieties, to screen silkworm varieties with better enrichment ability from the forage mulberry leaves, 10 mulberry varieties and 18 silkworm varieties were used as the materials for determination DNJ contents in whole body powder of day 3 silkworm larvae of the 5th instar by reverse-phase high-performance liquid chromatography-ultraviolet spectrometry(RP-HPLC-UV), optimization of extraction process using central composite design-response surface methodology(CCD-RSM), so as to exploring sericultural resources are used to functionnal fooding and medicine study for providing theoretical basis. The main results are as follows:
1. The method of RP-HPLC-UV. In potassium borate buffer (pH=8.5), FMOC-Cl was added to derive DNJ. The DNJ-FMOC was produced which can absorb ultraviolet radiation and then was tested with ultraviolet radiation inspector. The results showed that the peak area of DNJ-FMOC was correspond with the concentration of DNJ. All of the precision test, recurring test, stability test and the recovery test showed that the method was credible and stable, and it could be used as an effective method in determining the DNJ content in mulberry leaves, silkworm and other related health products.
2.The DNJ content in 10 mulberry varieties leaves in Spring and Autumn were determined by RP-HPLC-UV. The rusults showed that The DNJ content in Shansang305 mulberry leaves in Autumn was the highest, DNJ mass fraction was 0.3795%, had extremely significant difference with other mulberry varieties leaves(P<0.01), it indicated that the DNJ content had remarkable difference in different mulberry varieties. Determinating DNJ content in the same mulberry variety in different collection seasons, the rusults showed that the mulberry leaves of 707, Tengsang, Puxuanyihao, Shansang305, Shansang306, Shansang402 in Autumn had extremely significant difference with DNJ content in spring(P<0.01), it indicated that the DNJ content had remarkable difference in the same mulberry variety in different collection seasons. Determinating DNJ content in different genome number of mulberry varieties(diploid, triploid and tetraploid) the rusults showed that the DNJ content in triploid mulberry in Autumn was the highest, DNJ mass fraction was 0.3439%, had extremely significant difference with diploid and tetraploid(P<0.01), it indicated that the DNJ content had remarkable difference in different genome number of mulberry varieties.
3. The DNJ contents in whole body powder of day 3 silkworm larvae of the 5th instar in 18 silkworm varieties were determined by RP-HPLC-UV. The rusults showed that silkworm variety 797 of Chinese strain had the best enrichment result to DNJ from mulberry leaves, DNJ mass fraction in the whole body powder reached 0.4345%, had extremely significant difference with other silkworm varieties (P<0.01), it indicated that different silkworm varieties had remarkable different enrichment ability to DNJ from forage mulberry leaves. Determinating DNJ content in silkworm varieties different geographical strains(Japanese strains, Chinese strains and European strains), The rusults showed that Silkworm varieties of Japanese and European strains had significantly different enrichment ability to DNJ from forage mulberry leaves (DNJ mass fraction of the former was 0.3007% and that of the latter was 0.2469%). The average DNJ mass fraction in silkworm varieties of Chinese strain was 0.2880%, having no significant difference with those of Japanese and European strains. Silkworm varieties of different moltinisms (trimolter or tetramoulter) had no remarkable difference in their enrichment ability to DNJ from forage mulberry leaves. The correlation between DNJ mass fraction in the whole body powder and DNJ content in mulberry leaves were analyzed by feeding larvae of 6 silkworm varieties with mulberry leaves of different varieties. The result showed that the correlation coefficient was 0.8260. The combination with the highest DNJ content in the whole body powder was silkworm variety Wu2 and mulberry variety Shansang403, followed by silkworm variety Wugongtuzhong and mulberry variety Tiansang, silkworm variety 798 and mulberry variety Shansang403.
4. In order to optimize the DNJ extraction process from mulberry leaves, central composite design-response surface methodology was used to optimise DNJ extraction yield. Four independent variables, extraction concentration, temperature, time and liquid-solid ratio were investigated to obtain the optimum extraction yield. Established preliminarily the model of quadratic polynomial and definited the optimum conditions. The result showed that optimum conditions of DNJ extraction from mulberry leaves were: 70% ethanol, 70°C, 4h for extraction time and 32.5 at liquid-solid ratio. Under the optimized condition the predict DNJ extraction yield was 0.247%, the actual DNJ extraction yield was 0.263%, bias was -6.478%. It indicated that the model was predictive.
5. In order to optimize the DNJ extraction process from silkworm powder, central composite design-response surface methodology was used to optimise DNJ extraction yield. Four independent variables, extraction concentration, temperature, time and liquid-solid ratio were investigated to obtain the optimum extraction yield. Established preliminarily the model of quadratic polynomial, definited the optimum conditions. The result showed that optimum conditions of DNJ extraction from silkworm powder were: 80% ethanol, 75°C, 4h for extraction time and 32.5 at liquid-solid ratio. Under the optimized condition the predict DNJ extraction yield was 0.339%, the actual DNJ extraction yield was 0.327%, bias was 3.540%. It indicated that the model was predictive.
引文
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