米糠营养成分分析及其应用研究
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摘要
本课题的研究方向是米糠的营养成分分析及其应用研究。以米糠为研究对象,建立了米糠中水溶性维生素的分析测定方法。同时从米糠中提取了具有活性功能的米糠多糖,并建立了荧光法研究头发光损伤模型,应用该模型对米糠多糖抗紫外辐射活性进行了研究。
课题分为以下四部分进行研究:
第一部分,高效液相色谱法同时测定米糠中四种水溶性维生素VB_1,VB_6,烟酸,烟酰胺的含量。在Hypersil C_(18) (250 cm×4.6 mm i.d., 5.0μm)上,以体积比为88:12的0.1%磷酸水溶液-甲醇为流动相,以0.6 mL/min流速等度洗脱,在266 nm波长下检测。各组分在8 min能基线分离。四种维生素在0.6×10~(-6) g/mL-8.5×10~(-5) g/mL范围内呈线性关系,VB1、VB6、烟酸和烟酰胺的检出下限分别为0.43×10~(-6) g/mL、0.34×10~(-5) g/mL、0.32×10~(-6) g/mL、0.30×10~(-6) g/mL。经简单预处理,可以对米糠和淘米水中的这四种维生素的含量进行测定,平均回收率在96.0%-99.9%之间。该法简便、快速、准确、灵敏,对复杂体系水溶性维生素的测定具有重要的参考价值。
第二部分,研究了微波提取法,热水蒸煮提取法,超声波提取法提取米糠多糖。同时考察了三氯乙酸法,Sevage法去除蛋白质;一步消化法,两步消化法去除淀粉。选取了较优的微波提取法提取米糠多糖,采用改进的苯酚-硫酸法测定米糠多糖含量。实验结果:料液比为1:10在1000 W 50%微波火力条件下提取30 min,提取2次,三氯乙酸法去蛋白质,两步消化法去淀粉,30%醇沉,米糠多糖得率达1.41%。实验所得产品RBPa将被应用于其活性功能的研究中。
第三部分,建立了头发中色氨酸的荧光分析方法,并用该法研究了UVB辐射下头发中色氨酸的变化规律。头发样品以5.5 mol/L氢氧化钠在110℃下水解20 h,在pH10.5的磷酸二氢钠-氢氧化钠缓冲液中,激发波长为280 nm、发射波长为360 nm处测定色氨酸的荧光强度。方法线性范围8.0×10~(-8)-1.75×10~(-6) g/mL,检测限为1.6×10~(-8) g/mL,回收率在96.3-100.3%。头发样品中色氨酸的量随UVB辐射时间增加呈减少趋势,光照28天后,色氨酸的损失率达到18.8%。该法快速,简便,灵敏度高,可推荐作为衡量头发光损伤的定量指标。
第四部分,研究了微波提取法得到的米糠粗多糖RBPa,RBPb应用到头发中的抗UVB辐射效果。将米糠粗多糖-吐温-80模拟发胶喷洒到头发上,自然干燥,置于紫外灯下照射。头发样品以5.5 mol/L氢氧化钠在110℃下水解20 h,在pH10.5的磷酸二氢钠-氢氧化钠缓冲液中,激发波长为280 nm、发射波长为360 nm处测定色氨酸的荧光强度变化,加有多糖保护的头发较对照样品中色氨酸的受破坏量降低。表明米糠多糖具有一定的抗紫外辐射效果。
The research direction of this thesis is analysis of nutrient component in rice bran and studies on its application. Taken rice bran as the research object, HPLC method had been built up to measure water-soluble vitamins in rice bran; Rice Bran Polysaccharides had been abstracted in rice bran ; a study model of ultraviolet damnification of hair had been build and had usedthis model studying the activity Resisting UVB-Ultraviolet
In chapter1, High performance liquid chromatography (HPLC) has been applied for the simultaneous determination of four kinds of water-soluble vitamins including vitamin B1, vitamin B6, nicotinic acid, and nicotinamide in rice bran. The separation was performed on a Hypersil C18 column (250 cm×4.6mm i.d., 5.0μm) with a mobile phase of 0.1% H3PO4- methanol (88:12, V/V) at flow rate of 0.6 mL /min, and detection wavelength was 266 nm. Four vitamins had good linearity in the range of 6.0×10~(-7) g/mL - 8.5×10~(-5) g/mL, with detection limit of 4.3×10~(-7) g/mL, 3.4×10~(-6) g/mL, 3.2×10~(-7) g/mL and 3.0×10~(-7) mg/L for vitamin B1, vitamin B6, nicotinic acid and nicotinamide, respectively. Baseline separation of four vitamins can be achieved within 8 minutes. Without complicated pretreatment, four analytes in rice bran samples, as well as extracts from rice with water, were successfully determined with recoveries between 96.0% and 99.9%. The approach could be applied to determine water-soluble vitamins in complicated samples owing to simplicity, convenience, sensitivity and accuracy.
In chapter 2, extraction methods including microwave extraction, dedoction and ultrasonic extraction had been studied to extract polysaccharides in rice bran. The methods for removing proteins by trichloroacetic acid and Sevage method, as well as for removing amylum by one step assimilation and two steps were also investigatated. Microwave assistant extraction has been applied for extraction polysaccharides in rice bran. The content of polysaccharides were determined by improved vitriol -phenol method. The optimum technics were the material-water ratio of 1:10, microwave assistant extraction for 30 mins for two times under 50% firepower. It was proved thatα-amylase could wipe off amylum effectively by two steps, and trichloroacetic acid method was appropriate to wipe off albuminoid. And the ethanol concentration was 30%, the extraction rate of the water-soluble polysaccharides of rice bran was 1.41%.
In chapter 3, a fluorescence spectrometry method has been developed to determine tryptophan in hair, and applied to evaluate hair damnification under UVB ultraviolet. 5.5 mol L-1 NaOH solution containing 0.5% soluble starch was used for hydrolysis of the hair samples. In pH 10.5 buffer of NaH2PO4-NaOH, the fluorescent intensity of tryptophan was determined at 280 nm as excitation wavelength and 360 nm at emission wavelength. The linear range of tryptophan was from 8.0×10~(-6) g/mL to1.75×10~(-6) g/mL. The detection limit of 1.6×10~(-8) g/mL and the recoveries ranged from 96.3% to 100.3% showed that the method was sensitive and accurate. With the increase of the irradiation time with UVB ultraviolet, tryptophan in hair decreased accordingly. During 28 days irradiation, tryptophan in hair was lost by 18.8%. This method could be recommended to measure ultraviolet damnification of hair.
In chapter 4, rice bran polysaccharides were applied for protection hair from UV damnification. Tryptophan in hair was determined with fluorescence spectrometry method, the contents of tryptophan were applied to evaluate hair damnification due to UVB ultraviolet under protection of rice bran polysaccharides. For simulating fixature, hair was besprinkled with mixture solution of Rice Bran Polysaccharides and Tween-80 , naturally dryed, then radiated under UVB ultraviolet. It was shown that though the content of tryptophan in hair with protection of rice bran polysaccharides decreased, it was obviously higher compared with the hair besprinkled only with Tween-80, which prove rice bran polysaccharides might have effect on resisting UVB-Ultraviolet.
课题分为以下四部分进行研究:
第一部分,高效液相色谱法同时测定米糠中四种水溶性维生素VB_1,VB_6,烟酸,烟酰胺的含量。在Hypersil C_(18) (250 cm×4.6 mm i.d., 5.0μm)上,以体积比为88:12的0.1%磷酸水溶液-甲醇为流动相,以0.6 mL/min流速等度洗脱,在266 nm波长下检测。各组分在8 min能基线分离。四种维生素在0.6×10~(-6) g/mL-8.5×10~(-5) g/mL范围内呈线性关系,VB1、VB6、烟酸和烟酰胺的检出下限分别为0.43×10~(-6) g/mL、0.34×10~(-5) g/mL、0.32×10~(-6) g/mL、0.30×10~(-6) g/mL。经简单预处理,可以对米糠和淘米水中的这四种维生素的含量进行测定,平均回收率在96.0%-99.9%之间。该法简便、快速、准确、灵敏,对复杂体系水溶性维生素的测定具有重要的参考价值。
第二部分,研究了微波提取法,热水蒸煮提取法,超声波提取法提取米糠多糖。同时考察了三氯乙酸法,Sevage法去除蛋白质;一步消化法,两步消化法去除淀粉。选取了较优的微波提取法提取米糠多糖,采用改进的苯酚-硫酸法测定米糠多糖含量。实验结果:料液比为1:10在1000 W 50%微波火力条件下提取30 min,提取2次,三氯乙酸法去蛋白质,两步消化法去淀粉,30%醇沉,米糠多糖得率达1.41%。实验所得产品RBPa将被应用于其活性功能的研究中。
第三部分,建立了头发中色氨酸的荧光分析方法,并用该法研究了UVB辐射下头发中色氨酸的变化规律。头发样品以5.5 mol/L氢氧化钠在110℃下水解20 h,在pH10.5的磷酸二氢钠-氢氧化钠缓冲液中,激发波长为280 nm、发射波长为360 nm处测定色氨酸的荧光强度。方法线性范围8.0×10~(-8)-1.75×10~(-6) g/mL,检测限为1.6×10~(-8) g/mL,回收率在96.3-100.3%。头发样品中色氨酸的量随UVB辐射时间增加呈减少趋势,光照28天后,色氨酸的损失率达到18.8%。该法快速,简便,灵敏度高,可推荐作为衡量头发光损伤的定量指标。
第四部分,研究了微波提取法得到的米糠粗多糖RBPa,RBPb应用到头发中的抗UVB辐射效果。将米糠粗多糖-吐温-80模拟发胶喷洒到头发上,自然干燥,置于紫外灯下照射。头发样品以5.5 mol/L氢氧化钠在110℃下水解20 h,在pH10.5的磷酸二氢钠-氢氧化钠缓冲液中,激发波长为280 nm、发射波长为360 nm处测定色氨酸的荧光强度变化,加有多糖保护的头发较对照样品中色氨酸的受破坏量降低。表明米糠多糖具有一定的抗紫外辐射效果。
The research direction of this thesis is analysis of nutrient component in rice bran and studies on its application. Taken rice bran as the research object, HPLC method had been built up to measure water-soluble vitamins in rice bran; Rice Bran Polysaccharides had been abstracted in rice bran ; a study model of ultraviolet damnification of hair had been build and had usedthis model studying the activity Resisting UVB-Ultraviolet
In chapter1, High performance liquid chromatography (HPLC) has been applied for the simultaneous determination of four kinds of water-soluble vitamins including vitamin B1, vitamin B6, nicotinic acid, and nicotinamide in rice bran. The separation was performed on a Hypersil C18 column (250 cm×4.6mm i.d., 5.0μm) with a mobile phase of 0.1% H3PO4- methanol (88:12, V/V) at flow rate of 0.6 mL /min, and detection wavelength was 266 nm. Four vitamins had good linearity in the range of 6.0×10~(-7) g/mL - 8.5×10~(-5) g/mL, with detection limit of 4.3×10~(-7) g/mL, 3.4×10~(-6) g/mL, 3.2×10~(-7) g/mL and 3.0×10~(-7) mg/L for vitamin B1, vitamin B6, nicotinic acid and nicotinamide, respectively. Baseline separation of four vitamins can be achieved within 8 minutes. Without complicated pretreatment, four analytes in rice bran samples, as well as extracts from rice with water, were successfully determined with recoveries between 96.0% and 99.9%. The approach could be applied to determine water-soluble vitamins in complicated samples owing to simplicity, convenience, sensitivity and accuracy.
In chapter 2, extraction methods including microwave extraction, dedoction and ultrasonic extraction had been studied to extract polysaccharides in rice bran. The methods for removing proteins by trichloroacetic acid and Sevage method, as well as for removing amylum by one step assimilation and two steps were also investigatated. Microwave assistant extraction has been applied for extraction polysaccharides in rice bran. The content of polysaccharides were determined by improved vitriol -phenol method. The optimum technics were the material-water ratio of 1:10, microwave assistant extraction for 30 mins for two times under 50% firepower. It was proved thatα-amylase could wipe off amylum effectively by two steps, and trichloroacetic acid method was appropriate to wipe off albuminoid. And the ethanol concentration was 30%, the extraction rate of the water-soluble polysaccharides of rice bran was 1.41%.
In chapter 3, a fluorescence spectrometry method has been developed to determine tryptophan in hair, and applied to evaluate hair damnification under UVB ultraviolet. 5.5 mol L-1 NaOH solution containing 0.5% soluble starch was used for hydrolysis of the hair samples. In pH 10.5 buffer of NaH2PO4-NaOH, the fluorescent intensity of tryptophan was determined at 280 nm as excitation wavelength and 360 nm at emission wavelength. The linear range of tryptophan was from 8.0×10~(-6) g/mL to1.75×10~(-6) g/mL. The detection limit of 1.6×10~(-8) g/mL and the recoveries ranged from 96.3% to 100.3% showed that the method was sensitive and accurate. With the increase of the irradiation time with UVB ultraviolet, tryptophan in hair decreased accordingly. During 28 days irradiation, tryptophan in hair was lost by 18.8%. This method could be recommended to measure ultraviolet damnification of hair.
In chapter 4, rice bran polysaccharides were applied for protection hair from UV damnification. Tryptophan in hair was determined with fluorescence spectrometry method, the contents of tryptophan were applied to evaluate hair damnification due to UVB ultraviolet under protection of rice bran polysaccharides. For simulating fixature, hair was besprinkled with mixture solution of Rice Bran Polysaccharides and Tween-80 , naturally dryed, then radiated under UVB ultraviolet. It was shown that though the content of tryptophan in hair with protection of rice bran polysaccharides decreased, it was obviously higher compared with the hair besprinkled only with Tween-80, which prove rice bran polysaccharides might have effect on resisting UVB-Ultraviolet.
引文
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