牛蒡菊糖的制备、对双歧杆菌的增殖及应用研究
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摘要
牛蒡(Arctium lappa L.)是一种药食同源的两年生草本植物。目前,牛蒡的经济价值主要体现在牛蒡根作为一种出口的新、特蔬菜,但国际市场对出口牛蒡规格要求较高,造成等外根(次品)牛蒡资源的浪费。牛蒡的肉质根含有丰富的菊糖。菊糖(inulin)由果糖分子通过β-(2→1)键连接,聚合程度从2-70,一般平均为10。菊糖具有多种优良的功能作用,可作为质构改良剂、稳定剂、抗冻剂和抗老化剂应用于食品中。本论文利用等外牛蒡根为原料,研究牛蒡菊糖的提取分离技术,并对牛蒡菊糖的分子结构进行确定;研究牛蒡菊糖对双歧杆菌体的增殖作用;另外,对菊糖在低脂乳体系中的应用进行了探讨。主要研究内容如下:
牛蒡菊糖的提取和纯化工艺的研究。以等外牛蒡根为原料,通过响应面分析优化了牛蒡菊糖的提取工艺,确定了菊糖提取的最佳工艺条件,即:提取温度80℃,提取时间114min,固液比1:15,连续提取两次。在此条件下,菊糖得率可达93.86%。对牛蒡菊糖脱蛋白、脱色方法进行比较,结果表明采用离子交换树脂效果较好。牛蒡菊糖粗品经Sephadex G-50葡聚糖凝胶柱层析分离纯化后得到纯品,纯度为99.47%。
牛蒡菊糖的结构分析。通过气相色谱、红外光谱、~1H及~(13)C NMR波谱分析表明,牛蒡菊糖主要是由12个呋喃型的果糖以β-(2→1)糖苷键相连,末端1个吡喃型的葡萄糖以α-(1→2)糖苷键连接到果糖上的线性直链结构。经高效凝胶色谱分析表明,菊糖的相对分子量为2168,聚合度约为13。
牛蒡菊糖对双歧杆菌体外生长的促进作用研究。结果表明,牛蒡菊糖对双歧杆菌有明显增殖作用。牛蒡菊糖作为双歧杆菌培养基的碳源要有适宜的浓度,以添加1.0%的浓度增殖效果最好。对双歧杆菌生长曲线的研究表明,以牛蒡菊糖为碳源培养的双歧杆菌的生长速率比以乳糖为碳源培养的双歧杆菌生长速率快。双歧杆菌发酵牛蒡菊糖同时,使培养液的pH值降低。不同聚合度的果聚糖对双歧杆菌生长影响的研究表明,双歧杆菌能够利用短链的菊糖(牛蒡菊糖和菊苣菊糖)和低聚果糖,但是不能够利用高聚合度的菊糖(长链菊糖)。
牛蒡菊糖对肠道菌群影响的研究。小鼠体内实验表明,牛蒡菊糖对肠道益生菌-双歧杆菌和乳酸杆菌生长有增殖作用。研究表明,双歧杆菌能够利用短链的菊糖和低聚果糖,但是不能够利用长链菊糖。聚合度是决定双歧杆菌能否利用果聚糖的主要因素。在益生元(短链的菊糖和低聚果糖)增殖肠道益生菌的同时,对肠球菌和肠杆菌并没有显著的影响。
牛蒡菊糖在低脂乳混合体系中应用的研究。将不同聚合度的果聚糖(牛蒡菊糖、长链菊糖和低聚果糖)添加到脱脂乳体系中,结果表明,含有8%和10%(w/w)长链菊糖的样品中,可以看到轻微的沉淀,这是由于长链菊糖在溶液中形成的菊糖结晶聚集所产生的。对卡拉胶-牛奶-菊糖混合体系的流变研究表明,加入卡拉胶的体系,均为弱凝胶体系,即储能模量(G′)＞损耗模量(G″)。加入果聚糖后,对体系的流变特征也产生一定的影响。对玉米淀粉-牛奶-菊糖混合体系的研究表明,当淀粉浓度由3%增加到4%时,由于分散相中淀粉颗粒体积的增加,表观黏度显著增加。玉米淀粉-牛奶-菊糖混合体系的流变研究表明,低淀粉浓度,体系液体特征更明显;高淀粉浓度时,体系弱凝胶的特征更明显。但是长链菊糖在不同浓度淀粉体系中,对体系的影响与牛蒡菊糖有所不同。
Burdock(Arctium Lappa L.) is a kind of biyearly vegetable with food or medicine properties.At present,burdock's economic value depends on burdock root exported to Japan and other European countries as edible vegetable.While international markets rigidly control the quality of exported burdock root,so the substandard root would be discarded. Burdock root resource could not be made full use.The fresh root of burdock is rich in inulin. Inulin is formed with some fructose connected by the bondβ(2→1) which terminal is the unit of glucose.The degree of polymerization is from 2 to 70,generally,the average degree is 10.Inulin has many excellent functions and has been successfully applied in food industry as textural improver,stabilizer,antifreezer or anti-ageinger.This paper firstly studied the extraction technique and structure of burdock inulin,and then analyzed its effects on the growth of bifidobacteria in vitro and in vivo,finally discussed the potential of using burdock inulin into low-fat milk system.The main contents are as follows:
The extraction and purification techniques of burdock inulin by means of substandard burdock roots were studied.To optimize inulin extraction,various combinations of time, temperature,and solvent:solid ratio were used.The Response Surface Methodology(RSM) was used to analyze the data set.The optimal conditions were as follows:temperature at 80℃,extracting time for 114 min,solid:solvent ratio 1:15 m/V,times of continual extraction 2,the extraction rate was 93.86%.Deproteinized and decolored by ion-exchange resin(Amberlyste IRA-94 resin),separated and purified by Sephadex G-50 column chromatography from burdock substandard roots,pure sample was obtained.The purity was 99.47%.
The structures of the inulin had been characterized by several analytical methods:gas chromatography,FT-IR spectrum and ~1H NMR,respectively,as well as ~(13)C NMR analysis. The results showed that the burdock inulin were composed of fructose and glucose,which were confirmed by the composition of twelve fructose residues linked byβ-(2→1) glucoside bond and one glucose residue linked byα-(1→2) glucoside bond at the end of linear straight sugar chain.The average molecular weight(Mw) of inulin was 2168 Da and its degree of polymerization(DP) value was 13.
The growth-promoting action of burdock inulin(B-INU) on bifidobacteria was studied in vitro.The results showed that burdock inulin was an effective bifidobacteria growth-promoting factor,and the strain of tested bifidobacteria grew significantly better in burdock inulin-containing media with 1.0%B-INU as the carbohydrate resource than in sucrose-containing media with 1.0%sucrose as the carbohydrate resource(P＜0.05). Bifidobacteria is an obligate anaerobe.With this kind of germ,varieties of carbohydrate could be fermented to form acetic acid and lactic acid.As a result,the pH value of the media was decreased.Bifidobacteria strains studied utilized fructooligosaccharides(FOS) and B-INU,as well as chicory inulin(C-INU).Long chain inulin(L-INU) did not enhance the growth of bifidobacteria significantly.
To investigate the prebiotic potential of burdock inulin,the in vivo effects of B-INU on bacterial growth were studied.In vivo,B-INU significantly increased the number of lactobacilli and bifidobacteria(P＜0.05) in cecal content.Mice fed with B-INU,C-INU and FOS for 14 days had greater number of cecal beneficial bacteria population than those fed with L-INU for 14 days.The results indicated that utilization of inulin by bifidobacteria depended on the degree of polymerization of fructo-oligomeric chains.Feeding of a diet containing a prebiotic resulted in the stimulation on the growth of various bacterial groups, in particular acidogenic bacteria such as bifidobacteria and lactobacilli.Enterobacteria and Enterococci counts were not significantly affected by the different diets.
The effect of inulin on the flow behavior of low fat milk system was investigated.After cooling and storage in refrigeration during 24 h,no visual change in the samples was observed,except for the samples with 8%and 10%(w/w) L-INU.It was probable that the sedimentation observed in the above mentioned samples was due to a recrystallization of the inulin.Therefore,the inulin concentration of 6%was chosen.Flow behaviors of low fat milk-inulin system with or without K:-carrageenan consisted of different inulin type:FOS, B-INU and L-INU were studied.All of the samples withκ-carrageenan showed typical characteristics of a weak gel system:the storage modulus was higher than the loss modulus (G'＞G") throughout the whole range of frequency.Low fat milk-inulin-κ-carrageenan systems with different types of inulin also affected the flow behavior of the system.Flow behaviors of corn starch-milk-inulin(CMI) pastes consisted of different starch levels(3% and 4%) and inulin type:FOS,B-INU and L-INU during heating to 95℃were studied.The registered apparent viscosity values were higher with the high starch concentration(4%) than that with the low starch concentration(3%),due to the increase in the volumetric fraction of the starch granules in the dispersion.The CMI pastes showed shear-thinning flow behavior in both starch concentrations and various inulin types as well as throughout the shear rates studied,resulting from stress-induced breakdown of the network structure. When starch concentration was increased,B-INU containing CMI showed higher viscosity than L-INU containing CMI did.Therefore,increase in viscosity of CMI due to increase in starch concentration was also dependent on the different type of inulin.Higher starch concentration increased both storage modulus and complex viscosity values.
牛蒡菊糖的提取和纯化工艺的研究。以等外牛蒡根为原料,通过响应面分析优化了牛蒡菊糖的提取工艺,确定了菊糖提取的最佳工艺条件,即:提取温度80℃,提取时间114min,固液比1:15,连续提取两次。在此条件下,菊糖得率可达93.86%。对牛蒡菊糖脱蛋白、脱色方法进行比较,结果表明采用离子交换树脂效果较好。牛蒡菊糖粗品经Sephadex G-50葡聚糖凝胶柱层析分离纯化后得到纯品,纯度为99.47%。
牛蒡菊糖的结构分析。通过气相色谱、红外光谱、~1H及~(13)C NMR波谱分析表明,牛蒡菊糖主要是由12个呋喃型的果糖以β-(2→1)糖苷键相连,末端1个吡喃型的葡萄糖以α-(1→2)糖苷键连接到果糖上的线性直链结构。经高效凝胶色谱分析表明,菊糖的相对分子量为2168,聚合度约为13。
牛蒡菊糖对双歧杆菌体外生长的促进作用研究。结果表明,牛蒡菊糖对双歧杆菌有明显增殖作用。牛蒡菊糖作为双歧杆菌培养基的碳源要有适宜的浓度,以添加1.0%的浓度增殖效果最好。对双歧杆菌生长曲线的研究表明,以牛蒡菊糖为碳源培养的双歧杆菌的生长速率比以乳糖为碳源培养的双歧杆菌生长速率快。双歧杆菌发酵牛蒡菊糖同时,使培养液的pH值降低。不同聚合度的果聚糖对双歧杆菌生长影响的研究表明,双歧杆菌能够利用短链的菊糖(牛蒡菊糖和菊苣菊糖)和低聚果糖,但是不能够利用高聚合度的菊糖(长链菊糖)。
牛蒡菊糖对肠道菌群影响的研究。小鼠体内实验表明,牛蒡菊糖对肠道益生菌-双歧杆菌和乳酸杆菌生长有增殖作用。研究表明,双歧杆菌能够利用短链的菊糖和低聚果糖,但是不能够利用长链菊糖。聚合度是决定双歧杆菌能否利用果聚糖的主要因素。在益生元(短链的菊糖和低聚果糖)增殖肠道益生菌的同时,对肠球菌和肠杆菌并没有显著的影响。
牛蒡菊糖在低脂乳混合体系中应用的研究。将不同聚合度的果聚糖(牛蒡菊糖、长链菊糖和低聚果糖)添加到脱脂乳体系中,结果表明,含有8%和10%(w/w)长链菊糖的样品中,可以看到轻微的沉淀,这是由于长链菊糖在溶液中形成的菊糖结晶聚集所产生的。对卡拉胶-牛奶-菊糖混合体系的流变研究表明,加入卡拉胶的体系,均为弱凝胶体系,即储能模量(G′)＞损耗模量(G″)。加入果聚糖后,对体系的流变特征也产生一定的影响。对玉米淀粉-牛奶-菊糖混合体系的研究表明,当淀粉浓度由3%增加到4%时,由于分散相中淀粉颗粒体积的增加,表观黏度显著增加。玉米淀粉-牛奶-菊糖混合体系的流变研究表明,低淀粉浓度,体系液体特征更明显;高淀粉浓度时,体系弱凝胶的特征更明显。但是长链菊糖在不同浓度淀粉体系中,对体系的影响与牛蒡菊糖有所不同。
Burdock(Arctium Lappa L.) is a kind of biyearly vegetable with food or medicine properties.At present,burdock's economic value depends on burdock root exported to Japan and other European countries as edible vegetable.While international markets rigidly control the quality of exported burdock root,so the substandard root would be discarded. Burdock root resource could not be made full use.The fresh root of burdock is rich in inulin. Inulin is formed with some fructose connected by the bondβ(2→1) which terminal is the unit of glucose.The degree of polymerization is from 2 to 70,generally,the average degree is 10.Inulin has many excellent functions and has been successfully applied in food industry as textural improver,stabilizer,antifreezer or anti-ageinger.This paper firstly studied the extraction technique and structure of burdock inulin,and then analyzed its effects on the growth of bifidobacteria in vitro and in vivo,finally discussed the potential of using burdock inulin into low-fat milk system.The main contents are as follows:
The extraction and purification techniques of burdock inulin by means of substandard burdock roots were studied.To optimize inulin extraction,various combinations of time, temperature,and solvent:solid ratio were used.The Response Surface Methodology(RSM) was used to analyze the data set.The optimal conditions were as follows:temperature at 80℃,extracting time for 114 min,solid:solvent ratio 1:15 m/V,times of continual extraction 2,the extraction rate was 93.86%.Deproteinized and decolored by ion-exchange resin(Amberlyste IRA-94 resin),separated and purified by Sephadex G-50 column chromatography from burdock substandard roots,pure sample was obtained.The purity was 99.47%.
The structures of the inulin had been characterized by several analytical methods:gas chromatography,FT-IR spectrum and ~1H NMR,respectively,as well as ~(13)C NMR analysis. The results showed that the burdock inulin were composed of fructose and glucose,which were confirmed by the composition of twelve fructose residues linked byβ-(2→1) glucoside bond and one glucose residue linked byα-(1→2) glucoside bond at the end of linear straight sugar chain.The average molecular weight(Mw) of inulin was 2168 Da and its degree of polymerization(DP) value was 13.
The growth-promoting action of burdock inulin(B-INU) on bifidobacteria was studied in vitro.The results showed that burdock inulin was an effective bifidobacteria growth-promoting factor,and the strain of tested bifidobacteria grew significantly better in burdock inulin-containing media with 1.0%B-INU as the carbohydrate resource than in sucrose-containing media with 1.0%sucrose as the carbohydrate resource(P＜0.05). Bifidobacteria is an obligate anaerobe.With this kind of germ,varieties of carbohydrate could be fermented to form acetic acid and lactic acid.As a result,the pH value of the media was decreased.Bifidobacteria strains studied utilized fructooligosaccharides(FOS) and B-INU,as well as chicory inulin(C-INU).Long chain inulin(L-INU) did not enhance the growth of bifidobacteria significantly.
To investigate the prebiotic potential of burdock inulin,the in vivo effects of B-INU on bacterial growth were studied.In vivo,B-INU significantly increased the number of lactobacilli and bifidobacteria(P＜0.05) in cecal content.Mice fed with B-INU,C-INU and FOS for 14 days had greater number of cecal beneficial bacteria population than those fed with L-INU for 14 days.The results indicated that utilization of inulin by bifidobacteria depended on the degree of polymerization of fructo-oligomeric chains.Feeding of a diet containing a prebiotic resulted in the stimulation on the growth of various bacterial groups, in particular acidogenic bacteria such as bifidobacteria and lactobacilli.Enterobacteria and Enterococci counts were not significantly affected by the different diets.
The effect of inulin on the flow behavior of low fat milk system was investigated.After cooling and storage in refrigeration during 24 h,no visual change in the samples was observed,except for the samples with 8%and 10%(w/w) L-INU.It was probable that the sedimentation observed in the above mentioned samples was due to a recrystallization of the inulin.Therefore,the inulin concentration of 6%was chosen.Flow behaviors of low fat milk-inulin system with or without K:-carrageenan consisted of different inulin type:FOS, B-INU and L-INU were studied.All of the samples withκ-carrageenan showed typical characteristics of a weak gel system:the storage modulus was higher than the loss modulus (G'＞G") throughout the whole range of frequency.Low fat milk-inulin-κ-carrageenan systems with different types of inulin also affected the flow behavior of the system.Flow behaviors of corn starch-milk-inulin(CMI) pastes consisted of different starch levels(3% and 4%) and inulin type:FOS,B-INU and L-INU during heating to 95℃were studied.The registered apparent viscosity values were higher with the high starch concentration(4%) than that with the low starch concentration(3%),due to the increase in the volumetric fraction of the starch granules in the dispersion.The CMI pastes showed shear-thinning flow behavior in both starch concentrations and various inulin types as well as throughout the shear rates studied,resulting from stress-induced breakdown of the network structure. When starch concentration was increased,B-INU containing CMI showed higher viscosity than L-INU containing CMI did.Therefore,increase in viscosity of CMI due to increase in starch concentration was also dependent on the different type of inulin.Higher starch concentration increased both storage modulus and complex viscosity values.
引文
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