酵母微胶囊包埋茶多酚工艺研究

主要对酵母微胶囊包埋茶多酚的工艺展开研究。通过正交试验考察了芯材比、包埋温度、包埋时间和加水量等因素对微胶囊化茶多酚的影响。结果表明,以培养的酿酒酵母作包埋壁材,当芯材比4:1(茶多酚/酵母,g/g)、包埋温度30℃、包埋时间1 h,以1 mL蒸馏水为包埋介质时,包埋率最大,为35.71%;以市售的干酿酒酵母作包埋壁材,当芯材比为4:1(茶多酚/酵母,g/g)、包埋温度20℃、包埋时间2 h,以1 mL蒸馏水为包埋介质时,包埋率最大,为27.16%。光学显微镜观察显示酵母细胞包埋茶多酚后细胞的色泽发生变化;荧光显微镜观察显示微胶囊内有茶多酚的自发明亮绿色荧光;扫描电镜观察显示干瘪的酵母细胞包埋茶多酚后呈饱满的球形、表面光滑,表明茶多酚已被酵母细胞成功包埋。     

海藻酸钠-壳聚糖-海藻酸钠液芯微胶囊的传质及生物微囊化

制备海藻酸钠-壳聚糖-海藻酸钠(ACA)液芯微胶囊,考察了小分子物质从溶液向ACA液芯微胶囊内的传质性能并计算扩散系数;确定ACA液芯微胶囊膜的截留分子量。比较游离培养、固芯微囊化和液芯微囊化培养对酿酒酵母细胞生长的影响,并考察细胞在液芯微胶囊内的代谢情况。结果表明,小分子物质进入ACA液芯微胶囊的扩散系数(mm/min)为谷氨酸(0.0126)、L-苯丙氨酸(0.0110)、葡萄糖(0.0071)和乳酸(0.0049),膜的截留分子量为2000。液芯微胶囊中细胞的生长速度和密度均优于固芯微胶囊,液芯微囊化酵母细胞能够进行多批次连续培养,产量稳定。     

利用酵母细胞对薄荷油进行微胶囊化的研究

选用酵母细胞为微胶囊壁材,对制备薄荷油微胶囊的工艺条件进行了研究,采用单因素试验探索芯壁比、包埋温度、包埋时间、加水量等微胶囊化条件对薄荷油包埋率的影响,在此基础上采用响应面分析法优化微胶囊化条件,得到具有显著性的拟合回归方程。试验结果表明:酵母细胞对薄荷油进行微胶囊化的最优条件为:包埋温度48℃,包埋时间6 h,加水量13 m L/g。在此条件下得到薄荷油的最优包埋率为57.33%,与预测值十分接近,薄荷油已被包入酵母壁材内。     

酵母细胞微胶囊化丁香油的研究

以干啤酒酵母细胞为壁材制备丁香油微胶囊。通过正交试验考察了包埋温度、包埋时间、心材比等因素对微胶囊化丁香油的影响。结果表明,选择包埋温度70℃、包埋时间9h和心材比为1ml/g的条件比较适宜,微胶囊中丁香油包埋率可达41.26%。通过显微镜观察,微胶囊呈球形,大小均一,细胞质内充满亮亮的精油。通过馒头防腐实验,表明丁香油微胶囊具有一定的缓释抑菌效果。     

绿原酸酵母细胞微胶囊工艺研究

采用酿酒酵母的细胞壁包埋水溶性的绿原酸,通过正交试验考察包埋温度、包埋时间、芯材比和加水量因素对绿原酸微胶囊包埋率的影响。结果表明,在包埋温度40℃、包埋时间6h、芯材比3:1(g/g),以6mL 蒸馏水为包埋介质的组合条件下,包埋率最大为18.9%。微胶囊红外光谱分析发现绿原酸的特征官能团的振动消失;荧光显微镜观察显示微胶囊呈球形,微胶囊内的绿原酸自发明亮的荧光;高效液相色谱定性分析显示,绿原酸包埋前后保留时间及紫外扫描图谱相一致。研究结果表明绿原酸成功的包埋到酵母细胞壁内,且在包埋过程中没有发生任何化学变化。     

百里香黄酮微胶囊的制备及抑菌活性研究

利用酵母细胞制备百里香黄酮微胶囊,研究芯壁比、包埋温度以及包埋时间对百里香黄酮包埋率的影响,用Box-Behnken试验设计及响应面对百里香黄酮微胶囊的制备工艺进行分析优化。结果显示:百里香黄酮微胶囊的最佳制备条件为芯壁比3∶1,包埋时间6.5h,包埋温度36℃,百里香黄酮微胶囊包埋率平均值为(68.50±0.36)%(n=3)。百里香黄酮微胶囊具有较好的抑菌作用。     

猕猴桃籽油微胶囊在Caco-2单细胞层的吸收特性

以玉米肽为壁材,猕猴桃籽油为芯材,吐温-20为乳化剂,采用喷雾干燥法制备猕猴桃籽油微胶囊。MTT试验结果表明,当微胶囊浓度≤400μg/mL时,其对Caco-2细胞生长无明显抑制作用,且质量浓度为100~400μg/mL的微胶囊对细胞的生长有促进作用。对微胶囊在Caco-2单细胞层的吸收特性进行研究,结果表明,微胶囊在高、中、低3个浓度时,微胶囊的生物利用率均高于25%。当质量浓度为100μg/mL时,微胶囊的生物利用率达到最高,为41.70%±1.65%。微胶囊溶液从AP侧到BL侧的表观渗透系数Papp在1×10^-7~1×10^-6cm·s^-1之间,吸收水平较好。     

杏仁油的超临界CO2萃取及微胶囊的制备

采用超临界CO2萃取杏仁油,以萃取所得杏仁油为囊芯,探讨利用干酵母细胞作为囊壁材料制备微胶囊的可行性,通过正交试验考察了包埋温度、包埋时间、杏仁油与干酵母配比(芯壁材比)对微胶囊化杏仁油的影响.试验结果表明,在60℃和35 MPa萃取条件下,萃取率可达0.408 g/g杏仁;气相色谱分析结果显示,杏仁油中主要脂肪酸为油酸和亚油酸;在包埋温度75℃、包埋时间7 h和芯壁材比为1:1(w/w)的条件下,杏仁油包埋率达到45.76%,微胶囊化处理后杏仁油氧化稳定性显著增强.经最佳工艺制成的杏仁油微胶囊产品的颗粒外形较圆整,大小分布均匀,表面光滑.这种新型的微胶囊化方法,具有制备过程简单、包埋率高和不引入有机溶剂的优点.     

玉米醇溶蛋白微胶囊的制备及缓释性能

以玉米醇溶蛋白为壁材,5-氟尿嘧啶为芯材,用涂膜粉碎法和喷雾干燥法制备了微胶囊.研究了制备方法对制备的微胶囊形态和粒径大小的影响;探讨了制备方法(甘油添加量、制备温度)对包埋率和载药量的影响.结果表明,当壁材与芯材质量比为5:3时,涂膜粉碎法制备的微胶囊包埋率和载药量最大,分别为84.8％和31.8％,且大于喷雾干燥法制备的微胶囊.添加20％(质量分数)甘油制备的微胶囊的包埋率和载药量低于没有添加甘油制备微胶囊,但其缓释效果比没有加甘油的明显.此外,随着制备温度(60℃～80℃)的升高,微胶囊的包埋率和载药量也随着增加,80℃时的包埋率和载药量最高.经SEM观察涂膜粉碎法和喷雾干燥法制备的微胶囊粒径分别在100 μm～180 μm和7.μm～13μm之间.人工胃液和人工肠液中的缓释性能的研究发现,涂膜粉碎法制备的Zein微胶囊的累积释药量要比喷雾干燥法制备的微胶囊的释药量少,缓释效果好.人工胃液中Zein微胶囊壁材更容易裂解,缓释效果低于在人工肠液中的缓释.     

响应面法优化柑橘复合生物保鲜剂微胶囊工艺

利用响应面法对柑橘复合生物保鲜剂的微胶囊化工艺进行研究。探讨不同壁材、壁材配比、壁芯比和固形物质量分数对柑橘复合生物保鲜剂包埋率的影响,在单因素基础上,采用响应面分析方法,进一步研究各自变量及其交互作用对复合生物保鲜剂包埋率的影响,并对其工艺参数进行优化。结果表明:柑橘复合生物保鲜剂微胶囊的最佳工艺条件为壁材配比2.50 g/g、芯壁比3.00 g/g、固形物质量分数25.0%,在此最佳工艺条件下,柑橘复合生物保鲜剂的包埋率达87.21%。此微胶囊化的复合生物保鲜剂,能保护保鲜剂的活性物质,提高柑橘的保鲜时间和效果,有望用于柑橘贮藏保存和保鲜的工业化生产中。     

温度调节对克鲁氏假丝酵母海藻糖代谢的影响

研究了恒温条件和热冲击条件对克鲁氏假丝酵母海藻糖代谢的影响。结果表明,将指数生长期的克鲁氏假丝酵母细胞置于恒定的45℃和25℃,均不利于细胞生长及海藻糖的合成,而适宜的热冲击能够迅速促进细胞海藻糖的合成。在正常的生长状态下,处于指数生长期的克鲁氏假丝酵母细胞内只积累少量的海藻糖,但此时当细胞受到外界热冲击时海藻糖会大量积累。在热冲击结束后,海藻糖的含量又会恢复至对照水平。周期性热冲击实验表明,随着指数生长期的延续,这种应激性反应逐渐减弱,在3个冲击周期结束时细胞内海藻糖的含量分别是对照的3·9,3及1·6倍。热冲击的温度和持续时间对细胞生长和海藻糖的积累都有影响,过高的温度及过长时间的热冲击均会使细胞生长受到抑制。最佳的热冲击温度为45℃,最佳热冲击持续时间为1h。     

Microencapsulated Starter Culture During Yoghurt Manufacturing,Effect on Technological Features

The potential of living cell microencapsulation in sustaining cells’ viability, functionality and targeted release in gastrointestinal tract is relatively well documented. Differently, the effects exerted by the capsules on cell metabolic activities during fermentation of a food matrix as well as on cell physiology are poorly addressed. This paper aimed at studying the effects of chitosan-alginate capsules (matrix and core-shell) on metabolic activities of Streptococcus thermophilus and probiotic Lactobacillus delbrueckii during milk fermentation for yoghurt production. This food system has been used to monitor growth, acidification kinetics and strain proteolytic activity. Bacterial viability has been monitored during yoghurt storage at 4 °C for 28 days and an in vitro digestion to evaluate the protective effect exerted by the capsules. Furthermore, production of volatile metabolites associated with starter culture activity was monitored by headspace solid-phase microextraction-GC/MS to explore possible influence of microenvironment on cell metabolism. Results indicate that both kinds of capsules influenced at different extent cell functionalities (growth, acidification and proteolysis), while they improve cell viability during yoghurt storage and simulated gastrointestinal passage. The volatile pattern revealed that capsules influenced their production in yoghurt: 12 out of 28 volatiles recovered in yoghurt fermented by free and encapsulated starters had significantly different concentration. However, concentration of the main aroma constituents (e.g. acetaldehyde, diacetyl, acetoin) was not significantly affected. Due to the leakage of bacteria from microcapsules during fermentation, the final product resulted in co-existing of free and still encapsulated cells, with the main advantage of an increased viability during yoghurt storage and simulated digestion of the encapsulated counterpart.     

Effect of mixing time on taxoid production using suspension cultures of Taxus chinensis in a centrifugal impeller bioreactor

The effects of fluid mixing on the cell growth and secondary metabolite production of plant cells were investigated in a low-shear centrifugal impeller bioreactor (CIB) system. Suspension cultures of Taxus chinensis cells producing taxuyunnanine C (Tc), a physiologically active secondary metabolite, were used as a model system for this investigation. The mixing time (t(m)) and volumetric oxygen transfer coefficient (k(L)a) in the bioreactor were characterized at various cell densities and operating conditions. A constant t(m) of 5 s or 10 s was maintained during cultivation by adjusting the impeller agitation speed with no detrimental effect on the cultured cells. A higher cell density, Tc content and total Tc production were obtained under the shorter mixing time of 5 s. The favorable effect of more rapid mixing on Tc production was also confirmed when the Tc accumulation was significantly increased through culture elicitation using 100 microM methyl jasmonate (MJA). The lower Tc production at the longer t(m) of 10 s was mainly attributed to oxygen transfer limitation in the dead zones and larger cell aggregates resulting from poor mixing.     

Evaluation of attachment and growth of anchorage-dependent cells on culture surfaces with type I collagen coating

The effects of coating the culture surface with bovine type I collagen on the culture properties of anchorage-dependent cells were investigated. When human fibroblasts were cultured on a surface coated with collagen at 5.8 x 10(-3) mg/cm2, cell attachment and subsequent cell growth were both enhanced compared to the culture on an uncoated surface. The degrees of cell attachment and growth enhancement were numerically characterized using the time constant of cell adhesion (tau) and doubling time (t(d)) as kinetic parameters. These parameters applied to cultures of human keratinocytes and rabbit chondrocytes allowed the effects of collagen coating on the respective culture properties of both types of cells to be evaluated. In addition, the relative parameters R(tau) and R(t(d)) (defined as the ratios of the tau and t(d) values at a given collagen concentration against those without collagen coating, respectively) were employed to estimate the effects of collagen based on a standardized criterion. Similar R(tau) and R(t(d)) profiles were obtained for collagen concentrations ranging from 5.8 x 10(-13) to 5.8 x 10(-3) mg/cm2, whether the cells were fibroblasts, keratinocytes or chondrocytes. It was also revealed that coating the surface with collagen at a concentration over 5.8 x 10(-7) mg/cm2 led to reductions in both the R(tau) and R(t(d)) values, i.e. the promotion of cell attachment and growth, in the culture of each type of cells examined.     

紫苏细胞悬浮培养生产迷迭香酸条件研究

通过诱导紫苏下胚轴和子叶外植体产生愈伤组织,建立细胞悬浮培养体系,以提高细胞产量及细胞中迷迭香酸含量。结果表明,在MS液体培养上添加3.0mg/L 6-芐氨基嘌呤(6-BA)＋0.3mg/L萘乙酸(NAA)培养基上,下胚轴愈伤组织呈嫩黄色松散状态,出愈时间短,出愈率可达100%。紫苏细胞悬浮培养产生迷迭香酸的最佳条件为培养时间7d、接种量鲜质量浓度20g/L、摇床转速110r/min、蔗糖30g/L、L-苯丙氨酸0.15g/L,此条件下可获得高达2.283mg/g的迷迭香酸。     

大花金挖耳细胞悬浮系的建立及黄酮类化合物的产生

研究不同培养基、蔗糖质量浓度、pH 值、接种量、NAA、6-BA、VB1 及培养时间对大花金挖耳细胞生长和黄酮类化合物合成的影响。结果表明：NT 液体培养基在pH5.5、蔗糖质量浓度40g/L、接种量40g/L 鲜质量细胞、NAA 1.0mg/L+6-BA 0.2mg/L 时有利于细胞的生长和黄酮类化合物合成,向NT 培养基中添加1.0～4.0mg/LVB1 有抑制细胞褐化的作用,大花金挖耳悬浮细胞的生长及黄酮类化合物合成随培养时间的延长,表现出先升高后降低的趋势,在接种15～21d 后,细胞生物量可达23.54～24.51g/L,黄酮类化合物得率为1.19%～1.23%。     

Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels

Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 mum after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (mu). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.     

Pentanoic acid, a novel protein synthesis stimulant for Chinese Hamster Ovary (CHO) cells

Nine carboxylic acids were tested to evaluate their effects on recombinant fusion protein production and cell growth of Chinese Hamster Ovary (CHO) cells. Pentanoic acid was demonstrated to have the highest enhancement effect on the protein biosynthesis of CHO cells among the acids tested. Pentanoic acid also had less growth suppression effects compared with butyrate. The optimal induction time and concentration of pentanoic acid for a 120-h batch culture were 72 h and 1 mM, respectively. Apoptosis (programmed cell death) was observed in the serum-free batch culture of CHO cells using a cell death detection ELISA kit. The addition of butyrate accelerated the rate of apoptosis of CHO cells whereas the addition of pentanoate did not. These results confirmed that pentanoic acid was a better stimulant for protein biosynthesis in animal cell culture than butyrate.     

Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract

We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect blastocyst development. Overall, transfer of eight blastocysts produced using roscovitine-treated donor cells and our standard activation protocol yielded three pregnancies, of which two (25% of transferred embryos) resulted in delivery of viable foals. Flow cytometric evaluation showed that roscovitine treatment significantly increased the proportion of cells classified as small, in comparison to growth to confluence or serum deprivation, but did not significantly affect the proportion of cells in G0/G1 (2N DNA content). Transfer of one blastocyst produced using roscovitine-treated donor cells, with addition of roscovitine injection at activation, yielded one pregnancy which was lost before 114 days' gestation. Transfer to recipients of two blastocysts produced using confluent donor cells with addition of cycloheximide at activation gave no resulting pregnancies. We conclude that roscovitine treatment of donor cells yields equivalent blastocyst production after nuclear transfer to that for confluent donor cells, and that direct injection of roscovitine-treated donor cells, followed by activation using sperm extract, is compatible with efficient production of viable cloned foals.     

Cell behavior analysis to evaluate proliferative potentials of human lymphocytes expanded and activated for therapeutic use

The cluster designation 3-lymphokine activated killer (CD3-LAK) culture was conducted using human lymphocytes obtained from different donors. It was found that donor-dependent variances existed in terms of lag time and minimum doubling time, which were process parameters for comprehending the proliferative potentials of cells in an early phase with weak growth and in a subsequent phase with active growth, respectively. To correlate these variances with culture performances, cellular behaviors were estimated by constructing a custom-made observation tool that can capture and process images of culture surfaces. The tool enabled us to determine time-lapse changes in an average projected cell area and the frequency of cell aggregates during the culture in a noninvasive manner. It was found that linear relationships were obtained both between lag time and average projected cell area, and between minimum doubling time and formation rate of cell aggregates, irrespective of culture performance fluctuation depending on each donor state. It is concluded that the developed tool is helpful for operating CD3-LAK culture while monitoring the state of human lymphocytes.