重组大肠杆菌产β-胡萝卜素的发酵条件

对重组大肠杆菌产β-胡萝卜素发酵条件进行了研究．在M9培养基中添加Span-200．7g／L有利于细胞生长和β-胡萝卜素表达，初始pH值6．0、接种体积分数5％、发酵温度28℃、抗生素氨苄青霉素质量浓度80μg／mL和氯霉素质量浓度40μg／mL为最佳发酵条件，在7L发酵罐进行发酵动力学试验表明，重组菌最适发酵周期为22h，细胞干重最高可达1．55g／L，β-胡萝卜素表达量可达0．75μg／mL。     

L-丝氨酸产生菌的分离筛选及发酵条件

在含50mL／L的甲醇、5g／L的甘氨酸的基本培养基平板上，从土壤中分离出130多株细菌，测定了它们利用甘氨酸生产L-丝氨酸的能力，获得了一株产L-丝氨酸较好的菌株A3，L-丝氨酸产量最高为1．4g／L，分类学鉴定为微球菌科(Micrococcaceae)，对菌株A3利用甘氨酸发酵生产L-丝氨酸的发酵条件进行了初步研究，包括甘氨酸和甲醇添加量，不同碳源、氮源，初始pH值以及发酵时间对产酸的影响．结果表明，菌株A3在不添加甲醇、只以甘氨酸为惟一碳源的发酵培养基中也能产L-丝氨酸，培养基中较高质量浓度的甘氨酸存在对L-丝氨酸生产是必需的．添加葡萄糖、甘油等外源碳源对菌体生长有利但不利于L-丝氨酸的生产。     

阿片肽酿酒酵母工程菌摇瓶发酵工艺

研究了发酵培养基成分、补加方式及外源氨基酸对酿酒酵母发酵和表达的影响．结果表明，发酵前在液体发酵培养基中一次性补加酵母抽提物（YE）有利于目的产物的表达，发酵过程中补加YE不利于目的产物的表达，发酵中后期补加适量的葡萄糖有利于阿片肽的表达．添加外源氨基酸对酿酒酵母工程菌的生长有一定的促进作用，而且可以明显地影响阿片肽的表达．通过正交实验对酿酒酵母生长和表达条件进行优化，确定最优工艺参数为：培养基中酵母抽提物质量浓度为25g／L，C：N比2．8，培养基初始PH5．0，摇床转速220r／min．在最优条件下研究了酿酒酵母表达目的产物的情况，总蛋白质质量浓度为614．50mg／L．SDS-PAGE电泳和双波长凝胶扫描结果表明：目的阿片肽约占总分泌蛋白质的5％，其表达量为30．7mg／L，是优化前的1．49倍。     

羊肚菌液体深层发酵条件

以羊肚菌为材料，研究了发酵过程中碳源、氮源、无机盐、培养条件对菌丝体生物量、胞外多糖的影响，以及发酵过程中菌丝体生物量、胞外多糖、总糖及还原糖质量浓度、培养基PH值的动态变化，并在此基础上确定了羊肚菌液体深层发酵的最佳条件。结果表明：羊肚菌液体深层发酵的最优培养基配比为：玉米粉4．0g／dL、葡萄糖1．0g／dL、黄豆粉2．0g／dL、酵母粉0．3g／dL、KH2PO4 0．2g／dL、MgSO4 0．1g／dL、CaSO4 0．1g／dL；最优培养条件为：24℃，起始pH5．8，250mL的摇瓶装液量为100mL，接种量10mL，摇瓶转速140r／min，发酵时间为108h。     

法夫酵母产虾青素的补料发酵

以法夫酵母 (Phaffiarhodozyma)WSS FF6为产生菌进行产虾青素的补料发酵 ,在通气量为 2 5 0L/h、pH =6.0± 0 .5的条件下 ,先流加高糖浓度的培养基 ,后添加 0 .1%乙醇 ,进行分批补料发酵 ,经 130h发酵后 ,生物量与类胡萝卜素产量分别为 2 7.4mg/mL、2 6.12 μg/mL ,生长得率、产物得率及酵母色素质量分数分别为 0 .4 6、0 .4 4和 0 .95     

Thermobifida fusca WSH03-11突变株合成角质酶的发酵条件

以放线菌Thermobifida fusca WSH03—11——高产角质酶突变株为出发菌株，在摇瓶中考察了碳、氮源等条件以及接种量、装液量和初始pH值等环境条件对突变株细胞生长和产角质酶的影响。通过优化发酵条件，突变菌株的角质酶酶活达10．1U／mL，提高了15％。在此基础上，在5L发酵罐中进一步研究了碳源流加对突变株发酵产酶的影响，结果发现，分别在0、24、48h添加1％乙醇，角质酶酶活达到16．4U／mL，细胞干重（DCW）达到3．7g／L，而且发酵时间也由110h缩短到50h。     

一株曲霉果糖转移酶的发酵条件及转果糖基活性

对一株曲霉果糖转移酶菌株的产酶培养条件进行了研究。确定了最佳培养基组成：初始蔗糖质量浓度15～18g／dL，氮源为酵母膏，K2HPO4对果糖转移酶的产生具有明显的促进作用，添加0．2g／dLCMC能够使果糖转移酶活力提高到原来的1．3倍，在pH5．5，30℃条件下，果糖转移酶最高酶活力为30．42U／mL。HPLC分析结果表明，转糖基产物为总质量的55．8％。     

L-异亮氨酸菌种选育及发酵条件优化

以栖糖蜜棒杆菌（Corynedacterium melassecola）ATCC17965为出发菌株。根据代谢控制发酵原理。经硫酸二乙酯和60^Co诱变处理。定向选育出一株L-异亮氨酸产生菌131-5（SG^r＋LeuME^r＋Leu＋AHV^r＋Suc^g＋Eth^r）。在培养基未经优化的情况下产L-异亮氨酸14-15g／L．同时，考察了培养基及发酵每件对茼株产酸的影响，在优化的培养基和发酵条件下积累L-异亮氨酸19．2g／L．最高时可迭21．3g／L．     

黑曲霉原生质体的制备、再生及转化条件

为了建立原生质体介导的黑曲霉转化系统，研究了菌龄、酶系统、渗透压稳定剂、酶解时间对葡萄糖淀粉酶生产菌株黑曲霉CICIMF0410原生质体形成与再生的影响。结果表明，培养4d的幼嫩菌丝体最适于制备原生质体；综合考虑原生质体形成与再生的情况，作者选用1mol／L山梨醇为最适渗透压稳定剂，1g／dL蜗牛酶-1g／dL纤维素酶-0．1g／dL溶壁酶为最适裂解酶组合，30℃酶解2．5～3h，最适合的原生质体的再生培养基为含0．6mol／LMgSO4的TZ培养基。在PEG和CaCl2存在条件下，以潮霉素B为选择性标记，质粒pBC-Hygro转化原生质体，每微克DNA可获得4～5个转化予。     

放射型根瘤菌WSH2601生产辅酶Q10的摇瓶发酵条件

以放射型根瘤菌 (Rhizobusradiobacterium ,WSH2 6 0 1)作为辅酶Q10 的生产菌株 ,研究了氮源、碳源、接种量、溶氧、初始 pH、发酵温度及添加物等因素对细胞生长与产物辅酶Q10 合成的影响 .结果表明 :玉米浆和酵母膏是较好的氮源 ,葡萄糖与蔗糖是辅酶Q10 发酵的较好的碳源 ,接种量对辅酶Q10 发酵的影响不大 ,为 4 % .适宜的初始 pH值为 7,番茄汁能较好地促进细胞的生长 ;添加玉米浆、L 甲硫氨酸、番茄汁和异戊醇有利于产辅酶Q10 ;溶氧对细胞生长与产物辅酶Q10 合成的影响较显著 .通过正交试验初步确定了发酵条件 :碳源为 1.5 g/dL葡萄糖和 2 .5 g/dL蔗糖混合物 ,酵母膏 0 .8g/dL ,初始pH 7,每 5 0 0mL装液量为 5 0mL .最后经综合优化条件 ,在摇瓶发酵条件下 :菌体生长量 (以干重计 )为 13.8g/L ,发酵液中辅酶Q10 产量达到 2 2 .7mg/L ,比优化前分别提高 34%和 5 3% .     

Evaluation of different types of alginate microcapsules as bioreactors for producing endostatin

The use of nonautologous cell lines producing a therapeutic substance encapsulated within alginate microcapsules could be an alternative way of treating different diseases in a cost-effective way. Malignant brain tumors have been proposed to be treated locally using engineered cells secreting proteins with therapeutic potential encapsulated within alginate microcapsules. Optimization of the alginate capsule bioreactors is needed before this treatment can be a reality. Recently, we have demonstrated that alginate-poly-L-lysine microcapsules made with high-G alginate and a gelled core disintegrated as cells proliferated. In this study we examined the growth and endostatin secretion of 293-EBNA (293 endo) cells encapsulated in six different alginate microcapsules made with native high-G alginate or enzymatically tailored alginate. Stability studies using an osmotic pressure test showed that alginate-poly-L-lysine-alginate microcapsules made with enzymatically tailored alginate was mechanically stronger than alginate capsules made with native high-G alginate. Growth studies showed that the proliferation of 293 endo cells was diminished in microcapsules made with enzymatically tailored alginate and gelled in a barium solution. Secretion of endostatin was detected in lower amounts from the enzymatically tailored alginate microcapsules compared with the native alginate microcapsules. The stability of the alginate microcapsules diminished as the 293 endo cells grew inside the capsules, while empty alginate microcapsules remained stable. By using microcapsules made of fluorescenamine-labeled alginate it was clearly visualized that cells perforated the alginate microcapsules as they grew, destroying the alginate network. Soluble fluorescence-labeled alginate was taken up by the 293 endo cells, while alginate was not detected in live spheroids within fluorescence-labeled alginate microcapsules. Despite that increased stability was achieved by using enzymatically tailored alginate, the cell proliferation destroyed the alginate microcapsules with time. It is therefore necessary to use cell lines that have properties more suited for alginate encapsulation before this technology can be used for therapy.     

Shape retaining injectable hydrogels for minimally invasive bulking

PURPOSE: Particle migration, poor shape definition and/or rapid resorption limit the success of current urethral bulking agents. We propose that shape defining porous scaffolds that allow cell infiltration and anchoring, and may be delivered in a minimally invasive manner may provide many advantageous features. MATERIALS AND METHODS: Alginate hydrogels were prepared with varying degrees of covalent cross-linking and different pore characteristics. Dehydrated scaffolds were compressed into smaller, temporary forms, introduced into the dorsal subcutaneous space of CD-1 mice by minimally invasive delivery through a 10 gauge angiocatheter and rehydrated in situ with a saline solution delivered through the same catheter. Ionically cross-linked calcium alginate gel served as a control. Specimens were harvested at 2, 6, 12 and 24 weeks to evaluate implant shape retention and volume, cell infiltration and calcification, and the presence of an inflammatory response. RESULTS: A total of 90 scaffolds were implanted and 95% were recovered at the site of injection. All of these scaffolds successfully rehydrated and 80% recovered and maintained their original 3-dimensional shape for 6 months. Scaffold volume and tissue infiltration varied depending on the degree of alginate cross-linking. Highly cross-linked materials (20% and 35%) demonstrated the best volume maintenance with the latter facilitating the most tissue infiltration. The inflammatory response was minimal except with the 80% cross-linked material. Calcification was not observed in covalently cross-linked scaffolds. In contrast, 98% of calcium alginate implants were calcified. CONCLUSIONS: Shape retaining porous hydrogels meet many of the requirements necessary for a successful injectable bulking agent and offer advantages over currently used agents.     

海藻酸钠对酪氨酸酶的抑制作用

目的 研究海藻酸钠对酪氨酸酶及黑色素生成的抑制作用方法 开展酶抑制动力学实验，通过荧光光谱分析海藻酸钠对酪氨酸酶活性抑制效果，并研究海藻酸钠对黑素细胞抑制效果结果 海藻酸钠能够显著抑制酪氨酸酶活性，海藻酸钠浓度小于20 mmol/L时，浓度越高，抑制效果越好。超过20 mmol/L时， 海藻酸钠对酪氨酸酶活性抑制率65.45%，海藻酸钠浓度增加，对胞内黑色素生成抑制率随之上升，降低黑色素生成量；海藻酸钠与酪氨酸酶的相互作用对氨基酸残基疏水性环境产生破坏性，出现内源性荧光光谱红移；海藻酸钠对黑素细胞细胞活性超过90%，未产生明显毒性结论 皮肤相关疾病治疗药剂及美白化妆产品中可以使用海藻酸钠，效果良好。     

益生元-乳酸菌发酵活性成分对护肤功效的影响

研究益生元-乳酸菌发酵活性成分的抗氧化性能,分析其对护肤功效的影响。方法 采用传 统微生物发酵法对表皮葡萄球菌（SE）和植物乳杆菌（PC）进行发酵得到活性成分,之后通过1,1-二苯 基-2-三硝基苯肼（DPPH）抗氧化检测、羟基自由基清除、超氧阴离子清除等实验方法来检测活性成 分的抗氧化能力。结果 两种发酵液的活性成分含量在不同浓度益生元的影响下增长不同,最适宜SE生 长的低聚果糖益生元浓度为0.5%,最适宜PC生长的低聚果糖益生元浓度则为1.5%。结论 在2.5%的低聚 果糖浓度下培养出的PC羟基自由基清除率效果最佳,对SE更适宜的低聚果糖影响抗氧化性能的浓度为 1.5%~2.5%,此浓度下可以得到更高效的益生元-乳酸菌发酵活性成分,从而发挥护肤功效。     

用固定化生长微生物细胞改善氨基酸调味液品质

利用固定化生长的酵母细胞和黑曲霉菌丝体 ,对酸法水解的氨基酸水解液进行发酵 ,除去水解液异臭味 ,使调味液的口味和风味得到明显改善 ,产品既有氨基酸调味液的特点 ,又具有酱油的风味     

添加载银抗菌剂的藻酸盐印模材料抗菌性能及物理性能研究

目的:对添加磷酸锆载银抗菌剂的藻酸盐印模材料抗菌性能及物理性能进行测试和评价,为口腔抗菌印模材料的研制奠定实验基础。方法:以1%添加比将磷酸锆载银抗菌剂加入到藻酸盐印模材料,采用薄膜密着法,测试添加抗菌剂后印模材料的抗菌活性,并参照相关标准对材料的结固时间,压应变等物理性能进行测试和评价。结果:添加磷酸锆载银抗菌剂的藻酸盐印模材料对金黄色葡萄球菌和大肠杆菌的抗菌率分别为99.85%和99.83%。添加抗菌剂对藻酸盐印模材料主要物理性能没有显著影响(P>0.05)。结论:以1%的比例添加磷酸锆载银抗菌剂可赋予藻酸盐印模材料强抗菌性能。添加抗菌剂对藻酸盐印模材料的主要物理性能没有显著影响,可满足临床应用要求。     

骨髓基质细胞在藻酸盐凝胶中的生物学行为

目的：探讨骨髓基质细胞（Bone Marrow Stromal Cells,MSC)在海藻酸盐（Alginate)中的生物学行为，为在组织工程研究中选用海藻酸盐做为骨髓基质细胞的载体提供理论依据。方法：将地塞米松诱导后的骨髓基质细胞种植在1%海藻酸盐中，以HE染色，BMP-2免疫组化染色，扫描电镜观察骨髓基质细胞在凝胶中的生物学行为。结果：凝胶中细胞呈“悬浮”生长状态，可见到细胞分裂和核分裂相；细胞在凝胶中增殖，4d后可以见到基质分泌；海藻酸盐中培养组与常规培养组MSC的BMP-2阳性表达率无显著差异。结论：在海藻酸盐中骨髓基质细胞的生物学行为表现良好。此特征适宜作为骨组织工程的载体。     

Development of mechanically stable alginate/chondrocyte constructs: effects of guluronic acid content and matrix synthesis.

The purpose of this study was to investigate factors which enhanced the compressive properties of alginate/chondrocyte constructs. Firstly, we studied the effect of biochemical composition (high, mid and low guluronic acid content) and sterilization method on alginate properties. Secondly, we studied the biosynthetic characteristics of chondrocytes in three different alginate compositions and performed mechanical tests to determine whether the synthesis of cartilage matrix components could significantly enhance the compressive properties. 2% alginate solutions containing an initial cell density of 4 x 10(6) cells/ml were cast into cylinders and cultured for seven weeks. Compression tests, biochemistry, immunohistochemistry and electron microscopy were performed at fixed intervals during the seven-week culture period. The dynamic modulus, peak strain, and peak stress were maximum for alginate with the highest guluronic acid content. The presence of cells and their respective matrix components enhanced the equilibrium modulus of the constructs for all types of alginate, though this effect was small. Alginate containing the middle amount of guluronic acid resulted in constructs which were both mechanically stable and which promoted synthesis of cartilage matrix proteins. In experiments and applications in which the mechanical integrity of the alginate is important, the composition and purity of the alginate and its method of sterilization should be selected with care.     

Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension.

OBJECTIVE: To determine the influence of low oxygen tension on the redifferentiation and matrix production of dedifferentiated articular chondrocytes in monolayer and alginate bead culture. METHODS: Bovine articular chondrocytes were isolated enzymatically. After multiplication and dedifferentiation in a 2-week monolayer culture under 21% oxygen, the cells were subcultured in monolayer or alginate bead culture and subjected to 21% or 5% O(2)for 2 or 3 weeks in order to redifferentiate. Controls consisted of primary cultures in alginate. Matrix production was monitored immunocytochemically [collagen types I, II, IX, and GAGs (keratan sulfate, chondroitin-4- and -6-sulfate)] and collagen type II additionally assayed by Western blotting. Biosynthetic activity was measured by [(3)H]-proline incorporation and cell-viability by the trypan blue exclusion method. RESULTS: The cell number increased more than four-fold during dedifferentiation. Collagen type II was not produced by dedifferentiated chondrocytes under 5% or 21% oxygen in the monolayers or under 21% in alginate. However, dedifferentiated cells in alginate subjected to 5% oxygen exhibited a strong collagen type II expression indicating a redifferentiation. Additionally, collagen type IX and GAGs were also higher and [(3)H]-proline incorporation increased significantly. Primary cultures in alginate displayed a stronger collagen type II expression under 5% but no significant differences for other extracellular matrix components, or [(3)H]-proline incorporation. Viability was approximately 90% for all alginate cultures. CONCLUSION: A combination of alginate and high oxygen tension might not be suitable for redifferentiation or culturing of dedifferentiated chondrocytes. However, low oxygen tension promotes or induces a redifferentiation of dedifferentiated cells in alginate, stimulates their biosynthetic activity, and increases collagen type II production in primary alginate cultures.