毕赤酵母发酵ppt课件
巴斯德毕赤酵母(Pichia pastoris)表达系统综述27页PPT
营养要求简单，生长快速，适合高密度大规模培养， 很少产生有毒物质，毒性比细菌小，用甲醇不易染菌， 可以减少污染。
高的调控功能，可用于外源基因的表达调控。甲 醇营养型酵母表达系统以巴斯德毕赤酵母(Pichia pastoris)表达系统最为常用。
（3）裂殖酵母(Schizogenesis pombe) 表达系统
只能以分裂和产孢子的方式繁殖的一类酵母， 因此定名为裂殖酵母。与前面几种酵母相比， 它具有更多的与高等真核生物相似的特性： 线 粒体结构、 启动子结构、 转录机制和对蛋白2端 酰基化功能均更接近于哺乳类细胞， 因而正逐 渐成为研究真核细胞分子生物学的模式生物， 它作为外源基因表达系统也开始受到人们的关注。 目前， 已经有多种蛋白利用此系统进行了表达， 如人蛋白凝血因子)G、 细胞色素 V6:8、 人白细 胞介素 YL’D等。此系统表达的外源蛋白更接近 于它们的天然形式 。
的真核生物， 其全序列的测定已于 2019年完成 。 酿酒酵母难于高密度培养，分泌效率低，几乎不分泌分
子量大于30 kD 的外源蛋白质，也不能使所表达的外源 蛋白质正确糖基化，而且表达蛋白质的C 端往往被截短。 因此，一般不用酿酒酵母做重组蛋白质表达的宿主菌。 酿酒酵母本身含有质粒，其表达载体可以有自主复制型 和整合型两种。值得注意的是，酿酒酵母表达的外源蛋 白质往往被高度糖基化，糖链上可以带有40 个以上的 甘露糖残基，糖蛋白的核心寡聚糖链仅含有末端1, 3 甘 露糖，产物的抗原性明显增强。所以，酿酒酵母常常用 来制备亚单位疫苗(如默克乙肝疫苗、口蹄疫疫苗等)。
1993年，Philip Petroleum公司将毕赤酵母表达系统的专利卖给 Research Corporation Technologies公司，并委托Invitrogea公司 进行有关产品销售。
毕赤酵母发酵
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1.2过氧物酶体(Peroxisome)：遍布于真核生物的细胞器中, 用来去除有
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1. 3GFP-SKL（过氧化物酶体定位蛋白）,该载体含有融合了过氧化物酶体 定位信号1(PTS1)的绿色荧光蛋白报告分子GFP-SKL编码基因 成功地用于描 述不同 时间进程的AOX1启动子和过氧物酶体生物合成因素的特征
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2.3：补料分批培养条件
研究了三种不同发酵条件下的PpAOX1启动子（ YGLY29325 ）和过氧化物酶体 生物合成（ YGLY31884 ）的调控。 诱导阶段分批补料条件 ML:甲醇的起始供应速率为2.6gl/lh然后在0.0063/h的基础上以幂指的形式增加 SML:甲醇培养20h用50%的葡萄糖15g/h的速度培养8h作为一个周期共培养3个周期 OL:甲醇进料保持反应器中甲醇1％，每当DO迅速增加，这表明甲醇消耗。 DO级联 被打开，关闭和搅拌速度 被减小到实现氧气的限制
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2.4 GFP表达的定量和可视化： 约107固定的酵母细胞经PBS洗后用mounting solution重悬制作切片用 Axioscope 2 Plus 显微镜来观察OpenLAB软件来实现相机控制和图像收 集Volocity软件用来量化GFP在细胞中的强度
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2.5抗体效价的测定 在培养上清液中于280nm处抗体浓度用二极管阵列检测器和蛋白A亲 和柱与HPLC系统
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3.3毕赤酵母AOX1启动子（YGLY29325）根据三种不同的补料分批条件规制 - 甲醇 限制（ML），切换与葡萄糖和甲醇（SML）和氧气限制（OL）补料分批条件。 （a） GFP荧光表达图像（b）不同诱导时间的荧光蛋白相对密度。对于ML和OL，时间表示分 批阶段（T1），甘油补料分批阶段诱导前（T2）和诱导期（T3，10±2小时; T4中， 36±2小时; T5，62±2小时; T6，84±2小时）。对与SML条件下，细胞生长是在T2，T4 和T66/28/2014 葡萄糖阶段，诱导是在T3和T5甲醇阶段
毕赤酵母抗菌肽发酵工艺流程
毕赤酵母抗菌肽发酵工艺流程一、毕赤酵母是啥呀。
毕赤酵母可厉害啦，它就像是一个小小的魔法工厂。
这种酵母呀，和咱们平时做面包用的酵母有点不一样呢。
它是专门用来生产抗菌肽的小能手。
抗菌肽是个好东西呀，就像我们身体里的小卫士，可以对抗那些坏细菌。
毕赤酵母有它独特的本事，能够在合适的环境里，按照我们想要的方式生产出抗菌肽。
二、发酵前的准备。
1. 酵母的挑选。
这就像是挑选手下的精兵强将。
我们得选那些活力满满的毕赤酵母菌株。
这些菌株得是健康的、强壮的，这样才能在发酵过程中表现出色。
不能选那些病恹恹的或者是很弱小的酵母，不然它们可干不好生产抗菌肽这个大任务。
2. 培养基的制作。
培养基就像是酵母的食物和小窝。
我们要精心调配培养基，就像给酵母做一顿美味又营养的大餐。
里面要有合适的碳源，比如说葡萄糖之类的，这就像是给酵母的能量棒。
还有氮源，像酵母提取物、蛋白胨等，这就像是酵母的蛋白质补充剂。
而且呢，还要调节好培养基的酸碱度，让酵母在里面感觉舒舒服服的，就像我们喜欢住在温馨的小房子里一样。
三、发酵过程。
1. 接种。
把选好的毕赤酵母接种到准备好的培养基里，这就像是把小种子种到土里一样。
这个过程要很小心，就像对待娇嫩的小宝贝。
接种的量也很有讲究呢，不能太多也不能太少。
太多了的话，酵母们可能会因为空间太挤、食物不够而打架；太少了呢，它们又会觉得孤单寂寞，生产抗菌肽的速度就会很慢。
2. 温度和搅拌。
发酵的时候，温度就像一个魔法数字。
要把温度控制在毕赤酵母最喜欢的那个度数，一般是二十多度的样子。
这个温度下，酵母们就像在温暖的阳光下一样，活力满满地开始工作。
同时，还要进行搅拌呢。
搅拌就像是给酵母们做按摩，让它们在培养基里均匀地分布，这样每个酵母都能充分接触到食物，也能更好地呼吸新鲜空气，然后就能开开心心地生产抗菌肽啦。
3. 通气。
酵母们和我们一样，也需要呼吸新鲜空气呢。
所以在发酵过程中要给它们通气。
就像我们打开窗户让新鲜空气进来一样。
毕赤酵母发酵罐发酵
微生物发酵罐发酵（毕赤酵母）灭菌前：室温下校准PH电极，先校6.86零点再4.0斜率（若的发酵pH很长时间是酸性的（如酵母发酵）用6.86校正零点，4.0校正斜率；若你的发酵pH很长时间是碱性的（如某些细菌发酵）用6.86校正零点，9.18校正斜率）；室温下校准溶氧电极，1.0点在不接溶氧电极时候标定，100%点接上溶氧电极，放置在空气中较定；或2.0点在灭菌过程中，温度达到121度左右压力0.12mpa左右时候标定，100%在灭菌结束，降温至发酵温度并稳定，转速在发酵初始转速，通气量在发酵初始通气量时候标定灭菌：1.灭菌，先将各排气阀打开，将蒸汽引入夹套或蛇管进行预热，待罐温升至80~90℃，将排气阀逐渐关小。
接着将蒸汽从进气口、排料口、取样口直接通入罐中（如有冲视罐也同时进汽），使罐温上升到118~120℃，罐压维持在0.09~0.1Mpa（表压），并保持30min左右。
2.保温结束后，依次关闭各排汽、进汽阀门，待罐内压力低于空气压力后，向罐内通入无菌空气，在夹套或蛇管中通冷却水降温，使培养基的温度降到所需的温度，进行下一步的发酵和培养。
（注意压力：灭菌时，总蒸汽管道压力要求不低于0.3~0.35Mpa，使用压力不低于0.2Mpa。
）灭菌后:A.消耗甘油阶段1.灭菌后冷却30℃时：2.冷却至30℃时，开启搅拌(转速最大)和通气(0.1-1.0vvm),接通28%氨水(未稀释)调PH5.0;每升加4.35ml的无菌PTM1基础盐；3.从摇瓶中接种种子液，DO值为100%，开始培养后会消耗，导致DO值下降，通氧气以确保DO值超过20％，速率先为0.1vvm。
4. 发酵过夜甘油被完全消耗（18-24h），标志为DO值增加到100％。
【每天至少两次取样，测OD600，湿重，显微观察.将菌体和上清（离心后）在-80℃下保藏，用于后面的分析。
】5.这个阶段所期望达到的细胞产量为90-150g/L湿细胞。
发酵过程优化与控制PPT课件
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发酵过程技术原理
分批发酵 补料-分批发酵 半连续发酵 连续发酵
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分批发酵
几个重要参数：
为比生长速率,h-1； -qs 为比基质消耗速率,(g/g)/h； qp 为比产物形成速率,(g/g)/h 。
uX dX dt
q xX d S dt
补充养分，同时解除/消弱代谢产物的抑制。
不足：
丢失了未利用的养分和处于生长旺盛期的菌体；送去提炼 的发酵液体积更大；丢失代谢产生的前体物；利于非产生 菌突变株的生长。
实施：海洋微藻合成藻红素和EPA。
需要摸索最佳的培养基更新速率。
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连续发酵
发酵过程中一面补入新鲜的料液，一面以相同的流速 放料，维持发酵液原来的体积。（恒化培养）
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发酵过程优化与控制
发酵
狭义——厌氧条件下葡萄糖通过酵解途径生成 乳酸或乙醇等的分解代谢过程。
广义——微生物把一些原料养分在合适的发酵 条件下经特定的代谢途径转变成所需产物的过 程。
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发酵是一个很复杂的生化过程，其好坏涉及诸多因素： 菌种性能、培养基组成、原料质量、灭菌条件、种子 质量、发酵条件和过程控制等
pH变化会影响酶活，菌对基质的利用效率和细
胞结构，从而影响菌的生长和产物的合成。
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选择最适发酵pH的原则是获得最大比生产速率和
适当的菌量。
分阶段pH控制策略
如何控制发酵液pH？
基础培养基的配方；通过加酸碱或中间补料 例如，青霉素发酵，通过调节加糖速率来控制pH；链 霉素的生产，补充NH3来控制pH，同时为产物合成提 供氮源。
培养液pH可反映菌的生理状况：pH上升超过最适值，意 味着菌处于饥饿状态，可加糖调节；糖的过量又使pH下 降；用氨水中和有机酸需防止微生物中毒，可通过监测 培养液种溶氧浓度的变化来控制。
发酵工程在食品中的应用 PPT课件
绒毛状，网状、絮状菌丝体
1.产黄青 霉
2.紫红曲 3.点青霉 4.黑曲霉 5.米曲霉
部分霉菌菌落形态
泡菜发酵中的有益菌
乳酸菌 酵母和醋酸菌
酸奶、奶酪的乳酸发酵剂 乳酸菌发酵剂
乳杆菌、乳球菌、链球菌、明串珠菌、双歧杆菌
酵母发酵剂
乳糖发酵型酵母（开菲儿酸奶）
5-7d）→装坛→低温发酵→压榨→煮酒（灭菌）→装坛泥
封→堆置陈化（后熟）→出厂
淋饭酒 摊饭酒 喂饭酒
黄酒的 分类
以酒药、麦曲或米曲、红曲及淋饭
酒母为糖化发酵剂，进行自然的、
传统工艺黄酒 多菌种的混合发酵生产而成，发酵取代自然发酵，以大型发 酵生产代替小型手工操作，生产过程 简化，原料利用率高，去除了笨重的 体力劳动
长些，皮薄的时间短些）； ⑤压榨：液体进行后发酵（20-25℃，一周），澄清，取
上部液体，其中低挡的装瓶，较高档次的换桶（最好是 橡木桶，橡木能产生抑菌物质，同时具有一种天然植物 香）陈化三年（5℃）；渣进行蒸馏，调香，制成白兰 地（酒精多）。
葡萄酒的再加工
起泡葡萄酒：是一种含CO2的葡萄酒，以葡萄酒为基础 ，通过加糖发酵产生或人工压入CO2。
3.出芽短梗霉
4.多孢丝抱酵母
5.荚复膜孢酵
2.产肮假丝酵母 13.大型罗伦隐球酵母
红 酵
母1.酿酒酵母
12.玫红法佛酵母
6.解脂复膜孢
14.美极梅奇酵母
11.深红酵母
啤
酒 10.鲁氏酵母
15.浅红酵7母.季也蒙有孢汉逊酵
酵
母
8.碎囊汉逊酵母
9.卡氏酵母
（3）霉菌/丝状真菌
传统发酵食品 生产淀粉酶、蛋白酶、纤维素酶等 不利方面：食品发霉变质，引起病害
毕赤酵母生物发酵
Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitisCarolina Bellido,Silvia Bolado,Mónica Coca,Susana Lucas,Gerardo González-Benito,María Teresa García-Cubero ⇑Department of Chemical Engineering and Environmental Technology,University of Valladolid,Dr.Mergelina s/n,47011Valladolid,Spaina r t i c l e i n f o Article history:Received 20June 2011Received in revised form 29August 2011Accepted 30August 2011Available online 10September 2011Keywords:EthanolPichia stipitis Fermentation Inhibitory effect Wheat strawa b s t r a c tThe inhibitory effect of the main inhibitors (acetic acid,furfural and 5-hydroxymethylfurfural)formed during steam explosion of wheat straw was studied through ethanol fermentations of model substrates and hydrolysates from wheat straw by Pichia stipitis .Experimental results showed that an increase in ace-tic acid concentration led to a reduction in ethanol productivity and complete inhibition was observed at 3.5g/L.Furfural produced a delay on sugar consumption rates with increasing concentration and HMF did not exert a significant effect.Fermentations of the whole slurry from steam exploded wheat straw were completely inhibited by a synergistic effect due to the presence of 1.5g/L acetic acid,0.15g/L furfural and 0.05g/L HMF together with solid fraction.When using only the solid fraction from steam explosion,hydrolysates presented 0.5g/L of acetic acid,whose fermentations have submitted promising results,providing an ethanol yield of 0.45g ethanol/g sugars and the final ethanol concentration reached was 12.2g/L (10.9g ethanol/100g DM).Ó2011Elsevier Ltd.All rights reserved.1.IntroductionLignocellulosic materials are a promising alternative energy to fossil resources because they are the most abundant natural renewable organic material that exists on earth and there are con-cerns over CO 2emissions from fossil fuels (Prasad et al.,2007).Conversion process of lignocellulosic biomass to ethanol can be divided into five unit operations (Merino and Cherry,2007).(1)Size reduction to increase surface area and uniformity,(2)pretreatment to disrupt lignin and hemicellulose,reduce cellulose crystallinity and increase the porosity of the biomass,(3)enzymatic hydrolysis to convert sugar polymers to monomeric sugars,(4)fermentation to produce ethanol from those monomeric sugars and (5)ethanol recovery using distillation or other separation technologies.Lignocellulose is a matrix of cross-linked polysaccharide net-works,mainly cellulose and hemicellulose,which are tightly bound to lignin (Zaldivar et al.,2001).The efficient use of the sugar content of lignocellulosic biomass is the key for the economic feasibility of ethanol production.This implies that in addition to the glucose obtained from the cellulosic portion,all sugars released from the hemicellulose fraction,such as xylose and non-structural sugars,which may represent a significant percentage of the fermentable sugars,should be fermented (Díaz et al.,2009).Hence,the totalethanol production can be increased using an efficient xylose-fermenting yeast that can convert both hexose and pentose sugars (Nigam,2001).Among the xylose-fermenting yeasts,Pichia stipitis has shown the most promising results for industrial application,because it ferments xylose with a high ethanol yield and it is also able to ferment glucose.Besides,P.stipitis does not require vitamin addition for xylose fermentation and it is capable to ferment a wide range of sugars,including cellobiose (Agbogbo and Wenger,2006).On the other hand,some of the compounds formed during pre-treatment of lignocellulosic biomass such as sugar decomposition products and lignin degradation products may have a potential inhibitory effect on the fermentation process.Inhibitory com-pounds in lignocellulosic hydrolysates comprise aliphatic acids (i.e.acetic,formic and levulinic acid),furaldehydes (i.e.furfural and 5-hydroxymethylfurfural (HMF)),aromatic compounds (i.e.phenolics)and extractives (Martín and Jönsson,2003).The number and identity of these toxic compounds varies with the type of the raw material and pretreatment conditions.An extensive study about the effects of each pretreatment has been done recently (Hendriks and Zeeman,2009).In this study,steam explosion,a leading pretreatment for wheat straw biomass,was used as a del-ignification and hemicellulose solubilization method (Tomás-Pejóet al.,2008).The main objective of this work is to analyze the influence of inhibitors formed during steam explosion of lignocellulosic bio-mass on ethanol fermentation by P.stipitis.This study is focused on three inhibitors:acetic acid,2-furaldehyde (furfural)and0960-8524/$-see front matter Ó2011Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2011.08.128Corresponding author.Tel.:+34983423237;fax:+34983423616.E-mail address:maite@iq.uva.es (M.T.García-Cubero).5-hydroxymethylfurfural (HMF)in a concentration range of (0.5–3.5g/L),(0.5–2g/L)and (0.1–0.5g/L),respectively.Three dif-ferent approaches were used:first,the inhibitory effect of acetic acid,furfural and HMF in a model substrate medium,was studied.Then,the fermentation of wheat straw hydrolysates was analyzed.Finally,the effect of steam explosion liquid addition and the influ-ence of the presence of solid fraction during the fermentation pro-cess were also studied.2.Methods2.1.Microorganism and mediaP.stipitis DSM 3651was obtained from the German Collection of Microorganisms and Cell Cultures.The yeast was maintained on YEPX agar plates at 4°C in a refrigeration chamber containing 10g/L yeast extract,20g/L peptone,20g/L xylose and 20g/L agar.The inoculum medium was prepared as follows:a solution of 10g/L yeast extract and 20g/L peptone was sterilized at 120°C for 20min in an autoclave.Carbon source,20g/L xylose was then added after being sterilized (0.20l m Sterile Filters,Ministart Sartorius)and P.stipitis was supplemented from YEPX agar plates.Inoculum was grown aerobically on a rotatory shaker (WY-200,Comecta,S.A.)at 175rpm and 30°C for 24h.Yeast initial concentration was 0.5g/L in all experimental runs.2.2.Fermentation with model substrate mediaWhen inhibitor effect was studied individually,model fermen-tation medium was composed of 35g/L glucose,20g/L xylose,10g/L yeast extract,20g/L peptone,0.47g/L (NH 4)2SO 4,12.8g/L KH 2PO 4,0.51g/L Na 2HPO 4and 0.47g/L MgSO 4Á7H 2O.This medium was adjusted to pH 5with a buffer composed of two solutions (mL/L fermentation medium):250mL succinic acid 0.2M and 267mL NaOH 0.2M.The medium was autoclaved at 120°C for 20min,and sugars were added after being filtered (0.20l m Sterile Filters,Ministart Sartorius).Fermentation experiments were carried out in sterile 125mL serum bottles with cap and needle to remove CO 2in a rotatory sha-ker at 30°C and 175rpm for 168h.Experiments were performed with no oxygen supply,but they were not strictly anaerobic be-cause there was air before closing serum bottles.Each serum bottle was filled with 25mL of fermentation inoculated with 10%(v/v)growth culture.Inhibitor compounds (acetic acid,furfural and HMF)were added either separately to study their individual effect using typical inhib-itors concentrations for wheat straw hydrolysates according to the literature (Delgenes et al.,1996;Díaz et al.,2009)or as a mixture to simulate similar sugars and inhibitors concentration of wheatstraw hydrolysates.A fermentation experiment without inhibitors was also carried out as a control.Fermentation process was moni-tored for 7days by taking samples at 0,24,48,72,96,120,144and 168h for analyses.Table 1summarizes the initial sugars and inhibitors concentration for fermentations performed with model solutions.All the experiments were carried out in triplicate and the average data are shown.2.3.Wheat straw hydrolysates2.3.1.Raw materialWheat straw used in experiments was provided by the Institute of Technological Agriculture of Castilla y León.Wheat straw was milled using a laboratory sieve mill into small particles of 20mm prior to steam explosion pretreatment.Raw material was kept in an oven at 45°C until use.Wheat straw had the following compo-sition (%dry weight):cellulose,as glucose,32.4;hemicellulose,as xylose,19.1;acid lignin,21.3;ash,6.4;moisture,6.9.2.3.2.Wheat straw pretreatment and saccharificationSteam explosion was used as a delignification and hemicellu-loses release method.Pretreatment assays were carried out in a 5L stainless steel batch reactor equipped with a quick-opening valve and a controller for residence time and temperature.Wheat straw was exploded at 210°C for 10min.After pretreatment,the slurry was recovered and residual solid was separated by filtration.Liquid fraction was stored in a refrigeration chamber.Depending on the experiment,the whole slurry or just the solid fraction was used.After steam explosion,enzymatic hydrolysis was performed using a mixture of cellulase (NS50013)and b -glucosidase (NS50010),0.11g/g cellulose and 0.05g/g cellulose were added,respectively.Enzymes were kindly donated by Novozymes (Den-mark).Hydrolysis was carried out at 50°C for 72h in a 250mL stir-red tank at 175rpm with mechanical agitation (Heidolph RZR 2020).When solid fraction used for experiments,pretreated material was suspended in 0.05M citrate buffer (pH 5.0)with a solid content of 10%(w/v),while the whole slurry at the same solid loading was supplemented with a concentrated 0.5M citrate buffer to avoid sugars dilution.Depending on the experiment,the whole hydrolysate or just the liquid fraction (separated by vacuum filtration)were used for fermentation process.2.4.Fermentation of wheat straw hydrolysatesExperimental runs can be firstly divided into two groups of experiments after steam explosion.In order to analyze the influence of the addition of steam explosion liquid,two sets of experiments were carried out either with the whole slurry (WS)or just with the solid fraction (SF).In addition,to study the influence of solid fractionTable 1Initial sugars and inhibitors concentrations for synthetic fermentations.ExperimentInitial sugars concentration (g/L)Inhibitor concentration (g/L)GlucoseXylose Acetic acid Furfural HMF Control 36.521.0000HAc2.535.120.5 2.500HAc1.535.020.5 1.500HAc0.535.020.10.500F235.820.2020F135.121.4010F0.534.821.300.50HMF0.537.020.5000.5HMF0.136.720.2000.1Model FH-SF 22.3 5.80.500Model FH-WS21.411.11.50.150.05C.Bellido et al./Bioresource Technology 102(2011)10868–1087410869in fermentations,a second level of experiments were made after enzymatic hydrolysis step,using either the whole hydrolysate after enzymatic hydrolysis (WH)or the filtered hydrolysate (FH).By way of summary,four sets of experiments were performed:(1)WH-SF and (2)WH-WS experiments,where the whole hydrolysate was used after enzymatic hydrolysis and the solid fraction or the whole slurry was taken after steam explosion,respectively;and (3)FH-SF and (4)FH-WS experiments,where the filtered hydrolysate was used after enzymatic hydrolysis and the solid fraction or the whole slurry from steam explosion was utilized,respectively.For WH-SF and WH-WS experiments,substrate from enzymatic hydrolysis was directly inoculated with 10%(v/v)growth culture.Fermentations were carried out in a stirred tank at 30°C and 175rpm for 168h.The medium was pasteurized prior fermenta-tion process maintaining a temperature of 80°C for 30min.FH-SF and FH-WS experiments,solid fraction was separated by vacuum filtration.Liquid portion was fermented under similar con-ditions as model media experiments.Fermentation process was monitored for 7days and samples were taking at 0,24,48,72,96,120,144and 168h for analyses.Table 2summarizes the con-centration of inhibitors and fermentable sugars found in wheat straw hydrolysates after hydrolysis step.In all set of experiments,there was not sugar supplementation as it has been used by other researchers (Martín et al.,2006;Söderström et al.,2004).All the experiments were carried out in triplicate and the average data are shown.2.5.Analytical methodsSugar concentrations,inhibitor concentrations and ethanol pro-duction were determined by HPLC.The detector was based on the refractive index measurement.An HPX-87H column used,enabling quantification of glucose,xylose,acetic acid,furfu-ral,HMF and ethanol.Operational conditions were 0.01N H 2SO 4as mobile phase,at a flow rate of 0.6mL/min and 60°C.All the samples were centrifuged at 5000rpm for 5min and filtered before being analyzed.Cellular growth was determined by measuring optical density of cells (Hitachi U-2000)at 600nm and correlated with dry weight.All the analyses were performed in duplicate.3.Results and discussion3.1.Effect of inhibitors on model media fermentationsEffect of the main inhibitors produced during lignocellulosic biomass pretreatment has been studied in fermentation process.Acetic acid,furfural and HMF are three of the major inhibitors which appear in cereal straw hydrolysates in different concentra-tions depending on pretreatment and enzymatic hydrolysis condi-tions retention time,biomass load,enzyme ratio ...).The influence of such inhibitors on P.stipitis performance has been studied in terms of biomass growth,sugars uptake,ethanol yield and productivities.Inhibitors were added either separately or as a mixture using different initial concentrations.3.1.1.Acetic acidConcerning the individual effect of acetic acid,it was observed that inhibition increased whit acetic acid concentration and media containing 3.5g/L of acetic acid inhibited completely both growth and ethanol production.Ethanol theoretical yield,which is defined as the percentage of the total amount of ethanol that could be pro-duced from all sugars available,decreased when acetic acid was present in the medium from 76.5%corresponding to the control to 0%in HAc3.5,36.4%in HAc2.5,61.8%in HAc1.5and 63.8%in HAc0.5.Fig.1(a–c)show fermentation profiles for sugars consump-tion (glucose and xylose),ethanol production and biomass growth.In control experiment,after 24h,glucose was completely ex-hausted and xylose was gradually consumed in 168h after glucose depletion.As it can be seen in Fig.1(a),xylose consumption was much more affected than glucose consumption and the effect was higher when acetic acid concentration increases.Cellular growth and ethanol production were considerably affected when concentration increased to 2.5g/L.Ethanol concentration obtained in HAc2.5,HAc1.5and HAc0.5experiments were lower than that reached in the control,10.4g/L,17.5g/L,18.0g/L and 22.8g/L,respectively.In the control,maximum ethanol productivity was 0.61g L À1h À1after 24h of experiment,when all glucose was con-sumed.This value decreased to 0.14g L À1h À1at the end of the as-say (168h),when all xylose was depleted.In HAc1.5and HAc0.5,it can be observed the same effect but productivities were smaller,0.15g L À1h À1(24h)and 0.10g L À1h À1(168h)and 0.57g L À1h À1(24h)and 0.11g L À1h À1(168h),respectively.Ethanol yields,which are defined as the ratio between ethanol production and sugars consumption,were slightly higher than those obtained in the control.Despite of producing a lower ethanol concentration because of cellular growth inhibition,ethanol was produced with lower sugars uptake that caused an increase in that ratio.Ethanol yield in the control was 0.39g ethanol/g sugars (168h),while in HAc2.5,HAc1.5and HAc0.5experiments were 0.44g ethanol/g sugars,0.44g ethanol/g sugars and 0.39g etha-nol/g sugars (168h),respectively.Björling and Lindman (1989)reported complete inhibition of ethanol production by P.stipitis in synthetic medium containing 3.9g/L acetic acid at pH 4,while Díaz et al.(2009)observed cellular growth in fermentations by P.stipitis on 20g/L glucose and 15g/L xylose synthetic medium with 6g/L of acetic acid,but as far as ethanol yields are concerned,also reported an increase with acetic concentration in fermenta-tions performed with 3and 6g/L of acetic acid.3.1.2.FurfuralRegarding inhibitory effect of furfural,P.stipitis was inhibited by its presence,but the inhibition was not as strong that caused by acetic acid.In this case,the effect was the delay of sugars con-sumption and consequently a decrease on ethanol productivity during the first 24h increasing with concentration.Theoretical yield reached similar values about 73.4%,74.5%and 75.4%(F2,F1and F0.5,respectively)compared to 76.5%corresponding to the control.Fermentation profiles for sugars concentration,ethanol production and biomass growth are depicted in Fig.2(a–c).Glucose consumption and cellular growth reached the same value as in the control after 96h for all experiments when furfural was added,Table 2Initial composition of wheat straw hydrolysates for fermentation process.Fermentation experimentInitial sugars concentration (g/L)Inhibitor concentration (g/L)GlucoseXylose Acetic acid Furfural HMF FH-WS 23.7711.29 1.520.140.05FH-SF 22.64 5.840.4800WH-WS 23.2311.32 1.560.160.05WH-SF23.486.210.5210870 C.Bellido et al./Bioresource Technology 102(2011)10868–10874which means a decrease in sugar uptake rates.Inhibition of xylose consumption only was produced when furfural concentration reached2g/L.Ethanol concentrations in F2,F1and F0.5experi-ments were similar to that reached in the control,21.0g/L, 21.6g/L,21.6g/L and22.8g/L,respectively.Thus,the inhibitory effect of furfural is observed in terms of ethanol production rate and this effect increased with furfural concentration.Productivities decreased compared to the control,but the effect was more atten-In F1experiment,ethanol productivity decreased to the half value of the control,0.34g LÀ1hÀ1after24h.Values of0.24and 0.50g LÀ1hÀ1at24h were obtained for F2and F0.5experiments, respectively.Similar ethanol productivity,about0.11g LÀ1hÀ1 was observed at the end of the experiments with furfural as inhibitor,value close to0.14g LÀ1hÀ1obtained in the control. Cellular growth was slightly affected by the presence of furfural as it can be seen in Fig.3(c).Ethanol yield in the control was 0.39g ethanol/g sugars,very similar to F2,F1and F0.5experi-ments,0.40,0.39and0.39g ethanol/g sugars(168h),respectively. Palmqvist et al.(1999)did not observed any inhibitory effect infermentations by Candida shehatae in furfural concentrations upto2g/L.Díaz et al.(2009)reported a decrease in ethanol productiv-ity in fermentations carried out with2g/L of furfural and no sugarconsumption in experiments performed with4g/L furfural in fer-mentations with P.stipitis at initial sugar concentrations of20gglucose/L and15g xylose/L.At the end of the experiments,no furfural and HMF were found,which means that the microorganism assimilated these com-pounds before growth and ethanol production as it has been re-ported by other researchers(Tomás-Pejóet al.,2008;Liu et al.,2004;Taherzadeh et al.,2000).Furfuryl alcohol was not analyzed,but it should have been present as a product of fermentative C.Bellido et al./Bioresource Technology102(2011)10868–1087410871ence of0.5g/L HMF in the fermentation media,but the effect was not noteworthy compared to furfural and acetic acid.Ethanol the-oretical yields were75.1%and82.5%,which were slightly higher than that reached in the control for HMF0.1assay.Fermentation profiles are depicted in Fig.3(a–c).As it can be seen,glucose con-sumption is not affected by presence of HMF and ethanol concen-trations reached similar values in all the experiments.When HMF was added in a concentration of0.1g/L had a positive effect,cellu-lar growth was slightly higher compared to the control.At the end of the fermentation process,ethanol yields were similar to the con-trol,obtaining0.38and0.42g ethanol/g sugars for HMF0.5and HMF0.1experiments,respectively.Fermentation profiles were rather similar between control and HMF0.1experiment,even eth-anol concentration was higher,24.0g/L,only xylose consumption was slightly affected because there was not a total depletion when HMF concentration increased from0.1to0.5g/L.Delgenes et al. (1996)reported an inhibition effect when HMF was higher than 1g/L.Regarding to other microorganisms,Delgenes et al.(1996)re-ported the effect of inhibitory compounds on xylose fermentation by P.stipitis,C.shehatae,Zymomonas mobilis and Saccharomyces cerevisae.C.shehatae showed a good tolerance to acetic acid,but cell growth and ethanol production were almost completely inhib-ited when furfural appeared in a concentration of2g/L.In contrast to Z.mobilis,which showed a good tolerance to acetic acid,furfural and HMF,S.cerevisae was significantly inhibited by these com-pounds.The toxicity problem of furfural could be overcome by operating with high cell density in continuous fermentation with cell recycle,as a way to minimize the lag phase(Cantarella et al., 2004).3.1.4.Effect of ternary mixture of acetic acid,furfural and HMFIn order to study the behavior of P.stipitis when the three main inhibitors are present in fermentation media and to compare results between wheat straw hydrolysates and synthetic media, two set of experiments were performed:(1)fermentation of model solutions similar to FH-SF hydrolysate containing0.5g/L of acetic acid,and(2)fermentation of model solutions similar to FH-WS hydrolysate containing1.5g/L of acetic acid,0.15g/L of furfural and0.05g/L of HMF.The presence of the three inhibitors had an effect on sugar consumption,causing a complete inhibition of xylose utilization and consuming glucose at a lower rate in com-parison to the control experiment.In the same way as experiments performed with inhibitors added separately,ethanol yield was greater than in the control,0.46g ethanol/g sugars(168h).The process which represented FH-SF hydrolysate led to similar results to HAc0.5experiment,because the difference was only the initial concentration of total sugars.These data provided the basis for comparison with wheat straw hydrolysates,as explained in the following section.3.2.Fermentation of wheat straw hydrolysates.Influence of presence of solid fraction and steam explosion liquid addition in fermentation experimentsTwo set of experimental runs were performed with wheat straw hydrolysates using either the whole hydrolysate from enzymatic hydrolysis(WH-SF and WH-WS experiments)or thefiltered hydrolysate after this step(FH-SF and FH-WS experiments).Table 2shows the initial composition of media regarding sugars and inhibitors formed in the pretreatment step.Initial xylose concen-tration was higher when the whole slurry from steam explosion was enzymatically hydrolyzed because the steam explosion liquid contains the pentose sugars released during the pretreatment.On the other hand,only acetic acid was present when the solid frac-tion was used for enzymatic hydrolysis(FH-SF and WH-SF experi-ments),but furfural and HMF appeared when steam explosion liquid was added(FH-WS and WH-WS experiments).In thefirst group of assays,the whole hydrolysate from enzy-matic hydrolysis was utilized for fermentations.Fig.4illustrates fermentation profiles for these experiments.Experiment WH-SF offered an ethanol yield of0.45g/g and a theoretical ethanol yield of80.2%with a maximum ethanol concentration of12.2g/L(10.9g ethanol/100g DM),whereas experiment WH-WS showed a com-plete inhibition of P.stipitis growth and no ethanol was obtained. This result could be due to the synergic effect of the three inhibi-tors together with the presence of the solid fraction because these two situations separately did not produce absolute inhibition of10872 C.Bellido et al./Bioresource Technology102(2011)10868–10874fermentation process by P.stipitis.Effect of inhibitors as a ternary mixture was studied in FH-WS Model experiments when cellular growth was allowed and consequently ethanol was produced although the productivity was lower compared to the control. From WH-SF experiments,it can be concluded that the presence of the solid fraction in the fermentation process seems to show a good effect in ethanol production when steam explosion liquid was not added,obtaining the highest fermentation efficiency among the rest of the experiments.This effect could be caused by the release of sugars that still remained in the lignocellulosic biomass due to the presence of enzymes in the solid from the previous saccharification step.Arslan and Eken-Saraçoglu(2010)obtained a maximum ethanol concentration of16.8g/L in fermentations with P.stipitis of hydrol-ysates prepared from hazelnut shells delignified with3%NaOH at room temperature and detoxified with overliming and charcoal. They achieved an ethanol yield of0.449g/g from these hydroly-sates containing50g/L of total reducing sugars,when xylose was externally added(32.9g/L).Buaban et al.(2010)attained an etha-nol concentration of8.4g/L,corresponding to a conversion yield of 0.29g ethanol/g available sugars from undetoxified pretreated bagasse hydrolysate.In this case,pretreatment consisted of size reduction by ball milling for2h prior separate enzymatic hydroly-sis and fermentation at pH5.5,30°C for24h.Cho et al.(2011) obtained similar results with a hexose sugar-based ethanol yield between0.42and0.46g/g from acid hydrolysates based on the construction and demolition wood waste using P.stipitis.Regard-ing to other yeasts,Tomás-Pejóet al.(2008)reported inhibitory compounds such as acids(acetic,formic,coumaric and ferulic acid),aldehydes(furfural,HMF)and phenolic compounds(vanillin, syringaldehyde)affected the fermentation performance of F12 strain(engenieerically modified S.cerevisae to allow xylose consumption)on a steam-exploded wheat straw prehydrolysate (filtered fraction from steam explosion),resulting in a lag phase in cell growth and the sugar consumption was delayed.On the second group of assays,fermentations were carried out using only thefiltered portion from enzymatic hydrolysis(FH-SF and FH-WS experiments).Experiment FH-SF offered an ethanol yield of0.42g/g and a theoretical yield of77.3%with a maximum ethanol concentration of11.3g/L(10.1g ethanol/100g DM).Etha-nol concentration and theoretical yield were lower in FH-WS fermentation,9.1g/L(8.2g ethanol/100g DM)and50.9%,respec-tively.As it can be observed in Fig.5,xylose was not consumed in this experiment due to the presence of the three inhibitors.As previously stated,model fermentations offiltered hydrolysates were performed in order to compare fermentation profiles and P.stipitis performance between synthetic solutions and wheat straw hydrolysates at analogous initial sugars and inhibitors con-centration.A comparison between sugars and ethanol concentra-tions obtained infiltered hydrolysates(FH-SF and FH-WS)and synthetic solutions(FH-SF Model and FH-WS Model)at the end of the fermentation process is shown in Fig.6.In FH-WS experi-ment,acetic acid,furfural and HMF are present due to the addition of steam explosion liquid without solid fraction in the assay.Etha-nol concentration and productivity reached were similar compared to the model(FH-WS Model),thus indicates there was not other compounds in a considerable proportion that can inhibit the fer-mentation process in the steam explosion liquid.When the three inhibitors were present together with solid fraction,inhibition was complete in terms of cellular growth and ethanol production, thus P.stipitis can not perform the fermentation process when whole slurry from steam explosion is used.This effect could be solved by detoxification,as overliming or activated charcoal(Ku-had et al.,2010;Arslan and Eken-Saraçoglu,2010;Cantarella et al.,2004).When thefiltered portion from enzymatic hydrolysis was used,there was ethanol production and cellular growth was slightly affected whereas xylose consumption was not consumed. Best results referring to ethanol productivity and cellular growth correspond to WH-SF experiment,when only solid fraction from steam explosion was used and P.stipitis fermented the whole enzy-matic hydrolysate.4.ConclusionsRegarding inhibitors studied,acetic acid reduced ethanol pro-ductivity with increasing concentration and inhibited completelyGlucoseXyloseEthanolC.Bellido et al./Bioresource Technology102(2011)10868–1087410873。
毕赤酵母发酵罐发酵资料讲解
毕赤酵母发酵罐发酵微生物发酵罐发酵（毕赤酵母）灭菌前：室温下校准PH电极，先校6.86零点再4.0斜率（若的发酵pH很长时间是酸性的（如酵母发酵）用6.86校正零点，4.0校正斜率；若你的发酵pH很长时间是碱性的（如某些细菌发酵）用6.86校正零点，9.18校正斜率）；室温下校准溶氧电极，1.0点在不接溶氧电极时候标定，100%点接上溶氧电极，放置在空气中较定；或2.0点在灭菌过程中，温度达到121度左右压力0.12mpa左右时候标定，100%在灭菌结束，降温至发酵温度并稳定，转速在发酵初始转速，通气量在发酵初始通气量时候标定灭菌：1.灭菌，先将各排气阀打开，将蒸汽引入夹套或蛇管进行预热，待罐温升至80~90℃，将排气阀逐渐关小。
接着将蒸汽从进气口、排料口、取样口直接通入罐中（如有冲视罐也同时进汽），使罐温上升到118~120℃，罐压维持在0.09~0.1Mpa（表压），并保持30min左右。
2.保温结束后，依次关闭各排汽、进汽阀门，待罐内压力低于空气压力后，向罐内通入无菌空气，在夹套或蛇管中通冷却水降温，使培养基的温度降到所需的温度，进行下一步的发酵和培养。
（注意压力：灭菌时，总蒸汽管道压力要求不低于0.3~0.35Mpa，使用压力不低于0.2Mpa。
）灭菌后:A.消耗甘油阶段1.灭菌后冷却30℃时：2.冷却至30℃时，开启搅拌(转速最大)和通气(0.1-1.0vvm),接通28%氨水(未稀释)调PH5.0;每升加4.35ml的无菌PTM1基础盐；3.从摇瓶中接种种子液，DO值为100%，开始培养后会消耗，导致DO值下降，通氧气以确保DO值超过20％，速率先为0.1vvm。
4. 发酵过夜甘油被完全消耗（18-24h），标志为DO值增加到100％。
【每天至少两次取样，测OD600，湿重，显微观察. 将菌体和上清（离心后）在-80℃下保藏，用于后面的分析。
】5. 这个阶段所期望达到的细胞产量为90-150g/L湿细胞。
毕赤酵母发酵手册
毕赤酵母发酵手册总览简介：毕赤酵母和酿酒酵母很相似，都非常适合发酵生长。
毕赤酵母在有可能提高总体的蛋白质产量的发酵中能够达到非常高的细胞浓度，我们建议只有那些有过发酵经验或者能得到有经验的人的指导的人参与发酵。
因为发酵的类型很多，所以我们很难为您的个人案例提高详细的过程。
下面所给出的指导是基于Mut+和Mut s两种基因型的毕赤酵母菌株在15L的台式玻璃发酵罐中发酵而成。
请在您的发酵开始前先阅读操作员手册。
下面所给出的表就发酵参数：在整个发酵过程中监测和调控下列参数非常重要。
下面的表格描述了这些参设备推荐：下面是所推荐设备的清单：·发酵罐的夹套需要在发酵过程中给酵母菌降温，尤其是在甲醇流加过程中。
你需要一个固定的来源来提供冷却水（5-10℃）。
这可能意味着你需要一个冷冻装置来保持水的冷却。
·一个泡沫探针就像消泡剂一样不可或缺。
·一个氧气的来源——空气（不锈钢的发酵罐需要1-2vvm）或者纯氧（玻璃发酵罐需要0.1-0.3vvm）。
·添加甘油和甲醇的补料泵。
·pH的自动控制。
培养基的准备：你需要准确配置下列溶液：·发酵所需的基本盐类（第11页）·PTM1补充盐类（第11页）·75ml的50％的甘油每升初始发酵液，12ml的PTM1补充盐每升甘油。
·740ml的100％的甲醇每升初始发酵液，12ml的PTM1补充盐每升甲醇。
毕赤酵母生长的测定：在不同的时间点通过测OD600的吸光值和湿细胞的重量来检测毕赤酵母的生长。
培养的代谢速率通过通过观察溶氧浓度对应于有效碳源来测定。
溶氧的测定：简介：溶解氧的浓度时指氧气在培养基中的相关比例，溶氧100％是指培养基中氧达到饱和。
毕赤酵母的生长需要消耗氧气，减少溶解氧的满度。
毕赤酵母在生长时会消耗氧气，减少溶氧的程度。
然而，因为代谢甲醇的最初阶段需要氧气，所以将溶氧浓度维持在一个适当的水平（＞20％）来确保毕赤酵母在甲醇上的生长就至关重要。
毕赤酵母发酵 ppt课件
二：实验材料和方法
2.1：质粒的构建
表达GFP- SKl pGLY11245或pGLY13173 转化YGLY27355或YGLY31884 YGLY14836生产 单克隆IgG1抗体
表达Ub-R-GFP pGLY10148转化 YGLY19309或YGLY29325 生产单克隆IgG1抗体 2.2：转化：毕赤酵母感受态的制备和电转化的方法（参见nvitrogen公司的Pichia
• 最大量的生产重组蛋白高度依赖甲醇消耗速率
文章来源：Journal of biotechnology
• 1背景介绍 • 2实验材料和方法 • 3实验结果 • 4结论与分析
背景介绍
1.1醇氧化酶AOX1基因启动子作为甲醇诱导的强启动子,在Pichia pastoris表达系统生产重组蛋白中得到广泛应用。P.pastoris中的转录 因子通过特异的顺式作用元件与启动子相互作用而影响基因的转录,因此 通过调节AOX1基因启动子的活性可以控制下游基因的表达
2.6:细胞裂解
从毕赤酵母细胞释放细胞外的DNA使用 Quant-iT™ PicoGreen® dsDNA Assay Kit 双链DNA检测试剂盒检测
3实验结果与讨论 3.1:
通过荧光显微术 UB - R- GFP和GFP- SKL 被正确地定位于细胞 质和过氧化物酶体， 符合预期
3.2:YGLY29325的三种不同的补料
分批发酵条件的取样数据(a) 甲醇
限制 (ML); (b) 葡萄糖培养8h甲醇 培养(20 h) (SML); (c) 氧气限制并
额外增加1%甲醇（OL）DO (%) ：
黑色直线，甘油或葡萄糖：蓝色,甲 醇:红色。细胞湿重：黑色三角
3.3毕赤酵母AOX1启动子（YGLY29325）根据三种不同的补料分批条件规制 - 甲醇 限制（ML），切换与葡萄糖和甲醇（SML）和氧气限制（OL）补料分批条件。 （a） GFP荧光表达图像（b）不同诱导时间的荧光蛋白相对密度。对于ML和OL，时间表示分 批阶段（T1），甘油补料分批阶段诱导前（T2）和诱导期（T3，10±2小时; T4中， 36±2小时; T5，62±2小时; T6，84±2小时）。对与SML条件下，细胞生长是在T2，T4 和T6 葡萄糖阶段，诱导是在T3和T5甲醇阶段
酵母菌优秀PPT课件-优秀PPT文档
限公司
产品名称 瓜子仁
荣华花生
开心果
商 标
规格
吉 昌
散装
散装
旭 明
散装
生产 日期 200604-07
200603-24
200604-10
主要不合格 项目 酵母
酵母
霉菌
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4. 酱油、醋
酵母和霉菌嗜好酸性反应，因而能使醋 发生霉变。含一定盐分的食品，对一般细菌 生长是不利的，但酵母对盐则有抵抗性，而 且能够生长，所以酱油也会受到侵犯。
二是微生物指标不合格。微生物指标不合格主要是 霉菌、酵母超标。抽查中有个别产品微生物指标不 合格。其中有1种产品的酵母实测值为480cfu/g，是 标准规定值的近20倍
三是产品标签标注不规范。
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烘炒食品产品质量国家监督抽查部分 质量较差的产品及其企业名单
企业名称
广州番禺区 越秀食品厂
广州市番禺 钟村荣华 食品厂
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酵母菌的危害：
➢少数耐高渗的酵母菌和鲁氏酵母、蜂蜜酵母可 使蜂蜜和果酱等败坏； ➢有的酵母菌是发酵工业的污染菌，影响发酵的 产量和质量； ➢某些酵母菌会引起人和植物的病害，例如白假 丝酵母可引起皮肤、粘膜、呼吸道、消化道等多 种疾病.
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四、食品中常见的酵母属
（一）酵母属 酵母中最重要、应用最广泛的一类： 1.酒类的酿造、面包发酵 2.引起高糖食品（果酱等）变质 3.能抵抗高浓度食盐溶液
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作业题
1. 酵母菌的特征 2. 什么是冷链？
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3
（三）酵母菌菌落特征
啤酒酵母菌落
细菌菌落
4
（三）酵母菌菌落特征
啤酒酵母菌落
红酵母菌落
毕赤酵母的摇瓶发酵方法
毕赤酵母的摇瓶发酵方法：一、摇瓶发酵方法:毕赤酵母摇瓶发酵方法分为两个阶段，1、酵母菌株生长阶段；2、脂肪酶诱导表达阶段。
1、酵母生长阶段。
准备试剂：1000ml BMGY培养基，1000ml BMMY培养基，10X的甲醇，摇瓶1L（灭菌），温控摇床，50ml离心管（灭菌）。
紫外分光光度计，石英比色皿。
以下所有操作均在超净台内或者无菌条件下完成。
（1）往灭好菌的IL摇瓶中加入100mlBMGY培养基，然后加入约1ml 脂肪酶菌株（培养基：菌液=100：1），用透气膜封口（透气，但是细菌不能透过）。
置于温控摇床上，温度调至300C，转速为250-300rpm/min，使酵母生长，OD600=2.0-6.0，时间约为15-24小时。
（2）将发酵液转入50ml离心管，1500g-3000g离心5min。
去掉上清，用BMMY培养基将菌体浓度稀释至OD600=1.0，约有500ml左右。
将稀释后的发酵液分别加入到1L的药瓶中，每个摇瓶150ml 发酵液（绝不能超过200ml）。
（3）将摇瓶置于温控摇床上，温度调至300C，转速为250-300rpm/min，使酵母表达脂肪酶，每24小时加入一次5%的甲醇，使甲醇的终浓度为0.5%。
连续诱导表达48小时。
（4）将发酵液进行12000rpm/min离心5min，取上清（若上清仍混浊，可反复离心）；进行酶活分析和蛋白含量分析。
BMGY培养基的配制（1000ml）：20g蛋白胨（peptone）,10g酵母提取物（Yeast Extract）,加水至700ml；1210C高温灭菌20min。
然后分别在无菌条件下加入10X YNB 100ml，10X 磷酸钾缓冲液（PH6.0）100ml，10X甘油 100ml。
发酵工程在食品中的应用PPT课件
啤酒、葡萄酒、面包发酵菌种——酵母
酿造啤酒、酒精、饮料酒，发酵面包 提取核酸、麦角醇、细胞色素C、辅酶A等
啤酒酵母
卡尔斯伯酵母
啤酒酿造中的典型 可食用、药用、饲料用
啤酒酵母变种 由葡萄果皮分离而来，用于葡萄
酒酿造
产香——汉逊氏酵母
能产乙酸乙酯，用于食品增香 但却是酒精发酵工业的有害菌
酒精工业有害菌——毕赤酵母
①防治腹泻；②缓解不耐乳糖症状；③预防阴道感染； ④增强人体免疫力；⑤缓解过敏作用；⑥降低血清胆固 醇；⑦预防癌症和抑制肿瘤生长
常见益生菌
乳酸菌 双歧杆菌、乳杆菌、链球菌应用较多 酪酸菌（有芽孢） 可用于临床治疗肠道失衡 纳豆芽孢杆菌 抗菌、溶血栓（纳豆激酶）、产分解酶、抗癌等
（2）酵母
分布：酵母菌主要分布在含糖质较高的偏酸性环境，诸 如果品、蔬菜、花蜜和植物叶子上，特别是葡萄园和果 园的土壤中，因而称为糖菌。
液化酶生产
Bacillus subtilis 枯草杆菌
产品：中温液化酶
地衣芽孢杆菌
产品：高温液化酶，蛋白酶等
谷氨酸生产——诱变育种后的棒杆菌
棒状杆菌
产氨短杆菌
产品：谷氨酸 产品：氨基酸、核苷酸
益生菌
是指改善宿主微生态平衡而发挥有益作用，达到 提高宿主健康水平和健康状态的活菌制剂及其代 谢产物。
特点 （1）原料好：淀粉多，胚乳多。（2）水好 ：鉴湖水，秋天水呈青黛色。
2.加饭酒:蒸饭分批加入，保持高浓度的培养基，高浓度发酵。
3.善酿:进一步提高酒精度，用状元酒代替水冲缸，装坛。
4.香雪:黄酒蒸馏，酒浓度高。
5.花雕:雕花的缸装的状元红。
B.其他几种有特色的酒
①江阴黑酒（木酒），米煮熟后炒焦，用木头装 坛；
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Байду номын сангаас
• 1背景介绍
• 2实验材料和方法
• 3实验结果
• 4结论与分析
4/15/2020
2
背景介绍
1.1醇氧化酶AOX1基因启动子作为甲醇诱导的强启动子,在Pichia pastoris表达系统生产重组蛋白中得到广泛应用。P.pastoris中的转录 因子通过特异的顺式作用元件与启动子相互作用而影响基因的转录,因此 通过调节AOX1基因启动子的活性可以控制下游基因的表达
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4结论与分析
在这项研究中，我们调查AOX1启动子和过氧化物酶体如何被调 节以响应碳源（例如甲醇）和氧气分子的可用性 在蛋白质生产阶 段，以更好地了解在毕赤酵母发酵常用的工艺条件。
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1. 3GFP-SKL（过氧化物酶体定位蛋白）,该载体含有融合了过氧化物酶体 定位信号1(PTS1)的绿色荧光蛋白报告分子GFP-SKL编码基因 成功地用于描 述不同 时间进程的AOX1启动子和过氧物酶体生物合成因素的特征
1.4泛素蛋白（细胞液中的短周期荧光蛋白）泛素(ubiquitin)是一种存 在于大多数真核细胞中的小蛋白。它的主要功能是标记需要分解掉的蛋白质， 使其被水解。当附有泛素的蛋白质移动到桶状的蛋白酶的时候，蛋白酶就会 将该蛋白质水解
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3实验结果与讨论 3.1:
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通过荧光显微术 UB - R- GFP和GFP- SKL 被正确地定位于细胞 质和过氧化物酶体， 符合预期
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3.2:YGLY29325的三种不同的补料
分批发酵条件的取样数据(a) 甲醇
限制 (ML); (b) 葡萄糖培养8h甲醇 培养(20 h) (SML); (c) 氧气限制并
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2.5抗体效价的测定
在培养上清液中于280nm处抗体浓度用二极管阵列检测器和蛋白A亲 和柱与HPLC系统
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2.6:细胞裂解
从毕赤酵母细胞释放细胞外的DNA使用 Quant-iT™ PicoGreen® dsDNA Assay Kit 双链DNA检测试剂盒检测
酵母表达手册）
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6
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2.3：补料分批培养条件
研究了三种不同发酵条件下的PpAOX1启动子（ YGLY29325 ）和过氧化物酶体 生物合成（ YGLY31884 ）的调控。
诱导阶段分批补料条件
ML:甲醇的起始供应速率为2.6gl/lh然后在0.0063/h的基础上以幂指的形式增加
题目
Regulation of alcohol oxidase 1 (AOX1) promoter and peroxisom biogenesis in different fermentation processes in Pichia pastoris
毕赤酵母不同发酵进程中乙醇氧化酶启动子和过氧物酶体生物合 成 的调控规律
GFP荧光表达图像（b）不同诱导时间的荧光蛋白相对密度。对于ML和OL，时间表示分
批阶段（T1），甘油补料分批阶段诱导前（T2）和诱导期（T3，10±2小时; T4中，
36±2小时; T5，62±2小时; T6，84±2小时）。对与SML条件下，细胞生长是在T2，T4
和T64/葡15/萄202糖0 阶段，诱导是在T3和T5甲醇阶段
SML:甲醇培养20h用50%的葡萄糖15g/h的速度培养8h作为一个周期共培养3个周期 OL:甲醇进料保持反应器中甲醇1％，每当DO迅速增加，这表明甲醇消耗。 DO级联 被打开，关闭和搅拌速度 被减小到实现氧气的限制
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2.4 GFP表达的定量和可视化：
约107固定的酵母细胞经PBS洗后用mounting solution重悬制作切片用 Axioscope 2 Plus 显微镜来观察OpenLAB软件来实现相机控制和图像收 集Volocity软件用来量化GFP在细胞中的强度
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3.4：ML，OL，及（b）SML 过氧化物酶体的生物合成三种 不同的培养条件下比较。 SML： 切换葡萄糖和甲醇进料。在过 氧化物酶体中使用GFP-SKL （YGLY31884）进行定量测定。 数据点表示在两个独立的运行 平均值。星号（*）表示甲醇阶 段
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额外增加1%甲醇（OL）DO (%) ：
黑色直线，甘油或葡萄糖：蓝色,甲 醇:红色。细胞湿重：黑色三角
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3.3毕赤酵母AOX1启动子（YGLY29325）根据三种不同的补料分批条件规制 - 甲醇 -
限制（ML），切换与葡萄糖和甲醇（SML）和氧气限制（OL）补料分批条件。 （a）
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3.5三种不同的工艺条件下（YGLY29325）发酵特性 - ML， OL和SML。 （一）细胞的密度分布（gWCW/ L）; （二）细胞列 解指数（毫克 DNA/ L）; （三）抗体效价（毫克/升）; （d） OL条件下延长诱导时间的细胞密度和抗体效价。
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3.6 在甲醇补料阶段的代谢过程（a）甲醇限定（ML）（b） 氧受限（OL）条件下补料分批巴斯德毕赤酵母的培养。
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二：实验材料和方法
2.1：质粒的构建 表达GFP- SKl pGLY11245或pGLY13173 转化YGLY27355或YGLY31884 YGLY14836生
产单克隆IgG1抗体 表达Ub-R-GFP pGLY10148转化 YGLY19309或YGLY29325 生产单克隆IgG1抗体 2.2：转化：毕赤酵母感受态的制备和电转化的方法（参见nvitrogen公司的Pichia
1.2过氧物酶体(Peroxisome)：遍布于真核生物的细胞器中, 用来去除有 害物质. 它们用外表的单层膜与细胞的原生质分隔开来, 膜上有功能重要 的膜蛋白, 用以向细胞器中输入蛋白质和促进细胞分裂. 与溶酶体不同的 是, 过氧物酶体不是由分泌通路产生, 而是通过先涨大后分裂的自我复制 过程产生, 当然也有证据显示新的过氧物酶体可以直接产生. 过氧物酶体 是由比利时细胞学家德迪夫(Christian de Duve)在1965年发现的.