电容法在线活细胞量传感器在黑曲霉带渣发酵生产柠檬酸中的应用

以黑曲霉带渣发酵生产柠檬酸的培养过程为对象,探讨了用电容法在线活细胞量传感器测量复合培养基中可结团丝状菌的活细胞量的可能性。通过实验确定了仪器的最佳分析条件:低通滤波值为60,测定频率为双频(580,10 085 kHz)。实验结果表明,电容法活细胞量传感器的电容值与活细胞量之间有着较好的线性对应关系,其线性相关系数为0.994。在发酵培养液采用带渣发酵的情况下,考察了在线活细胞电容值与常规的离线测定菌浓方法,如细胞干重、波长600nm处吸光度OD600和菌体离心体积之间的关系结果表明,在0～20 h,各离线参数与电容值之间的线性相关系数最好的仅为0.91。同时利用在线活细胞传感器的电容值能较好地估计出比生长速率,比通过在线间接参数OUR、CER估计更准确。因此,电容传感器为黑曲酶带渣发酵提供了一个可靠的工具。     

High-cell-density fermentation for ergosterol production by Saccharomyces cerevisiae

The direct feedback control of glucose using an on-line ethanol concentration monitor for ergosterol production by high-cell-density fermentation was investigated and the fermentation parameters (e.g., pH, dissolved oxygen, ethanol concentration, oxygen uptake rate, carbon dioxide evolution rate and respiratory quotient) were analyzed. Controlling glucose feeding rate in accordance with ethanol concentration and adjusting pH with ammonia during the fermentation process were effective fed-batch methods for ergosterol production. The fermentation parameters well described the variation of the whole fermentation process. Cultivation in a 5 l fermentor was carried out under the following conditions: culture temperature, 30 degrees C; pH, 5.5; agitation speed, 600 rpm; fermentation time, 60 h; controlling ethanol concentration below 1% and keeping respiratory quotient (RQ) at approximately 1.0. Under these conditions, the yeast dry weight reached 120 g/l and the ergosterol yield reached 1500 mg/l.     

基于两阶段氨基酸添加的谷胱甘肽发酵高产方法

为提高产朊假丝酵母(Candida utilis SZU 07-01)发酵生产谷胱甘肽(glutathione,GSH)的产量,分别考察L-蛋氨酸和L-半胱氨酸在GSH分批发酵和流加发酵中的作用,结果发现L-蛋氨酸提升了酵母细胞在生长期的GSH合成能力,而L-半胱氨酸显著提高了细胞生长结束后的GSH产量。在此基础上,提出了两阶段氨基酸添加策略,即在分批发酵初始时(0 h)添加60 mmol/L L-蛋氨酸,在流加发酵过程中细胞干质量达到最大值时(第27小时)一次性添加30 mmol/L L-半胱氨酸。最终,GSH产量和胞内GSH含量进一步提高,分别达到1 247.1 mg/L和24.1 mg/g。该两阶段氨基酸添加策略在GSH发酵高产中的成功应用,对其他类似化合物的高效生产具有一定的借鉴意义。     

Maximizing yellow pigment production in fed-batch culture of Monascus sp

Yellow pigment production in exponential fed-batch cultivation of Monascus sp. was studied. Due to the difficulty of measuring the optical density for accurate determination of the cell concentration, a capacitance probe was employed on-line. The feed rate needed to keep the specific growth rate, mu, constant in fed-batch culture was determined on the basis of the cell concentration measured by the capacitance probe. Control of mu was improved by using updated information on the cell concentration compared with the simple feed-forward determination method using the initial cell concentration only. The highest specific pigment production rate was achieved with a mu of 0.02 h(-1) in the feeding phase. However, among several fermentation examined, the largest pigment production in the final step was obtained at a mu of 0.01 h(-1); in each case the same amount of substrates was used. An investigation of the optimal initial glucose concentration revealed that pigment production was maximum when the initial glucose concentration in the batch mode was 10 g/l and mu was 0.01 h(-1) in the fed-batch mode. It was also found that the pellet weight in the fermentation could be accurately estimated by image analysis. The ratio of the mycelium weight to the total cell weight estimated from information on the total cell weight and the estimated pellet weight was found to be more than 80%. However, no clear quantitative relationship could be discerned between the specific pigment production rate, rho, and the ratio of mycelium in the cell population.     

Taking advantage of the flow cytometry technique for improving malolactic starters production

In bioprocesses, the industrial use of lactic acid bacteria as starter cultures is directly related to the effectiveness of preservation technologies which must guarantee high cell survival and functionality rates through the processing stages and after storage and rehydration. In this work, flow cytometry (FC) technique in combination with fluorescent probes allowed to monitor, almost in real-time conditions, the physiological states of cider malolactic starter cultures (Lactobacillus hilgardii) throughout preservation processes (freezing or freeze-drying) and their further progress during the first stages of culture implantation in the fermentation medium. A subpopulation of non-culturable but metabolically active cells, which could contribute to further fermentation, was determined when comparing FC results and viable counts. Metabolically active and dead cell counts determined by FC and colony forming units determined by the standard plate counting method showed that reactivation of frozen L. hilgardii cells during 24 h, using apple must supplemented with YE as cultivation and rehydration medium, resulted in the more suitable procedure for obtaining malolactic starter cultures.     

耐高温α-淀粉酶高密度高表达发酵条件的优化

通过摇瓶实验对产耐高温α-淀粉酶的重组菌E.coli BL21的高密度高表达发酵条件进行研究。考察不同培养基和不同发酵条件对该菌株产耐高温α-淀粉酶的影响,并利用正交试验进行优化。结果表明:在葡萄糖0.5g/L,酵母粉15g/L,pH6.5,诱导时机为接种后发酵4h,诱导时间为6h,IPTG添加量为0.8mmol/L,接种量为体积分数3%的优化条件下,该重组菌发酵液菌体生物量为原来的1.29倍,酶活力达到8.754U/mL,是原来的1.55倍,目的蛋白的表达量也为原来的1.24倍。     

谷糠乳杆菌84-M-Y-7培养基配方和发酵工艺优化

以菌体量(OD600 nm值)为评价指标,通过单因素及响应面试验对谷糠乳杆菌(Lactobacillus farraginis)84-M-Y-7的培养基配方进行优化,应用正交试验对发酵条件进行优化,进一步以湿菌质量及活菌数为评价指标,对谷糠乳杆菌84-M-Y-7的菌体收集条件进行了优化。结果表明,最佳培养基配方为大豆蛋白胨3%、豆粕粉1.5%、乳糖3%、维生素B20.06%,在此优化培养基配方下,谷糠乳杆菌84-M-Y-7的菌体量比优化前提高了1.9倍;菌株最适培养条件为：接种量8%、发酵温度35℃、发酵时间24 h,在此培养条件下谷糠乳杆菌84-M-Y-7的菌体密度比优化前提高了2.3倍。最佳菌体收集条件为：4 000 r/min离心15 min,在此条件下,湿菌质量为0.012 g/mL,活菌数为1.05×10⁷ CFU/mL。     

海洋多粘类芽孢杆菌L_1-9菌株50L发酵罐发酵工艺

为了提高海洋细菌L1-9菌株液体发酵的生物量和抑菌活性,进行了50 L发酵罐的分批发酵和补料发酵工艺研究。结果表明:L1-9菌株在50 L发酵罐中分批发酵工艺为:接种量8%(菌龄24 h、浓度为109个细胞/mL)、初始搅拌速度250 r/min、通气量为3 L/min,发酵时间为11～12 h,生物量达2.40×1010CFU/mL;补料分批发酵工艺为:在分批发酵的条件下,发酵13 h开始流加60 g/L葡萄糖溶液,使还原糖浓度保持在0.4 g/L左右,发酵周期30 h,生物量达到4.63×1010CFU/mL,比分批发酵提高了78.07%;抑菌时效测定结果表明,发酵液对金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用伴随着生物量的增加不断加强,20 h抑菌活性达到高峰,抑菌带宽度为17.75 mm,抑菌活性与生物量呈正相关,为生长关联型。     

糯米发酵炸糕酵母菌的分离鉴定及优势菌株筛选

为探究天津耳朵眼炸糕工业化纯种或混种发酵中的优势酵母菌株,以耳朵眼炸糕的沥水米浆和发酵面团为原料,首先采用米浆固体培养基划线分离纯化的方法,获得6株酵母菌。其次对其进行WL培养基菌落形态观察、个体形态显微镜观察、生理生化特征测定和18S rRNA基因序列分析,发现Y2、Y3菌株为酿酒酵母（Saccharomyces cerevisiae）,Y4为西文毕赤酵母（Pichia occidentalis）,Y7-1、Y7-2为矮小假丝酵母（Candida humilis）,Y8为溶磷白地霉（Galactomyces geotrichum）。最后测定各菌株的产气量、碳源利用率和菌体生长速度,结果显示Y2发酵产气快且产气量最大,发酵结束时发酵液中的残糖含量最低为16.33 mg,快速生长期的OD600为0.149。由此认为Y2菌株为糯米炸糕发酵生产的优势酵母菌,具有作为纯种发酵菌剂生产的潜力,为耳朵眼炸糕的品质控制和工业化生产提供了理论依据。     

啤酒酵母浓度实时在线检测方法的研究(一)

酵母的存在会改变发酵液的电特性,发酵液在无线电频率范围内的电特性可用电容率和电导率充分地描述。实验证实发酵液在无线电频率范围内的电容率增量是测量频率和生物量浓度的函数。基于对发酵液电容率分布的研究,本文提出了测量酵母浓度的新方法。用本方法不用取样就能对发酵液中的酵母浓度进行实时在线测量,而且测得的是活的酵母浓度。本文制作的电极直接插入 发酵器中并满足高温蒸汽 灭菌条件。本方法在生化制药、食品发酵、啤酒酿造、污水检测等工业领域里有很好的推广应用前景     

培养基pH调控法高密度发酵唾液乳杆菌XH4B研究

本研究以MRS培养基为基础,通过优化碳、氮源及无机盐的配方和用量,再结合补料发酵,最终实现唾液乳杆菌XH4B高密度培养的目的。以乳酸菌的生物量为指标,同时考查发酵液pH值、乳酸含量等,最终确定酵母粉和蔗糖为最佳氮、碳源,同时增加乙酸钠用量至2%、磷酸二氢钠0.6%,可以对发酵液酸化时提供一定的缓冲作用。采用优化的PY-Suc培养基,唾液乳杆菌XH4B的生物量最高能达到6.91 g/L,明显高于MRS培养基的5.01~6.30 g/L(P0.05)。等量补料培养并且采用NaOH中和发酵液pH值时,乳酸最高积累速度可以达到5.958 g/(L·h),但是随着培养时间延长,积累速度迅速下降。发酵酸化较严重时(乳酸含量9~10 g/L),唾液乳杆菌XH4B的生物量积累变缓。结论:优化MRS培养基,并加大乙酸钠、磷酸二氢钠等能够缓冲发酵液的无机盐用量,结合补料发酵,可以实现唾液乳杆菌XH4B的高密度培养。     

用电容率频谱法测量啤酒酵母的浓度和活性

酵母的存在会改变发酵液的电特性,发酵液在无线电频率范围内的电特性可用电容率和电导率充分地描述。实验证实发酵液在无线电频率范围内的电容率增量是测量频率和生物量浓度的函数。基于对发酵液电容率分布的研究,提出了测量酵母浓度的新方法。用本方法不用取样就能对发酵液中的酵母浓度进行实时在线测量,而且测得的是活的酵母浓度。本研究制作的的电极直接插入发酵器中并满足高温蒸汽灭菌条件。本方法在生化制药、食品发酵、啤酒酿造、污水检测等工业领域里有很好的推广应用前景     

补料分批发酵生产谷胱甘肽的研究

考察5L 发酵罐中分批补加葡萄糖对发酵生产谷胱甘肽(GSH)的影响。采用20g/L 初糖质量浓度,在发酵12h 至27h 每隔3h 分别补加22、24、24、24、24g/L 和22g/L 葡萄糖,可以使酿酒酵母在发酵33h 时GSH 质量浓度达到72.49mg/L,细胞干质量浓度达到28.52g/L,分别为初糖20g/L 分批培养方式的2.86 倍和4.93 倍。补料分批发酵可以明显促进酿酒酵母生长和提高GSH 的合成。     

酵母浓度（生物量浓度）实时在线检测方法的研究

微生物的存在会改变发酵液的电物性,发酵液在无线电频率范围内的电特性可用电容率和电导率充分地描述。实验证实发酵液在无线电频率范围内的电容率增量是测量频率和生物量浓度的函数。基于对发酵液电容率分布的研究,本文提出了测量生物量浓度的新方法。此方法不用取样就能对发酵液中的生物量进行实时在线测量,而且测得的是活的酵母浓度。     

5L发酵罐高密度培养番茄红素工程菌及其发酵条件优化

本研究对产番茄红素的工程菌W-05进行发酵条件优化。首先单因素实验确定培养基种类;培养温度;培养基中的较优碳源、氮源以及无机盐。然后根据单因素实验结果,设置响应面实验确定各因素之间的交互影响,响应面实验结果表明培养基各组分为:蛋白胨10.00 g/L、酵母浸出粉5.00 g/L、甘油7.80 mL/L、硝酸铵3.30 g/L、KH2PO4 1.80 g/L、Na Cl 11.62 g/L时,番茄红素得率达理论值为3.42 mg/L。在5 L发酵罐中,使用优化后的培养基高密度培养工程菌W-05。实验结果显示工程菌W-05高密度培养较优发酵条件为:p H值为7.0,溶氧百分数为20%左右及指数流加补料。此条件对比普通分批培养条件,菌体的生物量和番茄红素产量显著提高(p0.05)。优化发酵29 h后的菌体干重达到16.55 g/L,番茄红素得率为19.93 mg/L。这说明改善培养基成分及发酵条件能大幅提高工程菌的番茄红素得率。     

Using Dimethyldicarbonate to Minimize Sulfur Dioxide for Prevention of Fermentation from Excessive Yeast Contamination in Juice and Semi-Sweet Wine

ABSTRACT: Sulfur dioxide (SO₂) at 0, 10, 25, and 50 mg/L and/or dimethyldicarbonate (DMDC) at 0, 50, 100, and 200 mg/L at different pH levels (3.0, 3.2, 3.4, and 3.6) in juice and semi-sweet wine were evaluated by monitoring yeast growth and visible fermentation at 20 °C after excessive yeast contamination (500 to 700 colony forming units/mL), using Saccharomyces bayanus. The treatments with the least preservatives that prevented yeast growth and visible fermentation at all pH levels in juice were 50 mg/L SO₂+ 100 mg/L DMDC and in wine were 10 mg/L SO₂+ 50 mg/L DMDC. DMDC and minimal SO₂ levels prevented fermentation in juice and semi-sweet wine with excessive yeast contamination.     

木糖浓度及补料发酵对树干毕赤酵母乙醇发酵的影响

探究不同浓度木糖及补料对树干毕赤酵母（Pichia stipitis）菌株1K-9发酵木糖产乙醇的影响,提高木糖产乙醇的发酵水平,为扩大规模发酵木糖产乙醇打下基础。结果表明,菌株1K-9先采用10%木糖进行乙醇发酵,36 h补加与10%木糖培养基等体积的20%木糖培养基继续发酵,发酵至108 h时菌数也达到了（12.16±0.07）×108个/mL,较未补料发酵时有所提高;发酵108 h时醪液中残留的木糖含量为（1.03±0.02）g/L,较未补料发酵时有所降低;乙醇含量达到了6.56%vol,较未补料时提高了1.85%vol。因此补料发酵是有效的。     

Production of 2-phenylethanol,a flavor ingredient,by Pichia fermentans L-5 under various culture conditions

Production of 2-phenylethanol by Pichia fermentans L-5 as affected by cultivation temperature (20–40°C), medium pH (4.0–11.0) and shaking speed (0–250 rpm) as well as the kinetics of 2-phenylethanol formation during fermentation were investigated in the present study. Results showed the optimum culture temperature, initial medium pH and shaking speed for the 2-phenylethanol production by test organism were 30–35°C, 8.5 and 250 rpm, respectively. Kinetics study revealed that production of 2-phenylethanol during P. fermentans L-5 fermentation increased almost in parallel with the increase in biomass or viable population during the initial stage of fermentation. During a 72-h fermentation period, level of phenylalanine in medium dropped coincidentally with increasing biomass and 2-phenylethyl derivatives, from the start of fermentation. A maximum 2-phenylethanol production of ca 505.5 mg/l with a molar yield of ca 0.74 was obtained after 16 h of fermentation.     

Design Considerations in Hybrid Neural Optimization of Fed-Batch Fermentation for PHB Production by <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

Mechanistic models are often inadequate for non-ideal fermentation processes. This has been a major limitation in the commercial success of fed-batch fermentations for poly-β-hydroxybutyrate (PHB), a biopolymer that is potentially better than many synthetic polymers. A previous study with Ralstonia eutropha has shown that optimization through neural networks in place of mechanistic models can help to enhance PHB production substantially. Hybrid models combine the strengths of mechanistic and neural models and minimize their weaknesses. However, there is no unique hybrid neural model for a given application. Therefore, three fundamental hybrid designs have been compared with the neural and mechanistic optimizations done earlier for a fed-batch bioreactor for PHB production. Non-ideal features were introduced by adding Gaussian noise in the two feed streams—glucose and NH₄Cl—and incorporating the optimum finite dispersion determined previously. All three hybrid designs were superior to the neural and mechanistic approaches. The best hybrid system, using a weighted combination of mechanistic and neural kinetic rates, generated 140% more of biomass and 330% more of PHB than conventional mechanistic optimization. This was achieved with reduced consumption of the two primary substrates. The supremacy of this hybrid system underlines the complexity of the PHB synthesis network and the merit in combining mechanistic and neural kinetics, and the performance enhancement achieved indicates the feasibility of such a system for an economically viable large-scale PHB fermentation.     

Alcoholic fermentation in winemaking: On-line measurement of density and carbon dioxide evolution

Linear correlations on the laboratory scale (15 litres) have been made between the concentrations of ethanol and sugars, and the volume of CO₂ released and the density of the fermentation medium. The on-line measurement of these variables in a 700-litre winemaking tank by corresponding sensors (flowmeter and pressure transducers) and application of resulting correlations have led to the real-time determination of the concentrations of ethanol and sugars.The proposed methods were validated by off-line measurements. The method using the volume of CO₂ released is easier to use. It is an accurate means for following alcoholic fermentation in winemaking.