Optimization of L(+)-Lactic Acid Production from Xylose with Rhizopus Oryzae Mutant RLC41-6 Breeding by Low-Energy Ion Implantation

In order to obtain an industrial strain with a higher L（＋）-lactic acid yield, the strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion beam implantation and the mutant strain Rhizopus oryzae RLC41-6 was obtained. An experimental finding was made in surprise that Rhizopus oryzae mutant RLC41-6 is not only an L（＋）-lactic acid producer from corn starch but also an efficient producer of L（＋）-lactic acid from xylose. Under optimal conditions, the production of L（＋）-lactic acid from 100 g/L xylose reached 77.39 g/L after 144 h fed-batch fermentation, A high mutation rate and a wide mutation spectrum of low-energy ion implantation were observed in the experiment.

Optimization of L-lactic Acid Production of Rhizopus Oryzae Mutant RLC41-6 by Ion Beam Implantation at Low-Energy

In order to obtain an industrial strain with a higher L（＋）-lactic acid yield, the strain Rhizopus oryzae RF3608 was mutated by means of nitrogen ion beam implantation and the mutant strain RLC41-6 was isolated. Under optimal conditions the yield of L（＋）-lactic acid produced in a shake-flask reached 133 g/L - 137 g/L after 36 h cultivation, indicating that the conversion rate based on glucose was as high as 88% - 91% and the productivity was 3.75 g/L.h. It was almost a 115% increase in lactic acid production compared with the original strain RF3608.

Improvement of L(+)-Lactic Acid Production of Rhizopus Oryzae by Low-Energy Ions and Analysis of Its Mechanism

The wild type strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion implantation （15 keV, 7.8×10^14 ～ 2.08 ×10^15 ions/cm^2） to find an industrial strain with a higher L（＋）-lactic acid yield, and two mutants RE3303 and RF9052 were isolated. In order to discuss the mechanism primarily, Lactate Dehydrogenase of Rhizopus oryzae was studied. While the two mutants produced L（＋）-lactic acid by 75% more than the wild strain did, their specific activity of Lactate Dehydrogenase was found to be higher than that in the wild strain. The optimum temperature of Lactate Dehydrogenase in Rhizopus oryzae RF9052 was higher. Compared to the wild strain, the Michaelis constant （Km） value of Lactate Dehydrogenase in the mutants was Changed. All these changes show that L（＋）-lactic acid production has a correlation with the specific activity of Lactate Dehydrogenase. The low-energy ions, implanted into the strain, may improve the specific activity of Lactate Dehydrogenase by influencing its gene structure and protein structure.

Optimization of L(+)-Lactic Acid Fermentation Without Neutralisation of Rhizopus Oryzae Mutant RK02 by Low-Energy Ion Implantation

In order to get an industrial strain which can yield a high concentration of lactic acid for ISPR （in situ product removal）, the original strain Rhizopus oryzae RE3303 was mutated by low-energy ion beam implantation. A mutant RK02 was screened, and the factors such as the substrate concentration, nitrogen source concentration, inoculum size, seed age, aeration and temperature that affect the production of lactic acid were studied in detail. Under optimal con- ditions, the maximum concentration of L（＋）-lactic acid reached 34.85 g/L after 30 h shake-flask cultivation without adding any neutralisation （5% Glucose added）, which was a 146% increase in lactic acid production after ion implantation compared with the original strain. It was also shown that RK02 can be used in ISPR to reduce the number of times of separation.

Rhizopus sp.052凝乳酶的部分酶学性质

Rhizopus sp．052在pH1～7范围内，4℃条件下稳定。凝乳酶的最适作用温度为40℃，55℃保持20min完全失去活性，与小牛凝乳酶性质相似，而热失活的温度低于小牛凝乳酶。Rhizopus sp．052凝乳酶对热不稳定的特性有利于干酪副产品乳清的加工处理。Rhizopus sp．052凝乳酶对酪蛋白的水解能力比蛋白酶低，比小牛凝乳酶高，可以用于干酪的生产。对N-末段15个氨基酸序列GTGSVPVTDYQNDVE进行检索，结果表明，Rhizopus sp．052凝乳酶与其他菌株相比不完全相同，尚未见有研究者对其进行研究和报道。

Enhancement of L(+)-Lactic Acid Production of Immobilized Rhizoous Orvzae Imolanted by Ion Beams

Immobilized Rhizopus oryzae culturing may be a solution to the inhibited production of L（＋）-lactic acid in submerged fermentation, which is caused by aggregated mycelia floc. In the present study, a R. oryzae mutant （RL6041） with a 90% conversion rate of glucose into L-lactic acid was obtained by N＋ implantation under the optimized conditions of a beam energy of 15 keV and a dose of 2.6 ×10^15 ions/cm^2. Using polyurethane foam as the immobilization matrix, the optimal L-lactic acid production conditions were determined as 4 mm polyurethane foam, 150 r/min, 50 g/L ～ 80 g/L of initial glucose, 38℃ and pH 6.0. 15-cycle repeated productions of L-lactic acid by immobilized RL6041 were performed under the optimized culturing conditions and over 80% of the glucose was converted into L-lactic acid in 30 hours on average. The results show that immobilized RL6041 is a promising candidate for continuous L-lactic acid production.

乳杆菌Lactobacillus sp.lxp发酵高产L-乳酸研究

筛选得到一株乳杆菌Lactobacillussp.,进行发酵生产高浓度L-乳酸的研究。考察了种龄、接种量、温度和不同pH调节剂对乳酸发酵的影响。结果表明:最佳种子培养时间为15 h;最佳接种量为15%;最适培养温度为42℃;与氨水和氢氧化钠相比,碳酸钙更适于作为发酵过程的pH调节剂;以葡萄糖为碳源,添加豆粕水解液和玉米浆作为辅料,2 L罐培养120 h,L-乳酸质量浓度可达202 g/L,糖转化率91.3%,L-乳酸占发酵液中总酸含量98%以上。

盐酸氨溴索联合莫西沙星、比阿培南治疗老年重症肺炎对患者肺泡灌洗液TLR4、PCT、hs-CRP水平的影响

目的:探讨盐酸氨溴索联合莫西沙星、比阿培南治疗老年重症肺炎(SP)的效果及对患者肺泡灌洗液中Toll样受体4(TLR4)、降钙素原(PCT)、超敏C反应蛋白(hs-CRP)水平的影响。方法:选取2023年1—12月在上饶市立医院接受治疗的80例老年SP患者,以随机数字表法分成对照组与观察组,各40例。两组均接受支气管肺泡灌洗治疗,对照组予以莫西沙星、比阿培南治疗,观察组在对照组基础上接受盐酸氨溴索治疗,两组均接受为期10 d的治疗。分析两组临床疗效、临床症状、血气指标及炎症因子,并记录不良反应发生率。结果:观察组临床总有效率高于对照组,咳嗽减轻时间、咳痰减轻时间、体温恢复正常时间均早于对照组,治疗后血乳酸、动脉血二氧化碳分压(PaCO_(2))、TLR4、PCT、hs-CRP水平均低于对照组,经皮动脉血氧饱和度(SpO_(2))、动脉血氧分压(PaO_(2))、氧合指数均高于对照组(P<0.05)。两组不良反应发生率比较,差异无统计学意义(P>0.05)。结论:盐酸氨溴索联合莫西沙星、比阿培南治疗老年SP的效果较好,可降低肺泡灌洗液中炎症因子水平,改善血乳酸及动脉血气指标。

Rhizopus oryzae LS1利用丢糟水解液发酵生产乳酸

研究了一株诱变米根霉Rhizopus oryzae LS-1利用丢糟水解液发酵生产乳酸的可行性。首先考察R.oryzae LS-1利用葡萄糖和木糖的糖代谢差异特性,并通过正交实验优化丢糟水解液发酵生产乳酸的工艺参数。结果表明,R.oryzae LS-1能代谢利用葡萄糖和木糖,且二者存在协同互补作用,有利于乳酸生成和糖酸转化,可用于木质纤维原料的乳酸生产。丢糟水解液发酵生产乳酸的实验表明,氯化铵是R.oryzae LS-1适宜的氮源。在接种量为3.0%、p H为6.5、发酵时间为96h、Ca CO3添加量为80g/L的条件下,乳酸生成浓度为13.27g/L,糖利用率为79.61%。说明诱变菌株R.oryzae LS-1具备发酵丢糟水解液制备乳酸的潜力。

米根霉发酵生产L(+)-乳酸研究进展

米根霉是生产绿色平台生物化学品L(+) 乳酸的理想菌种 ,目前集中在发酵工艺的优化、新型生物反应器的设计以及细胞固定化等方面的研究 ,通过控制米根霉菌体形态使之自聚集成为一定大小的球体进行乳酸发酵 ,操作简便、费用低。建议今后从应用代谢工程技术定向选育米根霉L(+) 乳酸高产菌 ,改进发酵设备、改良提取工艺 ,合理控制乳酸产品的构型等几个方面着手进行进一步研究 ,从而降低乳酸生产原料的成本 ,扩大L(+) 乳酸的应用领域。

根霉发酵L-乳酸

本文报道了选育一株产L-乳酸的根霉JSMI-R73,对该菌的性能及影响产酸的条件进行了研究,并进行了500升罐的扩大试验。当口服葡萄糖浓度为10％时产酸70g/l以上,13％时产酸101g/l,其中L-乳酸稳定在85％以上,最高达98％。该菌能适应玉米作培养基,当玉米浓度为12％时产酸70g/l以上,20％时产酸105g/l以上,其中L-乳酸在88％以上,最高可达99％,达到了与葡萄糖相似的发酵水平,这是较有实用意义的结果。

高效液相色谱法同时测定生物质乳酸发酵液中有机酸及糖类化合物

建立了高效液相色谱(HPLC)同时测定生物质乳酸发酵液中有机酸及糖类的分析方法。使用Bio-RadAminex HPX-87H色谱柱,以5 mmol/L的H2SO4为流动相,在柱温55℃,流速0.6 mL/min条件下,采用示差折光检测器进行检测。结果表明,该方法可在17 min内实现发酵液中各种有机酸和糖类化合物等的完全分离与定量,6种有机酸和3种糖类化合物在0.15～5.19 g/L范围内的线性关系良好,回归方程的线性相关系数在0.999 8以上。将该法用于米根霉发酵液的检测,两个水平的加标回收率为96.91%～103.11%,相对标准偏差(n=6)为0.81%～4.61%。该法适用于微生物发酵液中多种有机酸和糖类的快速、高效分离和定量测定。

反相高效液相色谱法测定米根霉乳酸发酵液中的乳酸

采用反相高效液相色谱法测定了米根霉乳酸发酵液中的乳酸。在Wakosil Ⅱ 5C18RS (4 6mmi d ×15 0mm ,5 μm)色谱柱上 ,以 0 0 1mol/L磷酸溶液 (pH 2 5 )作流动相 ,流速为 1mL/min ,紫外检测波长为 2 10nm。实验结果表明 ,该方法的相对标准偏差为 0 2 2 % (n =5 ) ,回收率为 99%以上。方法简便、快速。

L-乳酸发酵的研究

本文报导了L-乳酸产生菌筛选、发酵条件以及发酵产物鉴定的结果。从56株根霉中筛选出10株产L-乳酸较高的菌株,其中根霉R47产L-乳酸最高,产酸稳定。发酵条件试验结果表明,该菌最适发酵培养基组成(%):葡萄糖15,尿素0.2,KH_2PO_4 0.02,MgSO_4·7H_2O 0.025,ZnSO_4·7H_2O 0.0044,CaCO_3 6,7;pH6.7。在摇瓶培养条件下,35℃48小时,产L-乳酸达11.84g/100ml,对糖的重量转化率达78.9%。发酵液经离子交换等方法纯化,得到无色或微黄色透明糖浆状液体。经纸层析、比旋光度测定、紫外光谱和红外光谱分析证明确系L-乳酸。

L-乳酸高产菌株的选育和产酸条件的研究

为了获得更适于工业生产的产乳酸菌株 ,采用低能离子诱变方法 ,对出发菌米根霉(Rhizopusoryzae)PW35 2进行改良 ,获得高产L ( + ) 乳酸菌株RE330 3,其产酸比亲株提高75 %。用正交试验方法对突变株适宜产酸条件进行了研究 ,在最优条件下 ,其产酸量可达1 31～ 1 36g L ,最高可达 1 40g L ,糖转化率为 86%～ 90 %。

利用纤维废弃物生产L-乳酸的研究

为了使对环境造成污染的纤维废弃物得到资源化利用,采用纤维素酶对纤维废料进行酶解,并用米根霉发酵生产L-乳酸。研究结果表明,0.3 mol/L稀磷酸对纤维废弃物进行预处理后,纤维素酶酶解60h,取糖化液进行米根霉发酵,可得到较佳转化率;在固定摇床转速为120 r/min、种龄12 h的条件下,米根霉发酵生产L-乳酸的最佳条件为:装液量(250 mL瓶)50 mL,接种量8%;经稀磷酸预处理纤维废料分别糖化发酵时,糖对L-乳酸转化率高于未经稀磷酸预处理的,而在同时糖化发酵过程中,经稀磷酸预处理纤维废料发酵效果不明显。

L-乳酸高产菌株的诱变选育

以代谢控制发酵理论为依据,利用紫外线对NRRL-395进行诱变处理,然后用溴钾酚紫平板、高锰酸钾平板、高糖平板、高酸平板、纯乳酸平板、琥珀酸平板,筛选得到一株高产L-乳酸的正向突变菌株NAF-032,其平均产酸率为73.05g/L,对糖的转化率为68.3%。

机械搅拌生物反应器的CFD模拟及其在发酵生产乳酸中的应用

用计算流体力学(CFD)方法中的Mixsim模拟3L机械搅拌生物反应器中不同搅拌转速对搅拌流场的影响,结果显示Mixsim软件可以很好的模拟机械搅拌生物反应器工作过程中的流场变化。在高转速下,剪切速率较大,菌丝体98%以上为菌丝体小球,直径在0.1～0.3mm,菌体生长速度慢,产乳酸质量浓度较低;低转速下产生的剪切速率较小,菌体大部分为菌丝形态,产乳酸质量浓度较高,但是大量菌丝不利于后期发酵的进行。在400r/min下米根霉发酵产乳酸质量浓度最高,为106.7g/L,菌体80%以上为1.0～1.5mm左右的菌丝球,通过CFD模拟得到该条件下的剪切速率最大值为1.58m/s。

无载体固定化米根霉重复间歇发酵生产L-乳酸

通过研究影响米根霉菌丝体形态的培养基因素,初步构建了无载体固定化米根霉重复间歇发酵生产L-乳酸的工艺条件。研究结果表明,首批次发酵培养基采用120 g/L葡萄糖,3 g/L硝酸铵,K+和Na+浓度比为1:1,发酵72 h后,米根霉菌体形态为均匀的菌丝体小球,直径为1.0 mm^2.0 mm,此时L-乳酸产量可达100.8 g/L,葡萄糖转化率为84%。在此基础上,利用米根霉菌丝体小球重复间歇发酵16批次,每批次发酵24 h,此时葡萄糖转化率均高于75%,L-乳酸产量保持在60.0 g/L以上,米根霉菌丝体小球形态保持稳定。