发酵菌种对薏米乳品质及代谢产物的影响

分别利用保加利亚乳杆菌（Lactobacillus bulgaricus）、植物乳杆菌（Lactobacillus helveticus）和瑞士乳杆菌（Lactobacillus plantarum）为发酵菌株对薏米乳进行发酵。利用电子舌对得到的发酵薏米乳进行感官分析,利用非靶向代谢组学技术分析发酵薏米乳差异代谢产物,研究发酵菌种对薏米乳品质及代谢产物的影响。结果表明,植物乳杆菌发酵的薏米乳中三萜类化合物含量较高为26.19 mg/L,保加利亚乳杆菌发酵薏米乳中的多糖含量较高为34.17 mg/L,且具有较好的黏度（23.09 mPa·s）,其结构稳定性更高;瑞士乳杆菌发酵可以增加薏米乳的鲜味。非靶向代谢组学试验共检出947种代谢产物,其中植物乳杆菌发酵的薏米乳样品中检测到更多的短肽类化合物,还有吲哚乙酸、泛酸、亚油酸、β-柠檬酰-L-谷氨酸以及脂类等多种功能性成分。     

ε-聚-L-赖氨酸生产菌株的遗传改造与发酵优化控制

ε-聚-L-赖氨酸作为微生物合成的赖氨酸聚合物,具有良好的抑菌活性和生物相容性,在食品和医药等领域的需求量日增。然而因受微生物胞内代谢调控的制约,其合成量低。基于前体赖氨酸代谢途径的遗传改造,以及有效的发酵控制策略,已在一定程度上提高了ε-聚-L-赖氨酸的产率。对ε-聚-L-赖氨酸合成酶及降解酶的分子结构研究和产生菌遗传转化体系的构建,有望进一步提升ε-聚-L-赖氨酸的品质和产量。     

L-赖氨酸合成代谢中NADPH代谢的研究进展

L-赖氨酸是人类和动物所必需的但自身不能合成的氨基酸之一,它对平衡氨基酸组成、调节体内代谢平衡、提高体内对谷类蛋白质的吸收、改善动物营养、促进生长发育均有重要作用。L-赖氨酸的发酵不同于L-谷氨酸的发酵,每形成1 mol的L-赖氨酸需要消耗4 mol的NADPH,而每生产1 mol的L-谷氨酸只需消耗1 mol的NADPH,因此在L-赖氨酸发酵过程中,提高代谢途径中的NADPH量是增加L-赖氨酸产量的关键因素之一。作者从谷氨酸棒杆菌中L-赖氨酸生物合成途径、NADPH代谢以及与NADPH代谢相关的酶三个方面概述了L-赖氨酸合成代谢中NADPH代谢的研究进展。     

人工接种东北酸菜发酵过程中氨基酸类物质的代谢变化

氨基酸对人工接种发酵东北酸菜的风味形成有重要作用，该研究着重利用代谢组学对人工接种发酵东北酸菜发酵过程中的氨基酸转化途径进行了研究。在酸菜接种发酵过程中共检测出的638种代谢物以氨基酸，多肽及其类似物为主（25.43%），且在发酵中期显著增加。人工接种发酵酸菜代谢途径主要与氨基酸代谢、脂类代谢、其他次生代谢物生物合成、碳水化合物代谢、核苷酸代谢等途径有关，且代谢途径涉及的化合物以多肽类为主（33.33%）。在接种发酵过程中鉴定出12种对酸菜的酸味和鲜味具有重要贡献的氨基酸，并且20 ℃接种酸菜风味与滋味的形成集中在发酵前10天。此外氨基酸类代谢物显著富集的通路主要涉及精氨酸和脯氨酸代谢、半胱氨酸和甲硫氨酸代谢、谷胱甘肽代谢、丝氨酸代谢、组氨酸代谢以及赖氨酸降解。结果表明接种酸菜发酵过程氨基酸类物质代谢途径多变，并且对接种发酵东北酸菜的风味调节具有重要意义。     

Mn2+对植物乳杆菌影响的代谢组学分析

利用代谢组学研究分析技术,对比研究了锰离子缺乏对植物乳杆菌发酵过程中代谢途径变化的影响。采用气-质连用技术(GC-MS)测定菌体胞内代谢物,通过主成分分析(PCA)对不同处理下的乳酸菌胞内代谢组轮廓差异进行分析,采用偏最小二乘分析(PLS)识别不同处理的差异性代谢物。结果表明,锰离子缺乏对植物乳杆菌胞内能量代谢、脂肪酸代谢、氨基酸代谢等均有显著影响。富集分析和代谢途径分析也表明锰离子具有调节胞内糖代谢和氨基酸代谢的作用。锰离子缺乏在乳酸菌发酵前期和中期会显著提高胞内氨基酸的含量,同时胞内三羧酸循环的效率因锰离子的缺乏而大幅度下降,进而影响乳酸菌的发酵活力。     

链霉素发酵的代谢调控育种及研究进展

链霉菌在生产抗生素方面的特殊作用使它成为放线菌中遗传育种的核心,近年来的进展主要在于原生质体融合、脂质体的使用、质粒及其它载体的发现和克隆技术工业应用.本文综述了链霉素生物合成途径、代谢调节机制、链霉素发酵的代谢调控育种及其进展.     

发酵肠的风味物质及其来源

发酵肠风味物质的种类繁多，其来源也较复杂。碳水化合物发酵、脂肪代谢、含氟化合物代谢及添加的香料是风味物质产生的主要途径。本文对发酵肠风味物质及其主要来源进行概述。     

灰褐链霉菌与黄色短杆菌属间原生质体融合提高ε-聚赖氨酸产量

为提高灰褐链霉菌（Streptomyces griseofuscus LS-6）的ε-聚赖氨酸（ε-poly-lysine,ε-PL）发酵水平,从增加内源性前体赖氨酸角度考虑,将S. griseofuscus LS-6与赖氨酸生产菌株黄色短杆菌（Brebvibacterium flavum S62）进行属间的原生质体融合。以S. griseofuscus LS-6产黑色素作为遗传标记挑选到1 株融合子LS-32,其ε-PL摇瓶产量为2.82 g/L,是亲本菌株LS-6的1.73 倍;LS-32在补料分批发酵中的ε-PL产量达到56.4 g/L,比亲本提高了38.9%。随机引物扩增多肽DNA技术显示融合子LS-32中确实有来自B. flavum S62的基因;融合子ε-PL代谢途径中关键酶的活力（己糖激酶、天冬氨酸激酶、ε-PL合成酶等）和胞内氨基酸含量（赖氨酸、谷氨酸、精氨酸等）均高于亲本,是LS-32高产ε-PL的重要原因。本研究明确了内源性赖氨酸对于提升ε-PL产量的重要性,且属间融合为ε-PL产生菌的育种研究提供了新的参考途径。     

基于宏基因组技术分析自然发酵高粱菌群结构

由于高粱自然发酵过程中微生物菌群结构较复杂,代谢途径较难确定,使高粱自然发酵工艺较难控制,难以实现工厂化生产,通过高通量测序的宏基因学对高粱不同发酵时期的菌群结构、代谢通路的多样性进行分析,结果表明:自然发酵过程中的优势菌种为胚芽乳杆菌、乳酸乳球菌、费比恩毕赤酵母、醋酸杆菌、固氮葡糖醋杆菌等,高粱自然发酵的过程是酵母菌慢慢富集的过程,而乳酸菌的大量存在增加酵母菌的代谢潜能,在发酵后期醋杆菌属的大量富集使酵母菌丰富度下降,说明毕赤酵母等酵母菌耐酸性较差。通过宏基因组序列的代谢重建,揭示蛋白质和碳水化合物异养发酵的特征,通过碳水化合物代谢途径可知,有机酸的积累使整个发酵体系呈酸性,使优势菌醋杆菌属丰富度增加,说明自然发酵过程中的优势菌为乳酸菌和酵母菌,其中乳酸菌起主导作用。     

微生物发酵法生产鸟氨酸

本文介绍了鸟氨酸的生物合成途径及代谢控制育种理论，提出了鸟氨酸积累突变株应具有瓜氨酸或精氨酸缺陷及精氨酸结构类似等遗传标记，并介绍了鸟氨酸的发酵研究情况。     

Metabolic engineering--integrating methodologies of molecular breeding and bioprocess systems engineering

Metabolic engineering is an integrating methodology of analysis and synthesis for the improvement of flux distribution of metabolic pathways in complicated bioprocesses, which are highly multi-hierarchical systems to extend from macroscopic to microscopic levels. Recent progress in metabolic engineering methodologies to improve metabolic pathways in microorganisms was reviewed with many studies in this paper. Metabolic flux distribution was analyzed under different environmental conditions, using a metabolic reaction model. The physiological states of microorganisms were understood by interpreting metabolic flux analysis (MFA). This analysis was also used for development of process operation and control strategy. Cell capability to form a targeted product was analyzed with a metabolic reaction model and linear programming (LP). The use of a 13C-enriched carbon source and nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GCMS) analyses of intracellular and extracellular metabolites enabled determination of a metabolic flux distribution more accurately than the flux distribution determined only by the metabolic reaction model, which involves not only metabolite balances but also energy and redox balances. The comparison of metabolic flux distributions between before and after genetic modification of cells yielded information on the mechanism of regulation of metabolic flux in microorganisms. Finally, integration of bioinformatics and metabolic engineering is discussed, and cyclic modification of the complex bionetwork and process development were emphasized.     

基于代谢流量分析的黄色短杆菌TK0303合成L-亮氨酸发酵过程的优化

测定并计算了在发酵中后期L-亮氨酸等代谢物的胞外浓度和积累(或消耗)的速率。应用代谢流分析方法,通过MATLAB软件线性规划得到发酵中后期胞内代谢流分布及L-亮氨酸合成的理想代谢流分布。结果表明,在L-亮氨酸分批发酵过程中,有98.73%的葡萄糖进入糖酵解途径,仅1.27%进入HMP途径,55.10%的碳架进入TCA循环,25.21%用于合成L-亮氨酸。实验测定的合成L-亮氨酸的代谢流远低于理想代谢流(66.67)。根据代谢流分析结论,文中通过优化发酵过程控制如流加方式、溶氧水平等方面来减少副产物的生成,控制TCA循环代谢流,从而提高L-亮氨酸的产率。实验采用脉冲补料方式,控制溶氧浓度在20%左右,L-亮氨酸最高产酸达到38g/L。     

Metabolic control analysis for lysine synthesis using Corynebacterium glutamicum and experimental verification

Metabolic control analysis was carried out for the lysine biosynthetic pathway in Corynebacterium glutamicum ATCC 21253 using mechanism-based kinetic models developed for enzymatic reactions for this pathway. The rate-limiting steps during lysine production were determined with the aid of flux control coefficients, which indicated that the lysine synthesis flux is governed mainly by both aspartokinase and permease activity in the export system. Analyses indicated that an increase in the activity of aspartokinase would significantly alleviate its effect on the overall flux, and the limiting step is expected to shift to permease and other lysine synthetic enzymes. The influences of many participating precursors and cofactors on lysine synthesis flux were also investigated through the calculation of response coefficients for individual parameters. Although most parameters were not found to exert significant influences on lysine flux, extracellular lysine concentration was found to have a substantial effect on lysine production, and detailed analysis indicated that a decrease in the external lysine concentration will enhance the metabolite synthesis considerably and shift the control from export step to other steps. Although a decrease in external pH (pH0) seems to increase lysine flux, consideration of the export carrier concentration in response to external pH confirmed the optimum pH0 to be 7.0. Two types of recombinant strains overexpressing aspartokinase and dihydrodipicolinate synthase were constructed to verify the results obtained from metabolic sensitivity analysis.     

乳酸盐通量及其控制分析:食品功能的定量化评价方法

目的:食品对机体的影响主要表现为对代谢和代谢网络的影响,本实验旨在研究食品对机体代谢作用的定量化评价方法。方法:通过对志愿者外周血采样,首先研究4 种生理状态(基础状态、学习状态、运动状态和发烧状态)下,乳酸盐代谢通量的变化,同时研究中心代谢途径中的15 种酶对乳酸盐代谢通量的控制作用;采用相关分析和主成分分析相结合的方法,研究酶的表达(或合成)量对乳酸盐代谢通量的作用,确定主成分及各种酶的控制系数。另招募健康志愿者,采集其食用大米变性淀粉前后的外周血样,运用已建立的代谢通量模型及控制分析方法对变性淀粉的功能性进行评价。结果:4种状态下乳酸盐代谢通量各不相同,由小到大依次为:基础状态、学习状态、运动状态和发烧状态,4种生理状态下机体内的分解代谢依次增强,可见乳酸盐代谢通量实质上反映了机体分解代谢(氧化磷酸化供能)的强弱。丙酮酸激酶(pyruvate kinase,PK,Cp PK=0.221 6),丙酮酸脱氢酶复合物(pyruvate dehydrogenase complex,PDHC,Cp PDHC=0.206 4),乳酸脱氢酶(lactate dehydrogenase,LDH,Cp LDH=0.162 6),转酮酶(transketolase,TKL,Cp TKL=0.206 0)对乳酸盐代谢通量起到主要作用,其中,PK的控制作用最强。志愿者食用变性淀粉后,乳酸盐通量增强,分解代谢增强而合成代谢减弱;PK的基因表达(合成)量显著增加(P=0.010.05),PDHC的基因表达(合成)量极显著降低(P=0.0030.01),说明所测酶的表达量与所预测的乳酸盐通量之间的拟合程度较好。结论:本实验建立的评价方法可以通过采集适量的外周血对食用食品后的人体代谢作用进行定量化评价。     

Development of a kinetic model for L-lysine biosynthesis in Corynebacterium glutamicum and its application to metabolic control analysis

A mathematical model describing intracellular lysine synthesis by Corynebacterium glutamicum in batch fermentation was developed. The model is based on material balance equations of the key metabolites, and includes mechanistically based, experimentally matched rate equations for individual enzymes. From the measurements of the levels of intra- and extracellular metabolites during cultivation, the kinetic parameters in the model were identified through the decomposition of the network of reactions. The model predictions and experimental observations were in reasonable agreement. Using the model developed, metabolic control analysis was carried out to identify the rate-limiting steps, by evaluating the control on the overall lysine synthesis flux exerted by individual enzymatic reactions, which suggested how the control on lysine synthesis changes from aspartokinase to lysine permease as fermentation proceeded and indicated that lysine production could be enhanced by improving aspartokinase activity of this strain through genetic manipulation.     

Monitoring the ripening process of Cheddar cheese based on hydrophilic component profiling using gas chromatography-mass spectrometry

We proposed an application methodology that combines metabolic profiling with multiple appropriate multivariate analyses and verified it on the industrial scale of the ripening process of Cheddar cheese to make practical use of hydrophilic low-molecular-weight compound profiling using gas chromatography-mass spectrometry to design optimal conditions and quality monitoring of the cheese ripening process. Principal components analysis provided an overview of the effect of sodium chloride content and kind of lactic acid bacteria starter on the metabolic profile in the ripening process of Cheddar cheese and orthogonal partial least squares-discriminant analysis unveiled the difference in characteristic metabolites. When the sodium chloride contents were different (1.6 and 0.2%) but the same lactic acid bacteria starter was used, the 2 cheeses were classified by orthogonal partial least squares-discriminant analysis from their metabolic profiles, but were not given perfect discrimination. Not much difference existed in the metabolic profile between the 2 cheeses. Compounds including lactose, galactose, lactic acid, 4-aminobutyric acid, and phosphate were identified as contents that differed between the 2 cheeses. On the other hand, in the case of the same salt content of 1.6%, but different kinds of lactic acid bacteria starter, an excellent distinctive discrimination model was obtained, which showed that the difference of lactic acid bacteria starter caused an obvious difference in metabolic profiles. Compounds including lactic acid, lactose, urea, 4-aminobutyric acid, galactose, phosphate, proline, isoleucine, glycine, alanine, lysine, leucine, valine, and pyroglutamic acid were identified as contents that differed between the 2 cheeses. Then, a good sensory prediction model for “rich flavor,” which was defined as “thick and rich, including umami taste and soy sauce-like flavor,” was constructed based on the metabolic profile during ripening using partial least squares regression analysis. The amino acids proline, leucine, valine, isoleucine, pyroglutamic acid, alanine, glutamic acid, glycine, lysine, tyrosine, serine, phenylalanine, methionine, aspartic acid, and ornithine were extracted as ripening process markers. The present study is not limited to Cheddar cheese and can be applied to various maturation-type natural cheeses. This study provides the technical platform for designing optimal conditions and quality monitoring of the cheese ripening process.     

啤酒发酵中代谢工程的初步研究

将代谢工程理论应用于啤酒的发酵过程，通过构建啤酒发酵过程中酵母的代谢网络模型，对啤酒发酵过程进行代谢通量分析。研究不同压力下啤酒酵母代谢能力的变化，并对其进行了代谢通量分析，结果表明，高压可对酵母在啤酒发酵过程中的代谢产生一定的抑制作用。     

‘印度青’苹果合成与分解代谢通量研究及贮藏温度的优化

本研究以‘印度青’苹果为原料，研究在不同贮藏温度下合成与分解代谢网络通量，并在此基础上优化其贮藏温度。将‘印度青’苹果放置在7 个不同温度（0、2、5、10、15、20、40 ℃）条件下，测定‘印度青’苹果不同部位糖酵解途径、三羧酸循环、蔗糖合成途径、磷酸戊糖途径的代谢通量。结果显示：2 ℃时蔗糖有较高的合成通量，由外到内4 个部位分别为32、42、47和33，乳酸盐也在2 ℃时有较高的代谢通量，由外到内4 个部位分别为1.87、1.86、1.86和1.86，在此温度下有较低的分解代谢和较高的蔗糖合成代谢，因此，2 ℃是‘印度青’苹果较好的贮藏温度。通过不同温度下‘印度青’苹果的各个部位之间代谢通量上的变化，可以建立以乳酸盐与蔗糖通量为评价标准的定量化模型，这可为今后更好地研究延长水果的贮藏寿命提供新的研究方法和理论参考。     

温度对Zymomonas mobilis糖代谢关键酶活力和代谢流量的影响

以运动发酵单胞菌(Zynwmonas mobilis)ATCC31821为模式菌株,研究不同温度条件对其葡萄糖代谢关键酶活力的影响.采用全自动发酵罐,在整个发酵过程中通过充入氮气调节发酵液的溶氧量(DO)=0%,添加0.5mol/LNaOH溶液控制pH=5.5,发酵温度分别控制为25、30、35、40℃,发酵24h,测定其糖代谢网络中ED、HMP、TCA等途径的关键酶活力和代谢物成分.结果表明,在发酵温度为30～35℃时,乙醇脱氢酶(ADH)、葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC)、葡萄糖激酶(GK)、丙酮酸激酶(PK)和甘油醛-3-磷酸脱氢酶(GD-3-PDH)的活力较高,菌体的ED途径代谢活跃,碳素流量增加,乙醇生产量和糖转化率较高,而TCA途径的苹果酸脱氢酶(MDH)和异柠檬酸脱氢酶(ICDH)等活力较低,进入TCA途径的碳素流量明显减少;发酵温度为25、40℃时,TCA途径的酶活力升高,ED途径的酶活力减弱,生成乙醇的代谢流量减少,因此温度是z.mobilis发酵过程中调控菌体细胞生长和糖代谢的一个重要因素. 、葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC) 葡萄糖激酶(GK)、丙酮酸激酶(PK)和甘油醛-3-磷酸脱氢酶(GD-3-PDH)的活力较高,菌体的ED途径代谢活跃,碳素流量增加,乙醇生产量和糖转化率较高,而TCA途径的苹果酸脱氢酶(MDH)和异柠檬酸脱氢酶(ICDH)等活力较低,进入TCA途径的碳素流量明显减少;发酵温度为25、40℃时,TCA途径的酶活力升高,ED途径的酶活力减弱,生成乙醇的代谢流量减少,因此温度是z.mobilis发酵过程中调控菌体细胞生长和糖代谢的一个重要因素.葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC) 葡萄糖激酶(GK)、     

α-淀粉酶抑制剂生物合成途径和代谢流分析

为提高糖类的利用效率,加强糖类代谢向生成α-淀粉酶抑制剂的方向流动,提高α-淀粉酶抑制剂产量,对天蓝黄链霉菌合成α-淀粉酶抑制剂的代谢网络进行分析,找出影响α-淀粉酶抑制剂合成的代谢流量分配规律和关键节点,并且应用代谢流分析的方法研究了谷氨酸钠对α-淀粉酶抑制剂发酵中后期胞内代谢流分布的影响。结果表明：在α-淀粉酶抑制剂分批发酵过程中,未流加谷氨酸钠时合成α-淀粉酶抑制剂的代谢流量为1.84;在发酵培养基中流加谷氨酸钠使其质量浓度维持在4.0 g/L后,α-淀粉酶抑制剂生物合成的代谢流增长至3.18。因此发酵过程中流加谷氨酸钠能够改变α-淀粉酶抑制剂生物合成途径的关键节点6-磷酸葡萄糖、7-磷酸景天庚酮糖及谷氨酸的代谢流分布,提高α-淀粉酶抑制剂生物合成途径的代谢流量。