不同代数啤酒酵母对胞内代谢关键酶活性的影响

对不同代数啤酒酵母在丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、苹果酸脱氢酶、异柠檬酸脱氢酶、乙醇脱氢酶的酶活性特征方面的差异进行了比较分析,发现酵母在不断传代过程中,胞内代谢关键酶的活性均呈现一定变化,而丙酮酸激酶、葡萄糖-6-磷酸脱氢酶和乙醇脱氢酶变化最为明显,并且与酵母活力有着紧密的联系,当3种酶活性高时,酵母活力旺盛,代谢物质的量较为协调.     

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)、     

渗透压对酿酒酵母胞内代谢关键酶活性的影响

对耐高渗酵母与普通酿酒酵母在丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、苹果酸脱氢酶、异柠檬酸脱氢酶、乙醇脱氢酶的酶活性特征的差异进行比较分析,建立酶活力测定方法.发现耐高渗酵母在高渗环境的诱导下,其EMP途径、磷酸戊糖途径的关键酶活力都高于普通酿酒酵母.耐高渗酵母具有高活力的乙醇脱氢酶,其能高效地将丙酮酸转化成乙醇.并且三羧酸循环中的关键酶异柠檬酸脱氢酶和苹果酸脱氢酶的活性也得到加强.这些酶活力的增强维持了耐高渗酵母在高渗、高酒精环境下的生长需要与能量代谢的平衡.     

米根霉F1-ATPase活性降低突变株的筛选研究

本实验采用同步辐射软X射线Nk诱变米根霉,获得一株新霉素抗性突变株NA-2,该菌株F1-ATPase活性降低86.82%,胞内ATP含量下降18.99%,最终菌体浓度为出发菌株的79.22%,发酵周期缩短了12h,葡萄糖平均消耗速度和L-乳酸生产强度分别高出25.17%和32.57%,平均葡萄糖消耗比速(qs)、L-乳酸生成比速(qp)和细胞比生长速率(μ)分别提高86.42%、128.13%和60.0%,但胞内能荷变化不明显。进一步研究发现,突变株糖酵解途径中关键酶磷酸果糖激酶(PEK)、丙酮酸激酶(PK)和磷酸甘油醛激酶(GAPDH)的活性较原始菌株提高了58.34%、23.53%和11.29%,乙醇脱氢酶(ADH)活性降低了32.11%,乳酸脱氢酶(LDH)活性提高了42.27%。结果表明,降低米根霉F1-ATPase活性有效地提高了糖酵解关键酶活性而加速葡萄糖代谢,提高了L-乳酸生产强度。     

柠檬醛对指状青霉糖酵解的影响

本研究测定了不同浓度柠檬醛对指状青霉呼吸代谢及糖酵解途径相关指标的影响。结果表明,柠檬醛能明显抑制指状青霉的呼吸代谢,1.0μL/m L浓度下柠檬醛与糖酵解途径、三羧酸循环途径以及磷酸戊糖途径典型抑制剂(碘乙酸,丙二酸,磷酸钠)的叠加率分别为15.00%、12.60%和30.70%,且此结果显示糖酵解途径受到了柠檬醛的影响。经最小抑菌浓度(minimal inhibitory concentration,MIC,2.0μL/m L)和最小杀菌浓度(minimal fungicidal concentration,MFC,4.0μL/m L)柠檬醛处理后,指状青霉糖酵解途径重要的中间产物葡萄糖和丙酮酸的含量显著增加,己糖激酶(Hexokinase,HK)的活性下降,6-磷酸果糖激酶(Phosphofructokinase,PFK)和丙酮酸激酶(Pyruvate kinase,PK)的活性增加。本研究表明柠檬醛能够抑制指状青霉的呼吸代谢,改变指状青霉糖酵解途径关键酶的活性和重要中间产物的含量。     

镁、钼对野油菜黄单胞菌代谢关键酶的影响

野油菜黄单胞菌(Xanthomonas campestris)是一种利用碳水化合物为主要底物发酵产生酸性胞外杂多糖黄原胶的革兰氏阴性菌。本实验研究了通过添加不同浓度的Mg2+、MoO2-对野油菜黄单胞菌代谢过程中关键酶:葡萄糖-6-磷酸脱氢酶,丙酮酸激酶在不同发酵时期的活性变化。结果表明,Mg2+添加量为1ml即在发酵液中为0.5g/50ml时葡萄糖-6-磷酸脱氢酶活性明显提高,MoO2-在发酵液中浓度为0.20g/50ml时丙酮酸激酶活性提高,表明适量的镁、钼对上述酶活性有调控作用。     

乳酸杆菌糖酵解途径中关键酶基因的比较分析

通过比较乳酸杆菌属(Lactobacillus)中63株不同种的菌株的糖酵解途径中葡萄糖-6-磷酸异构酶(Glucose-6-phosphate isomerase,PGI)(EC 5.3.1.9)、磷酸丙糖异构酶(Triose-phosphate isomerase,TPI)(EC 5.3.1.1)、磷酸甘油酸变位酶(Phosphoglycerate mutase,PGM)(EC 5.4.2.1)和丙酮酸激酶(Pyruvate kinase,PYK)(EC 2.7.1.40)等关键酶基因的序列,构建这些菌株的系统发育关系,并与这些菌株的16S rDNA序列的系统发育关系进行比较,从而揭示糖酵解代谢途径的进化。     

冰温对鸡胸肉成熟过程中品质的影响

为研究冰温对宰后鸡胸肉成熟过程的影响,本研究以白羽鸡鸡胸肉为原料,对比冷藏(4℃)和冰温(-1.5℃)状态下鸡胸肉在成熟过程中pH、剪切力、乳酸以及糖酵解过程限速酶丙酮酸激酶和乳酸脱氢酶活力的变化。结果表明,冰温状态延缓了乳酸积累,剪切力和pH极限值的到达时间,并且延迟了糖酵解过程中关键酶丙酮酸激酶和乳酸脱氢酶的活力,与冷藏状态的鸡胸肉相比,冰温状态下鸡胸肉的最低pH被延缓了6 h,乳酸的积累延缓2 h,剪切力的最大值延迟了2 h,丙酮酸激酶和乳酸脱氢酶活力最大值被延迟了2 h,冰温可以延缓鸡胸肉成熟进程大约2~6 h,但在冰温和冷藏状态下各项指标的变化趋势相同,除乳酸脱氢酶外其余各项指标的极限值均无显著性差异(p>0.05)。     

冷冻干燥对乳酸菌代谢活力的影响

以保加利亚乳杆菌和植物乳杆菌ST-Ⅲ为研究对象,用15%蔗糖、10%海藻糖、15%蔗糖＋2%谷氨酸钠及10%脱脂乳为冷冻保护剂,与不加冷冻保护剂的样品进行冻干后菌种活力的对比,测定冷冻干燥后菌体中己糖激酶、丙酮酸激酶、乳酸脱氢酶的活力,胞外蛋白及胞内Ca2＋的浓度变化。结果表明：冷冻干燥对菌体的己糖激酶和丙酮酸激酶影响不显著,对乳酸脱氢酶具有显著性的影响。同时,不加保护剂菌体的胞外蛋白含量明显高于添加保护剂的,而胞内Ca2＋却远低于其他组。这一结果说明,冷冻干燥使代谢关键酶如乳酸脱氢酶失活,同时对细胞膜造成了损伤,从而引起细胞代谢活力的下降。     

乙醇胁迫对乳酸杆菌关键酶活力的影响

分别以面包乳杆菌(Lactobacillus crustorum D2-5)和植物乳杆菌(Lactobacillus plantarum D5-5)为研究对象,进行不同乙醇体积分数胁迫后菌体活力的对比,测定了乙醇胁迫后菌体中己糖激酶(HK)、丙酮酸激酶(PK)、乳酸脱氢酶(LDH)及ATP酶的活力变化,分析了乙醇胁迫后造成菌体失活的主要因素。结果表明:5%乙醇胁迫处理有助于提高菌体存活率,但是效果不显著;8%乙醇胁迫后的菌体存活率明显降低,面包乳杆菌D2-5和植物乳杆菌D5-5存活率依次仅为26.61%和23.50%,菌体的生长受到严重抑制,致使相关酶活力降低。乙醇胁迫对乳酸杆菌己糖激酶影响不显著,对其丙酮酸激酶、乳酸脱氢酶及ATP酶具有极显著的影响。这一结果说明,丙酮酸激酶、乳酸脱氢酶及ATP酶是影响乙醇胁迫损失的关键酶,酶活力变化与菌体活力相关。     

Regulation of lactate production in Streptococcus bovis: A spiraling effect that contributes to rumen acidosis

When Streptococcus bovis was grown in batch culture with 6 g/L glucose at pH 6.7, maximum specific growth rate was 1.47 h(-1), and lactate was the primary fermentation product. In continuous culture at pH 6.7 and growth rate equal to .10 h(-1), little lactate was formed, and formate, acetate, and ethanol accounted for most of the product. When extra-cellular pH decreased to 4.7, intra-cellular pH declined to 5.4, and organisms switched back to lactate production. Intracellular concentration of fructose 1,6-diphosphate of batch culture cells was greater than 12 mM, a concentration that allowed maximal lactate dehydrogenase activity. When Streptococcus bovis was grown in continuous culture at pH 6.7, intracellular fructose-l,6-diphosphate declined to .4 mM, a concentration which gave little lactate dehydrogenase activity at pH 6.5 or greater. Decreasing pH of continuous culture to 4.7 increased intracellular fructose-1,6-diphosphate concentration to .8 mM. This concentration was still limiting if lactate dehydrogenase was assayed at pH 6.5, but nearly maximal activity was obtained when enzyme was assayed at pH 5.5. The small increase in fructose-l,6-diphosphate and decreased requirement of lactate dehydrogenase for fructose-l,6-diphosphate under acidic assay conditions, accounted for increased lactate production during low pH (4.7) continuous culture. These and other aspects of lactate regulation by Streptococcus bovis are discussed as factors leading to rumen acidosis. This pattern of regulation also helps to explain why rumen acidosis is difficult to reverse.     

醋酸菌中乙醛脱氢酶的分离纯化及酶学性质

以实验室筛选得到的醋酸菌(Acetobacter pomorum)为实验菌株发酵产酶,通过细胞破壁,采用(NH₄)₂SO₄沉淀、透析、DEAE-Sepharose 离子交换层析及 Superdex G-75凝胶过滤层析分离纯化得到乙醛脱氢酶的酶液,并考察其酶学性质。该酶分子质量为221.60 kDa,单个亚基分子质量约为54.41 kDa,为四聚体结构;纯酶液比活力20.25 U/mg,纯化倍数为10.16倍,乙醛脱氢酶(aldehyde dehydrogenase,ALDH)的回收率为6.53%。酶学性质研究表明,ALDH促进乙醛分解的最适温度为50 ℃,40~50 ℃相对酶活力稳定性好;该酶的最适pH为7.0,当pH在5.5~7.5内酶活力表现稳定;金属离子对酶活性的影响实验表明,Na+、K+、Zn2+、Ba2+对该酶酶有不同程度抑制作用,而Mg2+、Ca2+、Al3+、Li+、Cu2+具有促进作用;ALDH的最适底物为乙醛,相对偏好直链醛类。ALDH活性较大,为后期表达和深入研究其生物学功能提供理论和数据支持。     

Myosin heavy chain isoform composition influences the susceptibility of actin-activated S1 ATPase and myofibrillar ATPase to pH inactivation

The objectives of this study were to determine the influence of pH and MyHC isoforms on myofibrillar and actin-activated myosin subfragment 1 (S1) ATPase activity and the protective effect of actin. Red (RST) semitendinosus and white (WST) semitendinosus myofibrils were incubated at pH 7, 6, or 5.5 with 0 or 2mM ATP. RST and WST S1 isolates were incubated at pH 7, 6, or 5.5 in the presence or absence of actin. Maximum calcium-activated myofibrillar and actin-activated S1-ATPase activity were then assayed at pH 7. Incubation of myofibrils with ATP caused ATPase activity of myofibrils to decrease (p<0.05) with the pH of the incubation. RST myofibrils maintained a higher (p<0.0001) relative activity than WST myofibrils after incubation at pH 6 with ATP. Myofibrils incubated without ATP exhibited higher (p<0.001) activities than those incubated with ATP following pH 5.5 treatments. WST myofibrils had a lower (p<0.05) relative activity than RST following incubation at pH 5.5 without ATP. S1 ATPase activities decreased (p<0.05) with incubation pH in WST samples, but not in RST samples. WST S1 activity was higher (p<0.01) in samples exposed to pH 6 and 5.5 with actin bound compared to those incubated without actin. RST S1 exhibited a higher (p<0.01) relative activity than WST samples following pH 5.5 treatment with bound actin. These data show that low pH inactivates myofibrils by altering actin-activated S1 ATPase. Furthermore, these results suggest that muscles with high proportions of fast fibers are more susceptible to pH inactivation of ATPase activity and that the protective effect of actin binding to myosin is less in fast fibers.     

Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase

The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.     

Elucidation of a basic protein, glyceraldehyde-3-phosphate dehydrogenase, as a contributing factor to raise Mg-ATPase activity of myofibrils during meat conditioning

We investigated the factor which increased the maximum value of the Mg-ATPase activity of myofibrils existing at low KCl concentrations during meat conditioning. On the treatment of myofibrils with the solution extracted with the buffer of pH 7.2 from muscle, the Mg-ATPase activity in the presence of 0–0.15 M KCl increased time-dependently. This change was most remarkable in the range of pH 5.6–7.0. Trypsin treatment of the extract abolished such effect, suggesting that the responsible factors were proteins. The fractionation of the extract with isoelectric focusing demonstrated that the factors were basic proteins (pI 8.3–9.6). The treatment of myofibrils with those basic proteins under various conditions suggested that the time-dependent adhesion of those basic proteins, through a denaturation at around pH 5.5, to myofibrils was assumed to raise the Mg-ATPase activity. Analysis of myofibrils prepared from rabbit muscles stored at 0°C for 12 days postmortem showed the appearance of the 35,000 Da protein, accompanied the increase in the Mg-ATPase activity. Therefore, the adhesion of this protein to myofibrils in situ probably caused the increase in the Mg-ATPase activity. Successive treatment with the basic protein and the crude cathepsin increased the dependency of the Mg-ATPase activity on KCl concentrations and the maximum value of the Mg-ATPase activity. Therefore, the coordinate action of a basic 35,000 Da protein and cathepsins was presumed to induce the changes in the Mg-ATPase activity of myofibrils during meat conditioning. The basic protein was concluded to be glyceraldehyde-3-phosphate dehydrogenase (its subunit molecular mass: 35,000 Da), since the incubation of myofibrils with its commercial preparation raised the Mg-ATPase activity of myofibrils.     

Characterizing endogenous and exogenous peroxidase activity for bleaching of fluid whey and retentate

The lactoperoxidase (LP) system may be used to achieve the desired bleaching of fluid whey with the addition of low concentrations (<50 mg/kg) of hydrogen peroxide. The addition of an exogenous peroxidase (EP) to whey may also be used to aid in whey bleaching when the LP system is not fully active. The objectives of this study were to monitor LP activity in previously refrigerated or frozen milk, fluid whey, and whey retentate (10% solids) and to evaluate peroxidase activity in fluid whey and whey retentate (10% solids), with and without additional EP (2, 1, or 0.5 dairy bleaching units), over a range of pH (5.5–6.5) and temperatures (4–60°C). Subsequent experiments were conducted to determine the relationship between enzyme activity and bleaching efficacy. Raw and pasteurized milk, fat-separated pasteurized whey, and whey retentate (10% solids) were evaluated for LP activity following storage at 4 or −20°C, using an established colorimetric method. A response surface model was applied to evaluate both endogenous and EP activity at various temperatures and pH in freshly manufactured whey and retentate. Refrigerated or frozen storage at the parameters evaluated did not affect LP activity in milk, whey, or retentate. In fluid whey, with and without added EP, as pH decreased (to 5.5) and temperature increased (to 60°C), peroxidase activity increased. Retentate with EP exhibited behavior similar to that of fluid whey: as pH decreased and temperature increased, activity increased. However, in retentate without EP, as pH increased and temperature increased, activity increased. Enzyme activity was negatively correlated to bleaching time (time for >80% norbixin destruction) in fluid whey but a linear relationship was not evident in retentate. When fluid whey is bleached enzymatically, if pH is decreased and temperature is increased, the rate of reaction increases (e.g., bleaching occurs in less time). When bleaching in retentate, a higher pH (pH 6.5 vs. pH 5.5) is desired for optimal bleaching by the LP system. Due to processing restraints, this may not be possible for all dairy producers to achieve and, thus, addition of EP could be beneficial to improve bleaching efficacy.     

Antimicrobial Activity and Hydrophobicity of Edible Whey Protein Isolate Films Formulated with Nisin and/or Glucose Oxidase

The use of edible antimicrobial films has been reported as a means to improve food shelf life through gradual releasing of antimicrobial compounds on the food surface. This work reports the study on the incorporation of 2 antimicrobial agents, nisin (N), and/or glucose oxidase (GO), into the matrix of Whey protein isolate (WPI) films at pH 5.5 and 8.5. The antimicrobial activity of the edible films was evaluated against Listeria innocua (ATCC 33090), Brochothrix thermosphacta (NCIB10018), Escherichia coli (JMP101), and Enterococcus faecalis (MXVK22). In addition, the antimicrobial activity was related to the hydrophobicity and water solubility of the WPI films. The greatest antibacterial activity was observed in WPI films containing only GO. The combined addition of N and GO resulted in films with lower antimicrobial activity than films with N or GO alone. In most cases, a pH effect was observed as greater antimicrobial response at pH 5.5 as well as higher film matrix hydrophobicity. WPI films supplemented with GO can be used in coating systems suitable for food preservation.     

Lactobacilli isolated from the Armenian fermented milk product matsoun: Growth properties,antibacterial and proteolytic activity and their dependence on pH

Growth, antibacterial and proteolytic activities of two new lactobacilli strains isolated from matsoun (Armenian traditional dairy product) and their pH dependence were studied. The results demonstrated the antibacterial activity of lactobacilli against Gram‐positive and Gram‐negative test strains. This activity was stable when pH of cell culture medium was adjusted to the value of 6.5. At pH 8, the antibacterial activity of only one strain was stable. Both strains were able to hydrolyse casein in pH range of 5.5–8 with maximal activity at pH 5.5. Controlled pH conditions were suitable for biomass yield, while noncontrolled pH was better for expression of antibacterial activity.     

氧化葡萄糖酸杆菌DSM 2003膜结合乙醇脱氢酶的纯化鉴定和性质研究

通过超速离心收集膜组分、表面活性剂溶解膜蛋白、CM- 纤维素柱层析纯化等步骤,从氧化葡萄糖酸杆菌DSM 2003 中获得电泳纯的具有1,2- 丙二醇脱氢酶活性的脱氢酶,此酶由两个亚基组成,其表观相对分子质量分别为80000 和50000。经MALDI-TOF MS-MS 质谱分析与肽质量指纹图谱检索鉴定,证明纯化得到的酶是乙醇脱氢酶。酶学性质分析表明,该酶的最适反应温度为30℃,最适pH 值为5.5～6.0;该酶能催化多种一元醇、二元醇,但对包含3 个以上羟基的多元醇基本无氧化活性;其催化活性随着底物碳链长度的增加而减小;大多数金属离子及抑制剂(Cu2+、Fe3+、Ca2+、EDTA 等)对此酶活性均有抑制作用。     

Study of benzoate, propionate, and sorbate salts as mould spoilage inhibitors on intermediate moisture bakery products of low pH (4.5-5.5)

A hurdle technology approach has been applied to control common mold species causing spoilage of intermediate moisture bakery products (Eurotium spp., Aspergillus spp., and Penicillium corylophilum), growing on a fermented bakery product analogue (FBPA). The factors studied included a combination of different levels of weak acid preservatives (potassium sorbate, calcium propionate, and sodium benzoate; 0-0.3%), pH (4.5-5.5), and water activity (a(w); 0.80-0.90). Potassium sorbate was found to be the most effective in preventing fungal spoilage of this kind of products at the maximum concentration tested (0.3%) regardless of a(w). The same concentration of calcium propionate and sodium benzoate was effective only at low a(w) levels. On the other hand, potassium sorbate activity was slightly reduced at pH 5.5, the 0.3% being only effective at 0.80 a(w). These findings indicate that potassium sorbate may be a suitable preserving agent to inhibit deterioration of a FBPA of slightly acidic pH (near 4.5) by xerophilic fungi. Further studies have to be done in order to adjust the minimal inhibitory concentration necessary to obtain a product with the required shelf life.