马克斯克鲁维酵母菊粉酶的分离纯化和酶学性质研究

马克斯克鲁维酵母产生的孢外菊粉酶经超滤浓缩、DEAE-Cellulose阴离子交换色谱、SephadexG-100凝胶色谱分离纯化,得到两个菊粉酶组分ExoⅠ和ExoⅡ,I/S值分别为0.0249和0.0253,均属外切菊粉酶。ExoⅠ经聚丙烯酰胺凝胶鉴定为均一组分,分子量为85kD。ExoⅠ最适pH值为4.0,最适温度为60℃,Mn2+和Mg2+对酶活力有促进作用,而Cu2+、Fe2+对酶活力有抑制作用,ExoⅠ水解菊粉溶液的产物为果糖和少量葡萄糖。     

马克斯克鲁维酵母固态发酵菊粉酶培养条件的优化

在单因素试验基础上,为进一步提高马克斯克鲁维酵母固态发酵产生的菊粉酶的活力,采用全因子试验设计和中心组合设计对影响的因素进行优化,运用响应面分析模拟得到二次多项式回归方程的预测模型,优化的培养条件为：菊芋粉3．62％,玉米浆干粉2．40％,培养温度28．13℃。模型预测的最大响应值为314．71U／gds,在优化条件下酶活为316．35U／gds,二者基本一致。     

Cloning and sequencing of the gene for apocytochrome b of the yeast Kluyveromyces lactis strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140)

The apocytochrome b genes from two strains of the yeast Kluyveromyces lactis, have been isolated and sequenced. The coding sequences in strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140) were identical but the upstream noncoding regions were slightly different. The sequences demonstrated the presence of a continuous open reading frame with no introns. The amino acid sequence, derived from the coding strand, showed 82% homology to the apocytochrome b of Saccharomyces cerevisiae strain D273-10B and only 58% homology to the protein from Schizosaccharomyces pombe strain 50. CUN and CGN codon families were absent from the K. lactis gene. Codon usage was very similar to that of other mitochondrial genomes with mostly U or A in the third position. There were two unusual features. All threonines were coded by ACA(U) and all arginines by AGA.     

A differential medium for the isolation of Kluyveromyces marxianus and Kluyveromyces lactis from dairy products

A selective and differential solid medium, called Kluyveromyces Differential Medium (KDM), is described for the isolation of Kluyveromyces marxianus and K. lactis from dairy products. Its discriminative potential is based on the detection of the enzyme beta-galactosidase, in the absence of lactose. Of the more than 95 strains tested, including yeasts, bacteria, and filamentous fungus, only the strains of K. marxianus and K. lactis produced blue colonies on the medium due to the presence of X-Gal/ IPTG. The bacterial strains were not able to grow in KDM. On this basis, the medium was very satisfactory when testing naturally or experimentally contaminated dairy food products. When quality assessment tests were performed, optimal values of productivity (growth and color) and selectivity were obtained for K. marxianus and K. lactis.     

Bioinformatic characterization of the extracellular lipases from Kluyveromyces marxianus

Lipases are hydrolytic enzymes that break the ester bonds of triglycerides, generating free fatty acids and glycerol. Extracellular lipase activity has been reported for the nonconventional yeast Kluyveromyces marxianus, grown in olive oil as a substrate, and the presence of at least eight putative lipases has been detected in its genome. However, to date, there is no experimental evidence on the physiological role of the putative lipases nor their structural and catalytic properties. In this study, a bioinformatic analysis of the genes of the putative lipases from K. marxianus L-2029 was performed, particularly identifying and characterizing the extracellular expected enzymes, due to their biotechnological relevance. The amino acid sequence of 10 putative lipases, obtained by in silico translation, ranged between 389 and 773 amino acids. Two of the analysed putative proteins showed a signal peptide, 25 and 33 amino acids long for KmYJR107Wp and KmLIP3p, and a molecular weight of 44.53 and 58.23 kDa, respectively. The amino acid alignment of KmLIP3p and KmYJR107Wp with the crystallized lipases from a patatin and the YlLip2 lipase from Yarrowia lipolytica, respectively, revealed the presence of the hydrolase characteristic motifs. From the 3D models of putative extracellular K. marxianus L-2029 lipases, the conserved pentapeptide of each was determined, being GTSMG for KmLIP3p and GHSLG for KmYJR107Wp; besides, the genes of these two enzymes (LIP3 and YJR107W) are apparently regulated by oleate response elements. The phylogenetic analysis of all K. marxianus lipases revealed evolutionary affinities with lipases from abH15.03, abH23.01, and abH23.02 families.     

Enzymatic Production of Ribonucleotides from Autolysates of Kluyveromyces marxianus Grown on Whey

After 20h fermentation of medium containing 5% (w/v) dehydrated whey, at 30°C, pH 4.5, yeast cells were harvested, diluted in 0.1M KH₂PO₄, and autolyzed at different pHs (6.5–7.5) and temperatures (45–55°C). Phosphodiesterase (0.2–1.0% w/v, 65°C, pH 6.5, 6h) and adenyl deaminase (0.5-1.0% w/v, 60°C, pH 5.5, 4h) were added to the autolysates. After heat treatment (100°C, 15 min), samples were analyzed by RP-HPLC and LC/MS. Production of 5′-ribonucleotides was maximized at 50°C, pH 6.5. Yields of 5′-AMP (800 μg/g of biomass) and 5′-GMP (2000 μg/g) increased considerably after addition of 1.0% phosphodiesterase. 5′-IMP increased only after addition of 1.0% adenyl deaminase.     

Comparison of volatile compounds produced in model cheese medium deacidified by Debaryomyces hansenii or Kluyveromyces marxianus

The aroma of a deacidified cheese medium is the result of the overall perception of a large number of molecules belonging to different classes. The volatile compound composition of (60%) cheese medium (pH 5.8) deacidified by Debaryomyces hansenii (DCM(Dh)) was compared with the one deacidified by Kluyveromyces marxianus (DCM(Km)). It was determined by dynamic headspace extraction, followed by gas chromatography separation and quantification as well as by mass spectrometry identification. Whatever the media tested, a first class of volatile compounds can be represented by the ones not produced by any of the yeasts, but some of them are affected by K. marxianus or by D. hansenii. A second class of volatile compounds can be represented by the ones produced by K. marxianus, which were essentially esters. Their concentrations were generally higher than their thresholds, explaining the DCM(Km) global fruity odor. A third class can be represented by the ones generated by D. hansenii, which were essentially methyl ketones with fruity, floral (rose), moldy, cheesy, or wine odor plus 2-phenylethanol with a faded-rose odor. The impact of methyl ketones on the DCMDh global flavor was lower than the impact of 2-phenylethanol and even negligible. Therefore, the global faded-rose odor of D. hansenii DCM can be explained by a high concentration of 2-phenylethanol.     

三种低聚果糖检测方法的比较

通过对纸层析法,高效液相色谱-示差检测法和高效液相色谱-蒸发光散射检测法三种低聚果糖检测方法进行对比研究。结果发现,高效液相色谱-蒸发光散射检测法分离度高,重现性和准确性好,操作相对简单易行,优于其它两种方法。该方法采用氨基柱,流动相为乙腈与水,采用梯度洗脱,流速设定1.0 m L/min。ELSD的漂移管温度设定85℃;载气为空气,流速2.0 L/min。低聚果糖各组分的回收率范围为97.73%~99.39%,日内精密度1.2%;日间精密度2.8%。高效液相色谱-蒸发光散射检测法简便快捷,准确性,重现性好,更适合于低聚果糖的检测。     

Transport of lactic acid in Kluyveromyces marxianus: evidence for a monocarboxylate uniport.

Lactic acid-grown cells of a strain of Kluyveromyces marxianus transported D- and L-lactic acid by a saturable mechanism that was partially inducible and subject to glucose repression, with the following kinetic parameters at pH 5.4: Vmax = 1.00 (+/- 0.13) mmol h-1 per g dry weight and Ks = 0.42 (+/- 0.08) mM. Lactic acid transport was competitively inhibited by pyruvic, glycolic, acetic and bromoacetic acids. The latter, a non-metabolizable analogue, was transiently accumulated, the extent depending on the extracellular pH. The pH dependence of the Ks values for undissociated lactic acid and for the lactate anion indicated that the latter was the transported species. Lactate uptake was not accompanied by the simultaneous uptake of protons, potassium ions or sodium ions excluding symport mechanisms. Initial lactic acid uptake led to transient membrane hyperpolarization as measured with a fluorescent dye excluding also an electroneutral anion antiport mechanism. It was concluded that lactate anions use a monocarboxylate uniport and that the counter anion, possibly bicarbonate, uses a separate channel, the coupling being electrical and loose.     

Application of multiple sensory evaluations to produce fermented beverages made from sole whey using Kluyveromyces marxianus

Various fermented whey-based beverages have been developed to reduce waste. However, approaches to increase the preference of whey-based beverages are required because of the low sensory acceptability of whey. Here, we identified the better starting material (whey type), between raw whey (RW) and demineralised whey (DMW), and determined the optimal initial concentration using multiple sensory evaluations to develop acceptable fermented beverages made from sole whey with pure cultured Kluyveromyces marxianus (i.e. without additional ingredients and processing methods). Acceptance tests showed that fermented beverages made from DMW were superior to RW as the starting material. The amounts of ethanol produced were 5.0%, 7.6% and 9.5% v/v from the different initial DMW concentrations of 10%, 15% and 20% w/v, respectively. We observed a significant positive correlation (r_s = 0.32, P < 0.05) between the assessment attributes, strength of taste as alcohol beverage and overall acceptability, indicating that higher concentrations of DMW yielded a more desirable product.     

藏灵菇源克鲁维酵母菌M3调节大鼠肠道菌群平衡的研究

利用从藏灵菇中筛选出的发酵性能优良且产胆盐水解酶的马克斯克鲁维酵母菌M3菌株,制备冻干菌粉作为受试物进行动物实验,探讨其对预防性高胆固醇血症大鼠肠道菌群平衡的作用。采用预防高胆固醇血症大鼠模型,将40 只无特定病原体（specific pathogen free,SPF）级3 周龄的大鼠分为高脂模型组、低剂量、中剂量和高剂量实验组进行实验。实验组分别灌胃低、中、高剂量的受试物,高脂模型组灌胃蒸馏水,连续7 周。结果表明,前5 周4 组大鼠粪便中乳酸杆菌（lactobacilli）、沙门氏菌（Salmonella）、产气荚膜梭菌（Clostridium perfringens）的数量处于平缓变化状态,且3 个实验组与高脂模型组相比无显著性差异;5 周时,3 个实验组大鼠粪便中乳杆菌的数量极显著高于高脂模型组（P＜0.01）,沙门氏菌的数量极显著低于高脂模型组（P＜0.01）,产气荚膜梭菌的数量显著低于高脂模型组（P＜0.05）;其中低剂量组的乳杆菌数量最高而沙门氏菌和产气荚膜梭菌数量最低,说明低剂量实验组优于中、高剂量实验组。显示藏灵菇源克鲁维酵母菌M3能够调节高胆固醇血症大鼠肠道有益菌的生长,并抑制有害菌的繁殖,具有良好的调节肠道菌群平衡的效果。     

吸附交联法固定化蔗糖异构酶及其在异麦芽酮糖制备中的应用

以壳聚糖为载体、戊二醛为交联剂,采用吸附交联法对重组短小芽孢杆菌来源的蔗糖异构酶进行固定化。以表观酶活力回收率为指标,对壳聚糖浓度、戊二醛加量、游离酶加量、固定化时间等条件进行了优化;并考察了温度、pH、固定化酶加量、反应时间以及底物浓度等因素对固定化蔗糖异构酶转化生产异麦芽酮糖的影响。结果表明,最佳固定化条件为：壳聚糖质量浓度3 g/dL、戊二醛加量（体积分数）0.75%、酶加量50 U/g、固定化时间16 h,此时固定化酶活力回收率达到70.3%;最佳转化条件为：温度30 ℃、初始pH 4.5、酶用量15 U/g,转化10 h,蔗糖质量浓度600 g/L,异麦芽酮糖最大产物得率达到87.8%。在最佳的转化条件下连续转化16次,产物得率仍保持在87.52%,显示该固定化酶具有良好的操作稳定性及较高的异麦芽酮糖合成能力。     

黄酮及维生素C的抗羟基自由基的比较研究

以大豆甙和芦丁作为黄酮化合物的代表，并与维生素C比较，研究其对羟基自由基的抑制能力，并以大豆、橙皮、柑皮和青椒提取物作羟基自由基清除剂，研究这些提取物对羟基自由基的清除作用。结果表明，大豆甙对羟基自由基的清除率最高，其次是芦丁，再次是维生素C；含黄酮化合物的提取物比含维生素C的提取物对羟基自由基的清除率大，其中以大豆撮以物最优。     

Comparison of Some One-, Two- And Three-Component Systems for Similarity To Meat Flavor Using Surface Response Methodology

The contribution of some compounds previously identified in the flavor volatiles from cooked beef was estimated by surface response methodology. In most cases, predicted maximum scores for mixtures were higher than scores predicted for single compounds tested alone. Synergistic and/or synosmic effects were often indicated since mixtures frequently gave much higher scores. Predicted scores for four out of eight compounds tested were not significantly different from actual mean scores. This indicated the usefulness of the method for predicting optimal combinations of meat flavor components. The best three-component mixture consisted of 2,5-dimethylpyrazine, furfural, and 1-butanol, which gave a score of 7.54, moderately resembling meat aroma.     

Structural and Rheological Properties of Succinoglycan Biogums Made from Low-Quality Date Syrup or Sucrose Using Agrobacterium radiobacter Inoculation

This study examines and compares the structural and rheological properties of succinoglycan exopolysaccharide made by fermentation of sucrose or date syrup at identical temperature, pH, and total soluble solid. Using thin-layer chromatography (TLC), Fourier transform infrared (FTIR) spectroscopy, and rotational viscometery structural and rheological properties of prepared exopolysaccharides were assessed. The variables under studies were biogum concentrations (0.5%, 1.0%, 1.5%, and 2.0% w/w), temperature (5, 25 and 40°C), and pH (2.5, 4.0, 7.0, and 10.0). Results revealed that the quantity of exopolysaccharide produced in the date syrup (DSE) medium was three to five times greater than that of sucrose (SE) medium. The biogums exhibited non-Newtonian, shear-thinning behavior, and the viscosity of DSE was higher than SE at all tested concentrations. The TLC and FTIR spectroscopy assessments designated that the biogum is composed of glucose and galactose units, carrying a carboxylic group and indicated the presence of succinoglycan. The outcomes of this study could be useful to food industries seeking low cost biogum.     

糖谱法比较不同产地竹荪多糖结构特征

利用微波辅助水提取长裙竹荪水溶性多糖,高效凝胶排阻色谱联用多角度激光光散射和糖谱法联用荧光辅助凝胶电泳分析比较不同产地长裙竹荪多糖结构特征。结果表明：不同产地长裙竹荪多糖的高效凝胶排阻色谱图、重均分子质量及其分布均相似,并均含有α-1,4-、α-1,6-、β-1,3-和β-1,4-葡萄糖苷键,以及α-1,4-半乳糖醛酸苷键和β-2,1-果糖苷键,不同产地长裙竹荪多糖具有较高相似性。     

均相碱催化大豆油制备生物柴油的比较研究

选用氢氧化钠和氢氧化钾碱性催化剂,以大豆油为原料催化制备生物柴油,通过正交试验得到了各自的最佳制备条件和影响因素次序.利用傅里叶变换红外光谱(FTIR)、气相色谱-质谱联用(GC-MS)和气相色谱(GC)等现代仪器对生物柴油进行了详细地定性和定量分析,结果表明反应均得到了较高纯度的生物柴油产品,并且氢氧化钾的催化活性大于氢氧化钠,所得生物柴油产率和纯度更高.