响应面法优化褐藻胶降解菌Cobetia sp.20发酵培养基

为了提高褐藻胶降解菌株Cobetia sp.20产褐藻胶裂解酶的能力,利用响应面法优化其发酵产褐藻胶裂解酶的培养基。首先利用单因素法分别对发酵培养基中的不同碳源、碳源添加量、不同氮源、氮源添加量以及氯化钠添加量、磷酸二氢钾添加量、硫酸镁添加量和pH进行探究,研究各因素对产酶的影响。在单因素实验的基础上,通过Plackett-Burman试验确定Cobetia sp.20发酵培养基中影响产酶的主要因素。通过响应面试验建立回归方程。研究结果表明,Cobetia sp.20最优发酵培养基配方为褐藻胶15.00 g/L、硫酸铵7.50 g/L、氯化钠15.00 g/L、硫酸镁0.50 g/L、磷酸二氢钾5.30 g/L、硫酸亚铁0.01 g/L、pH值7.58。优化后酶活为142.79 U/mL,比优化前提高了26.36%。褐藻胶裂解酶活的提高,为褐藻胶裂解酶的工业化生产提供了参考。     

产石杉碱甲内生真菌的复壮及产量提高研究

【目的】千层塔中分离得到的内生真菌胶孢炭疽Cg01可合成石杉碱甲(huperzine A, HupA)，但产量较低，且随着继代的增加，产量下降，菌株退化严重。研究表明，表观遗传修饰与次生代谢产物的合成密切相关。本研究旨在提高HupA的产量，改善退化菌株的品质，并从表观遗传修饰的角度探讨次生代谢产物合成的机理。【方法】通过改变培养基碳源、添加生物诱导子，根据胶孢炭疽Cg01的菌落形态、菌丝生长速度、生物量及HupA产量等筛选复壮培养基；添加不同浓度的组蛋白甲基化转移酶抑制剂，检测HupA的产量，筛选提高HupA产量的小分子抑制剂；检测相关表观遗传修饰基因的表达。【结果】添加同源刺激物千层塔茎叶汁，对胶孢炭疽Cg01的菌落形态、生长速度、形态特征及生物量无显著影响，但可提高HupA的产量，传代至第5代时为对照组的1.67倍(125.7 μg/L)。添加千层塔茎叶汁能显著降低组蛋白甲基化转移酶Cg12377、组蛋白去乙酰化酶Cg15620、DNA甲基化转移酶Cg02440基因的表达，提高组蛋白去乙酰化酶Cg02312基因的表达。UNC0224对内生真菌胶孢炭疽菌的HupA产量无显著影响；2‒15 μmol/L BRD4770能显著提高HupA的产量(169.57‒152.10 μg/L)。BRD4770组处理后，相关表观遗传基因Cg12377、Cg02440、Cg02312和Cg15620的表达量都显著下降。【结论】添加千层塔茎叶汁培养胶孢炭疽Cg01可维持其合成次生代谢产物的能力；添加组蛋白甲基化转移酶抑制剂BRD4770可提高HupA的产量。本研究为解决内生真菌大规模生产过程中的菌株退化问题提供了参考，并为组蛋白甲基化影响次生代谢产物的合成提供了依据。     

产辅酶Q10酵母的发酵条件研究

研究了豆油、豆粉、胡萝卜汁、西红柿汁、烟叶、β-胡萝卜素、桔子皮汁等自然物的添加对酵母发酵生产CoQ10的影响,结果表明它们均能大幅度提高酵母菌中CoQ10的含量。其中豆油、豆粉、西红柿汁、桔子皮汁是富含CoQ10。和胡萝卜素合成途经中的前体物质因而提高了CoQ10的产量;烟叶和β-胡萝卜素阻断了合成β-胡萝卜素的途经从而起到提高CoQ10合成的作用;胡萝卜汁的作用可能两兼而有之。因此可以得出以下结论,微生物中Co10的合成与β-胡萝卜素的合成密切相关。     

产辅酶Q1O酵母的发酵条件研究

研究了豆油、豆粉、胡萝卜汁、西红柿汁、烟叶、β -胡萝卜素、桔子皮汁等自然物的添加对酵母发酵生产CoQ₁₀ 的影响 ,结果表明它们均能大幅度提高酵母菌中CoQ10 的含量。其中豆油、豆粉、西红柿汁、桔子皮汁是富含CoQ₁₀ 和胡萝卜素合成途经中的前体物质因而提高了CoQ₁₀ 的产量 ;烟叶和 β -胡萝卜素阻断了合成 β -胡萝卜素的途经从而起到提高CoQ₁₀ 合成的作用 ;胡萝卜汁的作用可能两     

氮磷钾添加对罗汉松土壤微生物功能多样性的影响

为揭示罗汉松土壤微生物对不同氮磷钾养分水平的响应及规律,该研究以两年生罗汉松(Podocarpus macrophyllus)幼苗为试验树种,采用L9正交试验控制盆栽土壤的氮磷钾养分水平梯度,使用稀释平板涂布法和Biolog-ECO微平板法探讨不同土壤氮磷钾养分水平对罗汉松土壤微生物量和群落多样性及其对6种碳源的利用特征。结果表明:(1)随氮添加量的增加,土壤细菌(P<0.05)和放线菌数量(P<0.001)减少,真菌(P<0.001)及固氮菌数量(P<0.01)显著增加,土壤微生物群落的Pielou 指数(P<0.001)降低,Simpson指数(P<0.05)和McIntosh指数(P<0.001)升高,从而降低了土壤微生物对6种碳源的利用强度,特别是对难利用碳源胺类(P<0.001)、羧酸(P<0.001)、聚合物(P<0.001)及其他化合物(P<0.001)的利用强度显著降低。(2)磷添加量的增加显著降低了土壤微生物群落的Shannon指数(P<0.05)。(3)钾添加量的增加显著降低了土壤微生物群落的Shannon指数和Pielou指数及微生物群落对碳水化合物和氨基酸(P<0.01)两类易利用碳源的强度。综上所述,氮添加和钾添加是影响罗汉松土壤微生物群落功能多样性的主要因素,在罗汉松培育时应注意少量多次施肥,降低氮和钾的添加量,适当提高磷添加量,以促进罗汉松的生长及其可持续培育。该研究从微生物的角度为罗汉松施肥及管护提供了理论依据。     

类芽孢杆菌来源D-甘露醇氧化酶的性质及制备D-甘露糖的应用

D-甘露糖具有多种功能活性，在食品、医药、农业等行业应用广泛。D-甘露醇氧化酶可以高效地将D-甘露醇转化为D-甘露糖，在D-甘露糖的酶法制备中具有应用潜力。从类芽孢杆菌(Paenibacillus sp.) HGF5中发掘出一个D-甘露醇氧化酶(PsOX)，与天蓝链霉菌(Streptomyces coelicolor)来源的D-甘露醇氧化酶(AldO)氨基酸序列相似性为50.94%，分子量约为47.4 kDa，构建了重组表达质粒pET-28a-PsOX并在大肠杆菌BL21(DE3)中表达，PsOX对D-甘露醇的K_m、k_cat/K_m值分别为5.6 mmol/L、0.68 L/(s∙mmol)，最适pH和温度分别为7.0和35 ℃，在60 ℃以下保持稳定。PsOX对400 mmol/L D-甘露醇的摩尔转化率为95.2%。利用PsOX与AldO全细胞分别催化73 g/L D-甘露醇，PsOX反应9 h后反应完全，生成70 g/L D-甘露糖，相较于AldO具有更高的催化效率。PsOX作为新型D-甘露糖氧化酶为D-甘露糖的酶法制备提供了依据。     

两株解磷真菌的解磷能力及其解磷机理的初步研究*

从不同处理的水稻土壤中分离筛选出两株高效解磷真菌HP2、P5,研究了不同碳源条件对溶磷效果的影响,以及解磷菌株在不同的碳源培养条件下,溶磷量与培养介质pH值之间的相关性。结果表明,HP2菌株解磷能力在不同的测定时间内均高于P5菌株;不同碳源培养基的溶磷量顺序为蔗糖>葡萄糖>纤维素,且彼此差异显著;测定时间内,菌株的溶磷量与介质pH值之间存在极显著相关性(P<0.01)。     

红汁乳菇中一个聚酮合酶基因的鉴定和表达分析

聚酮化合物具有丰富的生物活性,为了解红汁乳菇(Lactarius hatsudake)中聚酮合酶基因,从红汁乳菇基因组中分离并克隆得到LhPKS1基因,通过生物信息学分析推测其功能,并通过RT-PCR验证该基因的表达量。结果显示,LhPKS1基因全长cDNA含有6 036 bp,编码2 011个氨基酸残基,结构域顺序依次为SAT-KS-AT-PT-ACP-TE,该蛋白无跨膜结构和信号肽,聚类分析显示LhPKS1蛋白与参与生物合成苔色酸的真菌PKS蛋白聚为一支。在以10%肌醇、2%和10%的山梨醇为碳源添加物及以番茄浸粉为氮源添加物时,该基因表达量较高。研究有助于通过LhPKS1基因的过表达及异源表达,为大量获取苔色酸类化合物及其骨架提供参考。     

鲍氏层孔菌培养条件的研究

本项研究对分离于野外的鲍氏层孔菌从不同的温度、pH值以及不同的碳源和氮源的营养成分三方面进行室内培养,其结果显示鲍氏层孔菌营养菌丝生长的最适温度为28℃;最佳碳源为甘露醇和葡萄糖;最佳氮源为蛋白胨,其次为牛肉膏;而pH值在6.0-8.0的范围内变化对其菌丝体生长影响不大。这些结果为将来大规模人工培植鲍氏层孔菌提供了很好的指导作用。     

益生菌体外增殖因子的初步研究

目的研究单糖、pH、温度及时间对青春双歧杆菌、长双歧杆菌和类干酪乳杆菌体外增殖的影响。方法用甘露糖、半乳糖、山梨醇及果糖代替MRS中的葡萄糖,筛选出每种细菌的最适碳源。以此为基础,选择其最佳初始pH、培养温度、碳源添加量及培养时间。结果青春双歧杆菌、长双歧杆菌和类干酪乳杆菌的最适碳源分别为葡萄糖、甘露糖和半乳糖;最佳初始pH为6.0、7.0和6.0;培养温度为42、30和30℃;碳源添加量为20、15和25 g/L;培养时间都为28-48 h。结论益生菌具有不同的最适增殖条件,本文研究结果为优化益生菌的生长条件提供了基础数据。     

Kinetic study of mannitol production using cashew apple juice as substrate

The use of agriculture excess as substrate in industrial fermentations became an interesting alternative to reduce production costs and to reduce negative environmental impact caused by the disposal of these products. In this work, a kinetic study of mannitol production using cashew apple juice as substrate was studied. The carbohydrates of cashew apple juice are glucose and fructose. Sucrose addition favored the yield of mannitol (85%) at the expense of lower productivity. The best results were obtained applying only cashew apple juice as substrate, containing 50 g L⁻¹ of total reducing sugar (28 g L⁻¹ of fructose), yielding 18 g L⁻¹ of mannitol with 67% of fructose conversion into mannitol and productivity of 1.8 g L⁻¹ h⁻¹.     

Post-harvest Changes in Sweet Sorghum II: pH, Acidity, Protein, Starch, and Mannitol

This experiment was conducted to evaluate the effect of four harvesting methods on juice quality and storability in sweet sorghum. Three cultivars (Dale, Theis, and M81-E) were harvested at 90, 115, and 140 days after planting. Stalks were stripped of leaves and topped at the peduncle, then divided into four treatments (whole stalk, 20- or 40-cm billets, or chopped). The sorghum was stored outside at ambient temperature in a shade tent, and juice was extracted from samples removed at 0, 1, 2, and 4 days after harvest. Changes in juice Brix and sugars were reported in an earlier paper (Lingle, Tew, Rukavina, Boykin, Post-harvest changes in sweet sorghum I: Brix and sugars, BioEnergy Research 5:158–167, 2012). In this paper, we report changes in juice pH, titratable acidity (TA), and protein, starch, and mannitol concentrations. Juice pH dropped rapidly after harvest in chopped sorghum, but changed little during 4 days of storage in whole stalks or billets. Similarly, TA increased with storage time in chopped samples, but was unchanged in whole stalks and billets. Protein concentration was highly variable, and no pattern with treatment or storage time could be discerned. In whole stalks and billets, starch content slowly decreased during storage, while in chopped samples starch appeared to increase. This was most likely a result of an increase in dextran synthesized by microorganisms in those samples, which was also detected by the enzymatic starch assay. The concentration of mannitol increased with storage time in chopped samples, but not in whole stalks or billets. Within a harvest date, pH was highly correlated with total sugar, while TA and mannitol were highly negatively correlated with total sugar. The results confirm that whole stalks and billets were little changed over 4 days of storage, while chopped sorghum was badly deteriorated 1 day after harvest. Changes in pH, TA, or mannitol could be used to measure deterioration in sweet sorghum after harvest.     

Fermentation of cashew apple juice to produce high added value products

The use of agriculture substrates in industrial biotechnological processes has been increasing because of its low cost. Cashew apples are considered an agriculture low cost product in the Brazilian Northeast because the cashew cultivation is done mainly to produce cashew nuts. About 90% of the cashew apples production is lost in the field after removing the nut. In this work, the use of clarified cashew apple juice as substrate for microbial cultivation was investigated. The results showed that cashew apple juice is a good source of reducing sugars and can be used to grow Leuconostoc mesenteroides to produce high added value products such as dextran, lactic acid, mannitol and oligosaccharides.     

Use of autochthonous lactic acid bacteria starters to ferment mango juice for promoting its probiotic roles

Strains of Leuconostoc mesenteroides, Pediococcus pentosaceus, and Lactobacillus brevis were identified from mango fruits by partial 16S rDNA gene sequence. Based on the ability of producing mannitol and diacetyl, Leuconostoc mesenteroides MPL18 and MPL39 were selected within the lactic acid bacteria isolates, and used as mixed starters to ferment mango juice (MJ). Both the autochthonous strains grew well in fermented mango juice (FMJ) and remained viable at 9.81 log cfu mL^?1 during 30 days of storage at 4°C. The content of total sugar of FMJ was lower than that of MJ, while the concentration of mannitol was higher than that of MJ, and the concentration of diacetyl was 3.29 ± 0.12 mg L^?1. Among detected organic acids including citric acid, gallic acid, lactic acid, and acetic acid, only citric acid and gallic acid were found in MJ, while all detected organic acids were found in FMJ. The concentration of lactic acid of FMJ was the highest (78.62 ± 13.66 mM) among all detected organic acids. The DPPH radical scavenging capacity of FMJ was higher than that of MJ. Total phenolic compounds were better preserved in FMJ. The acidity and sweetness had a noticeable impact on the overall acceptance of the treated sample.     

米曲霉两株营养缺陷型原生质体的形成和再生的条件实验*

采用1%溶壁酶加1%玛瑙螺酶(褐云玛瑙螺消化液的冷冻干粉)的混合酶,自米曲霉(Aspergillus oryzae)的两株营养缺陷型中获得了大量的原生质体,并比较了渗透压稳定剂、温度、菌丝体的培养基成分等因素对原生质体形成和再生的作用。无机盐类稳定剂(NaCl、KCl)获得了高产量的原生质体,而有机类(蔗糖、甘露醇、山梨醇)做为稳定剂不甚理想。对120和720菌株的原生质体在高渗再生培养基上进行再生试验,再生率分别为52%和65%。     

Some factors affecting the formation of protoplasts inAspergillus niger

The highest yield of protoplasts in the strainAspergillus niger K 10 was obtained from young, freshly harvested hyphae, grown on simple medium of sucrose-asparagine type on a rotary shaker. The residual cultivation medium has to be washed from the mycelial suspension with a solution of high osmotic pressure. Lyophilized snail digestive juice in concentration of 1 %, temperature 31° C, and incubation in Erlenmayer flasks on reciprocal shaker were optimal for the release of protoplasts. Good stabilizers of released protoplasts (in combination with CaCl₂) were for example galactose, mannitol, inositol and mixture of NaCl with glucose, sucrose or lactose.     

Preservation of potential fermentables in sweet sorghum by ensiling

Pressed and wilted samples of sweet sorghum [Sorghum bicolor (L.) Moench var. Rio] were ensiled for periods up to 155 days. A kinetic study of the biochemical changes which occurred during ensiling showed that in wilted sorghum ensilage invert sugars and mannitol levels collectively were maintained at 65% of the original ferment able sugar content of the sorghum. The acidic environment produced by ensiling also served as a pretreatment that resulted in enhanced yields of reducing sugar when the sorghum was contacted with cellulolytic enzymes. The quantity of sugar obtained from enzymatic hydrolysis more than compensated for carbohydrate used by organisms during the ensiling process. Both Saccharomyces uvarum and Clostridium acetobutylicum were able to ferment a medium constituted from pressed sorghum juice and the solution resulting from enzymatic hydrolysis of sweet sorghum ensilage.     

Identification and characterization of Leuconostoc fallax strains isolated from an industrial sauerkraut fermentation

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.     

Mannitol is required for asexual sporulation in the wheat pathogen Stagonospora nodorum (glume blotch)

The physiological role of the mannitol cycle in the wheat pathogen Stagonospora nodorum (glume blotch) has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene (Mdh1) was cloned by degenerate PCR and disrupted. The resulting mutated mdh1 strains lacked all detectable NADPH-dependent mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but, surprisingly, were still able to utilize mannitol as a sole carbon source, suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. To further our understanding of mannitol metabolism, a previously developed mannitol-1-phosphate dehydrogenase (gene mpd1) disruption construct [Solomon, Tan and Oliver (2005) Mol. Plant-Microbe Interact. 18, 110-115] was introduced into the mutated mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double-mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although, like the previously characterized mpd1 mutants, the strains were unable to sporulate in planta. These findings not only question the currently hypothesized pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.