Growth of Candida utilis on enzymatically hydrolysed cassava

Summary Enzymatically hydrolysed cassava starch was used for C. utilis cultivation. Highly efficient starch hydrolysis was achieved with a 92% DE syrup obtained after 15–20h. Cyanide content fell during cassava processing to very low levels in the hydrolysate. Comparison of biomass yields and protein of C. utilis using molasses and cassava hydrolysate as substrates demonstrates the potential of the latter for yeast production.     

Comparative studies on the glycolytic and hexose monophosphate pathways in Candida parapsilosis and Saccharomyces cerevisiae

Some enzymatic activities of the glycolytic and hexose monophosphate pathways of Candida parapsilosis, a yeast lacking alcohol dehydrogenase but able to grow on high glucose concentrations, were compared to those of Saccharomyces cerevisiae. Cells were grown either on 8% glucose or on 2% glycerol and activities measured under optimal conditions. Results were as follows: glycolytic enzymes of C. parapsilosis, except glyceraldehyde 3-phosphate dehydrogenase, exhibited an activity weaker than that of S. cerevisiae, especially when yeasts were grown on glycerol. Fructose-1,6 bisphosphatase, an enzyme implicated in gluconeogenesis and in the hexose monophosphate pathway, and known to be very sensitive to catabolite repression in S. cerevisiae, was always active in C. parapsilosis even when cells were grown on 8% glucose. However, the allosteric properties towards AMP and fructose-2,6-bisphosphate were the same in both strains. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, two other enzymes of the hexose monophosphate pathway, exhibited a higher activity in C. parapsilosis than in S. cerevisiae. Regulation of two important control points of the glycolytic flux, phosphofructokinase and pyruvate kinase, was investigated. In C. parapsilosis phosphofructokinase was poorly sensitive to ATP but fructose-2,60bisphosphate completely relieved the light ATP inhibition. Pyruvate kinase did not require fructose-1,6-bisphosphate for its activity, and by this way, did not regulate the glycolytic flux. The high glyceraldehyde-3-P-dehydrogenase activity, together with the relative insensitivity of fructose-1,6-bisphosphatase to catabolite repression and the high glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities suggested that in C. parapsilosis, as in other Candida species and opposite to S. cerevisiae, the glucose degradation mainly occurred through the hexose monophosphate pathway, under both growth conditions used.Abbreviations C. parapsilosis Candida parapsilosis - S. cerevisiae Saccharomyces cerevisiae - C. utilis Candida utilis     

Growth of Saccharomycopsis fibuliger and Candida utilis in mixed culture on pectic materials

Hydrolysis of pectin by Saccharomycopsis fibuliger and cell growth on the products of hydrolysis by Candida utilis require different incubation conditions. A three-stage sequential culture is described in which S. fibuliger was first grown under aerobic conditions to generate cell mass. The concentration of dissolved oxygen was then reduced to promote pectolytic activity and reduce the number of viable cells in the culture. Finally culture conditions were adjusted to promote the growth of C. utilis in mixed culture with S. fibuliger. The presence of C. utilis increased the rate of pectolytic activity by S. fibuliger. A yeast product, containing 98% C. utilis cells, was obtained from the mixed culture grown on 10 g l⁻¹ pectin. Cell yields using starch or an equal mixture of starch and pectin were similar to those reported in the Symba process, although lower cell yields were recorded using pectin alone.     

Culture of Candida in vinasse and molasses: Effect of acid and salt addition on biomass and raw protein production

Species of the genera Candida grown in vinasse and molasses were studied under the following conditions: agitation of containers, pH 4.6, culture time of 24 hours at 30°C. The greatest biomass production of C. krusei grown in vinasse was obtained with the addition of 0.1% H₃PO₄, and of C. guilliermondii and C. utilis with the addition 0.02% urea plus 0.03% H₃PO₄. Protein levels near 50% were found in C. utilis in vinasse supplemented either with molasses, with 0.05% MgSO₄, or with 0.02% urea plus 0.03% H₃PO₄.     

Production and characterization of raffinose-hydrolysing and invertase activities ofAspergillus fumigatus

Raffinose-type galactose oligosaccharides constitute a substantial part (40%) of the soluble sugars present in soybean seeds and are responsible for flatulence following ingestion of soybean and other legumes. Enzymic hydrolysis of these oligosaccharides would improve the nutritional value of soybean milk.Aspergillus fumigatus produces substantial raffinose-hydrolysing and invertase activities when grown on wheat straw. Three proteins displaying maximal activity at pH 4.5–5.5 and 55–60°C and having molar mass of 66.8, 50.3 and 30.2 kDa were purified. Raffinose and sucrose were hydrolyzed with equivalent affinities by each protein. Nevertheless, theK _m andV _lim values determined for hydrolysis of sucrose by the 66.8 kDa enzyme differed from those determined with the 50.3 kDa protein. Glucose was produced when sucrose was the substrate. The three proteins hydrolyzed also stachyose but not melibiose, maltose, inulin or 4-nitrophenyl α-d-galactopyranoside.A. fumigatus enzymes may be candidates for processing of soybean milk to reduce its flatulence potential.     

原产地和入侵地紫茎泽兰对泽兰实蝇的选择性、卵巢蛋白质含量与解毒酶活性的影响

[目的] 紫茎泽兰是我国危害严重的恶性入侵杂草。比较专一性天敌泽兰实蝇对该杂草入侵前后植株的适应性,是揭示外来植物入侵后适应性机制的重要科学问题之一。[方法] 比较泽兰实蝇对原产地和入侵地紫茎泽兰植株的寄主选择性,并测定寄生于2类植株的上泽兰实蝇卵巢蛋白质含量及乙酰胆碱酯酶、羧酸酯酶、谷胱甘肽S-转移酶活性。[结果] 泽兰实蝇对原产地和入侵地紫茎泽兰的选择无显著性差异;寄生在紫茎泽兰入侵地植株上的卵巢蛋白质含量较原产地植株上更高。解毒酶活力比较表明,入侵地紫茎泽兰上泽兰实蝇的羧酸酯酶活性低于原产地上的,但谷胱甘肽S-转移酶（雌虫）活性比较则相反,乙酰胆碱酯酶活性比较均无显著性差异。[结论] 紫茎泽兰入侵后,专一性天敌泽兰实蝇的适应性有所下降,丰富了外来植物入侵机制中天敌逃逸假说的内涵。     

Enzymatic hydrolysis of the Eisenia andrei earthworm: Characterization and evaluation of its properties

Some studies have carried out in order to retrieve proteins from the by-product of animal-processing industries. Earthworms are rich in protein and usually are used in animal feed. Thus, this study aimed to optimize the hydrolysis process of Eisenia andrei earthworms by employing Alcalase enzyme. Using the response surface methodology, we evaluated the following conditions: temperature, hydrolysis time, stirring speed, and enzyme/substrate ratio. The optimal conditions for the experimental design were determined through the analysis of the foaming and emulsifying properties, in vitro starch digestibility, and antioxidant activity. The results demonstrate that the highest degree of hydrolysis (i.e., 92%) was obtained under the following conditions: pH, 9.5; temperature, 25?°C; hydrolysis time, 2.25?h; stirring speed, 200?rpm; and enzyme/substrate ratio, 1.77%, using Alcalase enzyme. Evaluation of the amino acid composition under these conditions revealed higher concentrations of aspartic acid, glutamic acid, and leucine. The in vitro protein digestibility of the hydrolysate was approximately 73%. There were no significant improvements in either foam stability or emulsification after enzymatic hydrolysis. Additional studies on the antioxidant activity are required. This bioproduct could potentially serve as a promising supplementary food product.     

Characterization of ψM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg

We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase.However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.     

Efficient Preparation of Selenium/Glutathione-Enriched Candida utilis and Its Biological Effects on Rats

The main purpose of this study was to prepare selenium/glutathione-enriched Candida utilis and investigate its effect on growth performance, antioxidant capacity, and immune response in rats. The preparation of the selenium/glutathione-enriched yeast was conducted using fed-batch culture for high cell density. The optimal culture conditions for increased intracellular organic selenium and glutathione contents were as follows: the concentrated medium was fed beginning at 12?h using a polynomial feeding strategy until a total glucose concentration of 150?g/l was reached, and sodium selenite was continuously added together with glucose to a total concentration of 60?mg/l. As a result, 81?% of sodium selenite was assimilated and transformed into organic selenium by C. utilis under optimal conditions, which in turn resulted in greater glutathione accumulation and lower malondialdehyde cellular content in the yeast. To investigate and compare the effects of the prepared selenized C. utilis and other dietary supplements, 40 female rats were divided into five groups of eight rats each, following a randomized block design. Experimental feeding was conducted for a period of 6?weeks. Selenium supplementation with inorganic selenium (sodium selenite) and organic selenium (selenized C. utilis) showed better results than the control and other groups supplemented with yeast with or without glutathione. The body mass of rats, selenium deposition, and oxidative enzymes activities in both serum and liver samples, and immunity responses were all significantly improved by selenium supplementation, and between the two sources, organic selenium was more effective than inorganic selenium.     

Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass

The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.     

Stabilisation of some of the protein synthesis components in the thermophilic fungus,Humicola lanuginosa

The thermal stabilities of tRNA from the thermophilic fungus,Humicola lanuginose were compared with that from the mesophilic yeast,Candida utilis, by measuring the increase in the optical density with temperature. tRNAs from both the species were stable in the presence of millimolar quantities of magnesium chloride upto 50°C, the optimum growth temperature of the fungus. Aminoacyl tRNA synthetases were maximally active at 40°C under thein vitro assay conditions. They were fractionated and one species of valine tRNA synthetase was purified to homogeneity. The purified enzyme was protected against inactivation to varying degrees when preincubated with the substrates valine, tRNA and ATP as well as spermine. Protein turnover studies showed that the rate of turnover was higher at higher temperatures. It was concluded from these results that the protein synthesizing machinery of this fungus has no intrinsic stability but it is stabilised by intracellular factors. Higher rate of protein turnover also plays a role for growth at higher temperature.     

Expression of a New Cold Shock Protein of 21.5 kDa and of the Major Cold Shock Protein by Streptococcus thermophilus After Cold Shock

Streptococcus thermophilus is widely used in food fermentations; it commonly suffers diverse stress challenges during manufacturing. This study investigated the cold shock response of S. thermophilus when the cell culture temperature shifted from 42°C to 15°C or 20°C. The growth of cells was affected more drastically after cold shock at 15°C than at 20°C. The generation time was increased by a factor of 19 when the temperature was lowered from 42° to 20°C, and by a factor of 72 after a cold shock at 15°C. The two-dimensional electrophoretic protein patterns of S. thermophilus under cold shock conditions were compared with the reference protein pattern when cells were grown at optimal temperature. Two proteins of 21.5 and 7.5 kDa synthesized in response to cold shock were characterized. N-terminal sequencing and sequence homology searches have shown that the 7.5-kDa protein belonged to the family of the major cold shock proteins, while no homology was found for the new cold shock protein of 21.5 kDa. Received: 4 June 1999 / Accepted: 6 July 1999     

Are the proteinase inhibitory activities in lenticular tissues real?

The possibility of proteinase inhibitory activities in lenses measured with synthetic substrates being spurious, due to the effective competition of lens proteins as substrates for the target enzymes, was investigated. Goat, sheep and human cataractous lens proteins were found to be poor substrates for trypsin, elastase and papain compared to casein or bovine serum albumin. Further, the inhibition of elastase catalyzed hydrolysis of succinyl trialanyl p-nitroanilide by casein (500 μg, 53%) and albumin (500 μg, 49%) and of trypsin-catalyzed hydrolysis of benzoyl argininep-nitroanilide by albumin (1 mg, 24%) were significant only at high protein concentrations. These data indicated that the relatively high antielastase and antitryptic activities observed in human cataractous lenses were real. On the other hand, coincident lens protein hydrolysis elevating the true antitryptic and antielastase activities in goat and sheep lenses (that have low activities) could not be ruled out The lesser papain inhibitory activities observed in lenses when albumin was used as substrate compared to activities with benzoyl arginine p-nitroanilide as substrate, appeared to be partly due to lens protein hydrolysis masking the actual inhibition in the former method. Preincubation of goat, sheep and human lens extracts with trypsin for 1 h resulted in complete loss of antitryptic and antielastase activity except in the case of human lens antielastase activity which underwent 50% loss. Papain inhibitory activity was fully stable. Similar papain treatment caused loss of 80–100% of antielastase activity and 45–55% loss of antitryptic activity.     

Novel lignocellulolytic ability of Candida utilis during solid-substrate cultivation on apple pomace

Lignocellulolytic enzymes from conventional and non-conventional yeasts are not commonly studied, and they have never been described for Candida utilis species. After solid-substrate cultivation of C. utilis (CCT 3469) on apple pomace, degradation of cellulose, pectin and lignin fragments was observed. Production of the main lignocellulolytic enzymes by C. utilis was investigated and high activity for pectinase (239 U ml^–1) as well as a significant manganese-dependent peroxidase (19.1 U ml^–1) activity was found. Low cellulase (3.0 U ml^–1) and xylanase (1.2 U ml^–1) activities were also observed suggesting that C. utilis may have lignocellulose degradation ability.     

Molecular analysis of inorganic nitrogen assimilation in yeasts

Assimilation of nitrate and various other inorganic nitrogen compounds by different yeasts was investigated. Nitrate, nitrite, hydroxylamine, hydrazine, ammonium sulphate, urea and L-asparagine were tested as sole sources of nitrogen for the growth of Candida albicans, C. pelliculosa, Debaryomyces hansenii, Saccharomyces cerevisiae, C. tropicalis, and C. utilis. Ammonium sulphate and L-asparagine supported the growth of all the yeasts tested except D. hansenii while hydroxylamine and hydrazine failed to support the growth of any. Nitrate and nitrite were assimilated only by C. utilis. Nitrate utilization by C. utilis was also accompanied by the enzymatic activities of NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H: nitrite oxidoreductase (EC 1.6.6.4), but not reduced methyl viologen-or FAD-nitrate oxidoreductases (EC 1.7.99.4). It is demonstrated here that nitrate and nitrite reductase activities are responsible for the ability of C. utilis to assimilate primary nitrogen.     

RNA removal from Candida utilis by chemical and thermal treatments

The removal of the content of nucleic acids of fodder yeast (Candida utilis) by treatment with HCl or heat shock was investigated Acid concentrations between 5 and 15% (on dried matter basis) were used. A maximal removal of the content of nucleic acids of 88% was realized wheńn was worked with 15% HCl, 90 °C during 30 minutes. But under this conditions were observed high demanges of protein and also of some essential amino acids Good results for diminishing the content of nucleic acids were reached with the highest concentration of acid and a treatment during 20 minutes The experiments with heat shock were carried out at 68 °C, different times for heating and different contents of yeast In this way better results than for treatment with acid according to diminishing the content of nucleic acids and yield of protein and essential amino acids were reached A removal of over 80% of the content of nucleic acids was achieved in all cases.     

Precipitation of Trichoderma reesei commercial cellulase preparations under standard enzymatic hydrolysis conditions for lignocelluloses

Comparative studies between commercial Trichoderma reesei cellulase preparations show that, depending on the preparation and loading, total protein precipitation can be as high as 30 % under standard hydrolysis conditions used for lignocellulosic materials. ATR-IR and SDS-PAGE data verify precipitates are protein-based and contain key cell wall hydrolyzing enzymes. Precipitation increased considerably with incubation temperature; roughly 50-150 % increase from 40 to 50 °C and 800 % greater at 60 °C. All of the reported protein losses translated into significant, and often drastic, losses in activity on related 4-nitrophenyl substrates. In addition, supplementation with the non-ionic surfactant PEG 6,000 decreased precipitation up to 80 % in 24 h precipitation levels. Protein precipitation is potentially substantial during enzymatic hydrolysis of lignocelluloses and should be accounted for during lignocellulose conversion process design, particularly when enzyme recycling is considered.     

Pretreatment of cellulosic wastes to increase enzyme reactivity

The enzymatic hydrolysis of cellulose to glucose is generally a slow reaction. Different pretreatments, such as ball milling to a ?200 mesh or swelling in 1–2% NaOH are reported to increase the reactivity considerably. In this work a fiber fraction from cattle manure was treated in an autoclave for 5–30 min at temperatures ranging from 130–200°C. The reactivity of the cellulose, measured by incubating samples with a commercial cellulase preparation for one hour at 50°C and pH 4.8, was increased by a factor of 4–6 compared to NaOH treatment and 10–12 compared to untreated fiber. The increased reaction rate is probably mostly due to an increase in cellulose availability to enzymatic attack, as structural hemicellulose is hydrolyzed and removed during the treatment. Sugars, produced by hemicellulosis, hydrolysis, will react further to give caramelization products. These side reactions were shown to be suppressed by short treatment times. The treated fiber could support growth of a mixed culture of Trichoderma viride and Candida utilis only after washing, indicating the formation of water soluble inhibitory products during treatment. The treatment with high-temperature steam can probably be used also with other cellulosic materials to increase reactivity. This may be an attractive way to prepare low-valued wastes such as manure fibers, straw, stalks, or corn cobs for fermentation processes to increase the protein content or for use directly as ruminant animal feed.     

青刺及其护肤相关传统知识的民族植物学研究

青刺(Prinsepia utilis)为滇西北多民族地区药食同源的多年生落叶灌木,被该地区的社区群众广泛使用于传统文化、医药、食用等方面。随着现代社会的发展,青刺及相关的传统知识面临消失的危险。为探讨该地区青刺资源及其相关传统知识的保护与传承,该文基于民族植物学野外调研的基础上,采用天然药物化学与药理活性测试的方法,对青刺主要传统功效的物质基础及其护肤活性进行初步研究。结果表明:(1)在滇西北多民族聚居区,青刺被广泛用于围栏防护或防风固土、皮肤外伤的治疗、食用等多种传统用途;(2)从青刺不同部位的提取物中检测到没食子酸、槲皮素等10个有护肤活性的单体化合物;(3)青刺嫩叶提取物的总黄酮含量高于其发酵产物和青刺果提取物;(4)传统利用频率最高的青刺果油呈现出较好的DPPH自由基清除活性,不同产地和工艺之间抗氧化活性有差异。该研究结果初步验证了青刺传统护肤功效的物质基础及其相关活性之间的相关性,为青刺资源及相关传统知识的保护与传承及深度研发提供了参考。