Production and characterization of a highly active cellobiase from Aspergillus niger grown in solid state fermentation

Summary Aspergillus niger produced extracellular cellobiase when grown on different lignocellulosic substrates in solid state fermentation. The enzyme activity and yield were variable according to the carbon source. In Vogel’s medium, the cellobiase productivity was significantly higher on wheat bran, followed by Leptochloa fusca (kallar grass) straw augmented with corn steep liquor. Maximum yield of cellobiase/g wheat bran was significantly higher than the values reported on other potent fungi, bacteria and recombinants, harboring heterologous gene for cellobiase. This enzyme in the presence and absence of Trichoderma reesei and celluloclast, saccharified the biomass and the percentage saccharification as well as glucose yield from lignocellulosic biomass was doubled in its presence. The partially purified enzyme was thermotolerant as evidenced by melting temperature, activation energy demand for active catalysis, enthalpy and entropy of activation for reversible or irreversible thermal inactivation.     

Production of pectinases by Aspergillus niger in solid state fermentation at high initial glucose concentrations

Exopectinase (exo-p) and endopectinase (endo-p) production by Aspergillus niger CH4 in solid state culture was studied at initial glucose concentrations of 100, 250, 350 and 450 g/l. The highest activity of exo-p (35 U/g) was produced at 72 and 120 h in the medium containing 100 and 250 g glucose/l, respectively. The maximum endo-p activity (9 U/g) was produced at 72 h in the medium with 250 g glucose/l. The reduction in pectinase production at 350 and 450 g/l initial glucose concentration was due neither to repression of the synthesis of the enzyme nor to the glucose consumption rate of the strain but due to a drastic drop in pH of the medium.S. Solis-Pereyra, E. Favela-Torres, M. Gutiérrez-Rojas, G. Saucedo-Castañeda and G. Viniegra-González are with the Departamento de Biotecnologia, Universidad Autónoma Metropolitana, A.P. 55-535, C.P. 09340, México D.F., México; S. Roussos is with the Laboratoire de Biotechnologie, ORSTOM, B.P. 5045, 34032, Montpellier Cedex, France, and P. Gunasekaran is with the Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625-021, India.     

黑曲霉固态发酵生产单宁酶的条件优化

研究采用响应面法优化黑曲霉固态发酵生产单宁酶的培养条件。应用Plackett—Burman试验筛选出重要影响因子：五倍子粉含量、（NH4）2SO4浓度以及接种孢子量,最陡爬坡试验逼近最大响应区域。应用Box．Behnken响应面试验对重要影响因子进一步优化。得到最佳培养条件：每250mL三角瓶中装入1．0g五倍子粉、4．4g稻壳和0．5g麸皮、液固比（mL／g）2：1且营养盐溶液组成为（NH4）2s0421g/L、MgSO4·7H2O1g／L、NaCl1g／L,培养基pH自然,接种5．7×10^7个孢子后在30℃温度下培养4d。在此条件下,单宁酶产量从40U／g提高到114U／g,3次重复验证性试验平均值为115U／g,验证了模型的可靠性。     

Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid state systems

Production of pectinesterase and polygalacturonase by Aspergillus niger was studied in submerged and solid-state fermentation systems. With pectin as a sole carbon source, pectinesterase and polygalacturonase production were four and six times higher respectively in a solid state system than in a submerged fermentation system and required a shorter time for enzyme production. The addition of glucose increased pectinesterase and polygalacturonase production in the solid state system but in submerged fermentation the production was markedly inhibited. A comparison of enzyme productivities showed that those determined for pectinesterase and polygalacturonase with pectin as a carbon source were three and five times higher by using the solid state rather than the submerged fermentation system. The productivities of the two enzymes were affected by glucose in both fermentation systems. The membranes of cells from the solid state fermentation showed increased levels of C18:1, C16:0 and C18:0 fatty acids. Differences in the regulation of enzyme synthesis by Aspergillus niger depended on the fermentation system, favoring the solid state over the submerged fermentation for pectinase production. Received 12 May 1997/ Accepted in revised form 19 September 1997     

Influence of the glycolytic rate on production of citric acid and oxalic acid by Aspergillus niger in solid state fermentation

A study was performed to understand the physiology and biochemical mechanism of citric acid accumulation during solid state fermentation of sweet potato using Aspergillus niger Yang No.2. A low citrate-producing mutant was isolated followed by a comparative study of the fermentation process and selected physiological and biochemical parameters. In contrast with the parent strain, the mutant strain displayed lower concentrations, yields and production rates of citric acid, accompanied by higher concentrations, yields and production rates of oxalic acid. In addition, the mutant utilized starch at a lower rate although higher concentrations of free glucose accumulated in the cultures. Biochemical analyses revealed lower rates of glucose uptake and hexokinase activity of the mutant strain in comparison with the parent strain. It is proposed that, in common with submerged fermentation, over-production of citric acid in solid state fermentation is related to an increased glucose flux through glycolysis. At low glucose fluxes, oxalic acid is accumulated.     

Biomass estimation in solid state fermentation by modeling dry matter weight loss

The mathematical model introduced by Okazaki & Tanaka (J. Ferment Technol. 1980, 58: 471–476) for estimating fungal growth in solid state fermentations by CO2 evolution, was incorporated into a relationship developed to estimate biomass growth from dry matter weight loss. The proposed equation fitted experimental data very well (R2=0.987, P<0.0001) and allowed accurate predictions of fungal specific growth rate and maximal biomass in the solid substrate fermentation system.     

黑曲霉SL2-111复合酶固体发酵工艺研究

以酸性蛋白酶酶活为响应值,采用单因素搜索和正交试验对黑曲霉（Aspergillus niger）SL2－111固体发酵工艺进行优化,结果表明最适培养基的组成为：新鲜麸皮8．25g,米糠4．5g、豆饼粉1．5g、（NH4)2SO40.3g、K2HPO40．66g、CaCl20.075g、水8．6mL,pH5．5,变温培养,前30h28℃、后30h为23℃,培养时间为60h。采用最适培养基和优化工艺,在250mL三角瓶中进行验证实验,酸性蛋白酶酶活可达12586U/g,果胶酶和纤维素酶分别为16490U/g、9822U／g。     

Beneficial effect of protracted sterilization of lentils on phytase production by Aspergillus ficuum in solid state fermentation

Water addition to the solid substrate preceding autoclaving increased substrate porosity and phytase production in solid state fermentation. In comparison with dry sterilization, the phytase activity increased 6‐, 8.5‐, and 10‐fold when the autoclaving time was 20, 40, and 60 min, respectively. Autoclaving increased the void space of sterilized lentils, and the increase was 16% higher when water was supplemented to the lentils before sterilization. Image analysis of SEM pictures of the solid substrate showed that water supplementation presterilization portended greater micro‐fissure surface area, which also increased with increasing the sterilization time. SEM pictures of the fermentation product showed that fungal growth into the center of the solid substrate was ubiquitous when water was supplemented before sterilization but was absent when water was supplemented post sterilization. Similarly, spore formation on the substrate surface for the presterilization water supplementation samples far exceeded spore formation for samples that received supplementation poststerilization. This evidence suggests that improved mass transfer into the solid substrate resulting from additional pore volume and the formation of micro‐fissures on the substrate surface is responsible for the observed gains in phytase productivity in solid state fermentation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Phytase isozymes from Aspergillus niger NCIM 563 under solid state fermentation: Biochemical characterization and their correlation with submerged phytases

Aspergillus niger NCIM 563 produces dissimilar phytase isozymes under solid state and submerged fermentation conditions. Biochemical characterization and applications of phytase Phy III and Phy IV in SSF and their comparison with submerged fermentation Phy I and Phy III were studied. SSF phytases have a higher metabolic potential as compared to SmF. Phy I is tetramer and Phy II, III and IV are monomers. Phy I and IV have pH optima of 2.5 and Phy II and III have pH optima of 5.0 and 5.6, respectively. Phy I, III and IV exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. SSF phytase is less thermostable as compared to SmF phytase. Phy I and II show homology with other known phytases while Phy III and IV show no homology with SmF phytases and any other known phytases from the literature suggesting their unique nature. This is the first report about differences among phytase produced under SSF and SmF by A. niger and this study provides basis for explanation of the stability and catalytic differences observed for these enzymes. Exclusive biochemical characteristics and multilevel application of SSF native phytases determine their efficacy and is exceptional.     

Purification and characterization of glucoamylase produced by Aspergillus niger in solid state fermentation

Glucoamylases produced by Aspergillus niger grown on wheat bran in solid cultures were purified. Four different forms, GA I, GA I', GA II and GA III, were found having apparent molecular weights of 112 000, 104 000, 74 000 and 61 000 Da respectively. The enzymes are glycoproteins with a carbohydrate content of 16%, and optimal activity at 60C and pH 4.4. Activity was strongly inhibited by Hg²⁺ while Mn²⁺ and Fe²⁺ were stimulatory. The K_m values for the degradation of starch and maltose were 3.5 and 7.8 mg ml^-1, respectively.     

黑曲霉固态发酵生产酸性β-甘露聚糖酶

以黑曲霉WA301为生产菌固态发酵生产酸性β－甘露聚糖酶,较适的培养基组成和培养条件为：麸皮10g,魔芋粉0.4g,豆饼粉1.5g,硫酸铵0.2g,起始湿度55％,pH自然,发酵温度35℃,发酵周期约96h。在此条件下,WA301的酸性β－甘露聚糖酶产率为950U／g干曲左右。     

金龟子绿僵菌固态发酵产壳聚糖酶

对金龟子绿僵菌（Metarhizium anisopliae）AS3．4606产壳聚糖酶进行了固态发酵条件优化及酶学部分特征的研究。正交实验结果表明,以麸皮为碳源,日本根霉菌丝体粉末为氮源,在起始pH5．0,m（碳）：m（氨）为1：4,m（干重）：m（液体）为1：1．4,27℃下培养120h,壳聚糖酶活性可达35．08U／g（干培养基）。粗酶液的最适反应温度为40℃,最适反应pH为5．0,酶在pH5．0～6．0条件下稳定性最高。该酶不能水解固体甲壳素和纤维素粉末,最适底物为胶体壳聚糖。     

Extraction and purification of amyloglucosidase produced by solid state fermentation with Aspergillus niger

Summary Aqueous two-phase extraction was employed, as a new approach, for the recovery of amyloglucosidase (AMG) after solid state fermentation. In an aqueous two-phase system of PEG 6000 and potassium phosphate, 95% of the AMG was recovered in the bottom salt phase, with all visible particles from solid fungal bran in the interface, giving a purification factor of 11. Affinity purification of AMG was also done by adsorption onto crosslinked starch, in the presence of PEG or salt. Both were found to enhance the adsorption. In the presence of PEG 6000 (10% w/w), two fold increase in AMG adsorption, 85% of AMG recovery and 6.0 fold of purification were achieved/The purified AMG was found homogeneous by SDS polyacrylamide gel electrophoresis.     

Engineering aspects of solid state fermentation

Engineering aspects of solid state fermentation have not been available in spite of the recent interest in the technique and the appearance of a number of reviews on this subject. The present state of the art regarding substrate uptake, oxygen transfer, growth characteristics, growth estimation, control systems for maintaining parameters, mathematical models, design of fermenters and automation of fermentations does not provide a detailed insight. The work carried out at the Central Food Technological Research Institute, Mysore, on the development of a large-scale solid state fermenter, comparative cost estimation of SSF and submerged fermentation as well as development of know-how for production of a variety of food enzymes, has led to the commercial exploitation of the technology by industry. Future R&D needs in this area, neglected at present but yet so promising, are indicated.     

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g⁻¹ dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH₂PO₄ (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K⁺ and Zn²⁺, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243. Received 07 June 1999/ Accepted in revised form 18 December 1999     

利用黑曲霉固态发酵啤酒糟生产饲料复合酶的研究

以啤酒糟为主要基质,利用黑曲霉固态发酵生产酸性蛋白酶、木聚糖酶和纤维素酶等多种饲料复合酶,研究了黑曲霉固态发酵培养基组成对复合酶酶活的影响,确定最优培养基配方为:啤酒糟75%,麸皮25%,硫酸铵1%,KH_2PO_4 0.2%,MnSO_4 0.1%、ZnSO_4 0.2%,料水比1:2。在适宜的发酵条件下,经30℃发酵5 d,烘干后得到的复合酶制剂中,具有多种酶活性(以干基计)。其中酸性蛋白酶活力3 800 U/g,木聚糖酶活力12 00 U/g和纤维素酶活力18 U/g。     

Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs

The initial moisture content, cultivation time, inoculum size and concentration of basal medium were optimized in solid state fermentation (SSF) for the production of xylanase by an Aspergillus niger mutant using statistical experimental designs. The cultivation time and concentration of basal medium were the most important factors affecting xylanase activity. An inoculum size of 5 x 10(5) spores/g, initial moisture content of 65%, cultivation time of 5 days and 10 times concentration of basal medium containing 50 times concentration of corn steep liquor were optimum for xylanase production in SSF. Under the optimized conditions, the activity and productivity of xylanase obtained after 5 days of fermentation were 5,071 IU/g of rice straw and 14,790 IU l(-1) h(-1), respectively. The xylanase activity predicted by a polynomial model was 5,484 IU/g of rice straw.     

樟芝固态发酵产物抗氧化性分析

本文对樟芝谷物固态发酵产物抗氧化性能及活性成分进行了研究.在所选谷物中,樟芝青稞固态发酵产物的乙醇提取物总抗氧化性最好,较未发酵青稞提高了4.02倍.通过无水乙醇50℃水浴振荡提取80 min,其总抗氧化性达到了769.60 U/g.对其抗氧化性能分析发现,樟芝青稞固态发酵乙醇提取物为6 mg/L时,对DP P H自由基、羟基自由基以及超氧阴离子的去除率分别为91.9%、51.2%、61.3%,对铁离子的螯合能力为79.5%.相对于未发酵谷物,大米、小米、玉米以及青稞的樟芝发酵产物中总酚含量均有显著的提升,其中青稞乙醇提取和水提取物中总酚含量分别提高了2.36倍和4.23倍.通过HP LC分析可知,樟芝固态发酵产物含有丰富的活性化合物,包括马来酸衍生物(Antrodin)以及泛醌类衍生物(Antroquinonol),且各组分含量较为均衡;而樟芝液态发酵菌丝体乙醇提取物中主要活性成分为马来酸衍生物,不含有泛醌类化合物.     

Water and water activity in the solid state fermentation of cassava starch by Aspergillus niger

Summary During the solid state fermentation (SSF) of cassava starch by Aspergillus niger estimations were made of total water, consumed water and the residual water remaining in small quantities after 23 h. A theoretical calculation based on the Ross equation showed that the water activity (a _w) of the substrate decreased to 0.85 towards the end of the culture. Such low values were assumed to be inhibitory to growth. The a _w of the substrate was increased when sugarcane bagasse was used as a high water retention capacity support. Higher growth rates and substrate conversion to biomass were obtained with this system, confirming that water availability is a critical factor in the SSF of starch substrates.Abbreviations A, B Experimental constants - a _w Water activity - H₂O_c Consumed water - H₂O_R Residual water - H₂O_T Total water - IDW Initial dry weight - IMC Initial moisture content - OUR Oxygen uptake rate - S Substrate dry weight - Sc Substrate conversion: consumed substrate/initial substrate - S _H Amount of sugars hydrolysed - SSF Solid state fermentation - X Biomass dry weight - W ^* Amount of solids/g of water