Enzymatic Characterization of Pectinex XXL,a Pectinase Produced by Aspergillus niger,and Its Application in Fruit Juice Production

Pectinex XXL,a commercially prepared pectinase,was investigated for its potential application in the fruit juice industry.Polygalacturonic acid was used as the substrate for determining the enzymatic properties of Pectinex XXL using the DNS method.According to the results,the optimal pH for Pectinex XXL activity was 4.5,and the enzyme was stable in the pH range of 3.0~4.5.The optimal pH and pH stability range are consistent with those of some tropical and subtropical fruits.The optimal temperature for Pectinex XXL activity was 60℃,and the enzyme remained stable after one hour in a water bath set at 40℃.Additionally,the enzymatic activity was not inhibited in the presence of 1 mmol/L of Na^(+),Mg^(2+),Ba^(2+),Co^(2+),Zn^(2+),and Fe^(2+),whereas it was slightly inhibited in the presence of 2 mmol/L of K^(+)and Fe^(2+)and partially inhibited in the presence of 1 and 2 mmol/L of Ca^(2+)and Mn^(2+),demonstrating its good stability in acids and excellent thermal catalytic performance.Based on the above experimental results,depectinization experiments were performed on plantain and cherry tomato juices using different amounts of Pectinex XXL.After one hour reaction with 16 U/mL of the enzyme,the yields of the plantain and cherry tomato juices were substantially increased by 119.03%and 15.97%,respectively,while their light transmittance was remarkably enhanced by 37.65%and 12.35%,respectively.Furthermore,the enzyme reduced the viscosity of the plantain and cherry tomato juices by 88.29%and 29.50%,respectively.The juice production experiments confirmed that this enzyme can significantly improve the yield and light transmittance of plantain juice,while effectively reducing its viscosity.These findings indicate the potential of Pectinex XXL in the industrial production of plantain juice.

Pectinase Production by Aspergillus Niger P-6021 on Citrus Changshan-huyou Peel in Slurry-state Fermentation

The peel of Citrus changshan-huyou, coupled with wheat bran, could be utilized by Aspergillus niger P-6021 in slurry-state fermentation to produce pectinase with suitable enzyme composition for application in apple juice processing. The production of pectinase is improved by additional nitrogen source substances and mineral supplements. The ratio of carbon source substances to nitrogen source substances in the medium also has significant effect on the pectinase production by A. niger P-6021 in slurry-state fermentation. In the optimized medium composition, the maximal enzyme activity could reach 42 U.L^- 1 （polymethylgalacturonase）, 6.7 U.L^- 1 （polymethygalacturatesterase）, and 4.3 U.L^-1 （polymethylgalacturonate lyase）, respectively, after 3 days at 180 r.min^- 1 and 30℃. The crude pectinase shows significant effect to improve the yield and clarification of apple juice. Keywords Aspergillus niger, slurry-state fermentation, pectinase, Citrus changshan-huyou, apple juice

An Optimization Study of α-Amylase Production by Aspergillus niger ATCC 16404 Grown on Orange Waste Powder

In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of α-amylase by Aspergillus niger ATCC 16404. This statistical study consists of optimizing the factors that influence the production of α-amylase of A. niger ATCC 16404. Indeed, another statistical study has allowed the selection of 5 factors (pH, starch, yeast extract, “corn steep liquor”, CaCl2 and salts) affecting both the development of mould (biomass) and that of the enzyme production. The central composite design allows the determination of the optimum of these selected factors and a quadratic model explains the factor reaction. Thus, the “ridge analysis” method, has led to maximizing the experimental reaction. The results indicate that the production rate of α-amylase is maximized in the presence of starch at 8.97 g/l, yeast extract at 2.86 g/l, CaCl2 at 1.224 g/l, salts (composed of 25% FeSO4, 7H2O, 25% MnSO4 and 50% MgCl2, 6H2O): FeSO4, 7H2O, MnSO4 0.1518 g/l and MgCl2, 6H2O at 0.3036 g/l. As for the pH, it is maintained at the rate of 5.68.

响应面法优化Aspergillus niger X-6发酵生产菊粉酶工艺的研究

为提高Aspergillus niger X-6发酵生产菊粉酶的产率,运用Plackett-Burmen方法对麸皮、菊粉、蛋白胨、酵母膏添加量和发酵时间、温度、pH值、接种量8个影响因素进行考察,筛选出麸皮添加量、发酵时间和pH值为3个显著影响因素,然后采用Box-Behnken中心组合和响应面法对上述3个因素进行产酶条件的进一步优化,建立菊粉酶产率的二次多项式数学模型,并分析模型的有效性与因子间的交互作用。结果表明:黑曲霉发酵产菊粉酶的优化工艺参数为:麸皮添加量4.64%、发酵时间81.5h、pH6.0。在此条件下,产酶活力达20.42U/mL,与优化前相比提高了30.81%。

Inducing and Screening of High Producing Pectinase Aspergillus niger Strain

[Objective]The aim was to induce and screen the high producing pectinase Aspergillus niger Strain based on the original preservation strains.[Method]The original strain was induced by ultraviolet,and the highst enzyme activity and cultivated time were detected through the inspection of transparent circle and enzyme activity determination of flask fermentation.[Result] The enzyme activity of strain D1-4 achieved its highest after cultivated for 96 h in suitable conditions,which was 141.13 U/ml.[Conclusion] The induced strain D1-4 had the strong ability of producing pectinase.

生淀粉糖化酶产生菌Aspergillus niger(6#)的分离筛选及其产酶条件

从大曲中分离到了1株降解生淀粉能力较强的黑曲霉Aspergillusniger(6#).固体发酵酶活可达2461U/g,RDA值为21.47%;液体发酵酶活可达353U/mL,RDA值为20.30%.以6#菌为实验材料,进一步考察了氮源、碳源及pH对生淀粉糖化酶形成的影响.实验结果表明,无机氮源NaNO3较有机氮源蛋白胨更有利于生淀粉糖化酶的形成;pH是影响生淀粉糖化酶形成的重要因素,低pH会阻遏生淀粉糖化酶的形成;玉米粉和麦芽糖较其它碳源更有利于生淀粉糖化酶的形成.

Aspergillus niger FS6中β-1,3-1,4-葡聚糖酶cDNA片段的克隆及序列测定

通过 RT- PCR方法首次从真菌 Aspergillus niger FS6中扩增和克隆到 β- 1,3- 1,4 -葡聚糖酶 c DNA片段 .序列测定表明 :该片段全长 2 80 bp.经 Clustal W软件进行同源性分析后表明 ,该序列与枯草芽孢杆菌、水解淀粉芽孢杆菌、厌氧真菌、牛链球菌和热纤维梭菌中 β- 1,3- 1,4 -葡聚糖酶基因同源性很高 ,分别为 91% ,90 % ,70 % ,6 7% ,6 5 %

果胶酶高产菌株Aspergillus niger 6034的生理生化特性

诱变变异菌株黑曲霉Ａｓｐｅｒｇｉｌｕｓｎｉｇｅｒ６０３４，采用固体发酵法，最佳培养基组成为：麸皮、桔皮、硫酸铵和少量产酶促进剂。试验中某些碳源可促进产酶、某些氮源则抑制产酶。产酶的最佳条件为：培养温度２６℃，培养时间５１ｈ。以Ｓｉｇｍａ商品果胶为底物，该酶酶解作用的最佳条件为ｐＨ４２、温度５０℃，且酶热稳定性固态比液态好。６０℃保持１ｈ，固态酶保存８０％活力，而液态酶只有２０％的活力。

Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet

BACKGROUND The gluten-free diet(GFD)has limitations,and there is intense research in the development of adjuvant therapies.AIM To examine the effects of orally administered Aspergillus niger prolyl endopeptidase protease(AN-PEP)on inadvertent gluten exposure and symptom prevention in adult celiac disease(CeD)patients following their usual GFD.METHODS This was an exploratory,double-blind,randomized,placebo-controlled trial that enrolled CeD patients on a long-term GFD.After a 4-wk run-in period,patients were randomized to 4 wk of two AN-PEP capsules(GliadinX;AVI Research,LLC,United States)at each of three meals per day or placebo.Outcome endpoints were:(1)Average weekly stool gluten immunogenic peptides(GIP)between the run-in and end of treatments and between AN-PEP and placebo;(2)celiac symptom index(CSI);(3)CeD-specific serology;and(4)quality of life.Stool samples were collected for GIP testing by ELISA every Tuesday and Friday during run-ins and treatments.RESULTS Forty patients were randomized for the intention-to-treat analysis,and three were excluded from the per-protocol assessment.Overall,628/640(98.1%)stool samples were collected.GIP was undetectable(<0.08μg/g)in 65.6%of samples,and no differences between treatment arms were detected.Only 0.5%of samples had GIP concentrations sufficiently high(>0.32μg/g)to potentially cause mucosal damage.Median GIP concentration in the AN-PEP arm was 44.7%lower than in the run-in period.One-third of patients exhibiting GIP>0.08μg/g during run-in had lower or undetectable GIP after AN-PEP treatment.Compared with the run-in period,the proportion of symptomatic patients(CSI>38)in the AN-PEP arm was significantly lower(P<0.03).AN-PEP did not result in changes in specific serologies.CONCLUSION This exploratory study conducted in a real-life setting revealed high adherence to the GFD.The AN-PEP treatment did not significantly reduce the overall GIP stool concentration.However,given the observation of a significantly lower prevalence of patients with severe symptoms in the AN-PEP arm,further clinical research is warranted.

Aspergillus niger CGMCC No.6640全细胞生物转化制备蔗果低聚糖

蔗果低聚糖(FOS)作为一种益生元物质,由于其具有极好的生物和功能性质,受到人们极大的关注。具有独立知识产权的菌株Aspergillus niger CGMCC No.6640被发现能利用蔗糖制备FOS。为利用A.niger 6640的全细胞制备FOS,以蔗糖为底物,利用高效液相法对该菌株的全细胞生物转化参数进行了研究。利用色谱柱RezexRCM-Monosaccharife Cat的高效液相法能同时检测催化产物中蔗果四糖(或蔗果五糖)、蔗果三糖、蔗糖、葡萄糖和果糖的浓度,其保留时间分别为8.403 min,8.853min,9.705 min,11.473min和14.683 min。全细胞生物催化剂浓度和底物浓度对FOS产量有正面的影响。而CaCl2浓度对FOS含量有负面影响。当全细胞生物催化剂浓度,初始反应pH、反应温度、反应时间、蔗糖浓度分别为60 g/L、7.0、33℃、40 h和600 g/L的最佳条件下,FOS含量为314.60 g/L。总之,Aspergillus niger CGMCC No.6640全细胞生物催化剂能有效地制备FOS,为其工业化提供依据。

响应面法优化黑曲霉(Aspergillus niger)SW06产果胶酶发酵培养基条件

对果胶酶高产菌株黑曲霉(Aspergillus niger)SW06进行液体发酵工艺条件优化。首先采用单因素试验设计果胶酶发酵的最适温度和p H值,并筛选了最适的碳源、氮源和无机盐。在此基础上,采用中心组合设计进行回归分析及响应面分析,确定培养基的最佳组合。结果表明,果胶酶发酵的最适温度为28℃,最适p H值为5,最适碳源、氮源分别为果糖、蛋白胨。回归分析得到的优化发酵条件为:100 m L培养基中含果糖4.89 g、蛋白胨0.50 g、果胶0.60 g。在最优条件下发酵,果胶酶活最高可达4 175.65 U/m L。

Solid state fermentation of rapeseed cake with Aspergillus niger for degrading glucosinolates and upgrading nutritional value

Background： Rapeseed cake is a good source of protein for animal feed but its utilization is limited due to the presence of anti-nutritional substances, such as glucosinolates （GIs）, phytic acid, tannins etc. In the present study, a solid state fermentation （SSF） using Aspergillus niger was carried out with the purpose of degrading glucosinolates and improving the nutritional quality of rapeseed cake （RSC）. The effects of medium composition and incubation conditions on the GIs content in fermented rapeseed cake （FRSC） were investigated, and chemical composition and amino acid in vitro digestibility of RSC substrate fermented under optimal conditions were determined. Results： After 72 h of incubation at 34℃, a 76.89% decrease in GIs of RSC was obtained in solid medium containing 70% RSC, 30% wheat bran at optimal moisture content 60% （w/w）. Compared to unfermented RSC, trichloroacetic acid soluble protein （TCA-SP）, crude protein and ether extract contents of the FRSC were increased （P〈 0.05） 103.71, 23.02 and 23.54%, respectively. As expected, the contents of NDF and phytic acid declined （P〈 0.05） by 9.12 and 44.60%, respectively. Total amino acids （TAA） and essential amino acids （EAA） contents as well as AA in vitro digestibility of FRSC were improved significantly （P 〈 0.05）. Moreover, the enzyme activity of endoglucanase, xylanase, acid protease and phytase were increased （P 〈 0.05） during SSF. Conclusions： Our results indicate that the solid state fermentation offers an effective approach to improving the quality of proteins sources such as rapeseed cake.

Effect of agitation speed on the morphology of Aspergillus niger HFD5A-1 hyphae and its pectinase production in submerged fermentation

AIM: To investigate the impact of agitation speed on pectinase production and morphological changing of Aspergillus niger(A. niger) HFD5A-1 in submerged fermentation. METHODS: A. niger HFM5A-1 was isolated from a rotted pomelo. The inoculum preparation was performed by adding 5.0 m L of sterile distilled water containing 0.1% Tween 80 to a sporulated culture. Cultivation was carried out with inoculated 1 × 107 spores/m L suspension and incubated at 30 ℃ with different agitation speed for 6 d. The samples were withdrawn after 6 d cultivation time and were assayed for pectinase activity and fungal growth determination. The culture broth was filtered through filter paper(Whatman No. 1, London) to separate the fungal mycelium. The cell-free culture filtrate containing the crude enzyme was then assayed for pectinase activity. The biomass was dried at 80 ℃ until constant weight. The fungal cell dry weight was then expressed as g/L. The 6 d old fungal mycelia were harvested from various agitation speed, 0, 50, 100, 150, 200 and 250 rpm. The morphological changing of samples was then viewed under the light microscope and scanning electron microscope.RESULTS: In the present study, agitation speed was found to influence pectinase production in a batch cultivation system. However, higher agitation speeds than the optimal speed(150 rpm) reduced pectinase production which due to shear forces and also collision among the suspended fungal cells in the cultivation medium. Enzyme activity increased with the increasing of agitation speed up to 150 rpm, where it achieved its maximal pectinase activity of 1.559 U/m L. There were significant different(Duncan, P < 0.05) of the pectinase production with the agitation speed at static, 50, 100, 200 and 250 rpm. At the static condition, a well growth mycelial mat was observed on the surface of the cultivation medium and sporulation occurred all over the fungal mycelial mat. However with the increased in agitation speed, the mycelial mat turned slowly to become a single circular pellet. Thus, it was found that agitation speed affected the morphological characteristics of the fungal hyphae/mycelia of A. niger HFD5A-1 by altering their external as well as internal cell structures.CONCLUSION: Exposure to higher shear stress with an increasing agitation speed could result in lower biomass yields as well as pectinase production by A. niger HFD5A-1.

Optimization of Fermentation Conditions for Xylanase Production by Aspergillus niger NS-1

In this study, a xylanase-produeing Aspergillus niger strain, NS-1, was screened and isolated from agricultural and forestry wastes. Based on single-fac- tor experiments, the effects of different carbon sources, composite carbon sources, nitrogen sources, calcium carbonate concentrations, initial pH and surfactants on xylanase production by A. niger NS-1 were investigated. The results indicated that the most appropriate carbon source was corncobs ; the best composite carbon source was corncobs ＋ xylan, which was conducive to xylanase secretion; the most suitable nitrogen source was ammonium sulfate. Xylanase activity reached the highest in the medium added with 1.5% calcium carbonate and SDS as a surfactant with an initial pH of 5.0. This study provided the basis for the production of high-activity xylanase.

Aspergillus niger prolyl endopeptidase in celiac disease

We comment here on the article by Stefanolo et al entitled“Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet”,published in the World Journal of Gastroenterology.Celiac disease is a well-recognized systemic autoimmune disorder.In genetically susceptible people,the most evident damage is located in the small intestine,and is caused and worsened by the ingestion of gluten.For that reason,celiac patients adopt a gluten-free diet(GFD),but it has some limitations,and it does not prevent re-exposure to gluten.Research aims to develop adjuvant therapies,and one of the most studied alternatives is supplementation with Aspergillus niger prolyl endopeptidase protease(AN-PEP),which is able to degrade gluten in the stomach,reducing its concentration in the small intestine.The study found a high adherence to the GFD,but did not address AN-PEP as a gluten immunogenic peptide reducer,as it was only tested in patients following a GFD and not in gluten-exposing conditions.This study opens up new research perspectives in this area and shows that further study is needed to clarify the points that are still in doubt.

Pumpkin-like MoP-MoS_(2)@Aspergillus niger spore-derived N-doped carbon heterostructure for enhanced potassium storage

Biomass-derived carbon materials are widely applied in the energy storage and conversion fields due to their rich sources,low price and environmental friendliness.Herein,a unique pumpkin-like MoPMoS_(2)@Aspergillus niger spore-derived N-doped carbon(SNC)composite has been prepared via a simple hydrothermal and subsequent phosphorization process.Interestingly,the resulting MoP-MoS_(2)@SNC well inherits the pristine morphology of spore carbon,similar to the natural pumpkin,with hollow interiors and uneven protrusions on the surface.The special structure allows it to have sufficient space to fully contact the electrolyte and greatly reduces the ion transport distance.The theory calculations further demonstrate that the formed MoP-MoS_(2)heterostructure can enhance the adsorption of K ions and electronic couplings.With these unique advantages,the MoP-MoS_(2)@SNC anode for potassium storage shows a high reversible capability of 286.2 mAh g&(-1) at 100 mA g^(-1) after 100 cycles and superior rate performance.The enhanced electrochemical performance is mainly related to the unique pumpkin-like morphology of SNC and the construction of MoP-MoS_(2)heterostructure,as well as their perfect coupling.This study provides a feasible design idea for developing green,low-cost,and high-performance electrode materials for next-generation energy storage.

Improvement of xylanase production by Aspergillus niger XY-1 using response surface methodology for optimizing the medium composition

Objective: To study the optimal medium composition for xylanase production by Aspergillus niger XY-1 in solid-state fermentation (SSF). Methods: Statistical methodology including the Plackett-Burman design (PBD) and the central composite design (CCD) was employed to investigate the individual crucial component of the medium that significantly affected the enzyme yield. Results: Firstly, NaNO3, yeast extract, urea, Na2CO3, MgSO4, peptone and (NH4)2SO4 were screened as the significant factors positively affecting the xylanase production by PBD. Secondly, by valuating the nitrogen sources effect, urea was proved to be the most effective and economic nitrogen source for xylanase production and used for further optimization. Finally, the CCD and response surface methodology (RSM) were applied to determine the optimal concentration of each sig-nificant variable, which included urea, Na2CO3 and MgSO4. Subsequently a second-order polynomial was determined by multiple regression analysis. The optimum values of the critical components for maximum xylanase production were obtained as follows: x1 (urea)=0.163 (41.63 g/L), x2 (Na2CO3)=?1.68 (2.64 g/L), x3 (MgSO4)=1.338 (10.68 g/L) and the predicted xylanase value was 14374.6 U/g dry substrate. Using the optimized condition, xylanase production by Aspergillus niger XY-1 after 48 h fermentation reached 14637 U/g dry substrate with wheat bran in the shake flask. Conclusion: By using PBD and CCD, we obtained the optimal composition for xylanase production by Aspergillus niger XY-1 in SSF, and the results of no additional expensive medium and shortened fermentation time for higher xylanase production show the potential for industrial utilization.

Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity

The cellulase cocktail produced by marine Aspergillus niger exhibits a property of salt-tolerance,which is of great potential in cellulose degradation in high salt environment.In order to explain the mechanism on the salttolerance of the cellulase cocktail produced by marine A.niger,six cellulase components(AnCel6,AnCel7A,AnCel7B,AnEGL,AnBGL1 and AnBGL2)were obtained by directed expression.Studies on their enzymatic properties revealed that oneβ-glucosidase(AnBGL2)and one endoglucanase(AnEGL)exhibited an outstanding salttolerant property,and one cellobiohydrolase(AnCel7B)exhibited a certain salt-tolerant property.Subsequent study revealed that the salt-tolerant An EGL and AnCel7B endowed the cellulase cocktail with stronger salttolerant property,while the salt-tolerant An BGL2 had no positive effect.Moreover,after overexpression of AnCel6,AnCel7A,AnCel7B and AnEGL,the activity of cellulase cocktail increased by 80%,70%,63%and 68%,respectively.However,the activity of cellulase cocktail was not improved after overexpression of AnBGL1 and AnBGL2.After mixed-strain fermentation with cellobiohydrolase recombinants(cel6 a,cel7a and cel7b recombinants)and endoglucanase recombinant(egl recombinant),the the activity of cellulase cocktail increased by 114%,102%and91%,respectively.

Removal of cadmium and zinc ions from aqueous solution by living Aspergillus niger

The potential of living Aspergillus niger to remove cadmium and zinc from aqueous solution was investigated. Effects of pH, initial concentration, contact time, temperature and agitation rate on the biosorption of Cd(Ⅱ) and Zn(Ⅱ) ions were studied. The optimum adsorption pH value for Cd(Ⅱ) and Zn(Ⅱ) were 4.0 and 6.0. The best temperature and agitation rate were in the range of 25?30 ℃ and 120 r/min for all metal ions. Under the optimal conditions, the maximum uptake capacities of Cd(Ⅱ) and Zn(Ⅱ) ions are 15.50 mg/g and 23.70 mg/g at initial concentrations of 75 mg/L and 150 mg/L, respectively. Biosorption equilibrium is established within 24 h for cadmium and zinc ions. The adsorption data provide an excellent fit to Langmuir isotherm model. The results of the kinetic studies show that the rate of adsorption follows the pseudo-second order kinetics.

Removal process and mechanism of hexavalent chromium by adsorption-coupled reduction with marine-derived Aspergillus niger mycelial pellets

In order to remove hexavalent chromium(Cr(Ⅵ))from solutions efficiently,the mycelial pellets with a marine-derived fungus Aspergillus niger as a biosorbent were prepared.The effects of removal process parameters such as solution pH,initial Cr(Ⅵ)concentration and biomass concentration on Cr(Ⅵ)removal process were investigated.The results showed that Cr(Ⅵ)removal rate up to 100%could be achieved under optimized conditions,which indicated the excellent Cr(Ⅵ)removal performance of the Aspergillus niger pellets.As a more important point,the Cr(Ⅵ)removal mechanism was studied,and the results revealed that Cr(Ⅵ)removal was achieved in the adsorption-coupled reduction process.A little of Cr(Ⅵ)was reduced to less toxic trivalent chromium(Cr(Ⅲ))in solution,while some was absorbed on the surface of mycelial pellets.Then they may be reduced on the surface or transferred into cells and then be reduced.The marine-derived A.niger mycelial pellets show properties of easy preparation and separation and cost effectiveness,which are potential biosorbent and reductant in the treatment of trace chromate containing wastewater.