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.

Evaluation of Water Transfer Capacity of Poplar with Pectinase Treated under the Solar Interface Evaporation

Poplar wood,which was used as the absorption material for the solar-driven interfacial evaporation,was treated for 3 days,6 days and 9 days with the pectinase,and then was simulated for photothermal evaporation test at one standard solar radiation intensity(1 kW⋅m^(−2)).The effects of pectinase treatment on cell passage and water migration capacity of poplars were analyzed by the mercury intrusion porosimetry,the scanning electron microscope and fractal theory.It was found that the pit membrane and the ray parenchyma cells of poplar wood were degraded and destroyed after pectinase treatment.Compared with the untreated poplar wood,the evaporation rate of three sections of the specimen was changed.Especially the evaporation rate of radial and tangential direction was significantly increased.At the same time,based on the experimental data and fractal dimension deduction,fractal characteristics could be found in that the structure of poplars treated with pectinase.The porosity decreased with the increase of the fractal dimension in a certain range.It was shown that it is feasible to evaluate solar-driven water migration capacity by using fractal theory.

Adenosine cyclic phosphate with ultrasonic-assisted pectinase extraction alleviated allergic reactions in RBL-2H3 through inhibiting the influx of intracellular Ca^(2+)

Jujube contains abundant cyclic adenosine monophosphate(cAMP)and the ultrasonic-assisted pectinase extraction(UAPE)conditions for obtaining the maximum cAMP yield from jujube were optimized.Orthogonal array design was applied to evaluate the effects of 4 variables by UAPE on cAMP yield.The results showed that the optimal cAMP yield(783.0μg/g)was derived at ratio of liquid to solid 5 mL/g,ratio of pectinase to raw material 1.5%,time 60 min and temperature 40℃.Moreover,the effect of cAMP on the anti-allergic function of action induced by immunoglobulin E(IgE)and its meschanism was investigated through establishing the sensitized cell model in rat basophilic leukemia(RBL-2 H3)cells using dinitrophenylated(DNP)-bovine serum albumin(BSA)-IgE.The results showed that cAMP interfered with sensitized cells,effectively inhibited the occurrence of basophil degranulation in dose dependence,and significantly reduced the activity ofβ-hexosamindase(β-hex),at the optimal concentration of 50μg/mL.The level of anti-inflammatory factor interleukin-10(IL-10)was promoted and the content of pro-inflammatory factor tumor necrosis factor-α(TNF-α)was suppressed by cAMP.In addition,influx of intracellular Ca^(2+) was repressed effectively.Our results demonstrate that jujube cAMP regulated the cytokine balance in the allergy pathway through blocking the influx of extracellular Ca^(2+),with the prevention of allergy symptoms.

Study on Improving the Internal Quality of Tobacco Stems by Using Pectinase

[Objective] This study aimed to reduce the content of cell wall materials in tobacco stems and improve their internal quality. [Method] Pectinase was used to decompose cell wall materials in tobacco stems, followed by determination of the contents of four cell wall materials, six routine chemical components, as well as aroma constituents. [Result] Pectinase could effectively reduce the contents of cell wall materials in tobacco stems, with the largest decrease of 6.84%; after pectinase treatment,the content of reducing sugar in tobacco stems increased obviously, and the contents of total sugar, potassium ion, chloride ion and total nitrogen increased to varying degrees, of which the contents of potassium ion and reducing sugar displayed upward trends with the increase of pectinase concentration. Pectinase treatment significantly increased the contents of Maillard reaction products, with the most increase of 67.2%;the contents of carotenoid degradation products, phenylalanine degradation products and neophytadiene all increased to varying extents, and the contents of both Maillard reaction products and phenylalanine degradation products revealed ascending trends with the increase of pectinase concentration. [Conclusion] Pectinase treatment can effectively decompose cell wall materials in tobacco stems, improve routine chemical constituents, and increase the contents of aroma constituents.

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.

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.

Research Progress of Pectinase

Pectinase is a group of enzymes that can decompose complex pectin into small molecules such as galacturonic acid. It is mainly produced by microorganisms by liquid and solid fermentation and immobilized cell method. Its representative strain is Aspergillus niger,and yeast and actinomycete are also high-yield strains for screening pectinase. Pectinase can be used in many fields of life and production,such as clarification of fruit juice and wine,pulping of paper making industry,degumming of textile,extracting of traditional Chinese medicine components,animal feed and other industries,which is economic,green,and efficient and pollution-free. Moreover,pectinase is one of the four major enzymes in the world,and its practical application has very important significance,and the prospect of pectinase application is also important.

Effects of Neodymium on Growth, Pectinase Activity and Mycelium Permeability of Fusarium oxysporum

The diameter of the colony of Fusarium oxysporum in solid medium, and the mycelium growth, pectinase activity, and mycelium permeability of Fusarium oxysporum in liquid medium under varying concentrations of Nd^3＋ （0, 2, 4, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, and 400 mg·L^-1） were measured. The results indicated that the growth of Fusarium oxysporum was stimulated in solid medium when the concentration of Nd^3＋ ranges from 2 to 180 mg·L^-1, whereas it was inhibited when Nd^3 ＋ concentration was greater than 200 mg· L^-1. The colonies were fewer and smaller when Nd^3 ＋ was used in the solid medium. The growth of Fusarium oxysporum was inhibited in liquid medium when Nd^3＋ was used. The inhibition rate showed by the dry weight of mycelium ranged from 4.83% to 52.18% and inereased with Nd^3 ＋ concentration. The pectinase activity decreased compared with that of controls. When the concentration of Nd^3 ＋ was 10 and 400 mg· L^- 1, the pectinase activity decreased by 95 % at both concentrations. Mycelium cell membrane permeability increased when Nd^3 ＋ concentrations ranged from 10 to 400 mg· L^-1 but decreased when Nd^3＋ concentration was 2 mg· L^-1.

Optimization of Wickerhamomyces anomalus Fermentation Conditions for Pectinase Production Based on Wet Processing of Arabica Coffee in Yunnan Province

In order to improve the pectin-degrading efficiency in wet processing of Arabica coffee in Yunnan, Box-Behnken design and single factor experiment were used to optimize the fermentation conditions of five pectinolytic Wickerhamomyces anomalus strains from the fermentation broth of Arabica coffee in Baoshan, Yunnan during wet processing with pectase activity as an indicator. The results showed that the five strains all synthesized pectin lyase(PL), polygalacturonase(PG), and pectin methylesterase(PM).Among them, strain CAP5 had strong ability to produce PG and PL,while strain CAP4 secreted a large amount of PM. Under optimized conditions, the activity of PG, PL, and PM of the five strains came in at 250.17~411.20 U/mL, 12.98~16.55 U/mL, and 208.52~322.83 U/mL,respectively. The four factors of nitrogen source concentration,fermentation time, Mn2+ concentration, and pH value were optimized and the optimal pectinase-producing fermentation conditions for five strains were as follows: peptone 2.2 g/L, fermentation time 30 h, Mn2+ 1.5 mmol/L, and pH 4.3. After fermentation under the optimized conditions, the maximum PG activity of CAP5 amounted to 411.20 U/mL, 114.03% higher than that before optimization.Meanwhile, the PG activity of strains CAP3, CAP4, CAP8, and CAP10 increased by 86.74%, 114.55%, 65.79%, and 66.07%,respectively, and the activity of PL and PM of the five strains rose 150.35%~218.56% and 341.07%~418.52%, respectively. These findings suggested that W. anomalus strains could be used as coffee starter and had great potential for the lysis of pectin.

Isolation of Pectinase Producing Bacteria from the Rhizosphere of <i>Andrographis paniculata</i>Nees and 16S rRNA Gene Sequence Comparison of Some Potential Strains

Pectinases, the enzymes which break down pectic substances, have a wide range of applications in food, agriculture and environmental sectors. In the present study, attempts were made to isolate highly efficient pectinase producer from the rhizosphere of a medicinal plant, Andrographis paniculata Nees, known as the “King of bitters”. The total heterotrophic bacterial count of the rhizosphere soil of A. paniculata Nees ranged from 1.53 × 109 to 2.52 × 109 cfu/g. A total of 65 bacterial colonies were randomly selected from the nutrient agar plates, purified and assessed for pectinase activity. Out of the 65 isolates, 62 (95.38%) showed varying degree of pectinase activity in plate assay using pectin as a sole source of carbon. Among the pectinase producing strains, JBST36 showed best pectinase activity which is followed by the JBST22 and JBST27. Morphological characterization, biochemical tests and 16S rRNA gene sequencing were performed to identify the three most potential strains. Based on the morphological, biochemical and molecular data, JBST22 was identified as Bacillus flexus and the other two were identified as Bacillus subtilis. Furthermore, nucleotide sequences of the 16S rRNA gene of these 3 strains were compared and a phylogenetic tree was constructed. The study reveals that there are at least 66 base differences in the 16S rRNA gene sequences of B. flexus JBST22 and the B. subtilis JBST36.

Pectinases yeast production using grape skin as carbon source

Pectinases are used in Enology for some different utilities. Enzymatic preparations from moulds are a mixed of different enzymes with strong and unspe-cific activities. Some Saccharomyces cerevisiae pro-duce pectinases which can be used instead of com-mercial preparations. The objectives of this work were to study the enzyme secretion by one Saccharo-myces cerevisiae (CECT 11783) for growing on grape skin (industry oenological by-product) as carbon source. Preliminary experiments showed that the strain produced pectinases for growing on grape skin without any other carbon source. Statistical treat-ment (factorial design 25) was applied to evaluate the influences of related factors (agitation, temperature, presence of peptone and detergent in the medium and time of growth) Variables with the most significant interactions for pectinase production were agitation and nitrogen source concentration. Response surface methodology showed that a first order model was not adequate for results. Nevertheless, the built of a sec-ond order model offered a polynomial equation which surface predicted a maximum of activity (52.68 enzymatic units) for specific values of the studied variables (147.8 rpm of agitation and 15.9 g of pep-tone/ L culture medium).

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

Performance of a Pectinase from Bacillus Subtilis WSHB04-02 Used in Bioscouring of Cotton Fabrics

A pectinase produced by Bacillus subtilis WSHB04-02 isolated from soil with lyase activity operating at alkaline pH was studied. The Michaelis-Menten kinetic parameters of this newly isolated pectinase on different substrates, such as citrus pectin and polygalacturonic acid (PGA), were determined, and pectin proved to be the most suitable substrate. The effects of temperature and pH on pectinase activity and stability were also investigated. The optimal temperature for pectinase was 55℃ with a stable range of 45℃-55℃. In general, pectinase was pH insensitive and the stable pH ranged from 8.6 to 10.0. Ultimately the bioscouring effects of cotton fabrics using this pectinase were evaluated and some promising results were obtained.

The Role of Cellulase and Pectinase in Apricot Canker Caused by Hendersonula toruloidea and Phiaoacremonium aleophillium

Significant increasing in cellulase and pectinase activity were observed in response to infection with Hendersonula toruloidea and Phiaoacremonium aleophillium in apricot trees comparing with uninfected trees. Pectinase highest activity was recorded at the first week after inoculation with H. toruloidea （1.76 unit/g f.w.）, while cellulase highest activity was recorded after two weeks of inoculation with P. aleophillium （6.49 unit/g f.w.）. Peroxidase and polypolyphenol oxidase activity were significantly increased after inoculation with H. toruloidea and P. aleophillium, Peroxidase highest activity was recorded after 48 h of inoculation with H. toruloidea （1.77 unit/g f.w.）, while polypolyphenol oxidase highest activity was recorded after two weeks of inoculation with P. aleophillium （3.33 unit/g f.w.）. The result also showed that total phenol contents was significantly increased as a result to inoculation with H. toruloidea and P. aleophillium, highest total phenol contents was recorded after 48 h of inoculation with H. toruloidea （ 1.61 mg/g f.w. ）.

Bio-reduction of Sulphur Dyes with Alkaline Pectinase

Sulphur dyes are invariably applied on cotton to produce deep shades at cheaper cost possessing all-round fastness properties except against chlorine. Being water insoluble, these dyes are reduced and solubilised with sodium sulphide at boil to develop affinity for cotton. Application of sulphide has generated global debate because of its eco-unfriendly technology of dyeing. In this work, attempts were made to substitute sodium sulphide with alkaline pectinase. Obtained results suggested the ability of the latter to cause effective reduction and solubilisation of sulphur dyes. Stability of reduction baths as well as colour fastness was also reported to be in line with those obtained using sodium sulphide.

Preparation and properties of pectinase immobilized on chitosan support

Pectinase was immobilized onto chitosan support itsing glutaraldehyde as a coupling agent to obtain high activity and stability of pectinase.x A maximum residual activity of 55% was obtained with 0.4 mg proteirdg chitosan （w/w）, 5% （v/v） g/utara/dehyde, and 4℃ for the crosslinking reaction. The optimal pH and temperature for pectinase activity changed from 3.0 and 40℃ to 3.5 and 50℃, respectively, after immobilization The immobilized enzyme exhibited higher stability under varying conditions of pH and temperature and better reusability than the free enzyme.

Lucrative pectinase production by novel strain Pseudozyma sp. SPJ with statistical optimization techniques using agro-industrial residues

Production of high titers of an alkaline, extracellular and thermo-tolerant pectinase by a newly isolated yeast Pseudozyma sp. SPJ was carried out under solid state fermentation. Citrus peel, the inexpensive agro-industrial residue used as substrate, was experienced to be unsurpassed. Response surface methodology was conducted to optimize the culture conditions for Pseudozyma sp. SPJ for hyper production of pectinase. Plackett Burman design was applied to identify the most effective culture variables. Out of nine variables studied, incubation time, moisture content and ammonium sulfate were detected as most important. A full factorial Central Composite Design was used to optimize the levels of these variables, which resulted in 17-fold increase （71.19 IU/g to 1215.66 IU/g dry substrate） in the enzyme yield. The results of analysis of variance and multiple regression analysis implies that the effect of incubation time （p 〈 0.000） and moisture content （p 〈 0.018） is more than ammonium sulfate. And also the interaction of moisture content with ammonium sulfate （p 〈 0.002） is more significant.

Research on Production Conditions of Highgrade Wine from Northern Blueberry

In order to develop the best brewing condition for high-grade fruit wine from northern blueberry, on the basis of reviewing literatures about blueberry wine manufacturing process, this study put forward a new technological process, including raw materials-juicing-addition of auxiliary materials including pectinase-primary fer- mentation with yeast-post-fermentation-hot and cold treatment-sterilization-packaging- finished product, and sensory indexes, physicochemical indexes and hygienic index- es of the product were inspected according to corresponding national standards and industry standards. The results showed that for northern blueberry pulp, the optimal addition amount of yeast was 1.1 g/L, the fermentation temperature was 22 ℃, and the addition amounts of pectinase and sulfurous acid were 0.3 ml/kg and 100 ppm, respectively; the alcohol degree of the finished product was adjusted to 15.6°; and alternated cold and heat treatment used instead of conventional clarifying agent for removing colloid-like impurities resulted in the brewed product with good wine fra- grance, taste and color.

Technological Study on Instant Date Powder in Natural Ziziphus jujuba through Vacuum Freeze-drying

[Objective] The aim was to get optimized conditions for date powder with good color and taste.[Method] The pectinase enzymolysis and vacuum freeze-drying technology were used in the extract process of date powder.[Result] The production boasts of superior quality were with rich fragrance and uniform particle under certain process conditions.The optimized conditions for pectinase enzymolysis were:the amount of pectinase reached to 0.1% of that of date syrup,enzymolysis temperature and time were 50 ℃ and 50 min respectively,the pH value was 4.0.The optimized conditions for vaccum freeze-drying were:10% altodextrin,three times volumn of water with the thickness of 7 mm.[Conclusion] The optimized conditions were obtained in this study to produce instant date powder from Ziziphus jujuba.

In vitro Evaluation of Feed Enzyme Compound Produced by Aspergillus sulphureus in Solid-state Fermentation

ABSTRACT： Three trials were conducted to analyze a multi-enzyme compound produced by Aspergillus sulphureus in solid-state fermentation （SSF） as a po- tential feed additive. The results of the first trial showed that there were at least 5 non-starch polysac- charide enzymes： xylanase, 13-ghicanase, pectinase, mannase and carboxy methyl cellulase （CMCase） contained in the compound. Xylanase and fl-glucanase showed good activities at pH 2.5-7.0, which were in the range of 649-1046 U/g and 444-648 U/g, respec- tively. Pectinase showed good activity in acidic solu- tion （pH 2.5-3.0）,which ranged from 195 to 917 U/g. Mannase showed high activity of 235-298 U/g at pH 3.5-4.5 and the activity of CMCase was relatively constant at pH 2.5-7.0, which was in the range of 38.2-78.6 U/g. The second trial was aimed to test the stability of the enzymes in gastric liquor （pH 2.6） of finishing pigs and Na2 HPO4-gastric liquor （ pH 5.5 ）.After 6 h incubation at 40℃ in gastric liquor,the re- tained activity of xylanase, 13-glucanase, pectinase, mannase and CMCase was 26.3% ,65.0% ,71.0%, 74.8% and 85.6%, respectively. While after 6 h in- cubation at 40℃ in Na2I-IPO4-gastric liquor, the re- tained activity of xylanase, [3-glucanase, pectinase, mannase and CMCase was 87.9% ,91.1% ,92.3%, 95.0%, and 97.5%, respectively. The third trial was carried out in a jejunum liquor （ pH 5.8,200 mL）, which contained 0.2 g of the multi-enzyme compound and 10 g of soybean hull or wheat bran, respectively. After 8 h incubation at 40℃, 18.7% of soybean hull and 20.1% of wheat bran could be degraded to solu- ble saccharide, respectively. Compared with the tradi- tional methods for feed enzyme testing which involve feeding animals for 1-3 months, enzyme assay in this way was relatively convenient.