Biosynthesis of pectinolytic enzymes by Aspergillus on apple pulp

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The aim of this work was to develop a low cost process for apple pulp utilization. The apple pulp combined with corn flour and simple mineral salts by submerged production of pectinolytic enzymes by the fungus Aspergillus. Different concentration on
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    BIOSYNTHESIS OF PECTINOLYTIC ENZYMES BY ASPERGILLUSON APPLE PULP   Kiro Mojsov 1  , Meri Petreska 2  , Jugoslav Ziberoski 3   Abstract: The aim of this work was to develop a low cost process for apple pulputilization. The apple pulp combined with corn flour and simple mineral salts bysubmerged production of pectinolytic enzymes by the fungus  Aspergillus . Differentconcentration on apple pulp and different pH initial on the bases were studied, and allother process parameters were same. Results of different concentration on apple pulpgived maximal endo- PG with 1% apple pulp, during from 96 h, and the growth of themicroorganism showed maximum dry weight on initial pH on bases- 4, during from 48h. From results can be see that the apple pulp can be used as inexpensive base for industrial production on pectinolytic enzymes by Aspergillus . Key words : apple pulp, biosynthesis, pectinolytic enzymes,  Aspergillus Introduction The pressed apple pulp is actual waste from food and agriculture industry and because the aim of this work was to develop a low cost process for apple pulputilization. Accordingly this production of pectinolytic enzymes based on submergestate bioprocessing by Aspergillus niger of this actual waste, was developed.  Aspergillus niger  has been recognized as an industrially important fungus since theU.S. Food and Drug Administration (FDA) approved the GRAS status for manysubstances which were produced with it. These fungi produce several commercialenzymes, amongst which is also the pectinasis, its commercial value being about 20%of a USD billion annual sales of all industrial enzymes (Kashyap et al., 2001).The utilization of microbial enzymes has found broad technological application indifferent industrial processes. Fungal pectolytic enzymes are used in the food industryfor the production of fruit juices, olive oil and wine to increase yields and in theclarification of juices and wines (Fogarty and Kelly, 1983; Pilknik and Voragen, 1993).These enzymes are usually produced on solid or submerged fermentation(Schmidth et al., 1995). Submerged fermentations generally produce smaller quantities 1 Technological- Technical Faculty,University “Goce Delcev” Stip, Republic of Macedonia ( )  2 Ministry of Agriculture, Forestry and Water Economy, Jurij Gagarin 15,1000 Skopje, Republic of Macedonia ( 3 Faculty of Agricultural Sciences and Food,“Ss Cyril and Methodius University”, Skopje, Republic of Macedonia (    of secretory enzymes and solid fermentations are not susceptible to automation. For theindustrial production of pectinolytic enzymes it is important to improve thefermentation conditions, for better production of extracellular enzymes on inexpensivecarbon sources such as apple pomace, citric peels, pectin or other agricultural wasteswhich contain appreciable quantities of pectin (Alkorta et al., 1998; Larios et al., 1989).The most authors describe the use of an optimized medium composition to increase theenzyme content (Berovič   and Ostroveršnik  , 1997), Material and methods2.1 Micro-organism The microorganism used in this work was the fungus  Aspergillus niger MK-10 ,which was isolated from soil as a highly active producer of pectinolytic enzymes andwas maintained on slant agar according to Czapek with 2% pectin. Spores from 3 daysold agar slants were collected by adding sterile distilled water to each slant. The sporessuspension was adjusted to a final concentration in the culture medium of 6 . 10 6 sporesml-1. 2.2 Media and fermentation procedure The medium for   Aspergillus niger MK-10 was prepared by adding differentconcentration of apple pulp (1%; 2%; and 3%, w/v) and different pH initial (2-8) to the basic medium of the following composition : corn flour- 0,5% (v/v); (NH 4 ) 2 HPO 4 -0,7% (v/v); KH 2 PO 4 - 0,1% (v/v); MgSO 4 .7H 2 O- 0,05% (v/v); and KCl- 0,05% (v/v);.The base was previously sterilized by autoclaving at 121 o C for 30 min. The pressedapple pulp first are dry and after are mill to the ground apple pulp particles with thediameter under 0,315 mm.. The refuse apple pulp had the following content: moisture 10÷12 %, ashes 3÷5 %, proteins 6÷6,2 %, and pectin 9÷10 %.  The growth of the microorganism and synthesis of pectinolytic enzymes were performed in 500 ml flasks (100 ml base) with rotational shaking (200 rev min-1) on arotational laboratory shaker, at 30 o C within 120 h. 2.3 Enzyme assay Endo-pectinolytic activity (endo-PG), based on change in the viscosity of thereaction mixture (0.35% pectin solution, buffered at pH 4.5 in 0.1 mol l-1 citrate) at30 o C, was determined using Ostwald viscometer. The degree of degraded pectin (A)under known amount of filtrate(enzyme) was calculated with the formula: A= 100 ∙   (Ts-Tt)/(Ts-Tw) where Ts is the flow time of the substrate control. Tt is the flow time of thetest and Tw is the flow time of water. 1 U was defined as the amount of enzyme whichcatalyses hydrolyse of 1 g pectin per 1 h at 40 o C. 1 IU is the amount of enzyme which catalysees hydrolyse of 1 µmol pectin per 1 min at 40 o C. 2.4 Biomass production measurements    Biomass production was measured as dry weight (DW). After filtering, the retainedcell mass was dried at 100 o C to constant weight. Results and discussion Results of different concentration on apple pulp (Table 1), gived maximal endo- PG(480,5 U l -1 ) with 1% apple pulp, compared with endo-PG (398,4 U l -1 ) with 2% andendo-PG (190,5 U l -1 ) with 3% apple pulp during of cultivation 96 h.Results of different initial  pH  on bases (Table 2), gived maximal endo-pectinolyticactivity (endo-PG) (496,4 U l -1 ) on  pH= 4 compared with endo-PG (452,3 U l -1 ) on  pH= 3, endo-PG (420,2 U l -1 ) on  pH= 5 and endo-PG (353,9 U l -1 ) on  pH= 6.Tabela 1 Uticaj razlicitih concentracija jabukove pulpe na podloge na biosintezu pektinolitickih enzima Table 1 The influence of different concentration apple pulp on bases on biosynthesis of  pectinolytic enzymes. Koncentraciju jabukove pulpe,%(w/v) Concentration onapple pulp,%(w/v) Vreme kultivacije(h)  Duration of cultivation(h)  pH krajno   pH final   Endo-PG (U l -1 )  Endo-PG (U l  -1  ) Biomasa (g l -1 )   Biomass ( g l  -1  ) 1 0 4,0 001 24 3,25 53,524,51 48 2,58 289,432,31 72 2,64 455,221,31 96 2,96 480,515,61 120 3,10 398,211,92 0 4,0 0 02 24 3,24 44,215,82 48 2,43 232,4 24,52 72 2,46 304,713,22 96 2,83 398,412,42 120 2,84 334,8 11,23 0 4,0 003 24 3,31 23,3 12,93 48 2,49 54,621,23 72 2,43 98,212,23 96 2,57 190,5 11,33 120 2,66 176,410,9  Note: The values are average from 3 replicates    The growth of the microorganism (dry weight) (Table 1) showed maximum dryweight (32,3 g l -1 ) on 1% concentration of apple pulp and initial  pH  on bases- 4, duringof cultivation 48 h compared with maximum dry weight (24,5 g l -1 ) on 2%, and (21,2 gl -1 ) on 3% concentration of apple pulp. The growth of the microorganism (dry weight)(Table 2) showed maximum dry weight (31,2 g l -1 ) on  pH  -4, (23,5 g l -1 ) on pH  -3, (25,4g l -1 ) on pH  -5, (18,9 g l -1 ) on pH  -6.The results gived that the concentration of apple pulp and  pH  on bases had a pronounced effect on the biosynthesis of pectinolytic enzymes and growt by  Aspergillusniger MK-10. From results it became clear that on the biosynthesis of pectinoluticenzymes optimal concentration on apple pulp is 1% (w/v) and optimal  pH  =4 during of cultivation 96 h, and for the growth on microorganism optimal concentration on apple pulp is 1% (w/v) and pH=4 during of cultivation 48 h.The results presented here as optimal concentration on apple pulp and  pH  on themedium with a inexpensive refuse apple pulp as a carbon source for maximal enzyme production by  Aspergillus niger MK-10 will be of commercial importance for usingrefuse apple pulp.Tabela 2 Uticaj pocetne pH podloge na biosintezu pektinolitickih enzima Table 2 The influence of initial pH of the medium on biosynthesis of pectinolyticenzymes Pocetno pH  Inital pH    Endo-PG (U l -1 )  Endo-PG (U l  -1  )   Biomasa (g l -1 )   Biomass ( g l  -1  ) 2 293,8 12,9 3 452,3 23,5 4 496,4 31,2 5 420,2 25,4 6 353,9 18,9 7 289,3 14,3 8 210,8 10,5  Note: The values are average from 3 replicates Conclusion Microbial enzymes are routinely used in many environmentally friendly andeconomic industrial sectors. Pectinolytic enzymes play an important role in food processing industries and alcoholic beverage industries. The production of foodenzymes related to the degradation of different substrates. These enzymes degrade pectin and reduce the viscosity of the solution so that it can be handled easily. In this paper has been studied the effect of different concentration apple pulp on bases andinitial pH on biosynthesis of pectinolytic enzymes in laboratories trials, and all other  process parameters such as the type and concentration of nitrogen source, the growth    temperature, the aeration rate and the level of agitation were same and should beinvestigated in further research.The best carbon source on base for better production of pectinolytic enzymes wasthe pressed apple pulp with optimal concentration of 10.0 g l -1 . As this residue isrenewable and in an abundant supply, it represent a potential low cost material for microbial enzyme production. The results presented here will be of commercialimportance for using pressed apple pulp as a carbon source for production of  pectinolytic enzymes in submerged fermentation. References Alkorta, I., Garbisu, C., Liama, M.J, Serra, J.L. (1998), Industrial applications of pecticenzymes: a review . Process Biochemistry, Vol. 33, 21-28. Berovič, M. and Ostroveršnik  H. (1997), Production of   Aspergillus niger   pectolyticenzymes by solid state bioprocessing of apple pomace. Journal of Biotechnology,Vol. 53, 47-53,.Fogarty, W.M. and Kelly, C.T. (1983), Pectic enzymes.  In Microbial Enzymes and  Biotechnology . Fogarty,W.M. (ed.) 131-182. London : Elsevier Applied Science.Kashyap, R., Vohra, K., Chopra, S., Tewari, R., (2001), Applications of pectinases inthe commercial sector: a review. Bioresource Technology, Vol. 77, 215-227.Larios, G. , García, J.M. and Huítrón, C. (1989). Endo-polygalacturonase productionfrom untreated lemon peel by  Aspergillus sp. CH-Y-1043. Biotechnology Letters,   Vol. 11, 729-734.Pilknik, W. and Voragen, G.J.A. (1993). Pectic enzymes in fruit and vegetable juicemanufacture.  In Enzyme and Food Processing ed. Nagodawithana , T. and Reed, G. p. 363. London: Academic Press, 1993.Schmidth, O., Angermann, H., Frommhold-Treu, I., Hoppe, K.. (1995). Experimentaland theorical investigation of submerged fermentation and synthesis of  pectinolytic enzymes by  Aspergillus niger  . Applied Microbiology andBiotechnology, Vol. 43 , 424-430.  
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