*Corresponding Author: Farmohamadi Seifollah, University: Academic Center for Education, Culture and Research (ACECR) sharif university of Technology branch, 1.Address: P.O. Box 13145-1387 , Tehran, Iran ,Phone number:+989123431457 , telfax : +982166035177 ,Email Address: [email protected]
Pectin Esterase Production by Aspergillus Niger: Optimization of Fermentation Condition
Farmohamadi Seifollah1,2, Ghanbarov Khodaverdi2
1University: Academic Center for Education, Culture and Research (ACECR) Sharif University of
Technology Branch, 2University: Department of Microbiology, Baku State University, Azerbaijan
ABSTRACT Yeast, bacteria and a great deal of filamentous fungi are the main sources of pectinases enzymes, and the most relevant fungi is Aspergillus .pectin exists in the primary cell wall of the most plants, pectinases is an important enzyme which could proliferate hydrolysis pectin molecules, and also could increase the juice yield and its clarity ; due to fruit juices clarification and extraction of essential oils, pectinases are so useful in food industry. A two-step approach was applied for optimizing and studying the effect of seven factors including concentration of ammonium sulphate ,potassium dihydrogen phosphate and date pomace , PH ,total spores ,agitation speed and fermentation time in which all have been applied from date pomace through Aspergillus niger for pectin esterase enzyme production .According to the result of the first step ,total factors had positive effect on pectin esterase production in studied ranges .in the second step ,three variables such as Ammonium sulphate concentration (0.182-0.45 mg/ml) , PH (4.8-6.12) , fermentation time (48-90 h) were studied as effective factors on enzymes production ,applying RSM using central composite design (CCD).post optimization ,maximum enzyme activity (3.82u/ml) obtained in fermentation time ( 79.2 hour), Ammonium sulphate concentration(0.44 gr /ml), PH(6.18),total spore (10 푔푟/푚푙),date pomace (%6), potassium dihydrogen phosphate concentration (%0.8) and agitation speed (250rpm). KEYWORDS: Pectin esterase, Aspergillus niger, Date pomace, RSM method.
INTRODUCTION
Pectinases enzymes derived from Aspergillus niger ,are useful in the food industry ,as they classified as GRAS (generally regarded as safe), these enzymes include several types of polygalacturonases (PGs),pectin lyases (PLs) and pectin esterase (Warren et al ,2002).a great deal of agriculture wastes used as substrate sources for the Pectinases production which these wastes are as wheat bran ,sugar beet pulp ,citrus waste ,pumpkin oil cake ,grape pomace and orange peels ,etc .several Funguses like Fusarium moniliforme ,Botrytis cinerea, Rhizoctonia solani ,Aspergillus sp and etc ,can produce Pectinases enzymes in which Aspergillus niger is more useful in comparison with others . Pectin esterase is an important Pectinases enzyme that catalyses the hydrolysis of methylated carboxylic ester group in pectin into pectic acid and methanol, and it will produce through many plants and microorganism (Kuamar et al,2006).the aim of present research would be observation of several factors in pectin esterase enzyme activity in a date pomace substrate ,and also optimizing pectin esterase activity in the submerged fermentation production of this enzyme through Aspergillus niger. observed factors are included of fermentation time ,aeration rate , PH, Ammonium sulphate concentration, potassium dihydrogen phosphate, date pomace and total spore .according to obtained results in second step , PH in the range of 4.82 -6.12 had no effect on pectin esterase activity but it’s interaction with Ammonium sulphate affected enzyme production .also , increase of fermentation time until 75 hours caused the increase of pectin esterase activity ,and post this time the enzyme activity would decrease . Main body Substrate chemical composition
Date is a common agriculture production in Iran, and it could be used as a cheap substrate . So date pomace could be a useful substrate, thus its dried powder in 70°C could be used in culture medium. The chemical composition of date pomace were analyzed and determined per Iranian national standard.
Table 1- Substrate chemical composition
Ash(%) lipid(%) Fiber (%) Protein (%) Dry material 3.6+-0.38 1.5+-0.13 24.87+-0.37 7.9+-0.11 96.7+-0.21
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The culture medium composition (date pomace powder, Ammonium sulphate and potassium dihydrogen phosphate) scaled in a ml within fixed PH and autoclaved for 15 minute in 121°C and 15 psi pressure .in order to determine the effective factors on enzyme activity in the first step , several compositions of these factors as an experimental design were prepared for culture medium .according to table 2 , the factors including PH(3.5-5),total spore amount (10 − 10 푠푝표푟푒푠/푚푙), 퐾퐻 푃푂 (40-80 g/ml),(푁퐻 ) S푂 (10-30 g/ml),aeration rate (150-250rpm), date pomace (400-800 g/ml),and fermentation time (24-48).in the second step ,significant factors were selected and new compositions of these factors were prepared for culture medium as anew experimental design. (table 3) Enzyme activity assay
Two milliliters of culture medium filtrate were added to 10 ml of %0.5 pectin solution and NaCl molar. PH was adjusted in 4.5 through NaoH 0.1 molar ,then the mixture incubated in 30 °C for 60 minute .enzyme activity was determined per unbounded Carboxyl ‘s measurement through NaoH 0.02 normal titration . The pectin esterase activity unit defined as release amount of enzyme through a milli equivalent Carboxyl in a minute .enzyme activity specified as a unit in a milliliter culture medium filtrate (U/ml) (Maldonado et al ,1998). Optimizing methods
To specify interactions between multiple processes with fewer experimental trials, response surface methodology (RSM) could be used as a powerful statistical technique (Suresh et al, 2009).in the first step ,effects of seven factors were studied with a two-level fractional factorial selected for experimental design with 32 compositions (table 2).the results of first experimental design were analyzed with half normal plot method ,and the factors out of the curve were selected as significant factors that their variations were no random errors .factors with significant effect on pectin esterase enzyme activity which selected for second step were fermentation time ,PH ,and (푁퐻 ) S푂 .to determine the specific effect of these three factors and optimizing enzyme activity ,the RSM were used through a central composite design (CCD)with 24 experimental trials containing central points (table 3) .in CCD analysis ,the data fitted to a quadratic equation .regression method and lack of fit test used to valuate this model.
Table 2-establishing the experimental domain Symbols Independent
Several spoiled fruits like apple, orange , cucumber, apricot , strawberry, tomato, lemon and grapes were used to prepare microorganisms .growth of fungi in some fruits were so quick ,like orange and lemon whereas the growth was not observed in apple ,and the fungi was observed in orange and lemon in 48 hours .the mentioned funguses were cultured in PDA medium (PH=4.8-5.6)and incubated for five days at 25 ;the separated funguses were isolated in this medium .enzyme activity could be determined through adding %1 solution hexadecyl trimethyl bromide for 30 minute ,in this case the fungi would grow (Moyo et al ,2003) .
Aspergillus niger PG5 was selected among several microorganisms that were extracted from spoiled fruits. Morphological trait was used for determining Aspergillus niger spores under microscope in x40view. spectophotometer with 600nm wavelength was used to count the amount of Aspergillus niger spores ,and to regulate the amount of spores , in this case the suspension was diluted with physiological serum solution (Rezazadeh ,2002; ARAUJO ET AL ,2004).
Microflora of fruit surfaces have been the best source of antagonists against fungi causing postharvest decay of fruit. However, there is little information on microflora colonizing surfaces of fruits other than grape, apple, and citrus. We characterized bacterial microflora on nectarine fruit surfaces from the early stage of development until harvest. Pectinase is a general term for enzyme such as pectolyase, pectozyme and polygalacturonase, commonly referred to in brewing as pectin enzymes. These break down pectin, a polysaccharide substrate that is found in the cell walls of plants. It is useful because pectin is the jelly-like matrix which helps cement plant cells together and in which other cell wall components, such as cellulose fibrils, is embedded. Therefore pectinase enzymes are commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of fruit juice from fruit, including apples and sapota. Pectinases have also been used in wine production. The function of Pectinase in brewing is twofold, first it helps breakdown the plant (typically fruit) material and so helps the extraction of flavors from the mash. Secondly the presence of pectin in finished wine causes a haze or slight cloudiness, Pectinase is used to break this down and so clear the wine. Degradation of different pectins by fungi, and correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin Transfer to slant environment to keep the spor
Post assurance from the pectinase production of a mould, the obtained moulds have to be kept in particular conditions for accomplishment of upcoming stage of research .slant medium is the best environment
Moulds could be obtained in the hygenized condition from the medium ,and could be transferred to slant medium in order to be kept in this environment .in this environment , moulds would be incubated in 27 C° and 48 hours .post the growth of moulds , the lid could be covered with Parafilm.
MATERIALS AND METHODS
PDA medium Synthetic medium Ruthenium red Consumption (0.05) PH-Autoclave
Essential substances Critrion
Pectin 10 Fermentation juice 1.5
KH PO 2 KHPO 2
(NH ) SO 2 Agar 15
Funguses separation from different kind of spoiled fruits Moulds could be obtained from spoiled fruits. Mould could grow faster in fruits such as orange and lemon ,and
post 48 hours moulds could be observed in these fruits ; and growth of fungi in some fruits were so quick ,like
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orange and lemon whereas the growth was not observed in apple ,and the fungi was observed in orange and lemon in 48 hours. The moulds in vegetables such as tomato and bean were chosen for the test, in which we could measure the pectinase production. Firstly, PDA medium has to be provided in 4.8 and 5.6 PH, then moulds of fruits have to be cultured in the medium, finally they could be incubated for 5 days in 25 C°. Funguses separation Mediums including different types of moulds have to be cultured in PDA medium in order to obtain the single colony (subculture), and they would be incubated for 5 days in 25 C°. Keeping the isolated funguses Isolated funguses would be cultured in PDA medium which this would provide the long term keeping and less infection of funguses. Providing the medium within pectin Pectin or (퐍퐇ퟒ)ퟐ퐇S퐎ퟒ could be both deterministic in the medium, And PH of environment within adding the acid sulphuric could be adjusted in 4.2-4.6. The observation of pectinase enzyme Synthetic medium including following factors would be provided for the observation of pectinase enzyme (gr/lit) Pectin: 10 fermentation juice including 1.5 liter,
퐊퐇ퟐ퐏퐎ퟒ: 2, KH퐏퐎ퟒ: 2, (퐍퐇ퟒ)ퟐS퐎ퟒ: 2, and the medium has to be Adjusted in 4.8 and 5.6 .the isolated colony of funguses would be Cultured in this medium, and would be incubated for 7 days; then %0.05 Ruthenium red solution would be added to mediums for one hour, and Aquapura would be used for the decolorization. The isolated funguses from the moldy fruits
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As observed in image 1, the isolated moulds would be as follows : Geotricum Alternaria ,Penicillium ,Cladosporium , Cladosporiu ,Treichotecium ,Aspergillus , Geotricum ,Trichotecium .from the isolated moulds , most of the moulds were belonged to Aspergillus ,then they belonged to Penicillium. Isolated funguses from moldy fruits
Aspergillus Aspergillus
Aspergillus Treichotecium
Cladosporium Penicillium
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Geotricum Alternaria
Aspergillus Trichotecium
Aspergiilus Aspergiilus
Cladosporium Aspergiilus
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Alternaria Alternaria
Trichotecium Penicillium
Geothricum Alternaria
Trichotecium Penicillium
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Alternaria Aspergillus
Aspergillus Penicillium
Penicillium Alternaria
Alternaria Trichoteciu
The results of funguses separation
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To identify the moulds within high pectinase production, the ratio of corona diameter to mould diameter has to be obtained. Post measurement of corona diameter and moulds diameter, the results would be as following. The results show that the moulds of grape are more than other fruits. In next stages, it is attempted to utilize the moulds within high production from the fruits such as beet pomace for pectinase production, in which this fruit could be as an appropriate fruit for enzyme production .certainly, it is essential to point out that the fruit pomace are the wastage of industry and low costs have been provided for the supply. Table – the obtained results of the isolated samples from different fruits Fruit Sampling history The colony diameter of
The obtained results of the pectinase activity measurement from the isolated samples show that the most activity of Aspergillus in grape is within 34 mm corona diameter.
DISCUSSION AND RESULTS
According to the fractional factorial experimental results ,the effects of factors on pectinesterase activity could
be described through equation 3.the variables used in this equation are coded values of origin variables included fermentation time (X1), aeration rate (X2), PH(X3),(푁퐻 ) S푂 (X4),퐾퐻 푃푂 (X5),date pomace (X6),and total spore (X7).all variables have a linear effectr on enzyme activity in their used ranges .for this model ,푅 is almost 0.99 which this amount indicates the adaptability; ANOVA analysis for this model is presented in table 4. Model predictions were also compared with real values and this shows the significant correlation (fig 1).SAS software was used to analyze the results ,and the relative effects of all factors were compared with each other (fig 2).according to the Pareto chart ,the fermentation time had have the most effect on enzyme activity ,and fermentation time was followed through concentration of potassium dihydrogen phosphate , Ammonium sulphate ,aeration rate ,PH ,date pomace and total spore .
Table 4- ANOVA for selected factorial model Source Sum of Squares df Mean Square F Value P-Value
Figure 1- model prediction and real values of comparison
Figure 2- the Pareto chart shows comparison between effects of seven factors including A=fermentation time,
B=aeration rate, C=PH, D=(푁퐻 ) S푂 ,E=퐾퐻 푃푂 ,F=date pomace and G =total spore
Response surface method is used to show the relationship between their effects .increase of the ammonium sulfate
concentration in 0.1 – 0.3 gr / ml region would increase the enzyme activity (figure 3) and the highest pectin esterase activity occurs in 48 hours and %0. 3 concentration of ammonium sulfate. While funguses populations grow, they would need more nutrients, and at this time concentration of potassium dihydrogen phosphate would be effective. The increase of 퐾퐻 푃푂 from %0.4 to %0.8 would increase the pectin esterase activity in 24-48 hours of fermentation time, but it has the most effect in 48 hours .so, enzyme activity in 48 hours and %0.8 concentration of 퐾퐻 푃푂 would have the most value .as response surface plot in fig 5 shows ,agitation speed have direct effect in pectin esterase activity ,where its highest level occurs in high speed agitation (250rpm )and 48 hours .Palil showed positive effect of PH on pectinase activity (Palil et al , 2006) ; the result of present research was same ,and the response surface plot for PH (fig 6) shows that the increase of PH from 3.5 to 5 would increase the enzyme activity .the increase of date pomace from 4
Actual
Pre
dict
ed
Predicted vs. Actual
0.11
0.29
0.47
0.65
0.83
0.12 0.30 0.47 0.65 0.83
Pareto Chart
t-Val
ue o
f |E
ffect
|
Rank
0.00
2.49
4.99
7.48
9.97
Bonferroni Limit 3.74484
t-Value Limit 2.10982
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31
A
E
ABDEFFCGBF
ADAFCD
BC G
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to 8 gr/ml would have insignificant effect on enzyme activity ,and the interaction effect of date pomace and ammonium sulfate concentration on pectin esterase activity would be observed in fig 7.
Total spore also have insignificant effect on pectin esterase activity, and the effect of increasing the total spore from 10 푡표10 has been demonstrated as an interaction plot for total spore and fermentation effect in fig 8.highest enzyme activity would be within 10 spores per 100 ml in 48 hours fermentation time, and Palil reported the same results (Palil et al, 2006).
Figure 3- response surface plot for synchronic effect of fermentation time and concentration of ammonium sulfate on enzyme activity ,other factors kept in their central point ( PH :4.25 ,퐾퐻 푃푂 : %0.6 ,date pomace :%6 , aeration
rate : 200 rpm , total spore : 5.05 * 10 ).
Figure 4- response surface plot for synchronic effect of fermentation time and concentration of potassium dihydrogen phosphate on enzyme activity ,other factors kept in their central point ( PH :4.25 ,(푁퐻 ) S푂 = %0.2 ,
date pomace :%6 , aeration rate : 200 rpm , total spore : 5.05 * 10 ).
Figure 5- response surface plot for synchronic effect of fermentation time and aeration rate (agitation speed) on
enzyme activity ,other factors kept in their central point ( PH :4.25 ,(푁퐻 ) S푂 = %0. 2,퐾퐻 푃푂 : %0.6 , date pomace :%6 and total spore : 5.05 * 10 ).
24.00
30.00
36.00
42.00
48.00
0.10
0.15
0.20
0.25
0.30
0.22
0.325
0.43
0.535
0.64
Pec
tines
tera
se A
ctiv
ity(U
/ml)
A: Fermentation time(h) D: NH4SO4(gr/100ml)
24.00
30.00
36.00
42.00
48.00
0.40
0.50
0.60
0.70
0.80
0.21
0.3225
0.435
0.5475
0.66
Pec
tines
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se A
ctiv
ity(U
/ml)
A: Fermentation time(h) E: KH2PO4(gr/100ml)
24.00
30.00
36.00
42.00
48.00
150.00
175.00
200.00
225.00
250.00
0.23
0.325
0.42
0.515
0.61
Pec
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se A
ctiv
ity(U
/ml)
A: Fermentation time(h) B: Agitation speed(rpm)
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Figure 6- response surface plot for synchronic effect of fermentation time and PH on enzyme activity ,other factors kept in their central point ((푁퐻 ) S푂 = %0. 2,퐾퐻 푃푂 : %0.6 , date pomace :%6 ,aeration rate : 200rpm and
total spore : 5.05 * 10 )
Figure 7- date pomace and ammonium sulfate concentration interaction plot for their effects on enzyme activity
,other factors kept in their central point (fermentation time :36 h,퐾퐻 푃푂 : %0.6 , aeration rate : 200rpm , PH :4.25 , and total spore : 5.05 * 10 )
Figure 8- total spore and fermentation interaction plot for their effects on enzyme activity ,other factors kept in their
central point ((푁퐻 ) S푂 = %0. 2,퐾퐻 푃푂 : %0.6 , date pomace :%6 ,aeration rate : 200rpm and PH:4.25)
Second step (applying RSM using central composite design (CCD))
In this step, we have selected three factors among seven factors in which their effects have been studies in first step; RSM through central composite design (CCD) have to be applied in order to characterize the effects on pectin esterase activity and find optimized conditions.
24.00
30.00
36.00
42.00
48.00
3.50
3.88
4.25
4.63
5.00
0.2
0.295
0.39
0.485
0.58
Pec
tines
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se A
ctiv
ity(U
/ml)
A: Fermentation time(h) C: pH
F: Date pulp(gr/100)
0.10 0.15 0.20 0.25 0.30
Interaction
D: NH4SO4(gr/100ml)
Pect
ines
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ctiv
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/ml)
0.11
0.29
0.47
0.65
0.83
F-F+
G: Total Spore
24.00 30.00 36.00 42.00 48.00
Interaction
A: Fermentation time(h)
Pec
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/ml)
0.11
0.29
0.47
0.65
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G-G+
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In this step PH, ammonium sulfate concentration, and fermentation time were used in a five level experimental design that is depicted in table 4.according to results of first step, the selected fermentation conditions is the one in which enzyme activity would have the highest values;so the used date pomace is equal to 6 g/ml and others kept in their top levels .
ANOVA analysis of experimental results shows insignificant effect of PH on enzyme activity in its studied span (4.82 – 6.18). The only effect would be occurred while there is interaction with Ammonium sulphate concentration .the increase of Ammonium sulphate concentration up to %0.35 point in each level of PH would increase the enzyme activity, whereas post this point, the enzyme activity would decrease figure 9 although PH has insignificant effect on enzyme activity in 4.82 -6.18 regions, but it’s interaction with ammonium sulphate concentration would affect pectin esterase activity in %.25 of ammonium sulphate concentration, and the increase of PH from 4.82 to 6.12 would decrease the enzyme activity .while the ammonium sulphate concentration is equal to %0.45 ; in this case the increase of PH would have inverse effect ;thus the increase of ammonium sulphate concentration would not have any effect on enzyme activity and it would just affect the trend of PH effect . the response surface plot for effect of fermentation time on enzyme activity and it‘s interaction with ammonium sulphate concentration, has been depicted in figure 10 .as observed in ,the increase of fermentation time until 75 hours would increase the pectin esterase activity and post this time ,the enzyme activity would decrease .
Figure 9 - Response surface plot reveals the effect of pH and ammonium sulfate concentration on pectin esterase activity (date pomace=g/ml and other factors set in their top levels from first stage design)
Figure 10- response surface plot reveals the effect of ammonium sulphate concentration and fermentation time on pectinesterase activity (date pomace =g/ml and other factors have been set in their top levels from first stage design).
4.82
5.16
5.50
5.84
6.18
0.25
0.30
0.35
0.40
0.45
3.29
3.3525
3.415
3.4775
3.54
Pec
tines
tera
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ctiv
ity (U
/ml)
pH (NH4)2SO4 (gr/100ml)
56.88
62.94
69.00
75.06
81.12
0.25
0.30
0.35
0.40
0.45
2.76
3
3.24
3.48
3.72
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ctiv
ity (U
/ml)
Fermentation time(h) (NH4)2SO4 (gr/100ml)
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Optimization Desirability functions (numerical method)are such useful method for optimizing production process in the
industry .this method was presented by Harrington (Harrington ,1965), and the aim of this method is to find particular conditions of variables in which these variables could provide high desirability function for related function response .desirability value ranges from zero to one ,and it’s top value shows the best condition to achieve the aim .to reach maximum pectinesterase activity is the main aim ,and the desirability method has been used to find the best condition of culture medium and it’s combination .various conditions to reach maximum enzyme activity are illustrated in table 5 .high desirability and high enzyme activity could be observed in 79.2 hours of fermentation time in which PH =6.18 , ammonium sulphate concentration=0.44 gr /ml, pectinesterase activity in this condition is equal to 3.81686 U/ml and desirability is equal to 0.94492.
Table 5-the result of desirability function and optimized conditions
Number Time(h) pH (NH4)2SO4 Pectinesterase (gr/ml) (U/ml)
To reduce production costs ,the fermentation time reduction is a major aim ; so the best condition within high
enzyme activity and minimum fermentation time were optimized .results for desirability function have been shown in table 6 ,and the high pectinesterase activity in this condition is equal to 3.28 U/ml ,fermentation time =62.84 hour ,PH=4.28 and ammonium sulphate concentration =0.28 gr /ml ;the other factors within top level are based on first stage experimental design except date pomace which is equal to %6.
Table 6- the optimized conditions with minimum fermentation time
Number Time pH (NH4)2SO4 Pectinesterase gr/ml (U/ml)
To achieve optimized condition with fewer experimental trials, the statistical methods were used. The
optimized condition within maximum enzyme activity was determined through applying RSM, central composite design and desirability function. In optimum fermentation condition, pectin esterase activity were equal to 3.81686 U/ml, and the obtained optimum condition included the fermentation time (79.2hours), PH (6.18), ammonium sulphate concentration (0.44gr/ml), total spore (10 푔푟/푚푙), date pomace (%6), potassium dihydrogen phosphate concentration (%0.8), and agitation speed (250 rpm).
Conclusion
In pareto plot (fig 2), fermentation time in 24-48 hours period has the most important effect on pectin esterase activity, and the increase of fermentation time would increase enzyme activity .enzyme production depends on the kind of nitrogen source ,as many studies reported positive effect of ammonium sulfate concentration on pectinase activity (Loera et al ,1999 –Pandey et al ,2002).agitation affects enzyme activity because of its effect on heat and mass transportation and also on fungi morphology .the effect of agitation in 48 hours is more than 24 hours ,because as fermentation increases, fungi population grows as well ,and increase of agitation rate would produce more nutrient and oxygen for them . the optimum fermentation condition with minimum fermentation time and maximum enzyme activity also obtained while the maximum pectin esterase activity (3.28 U/ml ) occur in 62.84 hours of fermentation time ,and other factors included PH(4.28), ammonium sulphate concentration (0.28 gr/ml),total spore (10 푔푟/푚푙),date pomace (%6), potassium dihydrogen phosphate concentration (%0.8), and agitation speed (250 rpm).
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