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Screening, Optimization and Production of Phytase from

Aspergillus species Isolated from environmental samples.

BY

Leonard Oghenemaro ITABA

Matriculation Number: 187825

2

INTRODUCTION

Phytic acid is a major constituent of many cereals and oil seeds (Santos,

2011).

Traditionally phytate are considered by nutritionist as an anti-nutrient to

monogastrics that lacks microbes in their gut to break phytate-mineral

complexes (Lopez et al., 2002).

Phytases are phosphomonoesterases capable of hydrolyzing phytate to lesser

derivatives and other divalent elements (Qasim et al., 2016).

Phytases have been found in plants, microorganisms, and in some animal

tissues (Konietzny and Greiner, 2002).

The first commercial phytase products was launched into market in1991

(Haefner et al., 2005). And till date, only a little of commercial phytase

product are available, due to high cost of production (Qasim et al., 2016).

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STATEMENT OF PROBLEM

Even though phytases effectively improves nutritional quality while

decreasing phosphorus waste, microbiologist and ezymologiest are still

faced with challenges involving the cost of production.

JUSTIFICATION

The phytate hydrolytic activities of phytases makes their study unique

for nutritional improvement and biotechnology. Hence, other means of

reducing production cost and still have high yield is considered.

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AIMS AND OBJECTIVES

This research aims at producing phytase from fungi. isolated from diverse

phytate-rich sources.

• OBJECTIVES

• Isolation and identification of phytase producing microorganism from

environmental samples

• Screening and selection of fungi strains with highest phytase

production.

• Optimization of certain culture conditions on the production of

phytase.• Production of phytase from various isolates using organic and

inoganic substrates.• Purification and characterization of phytase enzyme

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MATERIALS AND METHODS

Collection of samples (Mittal et al., 2012)

Isolation and identification (Watanabe 2002; Awad et al., 2014)

Screening and selection phytase producing microorganism: was done

according to (Pikovskaya, 1948), Nautiyal, (1999) and (Qasim et al.,

2016)

Detection of Aflatoxin production: (Saito and Machida, 1999).

Production and Enzyme assay: Choi et al. (2001) and JECFA (2012)

Gunashree and Govindarajulu. (2015)

Optimization of the growth conditions for phytase production: was

done acording to the method of (Wang et al., 2004). Awad et al, (2014)

Phytase Purification and Characterization (Awad et al., 2014)

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Results

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Isolate code

Source Halo on PKV

Halo on PSM

Counter staining

Solubilizing index (%)

S1 Garden Soil + + - -

S2 Garden Soil + + + 55 ± 0.02

S3 Garden Soil - - - -


S5 Garden Soil + + + 68±0.01

S6 Garden Soil + + + 180±0.06


S8 Garden Soil + + + 44±0.03

P1 Poultry dropping - - - -

P2 Poultry dropping + + + 98±0.07

P3 Poultry dropping - - - -

P4 Poultry dropping + + + 157±0.04

Table 1: Source, solubilization index (SI) and Characteristics of fungi isolate on phytase. (-): No halo; (+): presence of halo

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P5 Poultry dropping + + - -P6 Poultry dropping + + + 185±0.4P7 Poultry dropping - - - -P8 Poultry dropping + + + 136±0.06P9 Poultry dropping - - - -

P10 Poultry dropping + + + 30±0.02C1 Cereal rich soil + + + 138±1C2 Cereal rich soil - - - -C3 Cereal rich soil + + + 194±0.05C4 Cereal rich soil + + + 151±0.07C5 Cereal rich soil - - - -C6 Cereal rich soil - - - -C7 Cereal rich soil + + + 60±0.04C8 Cereal rich soil + + - -C9 Cereal rich soil - - - -C10 Cereal rich soil + + + 55±0.03

TABLE 1 CONT’D

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A = magnification × 0.5 B = magnification × 0.4

Plate1: Phytase Production by Isolate S6 A= zone of hydrolysis before staining B= zone of hydrolysis after staining

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C1 C3 C4 C7 C10 S2 S5 S6 P2 P4 P6 P80

10

20

30

40

50

60

70

80

90%

Phy

tase

Act

ivity

U/L

Figure 1: Relative Percentage phytase activity of fungal isolates with 50% and above solubilizing index.

*Values are Means of two experiments, each with two replicates (n=4)

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Table 2: Screening of Aflatoxin production by selected fungal isolates

ISOLATE OBSERVATION Production of aflatoxin

C10

No colour change on the underside of the plate.

Negative

P2


Negative

P6

The yellow pigment on the underside of the plate turned plum-red.

Positive

S6


Negative

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Isolate Cultural characteristics on PDA Microscopic characteristis Identity

C10 White at first and eventually turn dark green with age.

Conidial heads: conidial heads were dark bluish green

Aspergillus fumigatus

P2 Colonies were colorless at first and then gradually turned orange-yellow to light brown with age.

Conidial heads varied greatly in size in same fruiting area, from more or less columnar

Aspergillus tamari

P6 Colonies were powdery, flat with radial grooves, yellow at First but later turned bright to dark yellowish green with age.

Conidial heads were radiate, splitting to form loose columns

Aspergillus flavus

S6 Colonies were flat and compact with yellow basal covered by dense layer of black conidial heads with powdery texture.

Conidial heads split into over 4 loose conidial columns with over 4 fragments apically.

Aspergillus niger

Table 3: Morphology, microscopic characteristics and Identity of Isolates.

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48hrs 72hrs 96hrs 120hrs 144hrs 168hrs0

200

400

600

800

1000

1200

1400

C10 P2 S6

incubation period (hours)

Phyt

ase

Prod

uctio

n U

/L

Figure 2: Time course for Phytase Production by (C10, P2 and S6) over 7days.


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Figure 3: Effect of medium pH of medium on phytase production

3 3.5 4 4.5 5 5.5 6 6.5 7 7.50

200

400

600

800

1000

1200

A. fumigatus

A. niger

A. tamari

pH

Phyt

ase

Prod

uctio

n U

/L


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Control Frutose Glucose Lactose Maltose Sucrose Starch0

100

200

300

400

500

600

700

A. fumigatus

A. niger

A. tamari

Carbon sources

Phyt

ase

Prod

uctio

n (U

/L)

Figure 4: Influence of Carbon sources on Phytase Production by A. fumigatus, A. niger and A. tamari


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control NaNO3 NH4Cl NH4NO3 (NH4)2SO4 peptone yeast extract0

100

200

300

400

500

600

700A. fumigatusA. nigerA. tamari

Nitrogen source

Phyt

ase

Prod

uctio

n (U

/L)

Figure 5: Influence of Nitrogen sources on Phytase Production by A. fumigatus, A. niger and A. tamari


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48hrs 72hrs 96hrs 120hrs 144hrs0

100200300400500600700800900

1000

A. fumigatus A. niger

A. tamari Series4

Inoculum age (hours)

Phyt

ase

Prod

uctio

n (U

/ml)

Figure 6: Effect of inoculum age on phytase production by A. fumigatus, A. niger and A. tamari


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Orange Peel Sesame Water melon PSM Phytate- free0

100200300400500600700800900

1000

A. fumigatus

A. niger

A. tamari

contol

Subtrates

Phyt

ase

Prod

uctio

n U

/L

Figure 7: Effect of different substrates on phytase production by A. fumigatus, A. niger and A. tamari


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PSM+OP PSM+S PSM+WM PSM PSM free phytate0

100200300400500600700800900

1000

A. fumigatus

A. niger

A. tamari

control

Substrates

Phyt

ase

Prod

uctio

n U

/L

Figure 8: Effect of supplementation of organic substrate on phytase production by A. fumigatus, A. niger and A. tamari.


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WORK IN PROGRESS

Monitoring the effect of different concentration of organic substrate

(dried milled sesame seed, orange peel and water melon seed)

Enzyme purification and characterization- SDS PAGE

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DISCUSSION/CONCLUSION

Several studies (Kim et al. (1998); (Sreedevi and Reddy, (2012); Qasim et

al. (2016)) have earlier reported phytase production in medium containing

wheat bran. Wheat bran is cheap agro source of phytate but still it is used

as fodder.

To the best of our knowledge this is one of the first work to utilize water

melon seed and orange peel for phytase production. This study has shown

that Aspergillus sp. (A. niger, A. fumigatus and A. tamari) offers possibility

for the industrial production of phytase under submerged fermentation

using cost effective Agro-materials. Consequently, solving the challenges

of agro-waste management, opening a new arena of research and reducing

the cost of production of phytase which is held as a major drawback to its

commercialization.

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REFERENCES

Awad, G.E.A., Helal, M.M.I., Danial, E.N. and Esawy, M.A. (2014). Optimization of phytase production by Penicillium purpurogenum GE1 under solid state fermentation by using Box–Behnken design. Saudi Journal of Biological Sciences. 21; 81–88.

Bae, H.D., Yanke, L.J., Cheng , K.J. and Selinger, L.B. (1999). A novel staining method for detecting phytase activity. Journal of Microbiological Methods. 39, 17–22.

Choi, Y.M., Suh, H.J. and Kim, J.M. (2001). Purification and properties of extracellular phytase from Bacillus sp. KHU-10. J Protein Chem; 20: 287-292.

Gunashree, B.S and Govindarajulu, V. (2015). Dephytinization of Cereals and Pulses by Phytase Producing Lactic Acid Bacteria. Int.J.Curr.Res.Aca.Rev. 3(12): 61-69

Lopez, H., Leenhardt, F., Coudray, C. and Remesy, C. (2002). Minerals and phytic acid interaction: is it a real problem for human nutrition? International Journal of Food Science and Technology 37: 727-739.

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REFERENCES CONT’D

Nautiyal, C.S. (1999). An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Letters, vol. 170, no. 1, pp. 265–270.

Pikovskaya, R.I. (1948). Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Microbiology, vol. 17, pp. 362–370.

Saito, M. and Machida, S. (1999). A rapid identification method for aflatoxinproducing strains of A. flavus and A. parasiticus by ammonia vapor. Mycoscience 40:205 -21 1.

The joint FAO/WHO expert committee on food additives (JECFA) (2012). Phytase from Aspergillus niger expressed in A. niger. FAO JECFA monographs 13. http://www.fao.org/ag/agn/jecf-additive/specs/monograph13/additive-528-m13.pdf retrieved Thursday June 16th 2016 8pm.

Wang, X., Upatham, S., Panbangred, W., Isarangkul, D., Summpunn, P., Wiyakrutta, S. and Meevootisomd, V. (2004). Purification, characterization, gene cloning and sequence analysis of a phytase from Klebsiella pneumoniae subsp. Pneumoniae XY-5. Sci. Asia 30: 383-390.

http://www.fao.org/ag/agn/jecf-additive/specs/monograph13/additive-528-m13.pdf

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