Optimization of process parameters for the production of naringinase by Aspergillus niger MTCC 1344

Process Biochemistry 40 (2005) 195–201

Optimization of process parameters for the production of naringinase by Aspergillus niger MTCC 1344 Munish Puri a , Anirban Banerjee b , U.C. Banerjee b,∗ b

a Department of Biotechnology, Punjabi University, Patiala 147002, Punjab, India Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, SAS Nagar, Mohali 160062, Punjab, India

Received 23 July 2003; accepted 12 December 2003

Abstract Aspergillus niger MTCC 1344 was used to produce extracellular naringinase in a complex (molasses, yeast extract and salts) medium. An initial medium pH 4.5 and cultivation temperature 30 ◦ C were optimal for enzyme production. Among various carbon and organic nitrogen sources used, molasses and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when metal ions were added to the medium. Fermentation conditions are described which produced a higher rate of enzyme synthesis. An increase in initial sugar concentration from 6 to 10 g l−1 in the fermentation medium produced decreased naringinase synthesis while cell mass growth increased with the increase of sugar concentration. At a higher sugar level (10 g l−1 ) the production of cell mass decreased. © 2004 Elsevier Ltd. All rights reserved. Keywords: Naringinase; Naringin; Aspergillus niger; Debittering; Enzyme production

1. Introduction The processing of citrus fruit juice has faced formidable problems in terms of bitterness and delayed bitterness, thereby affecting its consumer acceptability. All the processed citrus fruit juices contain naringin (4,5,7-trihydroxyflavonone 7-rhamnoglucoside) which attributes bitterness to the juices [1]. Naringinase is an enzyme which hydrolyses naringin into prunin which is then converted by the flavonoid ␤-d-glucosidase to naringenin (4,5,7-trihydroxyflavonone) [2]. Naringinase has become biotechnologically important due to its role in debittering citrus fruit juices [3,4], in the manufacture of rhamnose [5], preparation of prunin [6] and biotransformation of antibiotics [7]. For complete modification/degradation of bittering components, concerted efforts were made either to develop soluble enzymes or microbes capable of metabolizing naringin [8]. Suitable enzymes have been immobilized on many matrices and their application for the above purposes have been reported [9–11]. The use of enzymes with significant industrial application ∗ Corresponding author. Tel.: +91-172-2214682-86; fax: +91-172-2214692. E-mail address: [email protected] (U.C. Banerjee).

0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2003.12.009

is increasing but there are few reports available on the production of naringinase, most of them are either guarded secrets of the industry or are patented [12–14]. The debittering process could be more cost effective and economically viable if naringinase production is achieved industrially using microorganisms. In the present study, an attempt has been made to optimize the process parameters to increase naringinase production by Aspergillus niger MTCC 1344.

2. Materials and methods 2.1. Chemicals Naringin was obtained from Sigma, St. Louis, USA. Different growth factors and organic and inorganic nitrogen sources were obtained from Hi-Media, laboratories, Mumbai, India. All other reagents used were of analytical grade. 2.2. Culture medium and cultivation conditions A. niger MTCC 1344, was obtained from Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh and maintained on Czapeck-Dox medium.

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M. Puri et al. / Process Biochemistry 40 (2005) 195–201

The composition of the medium was (g l−1 ) NaNO3 2.0; KH2 PO4 1.0; KCl 0.5; MgSO4 ·7H2 O 0.5; FeCl3 0.1 and naringenin (4,5,7-trihydroxy flavonone) 0.05. Using A. niger MTCC 1344 as best naringinase producing strain, a range of media constituents were examined for enzyme production. Hundred millilitres medium was autoclaved in 500 ml Erlenmeyer flask for 15 min at 121 ◦ C and 15 psi pressure and the initial pH of the medium was adjusted to 4.5. The medium was inoculated with vegetative mycelia or spore suspension. The vegetative spores, taken from slants and suspended in 0.85% sterile sodium chloride, were inoculated in the medium. Flasks were incubated (28 ◦ C, 200 rpm) in a rotary shaker for 8–10 days. Samples were withdrawn aseptically at regular time intervals and analyzed for cell mass, naringinase activity and residual substrate. To study the effect on enzyme production, different carbon sources (1%, w/v), were added to the basal medium before autoclaving. Different organic (1%, w/v) and inorganic (0.5%, w/v) nitrogen sources were added to the medium to determine their effect on naringinase production. Different growth factors including yeast extract, beef extract, malt extract, meat extract and groundnut seed flour were used at a concentration of 1% (w/v) each in a medium containing molasses and peptone. The salts of different metal ions including Ca2+ , Co2+ , Cu2+ , Fe3+ , Mg2+ , Ni2+ , Mn2+ and Zn2+ were used in the culture medium. The effect of temperature and initial pH on growth and enzyme production was checked in 500 ml Erlenmeyer flask containing 100 ml medium. The effect of inoculum age and inoculum volume was checked at the shake flask level. Fermentation was carried out in a 7 l bioreactor (Chemap AG, Switzerland) with a working volume of 5 l at 30 ◦ C with aeration and agitation rates of 0.5 vvm and 350 rpm, respectively. Samples were withdrawn at regular time intervals and analyzed for cell mass, naringinase activity and residual substrate concentration.

3. Results and discussion

2.3. Assay methods

3.3. Effect of nitrogen source

Naringinase activity was estimated by determining residual naringin using the Davis method [15]. A typical assay mixture comprised of 1 ml 0.1% naringin dissolved in 300 ␮l 0.1 M sodium acetate buffer (pH 4) and 200 ␮l culture filtrate. The assay mixture was incubated at 50 ◦ C for 60 min after which 100 ␮l aliquot was added to 5 ml 90% diethylene glycol followed by the addition of 100 ␮l 4 N NaOH. Samples were kept at room temperature (28 ◦ C) for 10 min. The intensity of the resultant yellow colour was determined at 420 nm. One unit of naringinase activity was defined as 1 ␮mol of naringin hydrolyzed under the above assay condition. The dry weight of the A. niger mycelium was determined after filtering through Whatman (No. 1) filter paper, washing thoroughly (three times) with distilled water and drying overnight at 80 ◦ C. Extracellular protein was measured by the method of Lowry et al. [16]. Total sugars were determined with anthrone [17] method.

Six different nitrogen sources were used at a concentration of 10 g l−1 in a medium containing molasses (10 g l−1 ,

3.1. Media optimization for growth and naringinase production A. niger MTCC 1344 was grown on a salt medium with naringenin as an inducer. This organism produces significant amount of extracellular naringinase in the medium. To improve upon the yield and reduction in fermentation time, the medium composition was optimized in shake flasks. 3.2. Effect of carbon source Various carbon compounds were added at a concentration 10 g l−1 to the medium containing naringenin. Rhamnose and molasses (10 g l−1 ) exhibited highest naringinase (4.6 IU ml−1 ) activity after 8 days of fermentation (Table 1). Other carbon sources in the medium also showed naringinase activity but the yield was lower with respect to molasses. Repression of naringinase activity by glucose, sucrose, citrate, etc., was also reported by Bram and Solomons [18], although these carbon sources supported excellent growth. Mateles et al. [19] reported that rhamnose or plant meal containing rhamnose glucoside increased naringinase production. However, in this experiment, rhamnose was not selected as main carbon source and molasses was chosen for media formulation as it is easily available and cost-effective. As maximum enzyme activity was obtained with molasses as carbon source, for all subsequent experiments molasses was used. In order to determine the optimum concentration of the molasses for enzyme production, different concentrations (5–30 g l−1 ) of molasses were used in the medium. With increasing concentrations of molasses, naringinase activity increased up to 15 g l−1 molasses in the medium and thereafter declined. Maximal naringinase activity (5.4 IU ml−1 ) was obtained on the eighth day of fermentation (results not shown).

Table 1 Effect of carbon source on the production of naringinase by A. niger Carbon source (10 g l−1 )

Enzyme activity (IU ml−1 )

Relative activity (%)

Control Glucose Fructose Rhamnose Maltose Sucrose Molasses (sugar 5 g l−1 ) Starch Corn steep liquor Corn starch

2.1 2.8 1.8 4.6 4.1 2.5 4.6 3.6 3.2 1.8

46 61 39 100 89 54 100 78 70 39

Control did not have any carbon source; molasses contain 50% sugar.

M. Puri et al. / Process Biochemistry 40 (2005) 195–201 Table 2 Effect of nitrogen source on the production of naringinase by A. niger Nitrogen source

Enzyme activity (IU ml−1 )

Relative activity (%)

Control

4.6

100

Inorganic Ammonium sulphate Ammonium dihydrogen phosphate Sodium nitrate

2.0 1.10 4.6

43 24 100

Organic (10 g l−1 ) Peptone Tryptone Soyapeptone Soyabean meal Casein Cornsteep liquor

6.5 5.4 4.8 4.0 2.0 4.0

141 117 104 87 43 87

(5 g l−1 )

197

3.4. Effect of growth factors Five different complex materials (10 g l−1 ) were supplemented into the fermentation medium for the improvement in naringinase production. Enzyme activity was substantially lower for the medium supplemented with growth factors like yeast extract, beef extract, malt extract and groundnut seed flour compared to the control (Fig. 1). This suggests that growth factors are detrimental to the improvement of naringinase production. 3.5. Effect of inoculum age and size

having 5 g l−1 sugar). Among all the organic nitrogen sources used, peptone was the most effective in naringinase biosynthesis (Table 2). Soyabean meal and corn steep liquor, which are cheaper nitrogen sources than peptone, were relatively non-significant in naringinase production, but peptone gave consistent results. In order to determine the optimum concentration of peptone for naringinase production, different concentrations (2.5–20 g l−1 ) of peptone were used in the medium containing molasses. In terms of the enzyme yield, the optimum concentration of peptone was 5 g l−1 (results not shown). Higher concentrations of peptone in the fermentation medium did not significantly increase enzyme yield. For further experiments peptone (5 g l−1 ) was used in the cultivation of A. niger for the production of naringinase. Among the inorganic nitrogen sources used sodium nitrate did not have any inhibitory effect on naringinase production. Urea and diammonium hydrogen phosphate were inhibitory, presumably because of the release of ammonium ions.

A 72 h old seed culture when used as inoculum, gave maximum naringinase production in the fermentation medium (Fig. 2a). The inoculum level of 3–15% (v/v) was used in the cultivation medium to establish the effect of inoculum size on the enzyme production by A. niger. A 10% (v/v) inoculum (Fig. 2b) was optimal for growth as well as naringinase production and the lag phase was also minimal. With 3, 5 and 7% (v/v) inoculum size, the lag phase did not reduce significantly and maximal activity was obtained at a longer incubation time (9 days). A. niger grown on potato dextrose medium gave heavier sporulation. The vegetative mycelia bearing sporangiophore (containing spores) were used for inoculum preparation. On an average 5×105 spores per milliliter were used for the development of inoculum. The resultant spores were inoculated into the fermentation medium. 3.6. Effect of metal ions Different metal ions were used in the cultivation medium to determine the effects of metal ions on growth and naringinase production by A. niger. Cu2+ , Co2+ , Ni2+ were found to be inhibitory both to growth and naringinase production.

6

Control Yeast extract Beef extract Malt extract Meat extract Ground seed flour

Enzyme activity (IU/ml)

5

4

3

2

1

0 0

1

2

3

4

5

6

7

Fermentation time (day) Fig. 1. Effect of growth factors on the production of naringinase by A. niger.

8

198

M. Puri et al. / Process Biochemistry 40 (2005) 195–201 Table 3 Effect of metal ions on the production of naringinase by A. niger

Enzyme activity (IU/ml)

7

24 h 48 h 72 h 96 h

6 5 4

Metal ions (mM)

Enzyme activity (IU ml−1 )

Relative activity (%)

Control

6.5

100

7.4 8.0 6.0

114 123 92

Ca2+

3

5 10 30

2 1 0 3

4

5

6

7

8

9

10

Fermentation time (day)

(a)

Cu2+ 5

0

0

Co2+ 5

0

0

Fe2+

3%

7

5 10 30

5.5 3.2 0

85 49 0

Mg2+ 5 10 30

7.2 8.5 5.0

111 131 77

Mn2+ 5 10 30

4.8 5.0 5.2

74 77 80

1

Ni2+ 5

0

0

Zn2+ 5 10 30

4.0 0 0

5%

Enzyme activity (IU/ml)

6

7% 10%

5

15%

4 3 2

4

(b)

5

6

7

8

9

Fermentation time (day)

Fig. 2. Effect of (a) inoculum age and (b) size on the production of naringinase by A. niger.

Zn2+ at a concentration of 5 mM showed 38% inhibition of enzyme activity whereas Ca2+ (5–10 mM), Mg2+ (5–10 mM) stimulated naringinase synthesis (Table 3). In the case of Mg2+ , the maximum activity 8.5 IU ml−1 was observed at 10 mM which accounted for a 31% increase in enzyme activity. Above 10 mM enzyme activity decreased drastically. Ca2+ at 5–10 mM also supported maximal production (7.4 IU ml−1 ) of naringinase whereas at higher (30 mM) concentrations, a small decrease (6 IU ml−1 ) in enzyme activity was observed. This suggests that Mg2+ and Ca2+ ions are required for the production of naringinase by A. niger. Fe2+ and Mn2+ show an inhibitory action on growth and enzyme production by A. niger. 3.7. Effect of environmental factors on growth and naringinase production The effect of initial pH and temperature on growth and naringinase production by A. niger was observed in 500 ml Erlenmeyer flasks containing 100 ml medium (molasses, peptone, salts and naringenin). In the cases the fermentation was continued for 8 days, with periodic determination of enzyme activity. Varying the initial cultivation pH of the

0 62 0 0

medium between pH 4–7 with intervals of 0.5 units had an effect both on growth and naringinase production by A. niger. Maximal enzyme activity (5.4 IU ml−1 ) was obtained when the initial pH of the culture medium was adjusted to 4.5 (Fig. 3a). Varying temperature at 3 ◦ C intervals between 27 and 30 ◦ C had little effect on enzyme activity whereas at 37 ◦ C enzyme activity was drastically reduced (Fig. 3b). Room temperature (28 ◦ C) was used for further studies. 3.8. Bioreactor studies The effect of initial sugar concentration on growth and enzyme production was studied in the bioreactor. The initial sugar concentration was varied from 4 to 10 g l−1 and the bioreactor was operated at constant aeration (0.5 vvm) and agitation (350 rpm) at 28 ◦ C. Cell growth, sugar utilization and enzyme production were measured up to 144 h. The production profile of naringinase in a 7 l bioreactor when the initial sugar concentration in the reactor was maintained at 4 g l−1 is shown in Fig. 4. Enzyme production is growth associated in nature and on attaining the log phase of growth enzyme biosynthesis begins. Maximal enzyme activity (8 IU ml−1 ) was obtained after 120 h. Higher naringinase productivity was obtained in the bioreactor than in shake

M. Puri et al. / Process Biochemistry 40 (2005) 195–201

flasks. The dissolved oxygen (DO) concentration in the reactor decreased very sharply from the very beginning and from the mid of exponential phase of the growth the DO concentration increased. The pH remained constant up to 24 h of fermentation and then increased. The residual substrate concentration became zero within 96 h of fermentation. Increasing sugar concentration (6 g l−1 ) in the medium did not increase enzyme activity. On doubling the molasses concentration (sugar, 8 g l−1 ) in the media, increased cell mass formation was observed in the bioreactor, but enzyme activity was repressed (4.6 IU ml−1 ). The decreased enzyme activity at higher molasses concentration (sugar, 10 g l−1 ) is attributed to substrate inhibition. On extending the fermentation time from 144 to 168 h, no significant improvement in enzyme activity was observed (results not shown). An increase in initial sugar concentration from 6 to 10 g l−1 in the fermentation medium decreased naringinase synthesis while growth increased with the increase in sugar concentration. At higher sugar levels (10 g l−1 ) growth was also reduced (Table 4). A significant effect of repression by higher sugar concentrations up to 30 g l−1 was observed for naringinase production by A. niger MTCC 1344 (results not shown). In fact higher concentrations of sugar resulted in a fall in naringinase yield. At higher sugar concentrations in the medium, there was dense growth of cell mass at the bottom and upper portion of the reactor resulting in non-uniform mixing in the reactor. The impellers were clogged by cell mass and problems of submerged cultivation of fungus were encountered. The repression of enzyme activity is due to higher

pH(4.0) pH(4.5) pH(5.0) pH(6.0)

4 3 2 1 0 0

2

4

6

8

10

8

10

Fermentation time (day)

(a)

Enzyme activity (IU/ml)

6

27˚C 30˚C 37˚C

5 4 3 2 1 0 0

(b)

2

4

6

Fermentation time (day)

Fig. 3. Effect of (a) initial pH and (b) temperature on naringinase production by A. niger.

Cell mass (g/l) Residual substrate (g/l) Enzyme activity (IU/ml) pH DO (%)

Cell mass, Residual substrate (g/l), Enzyme activity (IU/ml)

9 8

120

100 7 6

80

5 60 4

DO (%), pH

Enzyme activity (IU/ml)

6 5

199

40

3 2

20 1 0

0 0

16

24

40

48

64

72

88

96

120

144

Fermentation time (day) Fig. 4. Course of production of naringinase by A. niger in a bioreactor (initial sugar concentration 4 g l−1 , pH 4.5, DO 100%).

200

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Table 4 Effect of initial sugar concentration on the growth and enzyme production by A. niger in bioreactor Initial sugar (g l−1 )

Maximum cell mass (g l−1 )

Maximum specific growth rate (µ, h−1 )

Maximum enzyme activity (IU ml−1 )

Sugar consumption (%)

Maximum specific enzyme activity (IU g−1 , dw)

4 6 8 10

5.1 7.4 9.8 11.7

0.15 0.17 0.20 0.23

8.2 4.2 3.6 2.3

94 95 95 94

1608 568 367 197

concentrations of sugar. Higher sugar concentrations in the medium did not improve enzyme yield. In fact enzyme yield in terms of sugar utilized was higher at lower concentration of sugar (Table 4). Maximum specific enzyme activity decreased at higher sugar concentrations. With 4 g l−1 sugar, the maximum specific enzyme activity was eight times more than that at 10 g l−1 sugar in the medium. An initial sugar concentration of 4 g l−1 was found to be the best choice in terms of naringinase production.

4. Conclusion The production of naringinase by A. niger MTCC 1344 when grown in a synthetic medium is repressed in the presence of glucose. Sucrose and starch similarly suppress enzyme synthesis, although they support excellent growth. Media containing rhamnose, molasses and corn steep liquor produced higher enzyme titres, probably because these media have low carbohydrate contents. Growth factors are not required for enzyme yield. The majority of the reports to date found naringinase to be an inducible enzyme [20]. Continuous or step-wise addition of inducer increases naringinase production [19]. Replacement of the inducer with other carbon sources supports growth but no enzyme is produced. Fukumoto and Okada [13] reported the use of hesperidin as inducer along with soybean residue and corn steep liquor for naringinase production by Penicillium sp. In the present study naringenin was essential for enzyme production and its presence increased enzyme yield. Enzyme production was little affected by pH change in the range 4–6, but yields were low at pH values below 4. Since the strain of A. niger used in this study, normally produces citric acid, the natural tendency of the culture was to become very acidic. Further studies are required to ascertain the role of sugars in inhibiting naringinase production. Secondly, fed batch studies involving slow feeding of the concentrated sugar in the cultivation medium would enable and understanding of the sugar control mechanism in naringinase production. The results presented here demonstrate that among many methods to improve enzyme activity and yield, optimization of medium components and cultivation conditions remains a facile and feasible way to enhance enzyme activity as well as yield.

Acknowledgements M. Puri is grateful to Department of Biotechnology, Govt. of India, New Delhi for awarding Biotechnology National Associate Award (1997–1999) to pursue this work at IMTECH, Chandigarh. A. Banerjee gratefully acknowledges the fellowship provided by CSIR, Govt. of India.

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