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Screening of Penicillium species and optimisation of culture conditions for the production of ergot alkaloids using surface culture fermentation process.

Introduction

Ergot alkaloids are tryptophan derived secondary metabolites with appreciably toxicological and pharmacological applications. They have been identified in different species of fungi of Ascomycota and in many families of higher plants (Peter and Shu-Ming, 2013). The toxicity of ergot alkaloids make them pharmacologically very useful. All classes of ergot alkaloids have been used in formulating different medicines to cure various diseases e.g., migraine and to reduce post partum bleedings (Moussa, 2003). Many of the ergot alkaloids have also been used in the induction of labour contractions and for the inhibition of lactation. It can also terminate pregnancy and inhibition of mammary tumors (Fleiger et al., 1997; Masurekar, 1991; Floss et al., 1973).

Previously, only the members of family Clavicipitaceae (Hypocreales) (Genus: Claviceps) were used in the synthesis of ergot alkaloids. Other than genus Claviceps, genus Balansia was also used for biosynthesis of these alkaloids. Researchers are now focusing their attention to other members of class Ascomycetes e.g., species of genus Penicillium (P aurantiovirens, P chermisinum, P. ciricavirde, P sizovae, P roquefortii, P. corylophyllum, P kapuscinkii, P. regulosum and P. concavoruglosum) (Kozlovsky et al., 2013; Flieger et al., 1997; Hong and Robbers, 1985; Rao et al., 1977). Aspergillusfumigatus and Penicillium commune of the order Eurotiales are also capable of producing ergot alkaloid (Katy et al., 2013; Wallwey and Li, 2011; Panaccione, 2010).

The ergot alkaloids profile of Hypocreales is quite different from Eurotiales (Penicillium and Aspergillus species) (Panaccione, 2010; Lorenz et al., 2009). Clavicipitaceae often have ergot alkaloids profiles including clavine alkaloids, simple amides of lysergic acid and ergopeptine alkaloids. Members of Eurotiales are also capable of producing clavine alkaloids but festucaclavine, pyroclavine and fumigaclavines A, B and C alkaloids are produced by Penicillium and Aspergillus species only (Wallwey and Li, 2011). A wide range of ergot alkaloids are being used in pharmaceuticals which has made research very interesting on these species and a strong need has been felt to explore new species capable of producing significant compounds.

The present study was focused on the screening for the best fungal strain and the optimisation of culture conditions for enhanced production of ergot alkaloids using surface culture fermentation process. The presence of ergot alkaloids was also confirmed by TLC method. This study was also focused on the use of Penicillium commune for the alkaloids production because this species is locally isolated and is present in the culture bank of University of the Punjab, Lahore, Pakistan. The ergot alkaloid production profile of this species is very significant, specially the ergotamine and ergocriptine. Both these alkaloids have commercial importance in pharmaceutical applications.

Materials and Methods

Fungal species. Different species of genus Penicillium i.e., Penicillium commune, Penicillium italicum, Penicillium oxalicum, Penicillium digitatum and Penicillium sp., IIB were obtained from Fungal Culture Bank, Institute of Agricultural Sciences, University of the Punjab, New Campus, Lahore and from the Institute of Industrial Biotechnology, GC University, Lahore, Pakistan.

Maintenance of the fungal cultures. The cultures were maintained on malt extract agar (2 g malt extract and 2 g agar) slants. Spores from 7-10 days old slant were transferred to the freshly prepared slants and placed in incubator at 25[degrees]C for 10 days for the growth of mycelium.

Screening of fungal species and fermentation medium. Species of genus Penicillium i.e., P. commune, P. italicum, P. oxalicum, P. digitatum and Penicillium sp., IIB were screened for ergot alkaloids production. For this purpose, different fermentation media Ml, M2, M3, M4 and M5 were designed for the growth and production of ergot alkaloids with each of the above mentioned fungal species as described in Table 1.

pH of all the fermentation media was adjusted to 5.2 with the help of 0.1N HCl and ammonia solution. All flasks were autoclaved at 121[degrees]C for 15 min. 5 mL of spore suspension ([10.sup.6-7] spores/mL) of each fungal species was transferred to the respective flasks containing the above mentioned fermentation media. After inoculation these flasks were incubated at 25[degrees]C for 21 days.

Optimisation of culture conditions for ergot alkaloids production. Fermentation medium was further optimised and following culture parameters were studied for the optimal growth of ergot alkaloids.

Effect of different carbon sources. In order to get the maximum production of ergot alkaloids by P. commune, different carbon sources were used e.g., glucose, fructose, maltose, sucrose, mannose and mannitol.

Effect of different nitrogen sources. Various organic and inorganic nitrogen sources were used to optimise the best nitrogen source for the maximum production of ergot alkaloids e.g., yeast extract, peptones, malt extract, meat extract, ammonium chloride and urea.

Effect of different concentrations of sucrose. Different concentrations of sucrose i.e., 10 g, 15 g, 20 g, 25 g, 30 g, 35 g and 40 g were used to optimise the best substrate concentration for the highest yield of ergot alkaloids.

Effect of different concentrations of yeast extract. Different concentrations of yeast extract i.e., 5 g, 10 g, 15 g, 20 g, 25 g and 30 g were used to optimise the best yeast extract concentration for the highest yield of ergot alkaloids.

Effect of different concentrations of phosphate. Different concentrations of K[H.sub.2]P[O.sub.4] were used i.e., 0.5 g, 1 g, 1.5 g, 2.0 g, 2.5 g and 3 g in selected medium to optimise the best concentration of K[H.sub.2]P[O.sub.4] for the highest production of ergot alkaloids.

Effect of different pH levels. The pH was adjusted to different pH values ranging from 3 to 8 by using ammonia solution and 0.1N HCl.

Effect of different inoculum sizes. Different inoculum sizes i.e., 5 mL, 10 mL, 15 mL, 20 mL, 25 mL and 30 mL were used to get the maximum production of ergot alkaloids in the fermentation medium.

Final batch of P. commune with optimised culture conditions. Ergot alkaloids production was also achieved from the final batch of fermentation medium when P. commune was grown on the above mentioned optimised fermentation conditions i.e., 35% sucrose, 30% yeast extract, 2 g K[H.sub.2]P[O.sub.4], 15 mL inoculum size and pH 5.

Estimation of ergot alkaloids. Preparation of supernatant extracts. The ergot alkaloid was estimated in fermented broth after centrifugation at 4[degrees]C and at 5000 rpm for 10 min. Mycelia of all the samples were also collected separately in petri plates. The cell biomass free fermented broth was then purified through rotary evaporator.

Preparation of extracts of dry cell biomass. The mycelia that separated from the fermentation broth were initially weighed and placed in oven for drying at 45[degrees]C for 24 h. Then weighed again to measure the dry weight of the mycelium. These dry mycelia were placed in methanol for 24 h and then subjected to cell lysis by sonication process for 5 cycles of 3 min at 200 rpm/min for every mycelium. After sonication, for further cell lysis, all the sonicated material was homogenised in a homogeniser for 3 cycles of 5 min so that all of the compounds of ergot alkaloids may be released from the mycelium of fungi (Naude et al., 2005). The mixture after homogenisation was again centrifuged and the supernatants were collected.

Assay of alkaloids. Chloroform extraction process was used for the extraction of ergot alkaloids made by two fungal species following method described by Moussa (2003). Ergot alkaloids were extracted 3 times in 50 mL of chloroform in a separating funnel by separating the layers. The chloroform extracts were evaporated to dryness at 40[degrees]C in a rotary evaporator. The residues containing ergot alkaloids were estimated after Moussa (2003). 1 mL of each of the culture filtrates was added to 2 mL of Van Urk Reagent in the test tubes and reaction mixture was incubated at 37[degrees]C for 30 min (Smith, 1930; Van Urk, 1929). The absorbance was measured at 590 nm by spectrophotometer. The amount of alkaloids present was compared with dihydroergotamine methane sulphonate salt and bromocriptine mesylate salt.

The residues were also analysed by thin layer chromatography on silica gel strips using the mobile phase of chloroform:methanol:ammonia solution in 80:20:0.5 proportion. Pure samples of methylergotamine maleate, dihydroergotamine methane sulphonate salt and bromocrptine mesylate were run as the reference to identify the possible alkaloids spectrum. Coloured spots were developed on the TLC plates by spraying Van Urk reagent according to the procedure of Stahl and Ashworth (1969).

Results and Discussion

Species of genus Penicillium i.e., Penicillium commune, Penicillium italicum, Penicillium oxalicum, Penicillium digitatum and Penicillium sp., IIB were obtained from Fungal Culture Bank, Institute of Agricultural Sciences, University of the Punjab, New Campus, Lahore and from the Institute of Industrial Biotechnology, GC University, Lahore, Pakistan.

Penicillium commune and M5 fermentation medium was recorded as the highest ergot alkaloids producer and best fermentation medium, respectively (1.36 mg/L) (Table 2).

Optimisation of some culture conditions for production of ergot alkaloids. Effect of various carbon sources. In order to obtain the maximum yield of ergot alkaloids, various carbon sources, i.e., glucose, fructose, sucrose, maltose, mannose and mannitol were used in the M5 fermentation medium. Sucrose was optimised as the most favourable carbon source for the mycelium growth (2.21 g/L) and ergot alkaloids production (2.60 mg/L) as indicated in Table 3.

Effect of various nitrogen sources. In order to obtain the maximum yield of ergot alkaloids, various nitrogen sources i.e., yeast extract, peptones, malt extract, meat extract, ammonium chloride and urea were used in the 100 mL of M5 fermentation medium. Yeast extract was the most favourable nitrogen source for the mycelium growth (2.56 g/L) and ergot alkaloids (2.90 mg/L) as given in Table 3.

Effect of different concentrations of sucrose. Various concentrations of sucrose i.e., 10 g, 15 g, 20 g, 25 g, 30 g, 35 g and 40 g were used in the 100 mL of M5 fermentation media and the best growth of mycelium (2.54 g/L). The highest yield of alkaloids production (2.72 mg/L) was obtained at 35 g of sucrose (Table 4).

Effect of different concentrations of yeast extract. Different concentrations of yeast extract i.e., 5 g, 10 g, 15 g, 20 g, 25 g and 30 g were used to optimise the best yeast extract concentration for the highest yield of ergot alkaloids in the selected optimised fermentation medium. 30 g of the yeast extract gave maximum yield of alkaloids (2.41 mg/L) and the highest growth of mycelium (2.15 g/L) (Table 4).

Effect of various concentrations of K[H.sub.2]P[O.sub.4]. Different concentrations of K[H.sub.2]P[O.sub.4] were used i.e., 0.5 g, 1 g, 1.5 g, 2.0 g, 2.5 g, and 3.0 g in selected M5 medium. 2 g of K[H.sub.2]P[O.sub.4] gave maximum production of alkaloids (1.53 mg/L) and fungus growth (2.50 g/L) as shown in the Table 5.

Effect of different pH levels. The pH of the fermentation medium had a great influence on the growth of fungal mycelia and the production of ergot alkaloids by Penicillium commune. pH of the medium ranged from 3-8. Ergot alkaloids production (2.13 mg/L) and the growth of fungal mycelium (2.22 g/L) was found maximum at pH 5 (Table 6).

Effect of different Inoculum sizes. Different sizes of inoculums i.e., 5 mL, 10 mL, 15 mL, 20 mL, 25 mL and 30 mL, were used in the M5 fermentation media and the best inoculum size 15 mL was optimised as the best alkaloids producing inoculum size (3.34 mg/L) fungal mycelium growth (2.94 g) (Table 6).

Final batch of P. commune. A yield of 4.32 mg/L of ergot alkaloids was achieved when P. commune was grown on the concluding fermentation medium containing all the above mentioned optimised culture condition (35% sucrose, 30% yeast extract, 2 g K[H.sub.2]P[O.sub.4], 15 mL inoculum size and pH 5).

Penicillium commune was screened as the best species amongst all the above mentioned species for the production of ergot alkaloids by surface culture fermentation process. Production of ergot alkaloids was also achieved by Penicillium concavorugulosum (Abe et al., 1969) and Penicillium chermesinum (Agurell, 1964) and many other species of genus Penicillium reported by Moussa (2003).

The optimum conditions for the growth of fungal mycelium and the yield of ergot alkaloids were (a) sucrose: 35 g (b) yeast extract: 30 g (c) carbon source (sucrose) (d) nitrogen sources (yeast extract) (e) pH 5, (f) K[H.sub.2]P[O.sub.4]: 2 g and 15 mL inoculum size.

The fungal organism must be provided with a healthy medium in which it can grow and sporulate well and can produce maximum amount of ergot alkaloids. Penicillium commune produced a high amount of ergot alkaloids when it was grown in M5 fermentation medium. Different fermentation media were also used for the production of ergot alkaloids when they were inoculated by species of Balansia described by Bacon et al. (1975).

In terms of sucrose and other carbon sources utilisation, sucrose was best amongst all the carbon sources for fungal species. It was seen that with the increase in the amount of sucrose there was a remarkable increase in the growth of mycelium and production of ergot alkaloids. Glucose, fructose, maltose suppressed the growth of fungus and yield of alkaloids in the fermentation medium. These results are in the harmony of conclusion given by Moussa (2003) that sucrose is the best carbon source for the production of ergot alkaloids giving a maximum yield of 0.800 mg/L in the fermentation medium. Similar results were also obtained by Socic and Gaberc-Porekar (1992) and Arcamone et al. (1961). On the other hand glucose and fructose had suppressed the yield of ergot alkaloids (Drew and Wallis, 1983).

The nitrogen level of the culture medium proved to be an important factor influencing the growth and yield of alkaloids. Therefore, why different nitrogen sources were also used in the experiment and they influenced the growth of the mycelium and the production of ergot alkaloids in the fermentation medium by surface culture fermentation process. Yeast extract showed a remarkable effect on the growth of the mycelium and on the yield of ergot alkaloids. Ammonium chloride and urea had a negative effect on the fungal mycelium production and ergot alkaloids production. Moussa (2003) and Taber and Vining (1958) also described a negative effect of ammonium chloride on growth of mycelium as well as on the yield of ergot alkaloids.

The pH value of the fermentation medium put a great influence on the growth of the mycelium and production of ergot alkaloids. It was shown that with the increase in pH value there was an increase in the growth of the fungus and yield of the alkaloids. The pH 5 was remarkably best amongst all pH values for the best fungal growth and production of alkaloids. Similar results were also described by Moussa (2003) and Mizrahi and Miller (1970), who reported that optimum pH 5 was proved to be the best for the maximum yield of alkaloids.

Variation of K[H.sub.2]P[O.sub.4] clearly affected the fungal growth and the production of ergot alkaloids. It was noted that with the increase in the phosphate concentration there was an increase in the mycelium growth and the production of ergot alkaloids but with the maximum increase in the phosphate there was a decrease in the fungal growth and the alkaloids production. Similar results were also achieved by experiments on Claviceps species by Rehacek et al. (1971). Taber and Vining (1958) studied the influence of low quantity of phosphate in the carbohydrate rich medium and best ergot alkaloids production was analysed but the production of ergot alkaloids was found to be inhibited with the increase in phosphate amount. Brady and Tyler (1960) also determined that phosphate is an important nutrient for alkaloids production. These results are also in harmony with the results of Taber (1967), Mary et al. (1965) and De Waart and Taber (1960). It can be described that the excess phosphate required for the production of protein and nucleic acids, prolongs the growth phase of the organism and during this phase amino acids e.g., tryptophane is used for the protein synthesis (Robbers et al., 1972; Weygand and Floss, 1963).

Different inoculum sizes in the form of spore suspension material markedly effect the production of ergot alkaloids and mycelial production in the fermentation medium. With the increase in the spores suspension size, there was an increase in the production of ergot alkaloids and when this was increased more in the fermentation media then there was a decrease in the production of alkaloids due to the limited nutrients availability in the fermentation media for the fungal spores. These results are similar with the results of Meyrath (1973) in which they described that with the increase in the inoculum size there was an increase in the alkaloids production.

Conclusion

Ergot alkaloids are pharmaceutically very significant natural alkaloids and their derivatives are very expensive. In the present study, ergot alkaloids were produced from Penicillium commune and ergotamine and ergocriptine alkaloids were identified which can be used to cure migraine and to stop post partum bleedings. This is a sustainable and a very cost effective way for the production of these useful alkaloids in the laboratory and this is the need of today in pharmaceutical industry.

References

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Memuna Ghafoor Shahid (a) * and Muhammad Nadeem (b)

(a) Department of Botany, GC University, Lahore, Pakistan

(b) Food and Biotechnology Research Center, PCSIR Laboratories Complex, Roomi Road, Lahore, Pakistan

* Author for correspondence; E-mail: memunaghafoorshahid@gmail.com

(received January 15, 2014; revised November 5, 2014; accepted December 8, 2014)
Table 1. Various fermentation media and their composition

                                   Fermentation medium
Ingredient
(conc. %)                M1       M2       M3       M4       M5

Sucrose                  -        5        5        -        5
Mannitol                 5        -        -        5        -
Peptones                 -       0.1       -        -        -
N[H.sub.4]OH             -       0.01      -        -        -
N[H.sub.4]Q              -        -        -       0.2      0.2
Succinic acid           0.5       -        -       0.54     0.5
Yeast extract            -        -       0.3       -       0.5
Tryptophane              -        -        -      0.025     0.5
Asparagine               -        -        -        -       0.5
[K.sub.2]HP[O.sub.4]     -       0.1       -        -        -
K[H.sub.2]P[O.sub.4]     -        -       0.1      0.5      0.5
MgS[O.sub.4].           0.01     0.15    0.001     0.03     0.03
  7[H.sub.2]O
FeS[O.sub.4]            0.01    0.001    0.001    0.001     0.01
ZnS[O.sub.4]           0.004    0.002    0.001    0.001    0.002

Table 2. Optimisation of fungal species and fermentation media for
the production of ergot alkaloids

Fungal                            Fermentation media
organism
                              M1                        M2

                  Supernatant    Mycelium    Supernatant   Mycelium
                   alkaloids     alkaloids    alkaloids    alkaloids
                     conc.         conc.        conc.        conc.

                                        (mg/mL)

P. commune            0.01         0.01         0.096        0.13
P. italicum          0.001         0.002        0.07         0.01
P. oxalicum          0.001         0.001        0.09         0.04
P. digitatum         0.010         0.01         0.096        0.05
Penicillium sp.       0.01         0.06         0.076        0.086
Strain IIB

Fungal                              Fermentation media
organism
                              M3                          M4

                  Supernatant     Mycelium    Supernatant     Mycelium
                   alkaloids     alkaloids     alkaloids     alkaloids
                     conc.         conc.         conc.         conc.

                                          (mg/mL)

P. commune            0.30          0.09          0.79          0.51
P. italicum           0.14          0.09          0.43          0.09
P. oxalicum           0.25          0.08          0.05          0.10
P. digitatum          0.96          0.08          0.09          0.14
Penicillium sp.       0.96          0.76          0.98          0.65
Strain IIB

Fungal                Fermentation media
organism
                              M5

                  Supernatant     Mycelium
                   alkaloids     alkaloids
                     conc.         conc.

                           (mg/mL)

P. commune            1.36          0.96
P. italicum           0.49          0.13
P. oxalicum           0.09         0.001
P. digitatum          0.14         0.095
Penicillium sp.       1.15          0.64
Strain IIB

Table 3. Effect of carbon and nitrogen sources on the growth of
P. commune and production of ergot alkaloids

Culture                   Supernatant       Mycelial       Mycelium
condition                 alkaloids conc.   alkaloids      growth
                          (mg/L)            conc. (mg/L)   (g/L)

Carbon source (5g/L)
  Glucose                 1.83              0.46           1.45
  Fructose                1.80              0.54           1.35
  Maltose                 1.72              0.61           1.90
  Sucrose                 2.60              1.89           2.21
  Mannose                 1.82              1.35           1.63
  Mannitol                2.15              1.60           1.84

Nitrogen sources (5g/L)
  Yeast extract           2.90              1.48           2.56
  Peptones                2.31              1.40           2.35
  Malt extract            1.00              1.04           2.01
  Meat extract            0.61              1.05           2.00
  Ammonium                0.81              0.34           1.21
  Chloride
  Urea                    0.13              0.14           1.01

Table 4. Effect of sucrose and yeast extract on the growth
of P. commune and production of ergot alkaloids

Culture                Supernatant     Mycelial        Mycelium
condition              alkaloids       alkaloids       growth
                       conc. (mg/L)    conc. (mg/L)    (g/L)

Sucrose (g/L)
  10                   0.14            0.03            1.60
  15                   0.38            0.31            1.41
  20                   0.70            0.68            1.92
  25                   0.96            0.96            1.86
  30                   1.66            1.23            1.68
  35                   2.72            1.68            2.54
  40                   2.33            1.34            1.99

Yeast extract (g/L)
  5                    0.43            0.28            1.16
  10                   0.66            0.51            1.34
  15                   0.80            0.61            1.60
  20                   0.81            0.91            2.11
  25                   1.91            1.24            1.80
  30                   2.41            1.80            2.15

Table 5. Effect of various concentrations of K[H.sub.2]P[O.sub.4] on
the growth of P. commune and ergot alkaloids production

                                   P. commune

K[H.sub.2]       Supernatant        Mycelial       Mycelium
P[O.sub.4]     alkaloids conc.   alkaloids conc.    growth
amount (g/L)       (mg/L)            (mg/L)         (g/L)

0.5                 0.27              0.15           1.13
1                   0.54              0.25           1.60
1.5                 0.96              0.45           2.34
2                   1.53              0.99           2.50
2.5                 1.34              0.43           2.00
3                   1.34              0.84           1.96

Table 6. Effect of pH and various inoculums sizes on
the growth of P. commune and ergot alkaloids production

Culture                Supernatant     Mycelial        Mycelium
condition              alkaloids       alkaloids       growth (g/L)
                       conc. (mg/L)    conc. (mg/L)

pH
  3                    0.81            0.62            1.60
  4                    1.72            0.96            1.54
  5                    2.13            1.95            2.22
  6                    1.74            1.56            1.96
  7                    1.90            0.95            2.15
  8                    1.34            0.43            1.88

Inoculum sizes (mg/L)
  5                    0.80            0.56            0.99
  10                   2.48            0.98            1.98
  15                   3.34            2.76            2.94
  20                   1.96            1.38            2.34
  25                   1.98            2.60            3.23
  30                   1.38            1.78            1.91
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Author:Shahid, Memuna Ghafoor; Nadeem, Muhammad
Publication:Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences
Article Type:Report
Geographic Code:9PAKI
Date:Mar 1, 2015
Words:4713
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