Antibacterial Activity of Culture Extracts of Penicillium--论文代写范文精选

青霉素,也许是最重要的治疗药物。β-lactam抗生素的抗菌作用是有效的，由不同类型的细菌产生β-内酰胺酶,虽然这些导致更便宜和有效的青霉素的可用性,进一步减少抗生素的生产成本。可以通过低成本发酵底物的使用。下面的paper代写范文进行讲述。

Abstract
Penicillium chrysogenum PCL501 produced β-lactam antibiotics when fermented with different agro-wastes: cassava shavings, corncob, sawdust and sugarcane pulp. In vitro antibacterial activity of the culture extracts was tested against four clinical bacterial isolates, namely, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. All the culture extracts and standard drug (commercial Benzyl Penicillin) inhibited the growth B. subtilis and E. coli; the potency varied with carbon source. Antibacterial activity of extracts from cultures containing cassava shavings and sugarcane pulp was comparable with that of the standard drug. The MIC against the susceptible organisms was 0.20mg/ml for the standard drug and ranged from 0.40 to 1.50mg/ml for the culture extracts. Neither the culture extracts nor the standard drug inhibited K. pneumoniae and P. aeruginosa; the bacterial strains produced β-lactamase enzymes. Cassava shavings and sugarcane pulp are indicated as suitable cheap carbon sources for the production of antibiotics by Penicillium chrysogenum PCL501. 
Key Words: Agro-wastes, Penicillium chrysogenum PCL501, Antibiotic production, Phenyl penicillin, Antibacterial activity, β- lactamase

Introduction
Penicillium chrysogenum (formerly, Penicillium notatum) is an important industrial organism due to its ability to produce several β-lactam antibiotics, particularly penicillins.1 The chance discovery of Penicillium notatum by Alexander Fleming and the production of the revolutionary drug, penicillin, is perhaps the most important finding in the history of therapeutic medicine.2 Two naturally occurring and commercially available penicillins are Benzyl penicillin (Penicillin G) and Phenoxy-methyl penicillin (Penicillin V). The R-group substituent of the penicillin nucleus can be substituted to give the molecule different antibacterial properties. The antibacterial effect of β-lactam antibiotics is effectively nullified by different types of bacteria which produce β- lactamase, an enzyme that breaks the β-lactam ring.3 Clinical isolates of extended-spectrum β-lactamase (ESBL)-producing bacteria have been reported in different regions of the world.4-6

Efforts at improving penicillin yields have centred on growth optimization, development of available strains of P. chrysogenum by classical mutagenesis procedures, and the search for better strains of the organism.7,8 Although these have led to the availability of cheaper and effective penicillins, further reduction in production cost of the antibiotics could be achieved by the use of low-cost fermentation substrates. Residual plant materials in urban refuse can serve as cheap carbon and energy sources for fermentation instead of refined sugars such as glucose and lactose.9,10 This could turn the recalcitrant waste plant biomass into a valuable resource and reduce the pollution problem caused by its accumulation in the environment.11,12 In Nigeria, agro-industrial wastes abound in the form of wood-wastes and crop residues such as cassava shavings, corncobs and sugarcane pulp.13

In this study, a strain of P. chrysogenum (PCL501) was fermented on four major waste cellulosic materials (cassava shavings, corncob, sawdust and sugarcane pulp) produced in Lagos, Nigeria and the culture extracts were tested for antibacterial activity against four clinical bacterial isolates (Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). The fungus thrives on sawdust and sugarcane pulp14 and produces hydrolytic enzymes such as cellulases15 xylanases16,17 and pectinases (unpublished data) in media containing agrowastes. The present results show that the strain of P. chrysogenum (PCL501) produces effective β-lactam antibiotics with antibacterial activity comparable to commercial benzyl penicillin (Retarpen, Sandox, Austria). Two waste plant materials, cassava shavings and sugarcane pulp, are indicated as suitable low-cost substrates for the production of antibiotics by the strain of P. chrysogenum.

Preparations of Agro-waste materials
Sawdust of Abora wood (Mitragyna ciliata) was collected from sawmills at Ikorodu, Lagos, Nigeria. Mature sugarcane (Saccharum offinarum) stems, fresh maize (Zea mays) and cassava shavings (Manihot esculenta) were purchased from a local market in Mushin, Lagos, Nigeria. The crushed sugarcane pulp was soaked overnight and washed repeatedly in distilled water until no trace of simple sugar was detected. Corncob was obtained by removing the maize grains and cut into small sizes. The materials were separately dried at 80oC to constant weight in the oven, and milled using Marlex Exceller grinder (Mumbai, India). Fine powder obtained after passing each through a sieve of 0.5 mm pore size was used as substrate in the fermentation media.

Growth culture and antibiotic production
The fungus was sub-cultured on PDA plates and incubated at 30oC for 3-5 days to obtain the spores used for antibiotics production. Spores were washed into a sterile beaker using 0.1% Tween 80 in 0.1M potassium phosphate buffer at pH 7.0. The spore suspension was standardized such that 1 in 10 dilutions has an Absorbance of 0.48 at 530nm. Fifty millilitres (50 ml) of the spore suspension was aseptically introduced into a litre of sterile fermentation media containing per litre of distilled water: 6.0g Ammonium acetate, 0.5g NaSO3, 0.02g ZnSO4.7H2O, 0.25g MgSO4.7H2O, 6.0g KH2PO4, 0.02g FeSO4.7H2O, 0.5g Phenylalanine, and 10.0g carbon source (glucose, lactose, cassava shavings, corncob, sawdust or sugarcane pulp). The pH was adjusted to 6.0. The flasks were incubated with intermittent shaking for 21 days after which the contents were sieved through cotton wool and filtrate centrifuged to remove cells. The pH of the supernatant was adjusted to 2.5 with chloroformphosphate buffer (20:1) and assayed for Antibacterial activity.

Antibacterial Sensitivity Testing
Antibacterial activity of the culture extracts was assayed by a modified method of Grau and Halliday.19 Nutrient agar plates were seeded with 0.1 ml of an overnight culture of each clinical isolate (equivalent to 107 – 108 CFU ml-1). A sterile cork borer of 8 mm diameter was used to cut three uniform wells on the surface of the agar after 24 hours of incubation. The wells on each plate were then filled with 0.3 ml of a particular culture extract and the effect on the growing "lawns" of each clinical isolate was monitored at intervals of 24 hours. This was repeated for each culture extract and the standard drug. Zones of clearance round each well means inhibition and the diameter; such zones were measured after a marked decline in the potency of the antibiotics to inhibit the growth of the test organisms was noticed.

Determination of MIC 
Minimum inhibitory concentration (MIC) was determined using the agar dilution guideline of NCCLS as described by Enwuru et al.20 The concentrations with inhibitory zone diameter of 10.0 mm were chosen for the assay. Different dilutions of the extracts were prepared to give final concentration in the range of 2.0, 1.0, 0.8, 0.6, 0.4 and 0.2 mg ml-1 were prepared from a stock solution of 5mg ml-1. One milliliter (1 ml) of each dilution was mixed with 18 ml of Mueller Hinton agar (MHA, Difco, France) and poured into Petri-dishes and allowed to set. The agar was streaked with an overnight broth culture of the clinical isolates (adjusted to turbidity equivalent to 0.5 McFarland standards) and incubated at 37°C for 24 hours. Controls containing only the nutrient agar and test organisms were set up. The plates were then examined for the presence or absence of growth. The MIC was defined as the lowest concentration of the extract inhibiting the visible growth of each organism.

Tests for β-Lactamase activity
The presence of β-lactamase enzyme in the clinical isolates was determined using β-lactamase identification sticks (Oxoid, Wesel, Germany) with nitrocefin as the substrate.21 Cells from a 24-hour culture of each isolate were collected into a test tube and lysed by sonication. This was centrifuged at 4000g for 15 minutes. The supernatant diluted 1 in 100 with nutrient broth and a β- lactamase identification stick with nitrocefin as the substrate was inserted into it and allowed to stand for 30 minutes. Presence of β-lactamase was indicated by a colour change from light to deep pink.

Test for β-Lactam antibiotics 
Culture extracts of P. chrysogenum PCL501 were tested to confirm if the antibacterial activity was due to the presence of β-lactam antibiotics. The supernatant containing β-Lactamase enzyme in the previous assay was used for the test. The supernatant (0.5 ml) was incubated with 0.5 ml of the culture extract and allowed to stand for 30 minutes at room temperature. The mixture was introduced into wells on agar plates streaked with β- Lactamase-free E. coli strain. The plates were observed for inhibition after 24 hours of incubation. Absence of noticeable zone of inhibition confirms the presence of β- lactam antibiotics.

Determination of Potency
The potency of the extracts was determined by a modification of Carter’s method as described below.21 Three plates were used for each sample solution. On each plate were two cylinders were filled with the reference dose of standard of known concentration, two cylinders with the sample solution, and two cylinders with an aliquot of the sample solution that has been treated with the supernatant containing β-Lactamase enzyme. The plates were incubated overnight at 30oC and the diameters of the zones of inhibition were measured. A positive test for the presence of an antibiotic residue is indicated by the production of zones of inhibition by the sample solution and the absence of any detectable zones of inhibition by the β-lactamase-treated portion of the sample solution. The mean responses for the sample and the reference dose of standard were determined and the concentration of sample per milliliter of solution was calculated.(paper代写)

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