Comparative Analysis of Penicillin Production Using Various Media Compositions: A Research Study

Background: Penicillin are bactericidal beta-lactam antibiotics that inhibit bacterial cell wall synthesis. A natural product, the penicillin structure has been modified to prepare a variety of semi-synthetic agents and used in pharmaceutical industries. The spectrum of antibacterial activity varies with each class of the penicillin family produced by penicillium species. Penicillin is generally well-tolerated, with hypersensitivity being the major adverse effect. Penicillin is used to treat a variety of conditions including skin infections, urinary tract infections, upper and lower respiratory infections, and endocarditis. Methods: Preparation of different media compositions to check the increased growth of Penicillium notatum and its ability to produce penicillin crystals using the varied compositions. The obtained penicillin from the extract we checked for the antibacterial activity against various microbial species. Result: We have successfully established that Penicillin can be useful drug for the treatment of serious Pseudomonas aeruginosa infections because of its enhanced activity against this organism. This indicates that Penicillin still could serve as medicinal drug and antimicrobial activity in pharmaceuticals for treatment of many pathogenic diseases. Conclusions: The above research work concludes that high yield of penicillin production can be obtained using sabouraud dextrose broth medium with wheat bran as the substrate in industrial means. It shows the efficacy of the Penicillin against various infection and can inhibit the growth of some organisms causing infection


INTRODUCTION
Penicillin is one of the most important and widely used antibiotics in modern medicine.It is a group of naturally occurring and synthetic antibiotics that are effective against a broad spectrum of bacterial infections.The discovery of penicillin marked a significant milestone in the history of medicine and revolutionized the treatment of infectious diseases.Penicillin was discovered by Sir Alexander Fleming, a Scottish bacteriologist, in 1928(BornsideGH, 1968).While working at St. Mary's Hospital in London, he accidentally observed that a mold called Penicillium notatum had contaminated a bacterial culture plate, leading to the inhibition of bacterial growth around the mold.He identified this mold as the source of a potent antibacterial substance, which he named penicillin (Bodey G P,1971).Penicillin is an antibiotic, which means it is a type of medication that kills or inhibits the growth of bacteria.It targets the bacterial cell wall, disrupting its formation and weakening the bacteria, ultimately leading to bacterial cell death.After Fleming's initial discovery, further research was conducted to isolate and produce penicillin in larger quantities.This involved the work of scientists Howard Florey, Ernst Boris Chain, and Norman Heatley at the University of Oxford during the 1940s ( Schatz et.al., 1944).They successfully developed methods to mass-produce penicillin, which played a crucial role in treating wounded soldiers during World War II.Penicillin and its derivatives have saved countless lives since their introduction into medical practice.Prior to its discovery, bacterial infections were a leading cause of death, and treatment options were limited.Penicillin was a breakthrough in the fight against bacterial diseases like pneumonia, strep throat, syphilis, and various skin infections (Landesman et.al., 1981) .Over time, various forms of penicillin have been developed to enhance their effectiveness against different types of bacteria and to address issues of resistance.The substrate used for penicillin production is typically a nutrient-rich medium that provides essential nutrients for the growth of the Penicillium mold, which is the microorganism responsible for producing penicillin.Penicillium fungi require a carbon source, nitrogen source, minerals, and other growth factors to thrive and produce penicillin (Rocha et. al.,2019).The primary substrate used for industrial-scale penicillin production is a liquid medium, often based on corn steep liquor or lactose as the carbon source and a nitrogen source such as corn steep liquor, soybean meal, or a combination of both.The medium may also contain various salts, vitamins, and other growth-promoting agents.The process of penicillin production involves fermentation, during which the Penicillium mold is grown in large fermentation tanks or bioreactors using the nutrient-rich substrate.
The mold secretes penicillin into the medium during its growth phase, and once the desired level of penicillin production is achieved, the fermentation is halted, and the penicillin is isolated and purified from the medium for further processing and use as an antibiotic.Penicillin is a highly effective antibiotic that exhibits potent antibacterial activity against a wide range of bacteria.It is particularly effective against Gram-positive bacteria.The antibacterial activity of penicillin is primarily due to its ability to interfere with bacterial cell wall synthesis ( Dede et.al., 2020) .
Around the world, antibiotics are essential for controlling infectious ailments that affect people, animals, cattle, and aquacultures.The potential hazard to all microorganisms in these habitats is created by the introduction of an increasing number of antibiotics into soils and streams (Vazquez-Munoz et al., 2019).It is crucial to create innovative antibiotic agents and therapies to control bacterial infections because the capacity of microorganisms to build resistance out competes the production of new and effective antibiotics (Cycon M, 2019).
In this study we have experimented with numerous media compositions in search of the simplest means to produce penicillin, checking the increasing production as we want.Penicillin primarily disrupts the production of the bacterial cell wall, which leads to an incorrect construction of the cell wall.This ultimately causes the cell to rupture by allowing water to enter the cell wall.The zone development surrounding the well with penicillin on a culture plate can be used to pinpoint the location (Girolami et.al., 1980).

METHOD 1. Isolation of Penicillium
10 gm of garden soil sample was collected and serial dilution was performed.Spread plate method was conducted in the Sabouraud Dextrose agar medium.The plates were incubated at room temperature for 7 days (Schwartzkroin et, al., 1977).

Staining Technique
After the incubation period the organism was stained using Lactophenol cotton blue stain.

Production Media Preparation
Three different mediums (Sabouraud Dextrose Broth, Rose Bengal Broth, Potato dextrose Broth) were prepared for 200ml and added 1g of wheat bran to each medium as substrate.The production media was sterilised in an autoclave at 121°C for 15 mins.After sterilisation, cool down the production media to room temperature.The isolated Penicillium sp. was Inoculated in the three different production media and kept for incubation for 25 days at room temperature.

Extraction of penicillin
The mat is taken out and crushed with the help of mortar and pestle using 50 ml of sterile distilled water.The remaining broth was filtered using Whatman no.1 filter paper.The crushed mat and the broth were centrifuged at 8000 rpm for 15 mins.The supernatant was collected in a separate tube and tested for antimicrobial activity (Sharifzadeh et. al., 2020).

Antimicrobial test
9 Muller Hinton agar plates were prepared and sterilised in an autoclave at 121 O C for 15 minutes.After the sterilisation the media were allowed to cool and poured into the respective sterile Petri plates and kept for solidification.After solidification of media four wells were cut by aseptic method in each plate (Lopez et. al., 2001).Using a sterile cotton swab 9 different organisms like Salmonella sp., Vibrio sp., Staphylococcus aureus, Staphylococcus cohnii, Rhodococcus sp., Klebsiella sp., Pseudomonas sp., Candida sp., Bacillus sp. were swabbed on each plate.Different concentrations (0.1, 0.3,0.5ml) of the filtrate was added to the well and one well acted as control without adding the filtrate.The plates were Incubated for 24 hours at 37°C.

RESULT
The findings of this study indicate that Penicillin has potential value in the management of many types of serious community-acquired infections.The organisms that cause these infections, with the exception of Staphylococcus aureus, are likely to be susceptible to Penicillin.Penicillin may also be a useful drug for the treatment of serious Pseudomonas aeruginosa infections because of its enhanced activity against this organism.These results indicate that Penicillin still could serve as medicinal drug and antimicrobial activity in pharmaceuticals for treatment of many pathogenic diseases.

DISCUSSION
Penicillium was isolated from the soil sample by performing serial dilution method and confirmed by lactophenol cotton blue staining observed under a 40X microscopic lens.Further using three different production media such as Potato Dextrose Broth (PDB), Rose Bengal Broth (RBB), Sabouraud Dextrose Broth (SDB) for penicillin production.After 25 days the yellow crystal penicillium mat was found only on the Sabouraud Dextrose Broth (SDB).From the extract of broth filtrate penicillin as a secondary metabolite was obtained and gave a positive result for the antimicrobial test by forming clear zones around the wells of plate inoculated with the test organisms (Dickinson et. al., 1978).This Data indicates the antagonistic property of Penicillin by antimicrobial analysis (Pisabarro et. al.,1986).The strain used is Penicillium notatum produced using 3 different types of production media where wheat bran is used as a substrate.Penicillin, a secondary metabolite of particular strain, can be found in sabouraud dextrose broth through industrial means of production.To demonstrate penicillinase activity in vitro method against various microorganisms strains like Salmonella sp., Vibrio sp., Staphylococcus aureus, Staphylococcus cohnii, Rhodococcus sp., Klebsiella sp., Pseudomonas sp., Candida sp., Bacillus sp..However, the penicillinase activity in the culture broth of Penicillium notatum, when minimal inhibitory concentration (MIC) procedures were tested in different concentrations (Khassaf et. al.,2009).
The antimicrobial behaviour of penicillin, as compared with test organisms, can best be illustrated by an examination of the respective in-vitro means, as presented in Table 1.Concentrated preparations, from the purification of the broth were used.Some strains of Bacillus and Candida species show resistance to Penicillin but that most of the negative organisms isolates are sensitive by checking MIC against Penicillin (Niksic et. al., 2018).
The majority of antibiotics that are now known to exist, such as penicillin and other mould compounds, work on bacteria.Numerous additional antibiotics have been developed as a result of the discovery of penicillin.One of the most significant and popular antibiotics in contemporary medicine is penicillin (Brook, 2016) Like penicillin, which has antagonistic qualities against a variety of organisms, these compounds' action towards organisms is quite selective.It is crucial to understand that penicillin only kills bacteria; it has no effect on human cells or some other types of microbes, such as viruses and fungi.Its safety and potency as an antibacterial therapy are aided by this specificity (Sabaa et. al.,2015) .

CONCLUSION
Penicillin is an excellent candidate to treat various infectious diseases but it is less effective against gram negative organisms, as well as bacterial resistance to the drug candidate, Where major development is required.The above research work concludes that high yield of penicillin production can be obtained using sabouraud dextrose broth medium with wheat bran as the substrate in industrial means.The filtrate obtained from broth after the incubation period possesses the antagonistic property which can inhibit the growth of bacterial test organisms.

Fig- 17 :
Fig-17: A graphical representation of in-vitro activity of Penicillin against various test organisms and form the zone of inhibition (mm).