Production of Antimicrobial Peptides
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To appreciate the importance of asceptic techniques with regard to bacterial cell cultivation, growth, purity and final harvesting by bench centrifugation in order to obtain a supernatant with extracellular bacterial by-products. The production of bacteriocins by S.warneri was measured on a150mm agar plate inoculated with Kocuria rhizophila (A Gram-positive bacteria) to determine antimicrobial activity using serial dilutions of a S. warneri sample and positive and negative controls. The anti-microbial activity was compared against the plotted data from the fermentation run in order to study any effects process variables such as pH, temperature biomass and dissolved oxygen may have. Profiles from the fermentation run were also studied to determine cell growth conditions and how local environmental changes affect the bacterial culture.
Fermentation is an energy-yielding anaerobic metabolic process in which microrganisms convert nutrients (carbohydrates) to alcohols and acids (lactic acid and acetic acid). The most commonly known fermentation process is the conversion of sugar to alcohol, using yeast under anaerobic conditions. In biotech industries, fermentation refers to the growth of microorganisms on food, under either aerobic or anaerobic conditions. Bioreactors are used for industrial fermentation processes and are made of glass, metal or plastic with automatic and/or manually controlled and settings to control aeration, stirring, temperature, pressure and pH and can be small enough for bench-top applications (5-10 L) or up to 10,000 L in capacity for large-scale industrial processes. Large bioreactors are used in the pharmaceutical industry for the growth of specialised pure cultures of bacteria, fungi and yeast, and the production of enzymes and drug sunder strictly controlled conditions. The study and practice of fermentation is called zymology or zymurgy. Louis Pasteur was one of the first zymologists and he referred to fermentation as "the result of life without air".
Lactic Acid Fermentation
The pyruvate sugar molecules from glucose metabolism known as glycolysis, may be fermented into lactic acid. Industrial bioreactors are used to convert lactose into lactic acid in the production of yogurt. Lactic acid is also produced muscletissue when the tissue is under stress and requires energy at a faster rate than oxygen can be supplied. The equation for lactic acid production from glucose is:
C6H12O6 (glucose) †’ 2 CH3CHOHCOOH
The production of lactic acid from lactose and water may be summarized as:
CH3CHOHCOOH + H2O †’ 4 CH3CHOHCOOH
Bacterial cultures are used for several industrial and research purposes including the production of microbial secondary metabolites such as enzymes, anti biotics and bacteriocins which are very like antibiotic and were originally classed as such. As such monitoring bacterial growth in bioreactors is important to exploit this secondary metabolism.
Bacterial growth in batch culture can be modelled in four different phases: a lag phase, an exponential or log phase followed by a stationary phase with a final death phase. The stationary phase is the growth-limiting phase because there is a depletion of nutrients with a corresponding rise of inhibitory products such as bacteriocins. In this phase of bacterial growth, the cell growth rate and death rate have the same values. At the death phase, the bacteria run out of nutrients and die. Batch culture is the most common laboratory growth method as it is ideally spatially unstructured and temporally structured. Batch culture kinetics indicates that the exponential growth phase slows down to give a deceleration phase due to the depletion of essential nutrients and accumulation of toxic by-products. As there is no net growth, bacteria will direct their metabolism to produce secondary metabolites. The bacteria produce several secondary metabolites including antibacterial toxins such as bacteriocins to help stave off any microbial competition for these scarce secondary nutrient sources. These bacteriocins can be cultured industrially and collected (1).
Bacteriocins are bacterial peptides that behave as toxins produced by bacteria to inhibit the growth of closely related bacterial strains. They are secondary metabolites produced during the first forty eight hours of the early stationary phase and are synthesised from precursor peptides that vary in their amino acid sequences (2). Most share common features such as low molecular weight, cathionic and hydrophobic, heat stablility and all are coded by structural genes that are translated into working peptides by microbial ribosomes. (2).They are also highly resistant to intestinal enzymes and can inhibit the growth of harmful bacteria and Candida species. Bacteriocins are cationic membrane active compounds that kill microorganisms by permeating the microbial membrane and impairing the cells ability to carry out anaerobic respiration. This is an important trait as bacteriocins are unlikely to face the same antimicrobial resistance mechanisms that limit current antibiotic pathways. Bacteriocins and antibiotics differ is that bacteriocins restrict their activity to strains of species related to the producing species and in particular to strains of the same species. Antibiotics display a far greater activity spectrum range and even when their activity is restricted, no preferential effect on closely related strains is observed (2).
They have been detected in abundance during the production of probiotics or natural antibiotics. Probiotics, such as enhanced yogurt are beneficial microorganisms that are introduced into food so that they can re-colonise the G I tract. These bacteria are usually a group known as lactic acid bacteria, especially species of Lactobaccillus. Lactic acid bacteria convert sugars to lactic acid in the absence of oxygen (1). They were first discovered in 1925 by Gratin who also went on to develop a range of antibiotics and discovered the bacteriophage. The first bacteriocin was called colicine, because it killed Escherichia coli (1). Colicins (From Gram-negative bacteria) were used as prototype bacteriocins from which all other bacteriocin analysis was compared to and are the most studied. Four classes of bacteriocins are described as follows:
Class 1 Bacteriocins: These are small peptide inhibitors such as nisin, produced by Lactococcus lactis, and subtilin, a nisin analogue and Streptococcus species which produce lantbiotics such as the widely studied, lactic acid bacteria (LAB).
ClassII Bacteriocins: These are the small heat stable peptides, usually <10kDa. There are five sub-classes. The class IIa bacteriocins are the largest subclass and are pediocin like bacteriocins, each containing a seven amino acid concensus sequence at the N-terminal while the C-terminal is responsible for species specific cell death, usually by permeating the cell wall and necrotic cell leakage.
Class IIb are so called two-peptide bacteriocins as two different peptides are required for activity. They includes the alpha enterocins and lactococcin G peptides which act as pore-forming toxins that permeates the cell membrane to give channels via a barrel-stave mechanism. This helps create an ion imbalance and leakage in the cell, leading to cell death.
ClassIIc include the cyclic peptides. Here the N- and C-terminals are covalently bonded to give circular bacteriocins. They effect membrane permeability and cell wall formation on target cells. Bacteriocin AS-48 which is produced by Enterococcus faecalis (a streptococcus bacterium) shows a range of antimicrobial mechanisms against both Gram-Negative and Gram-Positive bacteria. Bacteriocin AS-48 is encoded by a pheromone responsive plasmid, pMB2 and attacks the plasma membrane where it punches pores leading to an ion imbalance, leading to leakage and cell death. The globular structure of bacteriocin AS-48 consists of five alpha helices enclosing a hydrophobic core.
Class IId: These are the single peptide bacteriocins and display no post translational modifications or any pediocin like characteristics. One example is aureocin A53, which is stable under acidic pH conditions and is resistant to several proteases.
Class IIe: Aureocin A70 is encoded within an 8 kb plasmid, pRJ6, and is composed of four peptides with 30 or 31 amino acid residues without an N-terminal leader sequence. It is toxic against Listeria monocytogenes, a facultative anaerobic bacterium and dangerous virulent food-borne pathogen, with 20-30 % of clinical infections resulting in death.
Class III Bacteriocins: These are large heat labile bacteriocins of >10kDa. There are two subclasses.
Class IIIa: These bacteriocins kill other bacterial cells by cell wall degdaration leading to cell lysis. The best known is lysostaphin, a27kDa protein that lysises many of the staphylococcus species, especially S. aureus.
ClassIIIb: This subclass comprises of those bacteriocins that do not cause cell lysis. They destroy other bacterial cells by disrupting the cells membrane potential allowing for a net APT efflux.
Class IV Bacteriocins: They are large, heat labile complex bacteriocins with different lipid and carbohydrate functional groups.
Staphylococcus warneri Bacteriocin
Nukacin ISK-I is a linear, type A(II) lantibiotic produced by S. Warneri of molecular weight 2.96kDa and is encoded on plasmid pPI-1 on six separate genes. Nukacin ISK-1 contains three lanthioine molecules and/or three 3-methyllanthionine molecules, thus making it a lantibiotic. Type A lantibiotic proteins punch pores into the cytoplasmic membranes of sensitive cells leading to cell death as well as providing immunity proteins against self species produced lantibiotics by covalently binding free lantibiotic across the cell membrane (7).
Material and Methods
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