Technology used to preserve various forms of food such as fruit and vegetables can often rely heavily on natural resources such as the utilization of fossil fuels for energy consumption. Enzymes can be used as an alternative method to the pre-established technologies many of which are unsustainable in the food processing industry. Enzyme technology is an efficient method for food preservation as the enzymes can accelerate reactions such as the breakdown of chitin in cell walls of pathogens, these enzymes are known as chitinolytic enzymes (Silva 2019). The disintegration of the food spoiling pathogens can be sustainably achieved using certain types of enzymes. Protein in the structural cell walls and membranes of pathogens can be broken down efficiently by various types of lytic enzymes, breaking down the structural unit subsequently killing the fungal pathogen (Zhang et al 2012). This preserves fruit without harming the environment as the use of chemicals and natural resources is not required. The enzyme protease can be used for food preservation due to its capability of cell wall and membrane hydrolysis.
Protease enzyme
Protease is an enzyme that can be used to extend the shelf life of foods such as various types of fruits in a sustainable manner. The proteolytic activity of the enzyme can break down protein contained within the membrane and cell walls of fungal pathogens leading to the disintegration of the pathogen, preserving the food (Tavano 2013). The active site of the enzyme determines the specific way in which the substrate will bind to the surface; this is due to the residue around the active site (Tavano 2013). The proteins contained in the cell wall and membranes of the fungal cell are made up of peptide chains; the surface of the enzyme that specific peptide chains can bind to is called the subsite, this site allows only certain compatible chains to bind to the surface of the enzyme (Tavano 2013).
The peptide chain of the protein molecules that match with the specificity of the subsite bind to the surface of the enzyme. Once the substrate bonds to the sites of the protease enzyme, the enzyme breaks down the bonds resulting in amino acids (Tavano 2013). The breakage of proteins in the cell wall and membranes of pathogens leads to the disintegration of pathogens, preserving foods.
Hydrolysis of peptide bonds
There are numerous ways that the protease enzyme can interact with the peptide bonds of proteins and subsequently break the bonds. The endopeptidases can break the central bonds of the peptide chain (Tavano 2013). The exopeptidases can break peptide bonds at certain ends of the substrate (Tavano 2013). There are two forms of exopeptidases depending on the end of the peptide bond in which the protease enzyme hydrolyses the bonds. If the exopeptidases hydrolyse the peptide bond at the free amine group, then it is referred to as aminopeptidases and if peptide bond hydrolyses occurs at the free carboxyl group then it is a carboxypeptidase (Tavano 2013). Some protease enzymes are capable of hydrolysing peptide bonds at both end which are referred to as cathepsins (Tavano 2013). The breakage of the peptide bonds due to protease enzymes activity destroys the cell walls and membranes of pathogens leading to the disintegration and death of fungal pathogens. The graph below shows an endopeptidase, the enzymes works on the internal section of the chain breaking central peptide bonds.
Breakage of the centre peptide bond
The graphs below depict the two forms of exopeptidases. The graph on the left below shows an aminopeptidase as the protease enzyme is hydrolysing bonds at the positively charged H2-N, and the graph on the right shows a carboxypeptidase as it causes peptide bond hydrolysis at the negatively charged COOH end (Tavano 2013). Cathepsins can break bonds at both ends of the chain due to the enzymes ability to adapt to the most suitable charge for the end that it is hydrolysing.
Experiment to determine the effectiveness of Protease APL5 for food preservation:
Enzymes such as protease inhibit numerous pathogens from fruit spoilage. An experiment was conducted in which the fungus, Aureobasidium pullulans strain PL5 was used in order to obtain multiple protease PL5 gene (Zhang et al 2012). Various forms of analysis were undertaken for the examination of the deduced amino sequences from the cDNAALP5 gene such as BLAST a computer based technique (Zhang et al 2012). The analysis concluded that the protein APL5 was a serine protease and these types of protein have the capability to break peptide bonds, they are utilized for the disintegration of fungal pathogens (Zhang et al 2012). Information from the APL5 gene was utilized along with analytical techniques such as Western blotting to identify the desired protease protein, this process uses antibodies for detection and purification was also carried out for the recombinant protein (Zhang et al 2012). The processes carried out resulted in a uniform recombinant APL5 expressed in E. coli BL-21(Zhang et al 2012). This prohibited the growth of fungal pathogens that cause food spoilage especially to fruits as the hydrolysis resulted in the disintegration of the pathogen.
Results
The various parameters affecting the enzyme activity were investigated such as pH and temperature to determine the optimum operating conditions in which the enzymes exhibit optimum hydrolysis activity. The highest level of hydrolysation exhibited by the protease enzyme was at pH 10 and 50 degrees Celsius (Zhang et al 2012). In the laboratory 300 microlitre samples of the asexual spores of the fungi in Ringer solution were made up and to ensure there was no bacterial growth it was transferred to tubes containing potato dextrose broth, 300 microlitres of the enzyme was then added (Zhang et al 2012). A control was made up for comparison which contained no enzyme, sterile water was used instead of the protease enzyme. The graph below depicts a significant reduction in germination of the pathogen M. Laxa due to the proteolytic activity of the protease enzyme (Zhang et al 2012).
The germination of M.Laxa decreased by 48% due to the addition of APL5. This reduction in spore germination results in food preservation; there is a reduction in the number of spores resulting in a reduction in fungal pathogens, the pathogens were broken down by protease activity. In comparison to the control test tube the germ tube length was significantly reduced due to the proteolytic activity of the protease, the germ tube length decreased by 65 micrometres (Zhang et al 2012). The test concluded that the enzyme aided in preventing pathogens from spoiling fruit such as apples and peaches due to hydrolysis (Zhang et al 2012). This process does not alter the nutritional value or taste of the food.
Conclusion
Enzymes can be used as a sustainable technology for food preservation. Lytic enzymes such as the protease enzyme can break down the protein contained in cell walls and membranes of fungal pathogens. The peptide chain bonds to the subsite of the enzyme and the enzyme hydrolysis the peptide bonds resulting in the disintegration of the pathogen, extending the shelf-life of various foods. The peptide chains can be broken down various ways by different forms of lytic enzymes. Endopeptidases and exopeptidases and two ways in which the bonds can be broken (Tavano 2013). Chitinolytic enzymes can break down chitin in the cell walls of fungal pathogen aided in prolonging shelf life of food (Silva 2019). A test was carried out to determine if enzymes can preserve fruits. A serine protease gene APL5 was obtained from Aureobasidium pullulans strain PL5 (Zhang et al 2012). The protease gene reduced spore germination of the food spoilage pathogen M.Laxa by 48% and reduced germ tube length by 65 micrometres (Zhang et al 2012). It was concluded that the protease gene PL5 can preserve fruits such as peaches from food spoilage fungal pathogens without altering the taste or nutritional value of the food. This is a sustainable technology as no there is no utilization of natural resources required and no harmful chemicals are used.
References
Tavano, O.L. (2013) ‘Protein hydrolysis using proteases: An important tool for food biotechnology, Journal of molecular catalysis B: Enzymatic, 90, 1-11, available: https://ac-els-cdn-com.proxy.lib.ul.ie/S1381117713000192/1-s2.0-S1381117713000192-main.pdf?_tid=86b2ba71-069d-4132-a034-5ba3c2914c56&acdnat=1549307473_847fa023eacf29e8e8fd9085f039a373
Zhang, D., Spadaro, D., Valente, S., Garibaldi, A., Gullino, M.L. (2012) ‘Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens’, International Journal of Food Microbiology, 153, 453-464, available: https://ac-els-cdn-com.proxy.lib.ul.ie/S0168160511007288/1-s2.0-S0168160511007288-main.pdf?_tid=f67fc98a-3770-477f-932b-ee72bde721c7&acdnat=1549637061_d63e42699622e55efd944c70804408c9
Silva, R.R.D. (2019) ‘Enzyme technology in food preservation: A promising and sustainable strategy for biocontrol of post-harvest fungal pathogens’, Food Chemistry, 277, 531-532, available: https://ac-els-cdn-com.proxy.lib.ul.ie/S0308814618319605/1-s2.0-S0308814618319605-main.pdf?_tid=862170e4-1fb8-453e-88c9-cf1d7b53844a&acdnat=1550314424_6281429a7cf499cbd00c6048b38a5862