Using lysozyme as a preservative in cheese
Introduction
Food safety is one of the most important challenges in the food industry. Since the presence of synthetic preservatives in the last few years has caused concern to consumers, researchers and food manufacturers have recently conducted extensive research on the introduction and use of natural preservatives such as plant extracts and essences, bacteriocin have done antimicrobials.
Lysozyme structure
The natural enzyme lysozyme has antimicrobial ability and is widely used in various industries. Because lysozyme has low toxicity properties and is part of the human immune system, it can be considered a natural antimicrobial agent in the food industry. Based on the difference in amino acid sequence and structural properties, lysosomes are classified into three types: c, g, and i. This enzyme exerts antimicrobial activity against microorganisms, especially gram-positive bacteria, by hydrolyzing 1,4-β bonds between N-acetylmuramic and N-acetylglucosamine in the cell wall. Also, through chemical or physical interactions to modify lysozyme, this antimicrobial activity of lysozyme can be created against Gram-negative bacteria. Many factors contribute to cheese contamination, including bacterial and fungal contamination, uncontrolled and inappropriate processes, and contamination after pasteurization. As a result, lysozyme as a preservative (bio-preservative) in cheese production can be used alone or in combination with other preservatives.
Lysozyme is also known as muramidase or N-acetylmuramic hydrolase. This small and monomeric enzyme was accidentally discovered by Alexander Fleming that one drop of this nasal mucosa caused the lysis of bacteria. According to new findings, lysozyme is present in large quantities in tears, saliva, blood serum of human and cow’s milk, egg white of birds, and almost in most bacteria and bacteriophages. Egg white is the most accessible source of lysozyme which can generally be used as a preservative in various products such as fruits and vegetables, meat, milk, and dairy products.
Listeria monocytogenes, Salmonella, Escherichia coli, Staphylococcus aureus, and Bacillus cereus are among cheese’s most important contamination factors of food pathogens.
Another feature of lysozyme is the heat resistance of this enzyme, which is due to the presence of six-helix regions and disulfide bonds.
Antimicrobial activity of lysozyme
All these enzymes can hydrolyze β-(1-4) glycosidic bonds between peptidoglycan. In this way, the cell wall of gram-positive bacteria consists of peptidoglycan layers (about 40 layers) and without any outer membrane, where polysaccharides, lipoteichoic, and teichoic acids are attached to peptidoglycan. Therefore, they are susceptible to lysozyme attack. The presence of teichoic acids in the cell wall of gram-positive bacteria can affect their sensitivity to lysozyme, because the negatively charged acids can bind to lysozyme and reduce its activity.
Several factors affect the stability of the antimicrobial activity of lysozyme in food, including pH, temperature, salt concentration, and osmotic strength. The highest level of enzyme activity and changes in the structure of lysozyme was observed at pH=5 and the level of stability and activity was lower at pH <3.8.
Based on the information obtained about the sensitivity of food-related bacteria to lysozyme, some Gram-positive bacteria such as Micrococcus ssp., Sarcina spp., C. tyrobutyricum, Clostridium stearothermophilus, Bacillus Stearothermophilus, and Bacillus Coagulans were strongly attacked by the lysosome enzyme present in egg white.
On the other hand, the lysosome is ineffective against Gram-negative bacteria, because they have a thin layer of peptidoglycan without teichoic acids and are also covered with an outer membrane containing phospholipids, lipoproteins, and liposaccharides which act as barrier and limit the access of lysozyme to the peptidoglycan chain. However, this barrier can be overcome by membrane permeabilizing agents, such as carvacrol, organic acids, lactoferrin, and ethylenediaminetetraacetic acid (EDTA) or conjugation of lysozyme, and through binding with other compounds (polysaccharide, hydrophobic peptides, and fatty acids). It has been demonstrated that high hydrostatic pressure and a pulsed electric field can increase the sensitivity of Gram-negative bacteria such as E. Coli to lysozyme.
In addition to the enzymatic activity of lysozyme as an antibacterial agent, it has the activity of hydrolyzing bacteria related to their hydrophobic, structural, and cationic characteristics. As a cationic protein, this enzyme can act through electrostatic interactions between bacterial membrane components (phospholipids) and protein: It creates pores in the bacterial membrane and thus causes the membrane to leak without hydrolyzing peptidoglycan. Lysozyme also has weak activity against yeast, due to mannoprotein and fibrous β-(1-3) glucans being the main components of the yeast cell wall, but the chitinase activity of lysozyme is not very strong.
Lysozyme enzyme can also inhibit the growth of viruses and fungi such as Aspergillus niger and Penicillium. This enzyme has shown antiviral activity by forming complexes with the DNA of viruses.
Application of lysozyme in cheese
In the dairy industry, lysozyme can be used in cheese production to prevent the growth of spoilage bacteria that cause blowing, unpleasant flavors in the final product, and texture deterioration.
Based on the experiments, native lysozyme combined with dextran at the level of 400 μg/ml and glycosylated enzyme had a significant effect in reducing E. coli. On the other hand, lysozyme is easily absorbed due to hydrophobic material, such as Liposaccharide associated with the peptidoglycan thin layer of Gram-negative bacteria: It cannot penetrate the cell wall of Gram-negative bacteria hydrolyze them.
Lysozyme enzyme and (Na2-EDTA) can effectively prevent the growth of general forms and Pseudomonadaceae during 7 days of storage. It has also been reported that the functional microbiota of mozzarella cheese, such as lactic acid bacteria, was not affected by the addition of these active compounds. As a result, it can be said that the use of lysozyme and Na2-EDTA in conditions of brine, increases the shelf life of mozzarella cheese.
As a result, lysozyme or ethylenediaminetetraacetic disodium salt (Na2-EDTA) in combination with monoammonium phosphate (MAP), especially at the highest concentration of lysozyme (500 mg/kg) can be considered as an effective strategy against microorganisms of spoilage. Antimicrobial activity of lysozyme that inhibits the growth of bacteria, especially psychotropic bacteria that are responsible for the lipolysis and proteolysis of cheese during the storage period: prevents. This antimicrobial activity of lysozyme can be attributed to its ability to destroy the cell wall structure of bacteria and fungi.
In addition to the direct use of lysozyme enzyme in cheese or its brine, the use of lysozyme as an antimicrobial agent in packaging such as coating and film has also been investigated, and it was observed that composite layers and coatings consisting of Chitosan-lysozyme. It significantly reduced the growth of L. monocytogenes, Pseudomonas fluorscens, E. coli, and mold in mozzarella cheese compared to layers and multilayer coatings; Therefore, the antimicrobial properties of lysozyme can also be used for Gram-negative bacteria.
Conclusion
Lysozyme from HEWL is considered a major industrial source for practical applications due to its availability and cost-effectiveness. All lysozymes hydrolyze the β-(1-4) β-glycosidic bonds between N-acetylmuramic (NAM) and N-acetylglucosamine (NAG) peptidoglycan chains. They have antimicrobial activity, and this activity is influenced by various factors such as ionic strength, pH, water activity, the presence of lysozyme inhibitors, salt content, temperature, and the presence of chemical enhancers.
Different physical and chemical methods were used to improve the sensitivity of Gram-negative bacteria against lysozyme. This enzyme exists as a natural preservative in mammalian milk. As a result, it can be used for dairy products, including cheese, and according to studies, lysozyme in cheese production directly or in salt water can inhibit the growth of spoilage bacteria and improve texture, blowing, and unpleasant taste. Lysozyme enzyme can be used in packaging such as films and coatings to maintain its performance and antimicrobial activity during the storage and ripening period to prevent post-processing (secondary) contamination.
Finally, it can be stated that lysozyme as an alternative material can be used alone or as part of technology to improve food products, including cheese. It is also suggested that the use of lysozyme in combination with other antimicrobial enzymes and new technologies, such as cold plasma, high-intensity pulsed electric field, and radiation to increase its antimicrobial efficiency in cheese production, should be investigated in future research.
Reference:
Khorshidian N, Khanniri E, Koushki MR, Sohrabvandi S, Yousefi M. An overview of antimicrobial activity of lysozyme and its functionality in cheese. Frontiers in nutrition. 2022 Mar 9; 9:833618. DOI: https://doi.org/10.3389/fnut.2022.833618
