In the modern food industry, food preservation is extremely important. To this day the world still suffers from food contamination caused by bacteria, e.g. Vibrio cholerae, found in various types of seafood, or Listeria, associated with various ready-to-eat foods [1]. To solve the issue food industries are using synthetic preservatives or high amounts of salt or sugar. However, studies show that this type of conservation has caused harm to human health. Therefore, there is a need to find a substance that wouldn't cause bacterial resistance and would be naturally sourced. These requirements are met by bacteriocin-nisin.
Nisin is a small peptide made up of 34 amino acid residues, which is synthesized by the lactic acid bacteria Lactococcus lactis subsp. lactis. It aims to tackle both Gram-positive and certain Gram-negative bacteria due to its wide antimicrobial spectrum [2]. Nevertheless, nisin can be harmed by food-derived enzymes, which would result in a loss of antimicrobial activity. To ensure the stability of nisin, it could be coated with biopolymers [3]. In this work, pectin was used for particle preparation. Pectin is a complex anionic polysaccharide mainly composed of homogalacturonan (1→4 linked α-D-galacturonic acid and its methyl ester). According to its degree of esterification, pectin can be classified as high (> 50% esterified carboxyl groups) or low (< 50% esterified carboxyl groups) methoxyl pectin [4].
The aim of this study is to prepare nisin-loaded pectin particles and compare their proteolytic stability with free nisin. To encapsulate nisin three different types of anionic pectin biopolymer, i.e., high methoxyl pectin (HMP), low methoxyl pectin (LMP), and pectic acid (PecA) are used. The final concentration of pectin and nisin was 0.4 mg/ml. To test the proteolytic stability of the particles two distinct proteases were used, i.e., proteinase K and protease from Rhizopus sp. The first protease was dissolved in sodium dihydrogen phosphate dihydrate buffer solution at pH 7.5 and protease from Rhizopus sp. was dissolved in acetic acid buffer solution at pH 4. Nisin-loaded pectin and free nisin samples at pH 4 and 7 were prepared and were affected by proteinase K and protease from Rhizopus sp., respectively. Proteolysis was performed for 24 h at 37 °C. Finally, samples with proteolytic degradation products were analyzed by the capillary zone electrophoresis method using 7100 Capillary Electrophoresis unit (Agilent Technologies).
The appearance of new peaks in the electropherogram of proteolytically affected nisin was observed. If nisin-loaded pectin particles undergo proteolytic degradation, the profile of the electropherogram is the same as the sample containing only free nisin. The obtained results show that the area of peaks is obviously lower in nisin-loaded pectin particles compared with free nisin, especially when proteinase K is used. Thus, pectin could be successfully used to increase the resistance of nisin to the action of proteolytic enzymes.