What is new in lysozyme research and its application in food industry? A review

Accepted Manuscript What is new in lysozyme research and its application in food industry? -A review Tiantian Wu, Qingqing Jiang, Dan Wu, Yaqin Hu, Shiguo Chen, Tian Ding, Xingqian Ye, Donghong Liu, Jianchu Chen PII: DOI: Reference:

S0308-8146(18)31587-5 https://doi.org/10.1016/j.foodchem.2018.09.017 FOCH 23504

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Food Chemistry

Received Date: Revised Date: Accepted Date:

29 March 2018 4 August 2018 3 September 2018

Please cite this article as: Wu, T., Jiang, Q., Wu, D., Hu, Y., Chen, S., Ding, T., Ye, X., Liu, D., Chen, J., What is new in lysozyme research and its application in food industry? -A review, Food Chemistry (2018), doi: https:// doi.org/10.1016/j.foodchem.2018.09.017

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What is new in lysozyme research and its application in food industry? -A review Tiantian Wu1, Qingqing Jiang1,2, Dan Wu3, Yaqin Hu1*, Shiguo Chen 1, Tian Ding1, Xingqian Ye1, Donghong Liu1, Jianchu Chen 1 1

National Engineering Laboratory of Intelligent Food Technoklogy and Equipment, Key Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture, Key Laboratory for Agro-Products

Nutritional Evaluation of Ministry of Agriculture, Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China 2

Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan 3

Zhiwei Guan Foods Co., Ltd, Hangzhou 311199, China

Abstract: Lysozyme, an important bacteriostatic protein, is widely distributed in nature. It is generally believed that the high efficiency of lysozyme in inhibiting gram-positive bacteria is caused by its ability to cleave the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. In recent years, there has been growing interest in modifying lysozyme via physical or chemical interactions in order to improve its sensitivity against gram-negative bacterial strains. This review addresses some significant techniques, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), infrared (IR) spectra, fluorescence spectroscopy, nuclear magnetic resonance (NMR), UV-vis spectroscopy, circular dichroism (CD) spectra and differential scanning calorimetry (DSC), which can be used to characterize lysozymes and methods that modify lysozymes with carbohydrates to enhance their various physicochemical characteristics. The applications of biomaterials based on lysozymes in different food matrices are also discussed. Keywords: lysozyme; characterization; modification; Maillard reaction; food application

1. Introduction Lysozyme, also referred to muramidase or N-acetylmuramic hydrolase, is a small, monomeric protein stabilized by four disulfide linkages among the eight cysteine residues of its polypeptide chain (Fig. 1). The discovery of lysozyme is attributable to Alexander Fleming, who accidentally discovered that a drop of his nasal mucus could cause the lysis of bacteria present on the plate, which enabled him to detect a ‘remarkable bacteriolytic element’ that he later called lysozyme (Fleming 1922). Later, lysozymes were found in large quantities in human organs, tissues, and secretions (spleen, placenta, skin milk, tear, saliva, serum, etc.), and similar lytic enzymes were isolated from organs and secretions of various vertebrates, invertebrates, bacteria, and 1

even plants (e.g., Papaya latex), which make lysozymes widely distributed substances (Ogawa, Miyazaki, & Kimura, 1971). Based on different characteristics (e.g., structure, catalysis and immunization), lysozymes were divided into three dominant families: chicken-type (c-type), goose-type (g-type) and invertebrate-type (i-type). In addition, several other types of lysozymes, including phage-type, bacterial-type and plant-type lysozyme, have also been recognized (Callewaert, & Michiels, 2010; Cao et al., 2015). Generally, both cand g-type lysozymes are basic proteins due to their high isoelectric point (pI) values, while the pIs of i-type lysozymes are quite distinct and might be attributed to diverse functions of the protein (e.g., host defence, digestion) (Xue et al., 2004). The c- and i-type lysozymes (~11–15 kDa) are much smaller than the typical g-type lysozymes (~20–22 kDa) (Callewaert, & Michiels, 2010). The primary structure of c-type lysozymes consists of 129 amino acid residues with a molecular weight of 14.3 kDa, which contain four intact disulfide bonds (6Cys–127Cys, 30Cys–115Cys, 64Cys–80Cys, and 76Cys–94Cys) and six tryptophan (Trp), three tyrosine (Tyr), and three phenylalanine (Phe) residues (Cao et al., 2015; Chen et al., 2005). Among the six Trp residues, Trp-62 and Trp-108 are considered to be the major fluorophores in lysozyme (Shanmugaraj, Anandakumar, & Ilanchelian, 2015). Recently, hen egg-white lysozyme (HEWL), belonging to the c-type category, has attracted a great deal of attention in relation to its abundant resources and functional characteristics (Jiang et al., 2017; Wu et al., 2018). It is positively charged below the pKa (∼11) due to the presence of 17 positively charged (6 Lys, 11 Arg) and nine negatively charged residues (7 Asp, 2 Glu), making HEWL an ideal candidate to study interactions with compounds (e.g., drugs, metals) (Bijelic et al., 2015). Due to the ability to cleave the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of bacterial cell wall peptidoglycans, the antimicrobial efficiency of HEWL against gram-positive bacteria is incontestable and has been illustrated in numerous reports (Huopalahti, López-Fandino, Anton, & Schade, 2007). Until now, HEWL has been the only lysozyme allowed for use in the food industry (Silvetti, Morandi, Hintersteiner, & Brasca, 2017). In addition to killing bacteria, lysozymes from other sources are able to eliminate fungi. Two c-type lysozymes identified from the manila clam Venerupis philippinarum Pichia, encoding a polypeptide of 156 and 153 amino acid residues, respectively, showed higher inhibitory effects on pastoris GS115 and Pichia pastoris KM71 compared with HEWL (Yang et al., 2017). A lysozyme isolated from mung bean seeds demonstrated potent antifungal activity towards F. oxysporum, F. solani, S. rolfsii, P. aphanidermatum, and Botrytis cinera (Wang et al., 2005). However, when used alone, lysozyme exhibits weak inhibitory effects against gram-negative bacteria such as Enterobacteriaceae and Pseudomonadaceae, which are relatively common contaminants of meat-based products, as the presence of a protective lipopolysaccharide (LPS) on the bacterial cell wall makes the practical application of free lysozyme quite limited (Wu et al., 2017; Barbiroli et al., 2012); this will be further discussed in this paper. 2

To broaden the application of lysozyme, many methods have been developed. Examples include the modification of lysozyme with heat, chemicals and hydrolysis to enhance its beneficial biological properties (Carrillo, Spindola, Ramos, Recio, & Carvalho, 2016), the formation of complexes including encapsulation, micro/nano gels, edible films based on lysozyme and pectin, sodium alginate, starch, sodium carboxymethyl or cellulose to manufacture antimicrobial materials (Amara, Eghbal, Degraeve, & Gharsallaoui, 2016; Fuenzalida et al., 2016; Amara, Eghbal, Oulahal, Degraeve, & Gharsallaoui, 2016; Zhu et al., 2013), and the combination of ethylenediaminetetraacetic acid (EDTA) and lactoferrin with lysozyme to enhance the susceptibility of gram-negative bacteria to lysozyme (Branen, & Davidson, 2004). With these methods, lysozyme has been successfully applied in various food substrates to improve the quality and extend the shelf life of food, which remain to be summarized. In this review, a brief overview of the bacteriostatic mechanism of lysozyme will be outlined. Several main techniques for the characterization of lysozyme will be presented in detail. Finally, the modification of lysozyme with carbohydrates and its application in the food industry will be summarized at great length. 2. Significant techniques used to characterize lysozyme 2.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) With a molecular sieve effect, SDS-PAGE is widely used to separate proteins and oligonucleotides. The primary protein sequence can be monitored by SDS-PAGE. As depicted in Fig. 1, the SDS-PAGE pattern showed that the molecular weight of lysozyme from hen egg white was approximately 12 kDa. Usually, SDS-PAGE was conducted when lysozyme was conjugated or reacted with other chemicals to check whether the reaction had occurred and to investigate the change of the molecular weight of lysozyme influenced by the reaction. The conjugation of lysozyme to polysaccharides is a hotly discussed issue for many researchers, and SDS-PAGE should be used to check the conjunction process, which will be covered in greater depth in the next part. Moreover, in the extraction and purification process of lysozyme, SDS-PAGE could be used to check the purity of lysozyme, where the presence of only one band at approximately 14 kDa may demonstrate the successful preparation of lysozyme (Altıntaş, & Denizli, 2009). 2.2. Infrared (IR) spectra IR is a widely used method to determine the chemical structure of compounds as indicated by the functional groups. Lysozyme has three characteristic mid-infrared regions, including amide I (1700–1600 cm−1), amide II (1600–1500 cm−1) and amide III (1320–1230 cm−1). Among these three regions, amide I was believed to involve secondary structures, namely, the different protein structures assigned to specific ranges of the infrared spectra: 1650–1658 cm−1 for α-helix, 1620–1640 cm−1 and 1670–1695 cm−1 for β-sheet, 1640–1648 cm−1 for unordered conformation, and approximately 1670, 1683, 1688 and 1694 cm−1 for β-turns. Amide II is thought to not 3

be very sensitive to the conformation of the protein, which can be mainly assigned to in-plane N–H bending and C–N stretching vibration. However, the amide III signal was always ignored because of its weakness (Prosapio, Reverchon, & Marco, 2016). Moreover, the broad band approximately 3294 cm−1 arises from hydrogen bonding, which overlaps with the stretching band of –NH of the free amino groups. The absorption between 3100 cm−1 and 2980 cm−1 was characteristic of C–H stretching (Koshani, Aminlari, Niakosari, Farahnaky, & Mesbahi, 2015). The IR absorption spectra of lysozyme in the 3030–2830 cm−1 were also considered and were due to the existence of CH3 or CH2 (Barreca et al., 2014). The main characteristic peaks of lysozyme are summarized in Table 1. 2.3. UV-vis spectroscopy UV-vis spectroscopy is a fundamental and useful tool to explore the structural changes and to monitor complex formation between ligand and protein by detecting changes of the wavelength with an ultraviolet spectrophotometer. In the UV–vis spectra of lysozyme, there is a strong absorption peak near 200 nm that reflects the framework conformation as well as a relative weaker peak near 280 nm due to the presence of the aromatic amino acid residues (Trp and Tyr residues) of lysozyme (Shanmugaraj, Anandakumar, & Ilanchelian, 2015). 2.4. Fluorescence spectroscopy Fluorescence spectroscopy is a highly sensitive method for visualizing protein tertiary structural properties that can be used to determine the folding/unfolding process of proteins. The fluorescence characteristics of proteins arise from the aromatic residues, including tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). Once these residues are exposed to different solvent conditions, the fluorescence spectral characteristics of the protein will alter, providing a technique for tertiary structure determination. The fluorescence of lysozyme was mainly attributed to the Trp residues located at 62 and 108 of the amino acid sequence. The fluorescence spectroscopy assay often involves the use of Thioflavin T (ThT), a dye that can interact with the native, molten globule or unfolded protein molecules as well as with amorphous protein aggregates. For instance, HEWL was applied to study the nature of amyloid fibrillization with the purpose of identifying the various aspects of amyloid formation in humans, and ThT was found to interact with the assembled lysozyme amyloid fibrils with high cross-β structure content, resulting in significant increase of the fluorescence signal (Ponikova et al., 2017). Fluorescence spectra include fluorescence emission spectra and fluorescence excitation spectra. When the concentration of lysozyme is fixed and followed by the titration of different amounts of compounds, the fluorescence intensity of lysozyme decreases without appreciable transformation in the position of the fluorescence peak, which is called quenching. Generally, the wavelength of maximum emission (λmax) for lysozyme is approximately 340 nm (Antonov, Zhuravleva, Cardinaels, & Moldenaers, 2017), and the redshift of λmax usually indicates that a major unfolding of lysozyme has occurred (Kuramitsu, Kurihara, 4

Ikeda, & Hamaguchi, 1978). Amara, Degraeve, Oulahal, & Gharsallaoui (2017) noticed the conformational changes of lysozyme induced by pH (Fig. 2b), where an obvious redshift from 345.5 nm (pH 7 and pH 5) to 347.5 nm was detected at pH 9, presumably ascribed to the exposure of tryptophan residues to the polar solvent and amino acid deprotonation, while a slight blueshift of the λmax band and a reduction of fluorescence intensity was observed at pH 3 compared with pH 7 and pH 5. Quenching can be observed in various molecular interactions, including excited-state reactions, molecular rearrangements, energy transfer, ground state complex formation and collision quenching (Van, Andersen, & Frokjaer, 2004). 2.5. Nuclear magnetic resonance (NMR) 1

H NMR is characterized by a small range of chemical shifts. For lysozyme, organic spin labels or inorganic paramagnetic ions such as Gd3+ could be introduced to generate the broadening effect of electron spins on nuclear spin resonances for determining interatomic distances in solution (Allerhand, Childers, & Oldfield, 1973; Bradbury, & Brown, 1973). It had been established that structural alterations associated with denaturation of lysozyme are accompanied by obvious changes in the proton–magnetic–resonance spectra (Mcdonald, Phillips, & Glickson, 1971). The proton high-resolution magic angle spinning nuclear magnetic resonance (1H HRMAS NMR) was developed to determine the behaviour of hydrophilic interactions in hydrated lysozyme (the hydration level h=0.3), which could contribute to the better understanding of the physical processes that occur below the threshold of lysozyme thermal denaturation. The thermal region of the reversible folding/unfolding process essentially lies between TB=325K and TD=346K (Fig. 2c). At these temperatures, apparent spin–spin relaxation time, T2∗, of hydration water exhibits a maximum and a minimum, respectively. If the final temperature is maintained below the threshold TD, the folding and unfolding process of lysozyme is reversible (Corsaro, & Mallamace, 2011). Compared to protons, the range of 13C chemical shifts is much greater, and the assignment of non-protonated 13C NMR provides varied new markers for further investigations, some in hitherto “invisible” regions of lysozyme (Howarth, & Lian, 1984), probing its tertiary and quaternary structures. Allerhand, Childers, & Oldfield, 1973 reported the peaks observed in the aromatic region of the 13C spectrum of native HEWL. The results showed that the γ carbons of the six tryptophan residues resonate at 81.4, 82.1, 83.2, 83.8, and 85.2 ppm upfield from CS 2. The peak appearing at 85.2 ppm is attributed to a two-carbon resonance, and all six carbons resonate at approximately 83.8 ppm 13C chemical shifts upon denaturation with guanidinium chloride. NMR could be used to determine high-resolution 3D structure, ligand interactions, and dynamics of proteins, and the application of this technology depends on the observation of NMR nuclei such as 1H, 13C and 15N. Usually, the amount of isotopes such as 13C and 15N is quite limited, and they should be incorporated into proteins. Labelling techniques using a bacterial expression system could effectively incorporate 5

15

N- or 13C- labelled amino acids into lysozyme (Griffey, Redfield, Loomis, Dahlquist, 1985). In addition, the reductive methylation of lysozyme to produce monomethylamines is an available way to introduce sparse 13C to yield slightly higher lysyl pKa values compared with the unmodified form. Lysozyme has seven methylation sites: aLys1, eLys1, Lys13, Lys33, Lys96, Lys97, and Lys116, as six lysines and the N-terminal amine exist in lysozyme. The utility of dimethylamine NMR peaks of lysyl residue number and N-terminal amine of reductively methylated hen egg white lysozyme was adopted and developed by different researchers, and the major efforts from five research groups were reported in a previous article by Roberson, & Macnaughtan, (2014). 2.6. Circular dichroism (CD) spectra It is worth mentioning the extensive and extraordinary use of CD for the characterization of lysozyme. CD is a simple and fast method to elucidate secondary and tertiary structure of proteins by far-UV (190-250 nm) and near-UV CD, respectively (Liang et al., 2013). Many publications have demonstrated the detailed conformational perturbation of lysozyme when this molecule interacts with other compounds. Detailed descriptions of the basis of the CD approach and its application in the study of proteins have been given in a review article (Kelly, Jess, & Price, 2005). It can be observed from the CD spectra that lysozyme has α-helix, β-sheet, turns and unordered (random) secondary structures. Hydrogen bonds only exist in α-helix and β-sheet structures, which led to the rigidity of lysozyme, while β-turn and unordered structures contribute to the flexibility of lysozyme (Sheng, Wang, Huang, Xu, & Ma, 2016). It is generally considered that the n→π* transition peptide bond of the α-helical structure of lysozyme results in two characteristic negative peaks in the far-UV region at 208 and at 222 nm (Woody, 1995). A positive band near 195 nm was also observed, suggesting prominent α-helix content in lysozyme. The ellipticity at 220 nm is a standard calculation of the helical content of a protein. Melchiors et al., (2017) revealed that the treatment of pressurized fluids with lysozyme could result in a decrease in α-helix and an increase in β-sheets of lysozyme according to the calculation of molar ellipticities, where the corresponding values of native lysozyme were 34.2 ± 2.20% and 14.2 ± 1.60%, respectively. Sheng, Wang, Huang, Xu, & Ma, (2016) extracted lysozyme from fresh chicken egg, and CD spectra showed that the α-helical and β-turn declined from 34.72 to 20.35% and from 29.31 to 24.17%, respectively, while β-sheet and random coil increased from 11.72 to 18.96% and from 24.25 to 36.52%, respectively, after storage for 50 days, suggesting that lysozyme molecules became more flexible and disordered. CD combined with fluorescence spectroscopy was effectively used to elucidate the folding mechanism of canine milk lysozyme, which provided a key for understanding the molten globule (MG) state of lysozyme—a general intermediate of protein folding that was more stable and more native-like in structure than those of other lysozymes (Nakao, Arai, Koshiba, Nitta, & Kuwajima, 2015). 6

2.7. Differential scanning calorimetry (DSC) As summarized in previous reviews (Sanchez-Ruiz, 2010; Johnson, 2013; Ibarra-Molero, Naganathan, & Sanchez-Ruiz, 2016), DSC study has already become a useful technique for understanding protein stability and energetic profiles because its experimental output reflects the energetics of all conformations, yielding the melt transition temperature ™, enthalpy change (△Ηd), apparent heat capacity change (△ Cp), and Gibbs free energy change ( △ G), as well as pre-assumes equilibrium reversibility in some cases. According to different types of apparatus, DSC can be conducted for samples in two states—solution or powder. For solutions, the sample must be exhaustively dialysed against experimental buffer solution and degassed, while freeze-dried proteins only need to be weighed in aluminium pans and sealed before assay. To date, in lysozyme research, DSC has been extensively applied to understand the early stages of lysozyme crystallization, indicating the presence or absence of aggregates (Igarashi, Azuma, Kato, & Ooshima, 1999), to investigate the effects of ions and pH on thermodynamic stability during thermal denaturation of lysozyme (Blumlein, & Mcmanus, 2013), to evaluate the binding of other molecules to lysozyme that gives rise to changes of enzymatic activity or conformational and phase transitions (Burova, Grinberg, & Grinberg, 2006), to serve as a model molecule for evaluating the interaction between drugs and proteins (Nishimoto, et al., 2010) and to determine protein thermodynamics, folding pathway and unfolding mechanisms (Johnson, 2013). The commonly and mainly considered thermodynamic parameters are the mean denaturation temperatures (Td) and enthalpy change (△Hd) (Duan et al., 2017; Santos, & Garcia-Rojas, 2018). The Td value reflects the global stability of the enzyme and the △Hd value demonstrates the competition between endothermic unfolding and exothermic aggregation. If values of Td and/or △Hd decrease, there is a decrease in the heat stability of lysozyme. According to Santos, & Garcia-Rojas, (2018), there was an endothermic peak in the thermal denaturation profiles of lysozyme (see Fig. 2b), and Td and ΔHd of lysozyme were 72.3 °C ± 0.07 and 35.8 J/g ± 2.71, respectively. Despite the observation of △Hd and Td, if the sample can achieve a second heating cycle performed immediately after cooling from the first scan, and the difference in heat capacities △Cp between different states was independent of T, apparent heat capacity change (△Cp), refolding index (RI) and Gibbs free energy change (△G) could be the other useful indexes for analysing the denaturation process and the reversibility of the thermally induced unfolding process in depth. Based on this, the effect of five cations on reversibility and thermodynamic stability of lysozyme was determined, where the values of Tm, △Hd, △Cp, and RI for native lysozyme were 77.4 °C ± 0.10, 365.7 kJ/mol ± 18.3, 5.0 kJ/mol·K-1 ± 0.3 and 0.81 ± 0.06, respectively. The thermal transiti™(Tm) shift towards lower temperatures and RI increases as the concentration of the salts increases, and at high concentrations (150 mM) of added salt, a clear trend in terms of thermal destabilization of lysozyme was observed: Ca2+ > Mg2+ > K+, NH4+ > Na+. All ion concentrations have a negative 7

effect on the thermodynamic stability of lysozyme based on the calculation of △G (Stavropoulos, Thanassoulas, & Nounesis, 2017). However, to comprehensively study the properties of lysozyme, various techniques were used together to fulfil different purposes during scientific experiments. In protein chemistry, enzymology, crystallography and molecular biology, diffraction of electrons such as X-ray diffraction and small angle X-ray scattering (SAXS) could reveal detailed information about the crystal structure, chemical composition, and physical properties of materials and thin films based on lysozymes that act as model enzymes. There would be no changes in the width and intensity of diffraction peaks when the folding states of lysozymes do not change. Gdovinová et al. (2017) applied SAXS and AFM to study the structural properties of complex solutions of magnetic nanoparticles (MNPs) with lysozyme amyloid fibrils, which revealed the change in diameter and helical structure of the fibrils caused by MNPs. Moreover, online microspectrophotometry coupled with in situ X-ray irradiation and X-ray crystallography was combined to study disulfide damage in lysozyme crystals, which offered a comprehensive model of radiation damage that could potentially result in a combined computational and experimental approach to understanding when damage is likely to occur, to quantify it and to enable the recovery of the native unperturbed structure (Sutton et al., 2013). Table 2 provides a summary of the advantages and limitations of these techniques. 3. Modification of lysozyme with carbohydrates 3.1. Conjugation via the Maillard reaction Multiple reports on the modification of lysozyme and its possible use for eliminating foodborne pathogens as well as other bacteria are available. Modifications of lysozymes are meant to enhance their physicochemical characteristics to broaden their applications in different orientations. The general modification strategies typically include protein–polysaccharide conjugation to improve the activities of lysozyme and lysozyme polysaccharide complexes, involving the interaction between the carbonyl carbon of a reducing end of polysaccharides and amino group of lysozyme (Maillard reaction) (Hashemi, Aminlari, & Moosavinasab, 2014). The polysaccharide-conjugated lysozymes could be regarded as new, effective biopolymers that might have great potential to act as novel, natural antibacterial agents in the food industry. There are a variety of polysaccharides that could be conjugated with lysozyme under mild conditions. Tragacanthin (TRG) is the water-soluble component of tragacanth (TGC), a complex heterogenous anionic polysaccharide with high molecular weight obtained from the stems and branches of Asiatic species of Astragalus. Koshani, Aminlari, Niakosari, Farahnaky, & Mesbahi, (2015) revealed that under optimum conditions (pH = 8.5, 60 °C, RH = 79%, 8 days), approximately 2 TRG molecules were attached to one lysozyme molecule. Maillard reactions (Fig. 3a) between TRG and lysozyme were proved by SDS-PAGE together with FT-IR spectroscopy. Although the enzymatic activity of lysozyme showed a decrease of approximately 23% when the denaturation 8

temperature was increased by 6.35 °C, enhancement of solubility and emulsion properties was observed for the conjugated lysozyme compared to those of native lysozyme. Conjugating with TRG significantly improved the inhibitory efficiency of lysozyme on the growth of bacteria, where the reduction rates of Staphylococcus aureus, Bacillus cereus, Escherichia coli and Salmonella typhi treated by 4000 mg/ml TRG-conjugated lysozyme were 90%, 80%, 50% and 40%, respectively. Interestingly, the research group later found that ultrasonic-treated tragacanth (US-treated TGC) could also crosslink with lysozyme under mild Maillard reaction conditions, where one of the free amino groups of lysozyme was blocked by TGC under optimum conditions (pH = 8.5, 60 °C, RH = 79%, 8 days). The conjugation was beneficial to the improvement of the emulsifying and foaming properties of lysozyme (Koshani, & Aminlari, 2017). Pullulan, produced by the yeast-like fungus Aureobasidium pullulans, is a linear homopolysaccharide with glucosyl residue reduplicative units linked by α-1,4-linkages and α-1,6-linkages. Owing to its outstanding physicochemical properties, pullulan and its derivatives play a vital role in various food, pharmaceutical and biomedical applications (Singh, Kaur, Rana, & Kennedy, 2017). A recent study by Sheng et al. (2017) revealed that pullulan was conjugated to lysozyme under controlled Maillard reaction (60 °C at pH 7.5 for 5 days with a lysozyme to pullulan molar ratio of 1:8). The covalent attachment of pullulan to lysozyme was proved by SDS-PAGE, and remarkable changes in the structure and conformation was observed by fluorescence and CD spectrum analysis. SDS-PAGE showed that the molecular weight of lysozyme was approximately 14 kDa, which was the feature band of native lysozyme. A new band at approximately 28 kDa was observed for the heated lysozyme because of the formation of dimers, while the lysozyme band was significantly diminished and new bands appeared for the heat-treated lysozyme-pullulan mixture, indicating that large molecular polymers had associated into lysozyme. Meanwhile, the fluorescence signal of tryptophan residues of lysozyme decreased due to the steric-hindrance effect caused by the behaviour of pullulan molecules acting as flexible random coils in water, and the CD spectrum suggested that the structure of lysozyme had been changed after conjugation, where a significant reduction in α-helix from 33.4±1.7 to 24.3±0.5%, a slight decrease in β-sheet from 35.1±1.6 to 33.5±1.0%, and increases in β-turn from 1.7±0.4 to 9.1±1.1% and random coil from 28.8±1.3 to 33.1±0.8% were observed. Most importantly, the solubility and antimicrobial activity of conjugates were better than those of native lysozyme. Guar gum is another hydrophilic polysaccharide that can conjugate with lysozyme. It is extracted from the seeds of Cyamopsis tetragonolobus with significant antioxidant properties according to its ability to chelate metal ions and scavenge DPPH radicals. Hamdani, Wani, Bhat, & Siddiqi, (2018) prepared guar gum-conjugated lysozyme in 0.1 M sodium phosphate buffer (pH 8.5), and the results obtained by gel permeation chromatography showed that after conjugation, the average molecular weight of modified lysozyme was 535.0 KDa, whereas the unmodified one was 14.65 KDa, 9

indicating that two moles of guar gum bind to lysozyme. The study also demonstrated that gum conjugation increased the thermodynamic stability and the inhibitory ability of the free DPPH radical scavenging and reducing power of lysozyme. The antibacterial properties were observed to increase for conjugated lysozyme according to the inhibition zones both for gram-negative (Salmonella and Escherichia coli) and gram-positive (Staphylococcus aureus and Enterococcus) bacterial strains. Dextrans are extracellular bacterial homopolysaccharides produced by extracellular dextransucrase released from LAB of the genera with a linear backbone of α-D-pyran glucose units repeating units. Due to the well-known properties such as viscosifying, emulsifying, texturizing, and stabilizing dextrans showed extensive applications in the field of bakery and other food industries (Kothari, Das, Patel, & Goyal, 2015). Early in 1991, Nakamura et al. reported that dextran-conjugated lysozyme mediated by the Maillard reaction provided lysozyme with a novel bifunctional effect on both gram-positive and gram-negative bacteria strains despite the loss of 20% in lytic activity compared to the native lysozyme (Nakamura, Kato, & Kobayashi, 1991). A study by Scaman, Nakai, & Aminlari, (2006) reported that the optimum condition for glycation of lysozyme with dextran was pH 8.5 and 60 °C with a lysozyme to dextran molar ratio of 1:5, resulting in three moles dextran attached to one mole lysozyme. Detection using ion exchange chromatography and SDS-PAGE demonstrated that a combination of high pH values and temperatures are adverse to glycation, and these conditions should be avoided in foods when protection of protein by conjugation with polysaccharides is desired. As expected, dextran-conjugated lysozyme showed improved heat stability, pH and heat solubility and better emulsifying property compared with the native lysozyme, making the modified lysozyme more suitable for food and pharmaceutical applications. Dextran sulfate (DS), a polyanionic derivative of dextran prepared by sulfating a selected fraction of dextran, could also be conjugated with lysozyme. Alahdad, Ramezani, Aminlari, & Majzoobi, (2009) confirmed that a Maillard-type reaction occurred between the ε-amino group of lysine of lysozyme and the reducing end carbonyl group of DS. Conjugation contributed to the appearance of diffused bands, indicating a wide distribution of molecular weights of the products of the reaction, and the formation of multiple derivatives might belong to lysozyme with one to seven DS molecules. DS-conjugated lysozyme contributed to the improved solubility at alkaline pH values and different temperatures, enhanced heat stability and emulsifying characteristics compared with the native lysozyme. Although this glycosylation of lysozyme led to a decreased antimicrobial effect of S. aureus, it did extend the antimicrobial spectrum of lysozyme towards E. coli as representative of gram-negative bacteria, which may related to the surfactant properties of DS-lysozyme conjugate. Xanthan gum (XG), an anionic extracellular polysaccharide secreted by the microorganism Xanthomonas campestris, is widely used as a stabilizer, thickener or emulsifier in food products. Hashemi, Aminlari, & Moosavinasab, (2014) prepared xanthan gum-conjugated lysozyme under pH 8.5 and 60 °C for 10 days. The disappearance of FTIR absorption bands at 3500 and 3400 cm-1 in the conjugated 10

spectra demonstrated the involvement of amino groups of lysozyme in a Maillard reaction. The conjugation contributed to better solubility at acidic pH values and at different temperatures and improved heat stability with enhanced emulsion and foaming properties. Lysozyme-XG conjugate at 400 μg/ml could reduce the number of E. coli and S. aureus by 3 and 2 log, respectively while the value for unmodified lysozyme at same concentration was only approximately 0.5 and 1.7 log, respectively. Galactomannan (GM) is a polysaccharide consisting of β-(l–4)-mannose backbone with single ɒ-galactopyranosyl units attached via α-(l–6) linkages as side branches, which has efficacy in immune modulation and reducing inflammation (Barak, Mudgil, 2014). Yang, Chun, Kim, Choi, & Lee, (2017) revealed that in the conjugation process, approximately 1 mol of GM was linked to 1 mol of lysozyme based on the binding weight ratio. After the 7-d incubation at 60 °C and 79% RH, there was 28% loss of free amino groups for lysozyme, where the reducing end of GM and the ε-amino residue of lysozyme were expected to form a covalent bond, and thus a high-molecular-weight compound was obtained. The solution of GM-conjugated lysozyme exhibited heterogeneous shapes with a mean size of 337 nm, and GM conjugated to Lys115 on lysozyme, which strengthened the immune-enhancing activity of lysozyme. Scaman, Nakai, & Aminlari, (2006) also reported the ability of GM conjugated to lysozyme to improve the activities of lysozyme and make it more available for application in foods and pharmaceuticals. 3.2. Complex formation via electrostatic attractions and/or other physical interactions The complex formation between proteins and polysaccharides, which could be widely utilized to control release of proteins and drugs, has been the other active field of research in the past decade due to simultaneous uses in a variety of food products (Schmitt, & Turgeon, 2011). The formation of protein-polysaccharide complexes is mainly driven by electrostatic interactions requiring the control of several physicochemical parameters influencing the overall and local charge of the protein and the polysaccharide, namely, pH, ionic strength, protein to polysaccharide ratio and total biopolymer concentration, biopolymer molecular weight and flexibility, charge density, stirring, pressure and temperature (Antonov, Zhuravleva, Cardinaels, & Moldenaers, 2017; Schmitt, & Turgeon, 2011). The pH values are required to be between the isoelectric point (pI) of the protein and the pKa value of the anionic groups of the polysaccharide, which stimulates the formation of electrostatic complexes. An assessment of protein surfaces revealed that positive charges were almost evenly distributed along lysozyme (Van, Andersen, & Frokjaer, 2004). The functional characteristics of lysozyme, including solubility, surface activity, enzyme activity, conformational stability, antimicrobial activity, emulsifying and foaming properties, varied upon interaction with different polysaccharides. Alginates, composed of β-d-mannuronic acid (M block) and α-l-guluronic acid (G block) units acid (M block), are the commonly used natural, hydrophilic and biocompatible polysaccharides that form complexes with proteins via electrostatic interactions. Fuenzalida et al., (2016) prepared electrostatic self-assembled 11

nanocomplexes between oppositely charged alginate and lysozyme, where the increase in M/G ratio and molecular weight (Mw) of alginate or adding calcium ions enhanced the capacity of alginate to crosslink lysozyme molecules, and these molecules participated in the electrostatic interaction and hydrogen bond formation. A study by Amara, Eghbal, Oulahal, Degraeve, & Gharsallaoui, (2016) demonstrated that complexes were formed between lysozyme (0.714 g/L) and different concentrations of sodium alginate (from 0 to 4 g/L) at pH 7. The aggregation process as well as the electrostatic interactions were confirmed by turbidity and ζ-potential measurements. Complex formation led to the decline of the enzymatic activity of lysozyme, while the addition of CaCl2 contributed to the recovery of this activity, indicating the predominance of electrostatic interactions. Another negatively charged polysaccharide—low methoxyl (LM) pectin (degree of esterification: 22-28%, degree of acetylation: 20-23%) could also form complexes with lysozyme via electrostatic attractions, and was able to control the release of lysozyme from an edible antimicrobial film (Bayarri, Oulahal, Degraeve, & Gharsallaoui, 2014). A recent study by Souza, Da, Souza, Tosin, & Garciarojas (2017) revealed that high-methoxyl (HM) pectin (Galacturonic acid ≥74.0%; Mw 136,80 kDa) could also form a complex with lysozyme under a large pH range of 2.0–7.0, where hydrophobic interactions were involved in the complex formation process among the hydrophobic regions of lysozyme and ester methyl groups (-COOCH3) of HM pectins. Xu et al., (2015) revealed that interpolymeric complexes mediated by heat-induced self-aggregation can be obtained from lysozyme and κ-carrageenan (CRG, a sulfated linear polysaccharide) via physical interactions, including electrostatic attraction, hydrogen bonding, and hydrophobic interactions, which could enhance the thermal stability of lysozyme. Lysozyme/CRG complexes easily self-aggregated into structures with large size and low ζ-potential induced by pH. Lysozyme was also reported to form complexes with sodium caseinate (SC) and micellar casein (MC) (see Fig. 3b). The morphology of the prepared complex particles was influenced by the charge ratio (ChR) between lysozyme and SC or MC, as shown in Fig. 3b, which was induced by two types of driven forces acting in opposite directions; hydrophobic forces contributing to aggregation and weak polarization effects ascribed to electrostatic forces leading to dissociation of aggregates (Antonov, Moldenaers, & Cardinaels, 2017). 3.3. Immobilization via binding The nonspecific systematic distribution of proteins or polypeptides and the low concentration of these materials at the site are the major drawbacks of conventional drug delivery systems. Thus, a carrier system that protects and delivers such proteins or polypeptides to the targeted position is necessary. Different systems have been developed to immobilize proteins. Zhang, Tao, Niu, Li, & Chen (2017) prepared microgels based on starch to guard against the early release of lysozyme in the stomach and deliver targeted objects to the intestine. The formation of microgel-lysozyme complexes did not destroy the secondary structure of lysozyme, suggesting that the microgels could potentially be used as a carrier system to protect 12

lysozyme. Additionally, charged core–shell microgels could act as superior protein carriers with high loading efficiency and the ability to enhance the enzyme activity. Welsch, Becker, Dzubiella, & Ballauff (2012) developed core–shell microgels based on N-isopropylacrylamide-co-acrylic acid (pNiPAm) that presented ‘‘smart’’ colloidal carriers for lysozyme (see Fig. 3c). The binding of lysozyme to the negatively charged microgels was due to hydrophobic interactions and electrostatic forces that play key roles at low ionic strength. Moreover, the immobilization of lysozyme using sulfonic acid functionalized silane grafted copolymer via an adsorption process that was exothermic and spontaneous with maximum entropy was reported by Anirudhan & Rauf, (2013); where the optimum pH for maximum adsorption was 7.0, the adsorption equilibrium was reached within 3 hours and the Sips isotherm model perfectly fitted the process with a maximum adsorption ability of 37.68 mg/g. Due to the presence of hydroxyl (-OH) and carboxyl groups (-COOH) that are suitable for binding enzymes, hydroxypropyl methylcellulose acetate succinate (AS-L) can be used as the polymeric support to immobilize lysozyme, and the immobilized lysozyme has much higher specific activity than the native lysozyme (Chen, Yen, Wang, & Wang, 2003). Other systems based on carbohydrates aimed to improve the activity of enzyme molecules were developed by Jiang et al., (2017); Wu et al. (2018), where chitin nanowhiskers or chitosan hydrogels were applied to deliver lysozyme, resulting in superior enzymatic or antimicrobial activities. 4. Bacteriostatic mechanism of lysozyme 4.1. Lytic mechanism for the antibacterial activity of lysozyme It is generally believed that lysozyme can hydrolyse the β-l,4-glycosidic linkage of peptidoglycans, and the muramidase activity leads to breakdown of the murein layer and decrease of the mechanical strength of the bacterial cell wall, finally resulting in the death of the bacteria (Wang et al., 2005). This enzymatic property is evident when at least two of the disulfide linkages remain intact, as depicted in Fig. 1, which enables lysozyme to serve as a non-specific innate immunity molecule against the invasion of bacterial pathogens (Jollès, & Jollès, 1984). From the crystal structure, the β-1,4 glycosidic (C—O) bond of N-acetylmuramic acid and N-acetylglucosamine has been observed to be in the close proximity to the two potentially catalytic residues, Glu35 and Asp52 of lysozyme, which contributes the disintegration of the glycosidic bond, although the understanding of the atomistic details of lysozyme enzyme mechanism were not comprehensive over the last decades (i.e., whether the cleavage was mediated by SN1 or/and SN2 pathway) (Kirby, 2001). However, the bactericidal effect of lysozyme is limited to some gram-positive bacteria, as the main component on the outer surface of gram-negative bacteria is a protective lipopolysaccharide (LPS) layer along with proteins and phospholipids, where the access of lysozyme to the peptidoglycan layer of the cell wall is retarded by the outer LPS layer (Ibrahim, Kato, & Kobayashi, 1991). While lysozyme was used in combination with potentiating chemicals (e.g., EDTA, DL-lactic acid), lysis of several of the pathogenic and spoilage bacteria could be promoted and continuous, as these chemicals could allow partial removal of some cell wall components of the outermost surface (outer 13

membrane), and thus the penetration of lysozyme into the site of its action (peptidoglycan) was improved (Hughey, & Johnson, 1987). 4.2. Non-lytic mechanism for antibacterial activity of lysozyme Another mechanism of the bactericidal action adopted for lysozyme is independent of its muramidase activity but attributed largely to its structural factors, cationic and hydrophobic properties. This theory was supported by the fact that partially or completely denatured lysozymes lacking enzymatic activity could still work against both gram-positive and gram-negative bacteria (Ibrahim, Aoki, & Pellegrini, 2002; Masschalck, Houdt, Haver, & Michiels, 2001). Ibrahim, Matsuzaki, & Aoki, (2001) revealed that the antibacterial action of lysozyme was due to structural factors, which involved the existence of microbe-associated targets of lysozyme, rather than muramidase activities. In their study, the catalytic residue aspartic acid at position 52 of hen egg white lysozyme was substituted with serine (D52S-Lz), and the mutant cDNA was inserted into a yeast expression vector, pYES-2, to abolish the catalytic activity of lysozyme. The results showed that the mutation did not affect its ability against Staphylococcus aureus and Bacillus subtilis, while at the same time there was no change in the structure of D52SLz compared to that of wild-type lysozyme, as revealed by circular dichroism (CD) and intrinsic fluorescence emission spectra. A further heat treatment leading to enzyme inactivation also had no effect on the bactericidal activity of either wild-type or mutant D52S-Lz lysozyme. This can be regarded as the direct genetic evidence that the antimicrobial activity of lysozyme against these typical gram-positive bacteria is independent of its muramidase activity, and the antimicrobial activity would be ascribed to structural features. Pellegrini, Thomas, von Fellenberg, &Wild, (1992) reported that chicken egg white lysozyme killed gram-positive as well as gram-negative bacteria without addition of complement or EDTA. However, when lysozyme was denatured by dithiothreitol, it did not lose its bactericidal potency, and a further investigation of the morphology of E. coli treated by lysozyme indicated the gradual disintegration of bacterial cytoplasm, which could be ascribed to the cationic and hydrophobic properties of lysozyme. Ibrahim et al., 1996 also observed that the antimicrobial activity of lysozyme can be switched to include gram-negative strains by different levels of denaturation of this enzyme through heating at proper pH values. Lysozyme heated at 80 °C and pH 6.0 (retaining 50% residual enzymatic activity with the improved hydrophobicity) exhibited stronger binding affinity than the native lysozyme to the lipopolysaccharide of E. coli K12 and stimulated agglutination and an obvious decrease in colony-forming ability. Further assays including agglutination, killing and membrane integrity of E. coli all indicated that the denatured lysozyme could disrupt the cell membranes and inhibit the transport and incorporation of precursor molecules into peptidoglycans due to the proper acquisition of the hydrophobic pocket to the surface of the enzyme molecule rather than the effect caused by muramidase activity (Ibrahim et al., 1994). A study by Düring, Porsch, Mahn, Brinkmann, & Gieffers, (1999) revealed that heat-denatured T4 lysozyme and HEWL without enzymatic activity exerted bactericidal and fungistatic activities, 14

which were due to the observation that the amphipathic helix stretches in the C-terminus of lysozymes mediated their membrane-disturbing activities. Additionally, the decrease in lysozyme disulfide bonds could induce a great reduction in its lytic activity, enable the enzyme molecule to interact with bacterial membranes and thus promoted its antimicrobial activity towards gram-negative bacteria (Touch, Hayakawa, Saitoh, 2004). The findings that partial or complete denaturation of lysozyme by site-directed mutagenesis, heat or dithiothreitol, which induce changes in the secondary or tertiary structure of lysozyme, did not abolish its bactericidal action indicted that the bactericidal activity of lysozyme was not only attributed to the muramidase activity. 5. Application of lysozyme in the food industry Lysozyme has been widely used against bacterial, viral and inflammatory diseases in the pharmaceutical industries. Thanks to its unique characteristics (higher natural abundance, stability, small size and drug binding ability), lysozyme has been used as a model protein for investigating the interaction with different small molecules, including metal ions (Wang, Tan, Chen, Yue, & Song, 2009), dyes (Peng, Ding, Peng, Jiang, & Zhang, 2013), and several pharmaceutical drugs (Ding, Zhao, Huang, Sun, & Zhang, 2009; Liang et al., 2013), as well as in studying the relationship between protein folding and dynamics, structure-function relationship, and cell-to-cell interaction (Nakao, Arai, Koshiba, Nitta, & Kuwajima, 2015; Kiran, Prasad, & Prakash, 2017; Abey et al., 2016). It is noteworthy that the binding properties of lysozyme to food additives (e.g., food dyes, food antioxidants) offer a more comprehensive profile of the essence of toxicity evaluation and provide important insight into the metabolic mechanism of food additives (Peng, Ding, Peng, Jiang, & Zhang, 2013; Wu et al., 2015). More importantly, in proteomics studies involving complex protein systems, lysozyme could act as a useful tool. First, it has a negligible effect on the establishment of adsorption models that are essential to proteomics studies and the pharmaceutical industry. In this case, lysozyme and other proteins with different extinction coefficients present competitive adsorption onto ordered mesoporous materials, which could be used to incorporate adsorption isotherms into mathematical descriptions of chromatographic processes to assess complex protein separation in the column (Darwish, Robie, Desch, & Thiel, 2015). Due to its ability to breakdown the peptidoglycans of the cell wall to release the associated proteins, lysozyme could digest the proteins exterior to the cell wall in a methodical way, which can then be subsequently analysed by two-dimensional electrophoresis (2-DE), giving a complete picture of some portion of the proteome (Wright et al., 2005). Additionally, lysozyme could act as an unintrusive internal standard or a model protein, which plays a vital role in monitoring digestion to mass spectrometry of a proteomics process (Riter, Hodge, Gooding, & Jr, 2005; Aslebagh, Pfeffer, Fliesler, Darie, 2016). However, from our perspective, in this review we only emphasized its application in the food industry, acting as an antimicrobial substance or indicator in fish, meat, dairy, 15

fruits, vegetables and wines or serving as the active component integrated into food packaging systems as well as other active functions in the food matrix. Every year, the deterioration of food products results in great economic losses all over the world, and the contamination of food by microorganisms is the main cause. The development of methodology used to maintain the quality and extend the shelf life of foods is a hotly discussed issue. Therefore, the anti-microorganism properties of lysozyme give it extensive applications in food industry. For instance, lysozyme exerted an inhibitory effect against the growth of lactic acid bacteria (LAB) involved in curd acidification and cheese ripening (D'Incecco et al., 2016). Lysozyme peptides (LP) derived from hen egg lysozyme presented only 11% of the lysozyme lytic activity, while LP at a concentration of 100 μg·ml−1 completely inhibited Bacillus species that caused food contamination, including B. subtilis, B. licheniformis, B. megaterium, B. mycoides, B. pumilus, B. coagulans, B. amyloliquefaciens, B. polymexa and B. macerans (Abdou, Higashiguchi, Aboueleinin, Kim, & Ibrahim, 2007). Chitosan films incorporated with 60% lysozyme (w lysozyme/w chitosan) exhibited enhanced inhibition efficacy against bacteria, where 3.8 log cycles reduction in S. faecalis and 2.7 log cycles decrease in E. coli were observed, showing great potential in ensuring food quality and safety (Park, Daeschel, & Zhao, 2004). Lysozyme together with nisin clearly showed synergistic inhibition effect against food spoilage lactobacilli, even in the presence of high salt conditions. Lysozyme at 500 mg/ml showed very slow bacterial killing of L. curvatus 845; a 3:1 combination of lysozyme: nisin at the same concentration led to an almost identical level of killing as 500 mg/ml nisin after 60 min, but a much stronger killing after 2 hours (Chung, & Hancock, 2000). The control of malolactic fermentation is crucial to the quality of wine, where the conventional method is based on the use of sulfur dioxide that causes a health concern to sulfite-sensitive asthmatic consumers. Hen egg white lysozyme (HEWL) could be a suitable alternative due to its ability to control the spontaneous gram-positive bacterial growth that often causes spoilage or stuck fermentation. Cappannella et al. (2016) developed a system based on the immobilization of lysozyme into microbial chitosan beads to achieve the continuous, efficient and food-grade enzymatic lysis of lactic bacteria (Oenococcus oeni) in white and red wine. Moreover, recent decades have witnessed the effectiveness of antimicrobial packaging systems, referred to as migrating or non-migrating, with the distinction relying on the antimicrobial agent applied and on its interactions with the packaging and food matrix (Irkin, & Esmer, 2015). Packaging based on immobilization of antimicrobial enzymes provides a promising form of active packaging system applicable in food processing. The merit of antimicrobial packaging is that this form could protect the activity of antimicrobials to some extent, as it can avoid the direct contact of antimicrobials with other food components (e.g., lipids, proteins) (Mauriello, De, La, Villani, & Ercolini, 2005). Min, Rumsey, & Krochta, (2008) reported that the advantages of immobilization of antimicrobial lysozyme in whey protein isolate films lies in the observation that lysozyme can maintain a minimum inhibitory concentration at the 16

film (coating) outer surface and/or film–salmon interface much longer than if an equivalent amount of lysozyme is simply spread or sprayed on the fish surface. The formation of a complex between lysozyme and polyacrylic acid complexes integrated into hydrophilic whey protein isolate (WPI) film could contribute to the slow, sustained release of lysozyme, maintaining long-term antimicrobial effect and showing a great potential for food preservation (Ozer, Uz, Oymaci, & Altinkaya, 2016). Recent advances of applications of lysozyme and its derivatives in various foods are summarized in Table 3. 6. Conclusions and Future perspectives 6.1. Conclusions The anti-bacteria mechanism of lysozyme could be due to its muramidase activity, which hydrolyses the β-l,4-glycosidic linkage of peptidoglycans, to its the ability to bind to nucleic acids in microorganisms and cause bacterial genetic material to mutate or disintegrate, or to its non-lytic activity, including structural factors, cationic and hydrophobic properties. Extensive methods are used to characterize lysozyme, via which the primary, secondary and tertiary structure could be assessed in detail. Generally, the modification of lysozyme with polysaccharides via the Maillard reaction showed advantages in many aspects, such as improved solubility, stability, emulsion property, emulsifying characteristics and antimicrobial activity. The antimicrobial activity makes lysozyme an ideal candidate in food matrices, especially in the application of fish, meat, dairy, fruits, vegetables and wines. 6.2. Future perspectives i.

Lysozyme has great potential in the food preservation industry due to its peculiar characteristics, especially the strong bacteriostatic activities against gram-positive bacteria, and lysozyme is considered to be a safe food additive in some parts of the world. However, studies on the cellular toxicity of lysozyme and its residues in the body, as well as its corresponding potential risks to humans, should be reinforced.

ii.

Despite the numerous studies that have focused on the antimicrobial activities of lysozyme on bacteria, few of them exerted antifungal properties, and thus the extraction of other types of lysozyme that show this property is of great commercial importance, which could inhibit the growth of some fungi that lead to foodborne diseases.

iii.

The mass production and wide application of lysozyme is hindered by its high cost, and several attempts should be made to produce lysozymes from low-cost methods. Acknowledgements This work was supported by project 2017YFD0400403 and NSFC31671918, and by grants from the China Scholarship Council 17

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BBA - Proteins and Proteomics, 1834(2013), pp. 2064-2070 Burova et al., 2006 T. V. Burova, N. V. Grinberg, V. Y. Grinberg Conformational and phase transitions in lysozyme-thermosensitive polyelectrolyte complexes Polymer Science, 48(2006), pp. 294-301 Branen and Davidson, 2004 J. K. Branen, P. M. Davidson, Enhancement

of

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activities

by

ethylenediaminetetraacetic acid and lactoferrin International Journal of Food Microbiology, 90(1), pp. 63-74. Bradbury and Brown, 1973 J. H. Bradbury, L. R. Brown Determination of the dissociation constants of the lysine residues of lysozyme by proton-magnetic-resonance spectroscopy Febs Journal, 40(1973), pp. 565-576 Callewaert and Michiels, 2010 L. Callewaert, C. W. Michiels Lysozymes in the animal kingdom Journal of Biosciences, 35(2010), pp. 127-160 Cao et al., 2015 D. Cao, H. Wu, Q. Li, Y. Sun, T. Liu, F. Jing, Y. Zhao, S. Wu, X. Hu, N. Li Expression of recombinant human lysozyme in egg whites of transgenic hens Plos One, 10(2015), pp. e0118626 Carrillo et al., 2016 W. Carrillo, H. Spindola, M. Ramos, I. Recio, J. E. Carvalho Anti-inflammatory and anti-nociceptive activities of native and modified hen egg white lysozyme Journal of Medicinal Food, 19(2016), pp. 978-982 Cappannella et al., 2016 E. Cappannella, I. Benucci, C. Lombardelli, K. Liburdi, T. Bavaro, M. Esti Immobilized lysozyme for the continuous lysis of lactic bacteria in wine: bench-scale fluidized-bed reactor study Food Chemistry, 210(2016), pp. 49-55 Cegielska-Radziejewska and Szablewski, 2013 R. Cegielska-Radziejewska, T. Szablewski Effect of modified lysozyme on the microflora and sensory attributes of ground pork Journal of Food Protection, 76(2), pp. 338-342 Chaturvedi et al., 2016 S. K. Chaturvedi, J. M. Khan, M. K. Siddiqui, P. Alam, R. H. Khan Comparative insight into surfactants mediated amyloidogenesis of lysozyme International Journal of Biological Macromolecules, 83(2016), pp. 315-325 Chen et al., 2013 S. H. Chen, Y. H. Yen, C. L. Wang, S. L. Wang Reversible immobilization of lysozyme via coupling to reversibly soluble polymer Enzyme & Microbial Technology, 33(2013), pp. 643-649 20

Chen et al., 2005 X. Chen, F. Niyonsaba, H. Ushio, D. Okuda, I. Nagaoka, S. Ikeda, K. Okumura, H. Ogawa Synergistic effect of antibacterial agents human β-defensins, cathelicidin LL-37 and lysozyme against Staphylococcus aureus and Escherichia coli Journal of Dermatological Science, 40(2005), pp. 123-132 Chung and Hancock, 2000 W. Chung, R. E. W. Hancock, Action of lysozyme and nisin mixtures against lactic acid bacteria International Journal of Food Microbiology, 60(2000), pp. 25-32 Conte et al., 2011 A. Conte, I. Brescia, M. A. Del Nobile Lysozyme/EDTA disodium salt and modified-atmosphere packaging to prolong the shelf life of burrata cheese Journal of Dairy Science, 94(2011), PP. 5289-5297 Corsaro and Mallamace, 2011 C. Corsaro, D. Mallamace, A nuclear magnetic resonance study of the reversible denaturation of hydrated lysozyme Physica A Statistical Mechanics & Its Applications, 390(2011), pp. 2904-2908 Darwish et al., 2015 A. Darwish, T. Robie, R. J. Desch, S. W. Thiel Competitive adsorption of lysozyme and myoglobin on mesostructured cellular foam silica Microporous & Mesoporous Materials, 210 (2015), pp. 101-109. Ding et al., 2009 F. Ding, G. Zhao, J. Huang, Y. Sun, L. Zhang Fluorescence spectroscopic investigation of the interaction between chloramphenicol and lysozyme European Journal of Medicinal Chemistry, 44(2009), pp. 4083-4089 D'Incecco et a., 2016 P. D'Incecco, M. Gatti, J. A. Hogenboom, B. Bottari, V. Rosi, E. Neviani, L. Pellegrino Lysozyme affects the microbial catabolism of free arginine in raw-milk hard cheeses Food Microbiology, 57(2016), pp. 16-22 Duan et al., 2017 X. Duan, J. Li, Q. Zhang, T. Zhao, M. Li, X. Xu, X. Liu Effect of a multiple freeze-thaw process on structural and foaming properties of individual egg white proteins Food Chemistry, 228 (2017), pp. 243-248 Düring et al., 1999 K. Düring, P. Porsch, A. Mahn, O. Brinkmann, W. Gieffers The non-enzymatic microbicidal activity of lysozymes Febs Letters, 449(1999), pp. 93-100 Fleming, 1922 A. Fleming 21

On a remarkable bacteriolytic element found in tissues and secretions Proceedings of the Royal Society of London, 93(1922), pp. 306-317 Fuenzalida et al., 2016 J. P. Fuenzalida, P. K. Nareddy, I. Moreno-Villoslada, B. M. Moerschbacher, M. J. Swamy, S. Pan, M. Ostermeier, F. M. Goycoolea On the role of alginate structure in complexing with lysozyme andapplication for enzyme delivery Food Hydrocolloids, 53(2016), pp. 239-248 Gdovinová et al., 2017 V. Gdovinová, N. Tomašovičová, I. Batko, M. Batková, L. Balejčíková, V. M. Garamus, V. L.

Petrenko, M. V. Avdeev, P. Kopčanský

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Howarth and Lian, 1984 O. W. Howarth, L. Y. Lian Hen egg white lysozyme: carbon-13 nuclear magnetic resonance assignments and dependence of conformational flexibility on inhibitor binding and temperature Biochemistry, 23(1984), pp. 3522-3526 Hughey and Johnson, 1987 V. L. Hughey, E. A. Johnson Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease Applied & Environmental Microbiology, 53(1987), pp. 2165-2170

22

Huang et al., 2012 W. Huang, H. Xu, Y. Xue, R. Huang, H. Deng, S. Pan Layer-by-layer immobilization of lysozyme–chitosan–organic rectorite composites on electrospun nanofibrous mats for pork preservation Food Research International, 48(2012), pp. 784-791 Huopalahti et al., 2007 R. Huopalahti, R. López-Fandino, M. Anton, R. Schade Bioactive egg compounds. Fandiño Ibarra-Molero et al., 2016 B. Ibarra-Molero, A. N. Naganathan, J. M. Sanchez-Ruiz Modern analysis of protein folding by differential scanning calorimetry Methods in Enzymology 567(2016), pp. 277–314 Ibrahim et al.,1991 H. R. Ibrahim, A. Kato, K. Kobayashi Antimicrobial effects of lysozyme against gram-negative bacteria due to covalent binding of palmitic acid Journal of Agricultural & Food Chemistry, 39(1991), pp. 2077-2082 Ibrahim et al., 1994 H. R. Ibrahim, M. Yamada, K. Matsushita, K. Kobayashi, A. Kato Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C-terminus J. Biol. Chem. 269(1994), pp. 5059-5063 Ibrahim et al., 1996 H. R. Ibrahim, S. Higashiguchi, M. Koketsu, L. R. Juneja, M. Kim, T. Yamamoto, Y. Sugimoto and T. Aoki Partially unfolded lysozyme at neutral pH agglutinates and kills Gram-Negative and Gram-Positive bacteria through membrane damage mechanism Journal of Agricultural & Food Chemistry, 44(1996), pp. 3799-3806 Ibrahim et al., 2001 H. R. Ibrahim, T. Matsuzaki, T. Aoki Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function Febs Letters, 506(2001), pp. 27-32 Ibrahim et al., 2002 H. R. Ibrahim, T. Aoki, A. Pellegrini Strategies for new antimicrobial proteins and peptides: lysozyme and aprotinin as model molecules Current Pharmaceutical Design, 8(2002), pp. 671-693 Igarashi et al., 1999 K. Igarashi, M. Azuma, J. Kato, H. Ooshima The initial stage of crystallization of lysozyme: a differential scanning calorimetric (DSC) study Journal of Crystal Growth, 204(1999), pp. 191-200 Irkin and Esmer, 2015 R. Irkin, O. K. Esmer Novel food packaging systems with natural antimicrobial agents Journal of Food Science & Technology, 52(2015), pp. 6095-6111 23

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Aminlari

Physicochemical and functional properties of ultrasonic-treated tragacanth hydrogels cross-linked to lysozyme International Journal of Biological Macromolecules, 103(2017), pp. 948–956 Kothari et al., 2015 D. Kothari, D. Das, S. Patel, A. Goyal Dextran and Food Application. Springer International Publishing Kulsing et al., 2016 C. Kulsing, A. Z. Komaromy, R. I. Boysen22, M. T. Hearn, On-line determination by small angle X-ray scattering of the shape of hen egg white lysozyme 24

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25

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of

lysozyme

amyloidogenesis

by

phospholipids.

dimyristoylphosphocholine Biochimica et Biophysica Acta, 1861(2017), pp. 2934-2943 Prosapio et al., 2016 V. Prosapio, E. Reverchon, I. D. Marco 26

focus

on

long-chain

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Pharmaceutical Research, 21(2004), pp. 2354-2359 Wang et al., 2005 S. Wang, T. B. Ng, T. Chen, D. Lin, J. Wu, P. Rao, X. Ye First report of a novel plant lysozyme with both antifungal and antibacterial activities Biochemical & Biophysical Research Communications, 327(2005), pp. 820-827 Wang et al., 2017 Z. Wang, S. Hu, Y. Gao, C. Ye, H. Wang Effect of collagen-lysozyme coating on fresh-salmon fillets preservation LWT - Food Science and Technology, 75(2017), pp. 59-64 Wang et al., 2009 Z. Wang, X. Tan, D. Chen, Q. Yue, Z. Song Study on the binding behavior of lysozyme with cephalosporin analogues by fluorescence spectroscopy Journal of Fluorescence, 19(2009), pp. 801-808 Welsch et al., 2012 N. Welsch, A. L.Becker, J. Dzubiella, M. Ballauff Core-shell microgels as ``smart'' carriers for enzymes Soft Matter, 8(2012), pp.1428-1436 Woody, 1995 R. W. Woody Circular dichroism Methods in Enzymology, 246(1995), pp. 34-71 Wright et al., 2005 A. Wright, R. Wait, S. Begum, B. Crossett, J. Nagy, K. Brown, N. Fairweather Proteomic analysis of cell surface proteins from Clostridium difficile Proteomics, 5(2005), pp. 2443–2452 Wu et al., 2018 T. Wu, J. Huang, Y. Jiang, Y. Hu, X. Ye, D. Liu, J. Chen Formation of hydrogels based on chitosan/alginate for the delivery of lysozyme and their antibacterial activity Food Chemistry, 240(2018), pp. 361-369 Wu et al., 2017 T. Wu, C. Wu, S. Fu, L. Wang, C. Yuan, S. Chen, Y. Hu Integration of lysozyme into chitosan nanoparticles for improving antibacterial activity Carbohydrate Polymers, 155(2017), pp. 192-200 Wu et al., 2015 D. Wu, J. Yan, P. Tang, S. Li, K. Xu, H. Li Binding properties and structure-affinity relationships of food antioxidant butylated hydroxyanisole and its metabolites with lysozyme Food Chemistry, 188(2015), pp. 370-376 Xue et al., 2004 Q. G. Xue, K. L Schey, A. K. Volety, F. L. Chu, J. F. La Peyre Purification and characterization of lysozyme from plasma of the eastern oyster (Crassostrea virginica) 29

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30

Fig.1. Structure of lysozyme (modified from Hamdani, Wani, Bhat, & Siddiqi, 2018).

31

c

Fig. 2. (a) SDS-PAGE of lysozyme from hen egg white. C (lysozyme)= 2.0 mg/ mL; (b) Thermogram generated by DSC analysis of lysozyme (Santos, & Garcia-Rojas, 2018); (c) T2∗ determined by 1H

HRMAS NMR as a function of temperature. Vertical lines indicate the border between the native and the intermediate region (TB, dotted line), and between the intermediate and the unfolded region (TD, solid line) (Corsaro, & Mallamace, 2011)

32

(a)

(b)

(c) 33

Fig. 3. (a) Schematic illustration of the reaction mode of TRG arabinogalactan with lysozyme via Maillard reaction (Koshani, Aminlari, Niakosari, Farahnaky, & Mesbahi, 2015); (b) Morphological changes in lysozyme/SC and lysozyme/MC mixtures as a function of charge ratio ChR. Where, concentrations of SC and CM are both 0.02 wt%, concentration of lysozyme in the mixtures is varied. pH 7.0, I = 0.01, 23°C; (b) Schematic illustrating the complex structure as a function of charge ratio for sodium caseinate (SC) and micellar casein (MC) (Antonov, Moldenaers, & Cardinaels, 2017); (c) Top: interaction between a positively charged protein carrying charged patches and the negatively charged microgel. The dashed line indicates the dimensions of the gel network. The protein is protonated by the electrostatic potential of the polymer network when it is closed to the core–shell particle. The protein binds to the charged network because of electrostatic and hydrophobic interactions which lea to microgel deswelling. Bottom left and right represents structure of the cross-linked p(NiPAm-co- AAc) shell and chicken egg-white lysozyme (PDB: 193L), respectively. The amino acids are colored as follows: positive (blue), negative (red), polar (yellow), and hydrophobic (grey) (Welsch, Becker, Dzubiella, & Ballauff, 2012)

34

Table 1 The main characteristic peaks of lysozyme in infrared (IR) spectra Range of bands (cm−1)

Chemical bonds or conformation

Around 3294

Hydrogen bonding, stretching band of–NH

∼3100–2980

C–H stretching

Amide I

1650–1658

α-helix

(1700–1600 cm−1)

1620–1640 & 1670–1695

β-sheet

1640–1648

Unordered conformation

Around 1670, 1683, 1688 and 1694

β-turns

1600–1500

In-plane N–H bending and C–N stretching

Amide II

vibration (not sensitive to conformation) Amide III

1320–1230

Very weak, ignored

35

Table 2 Comparison of different techniques for determining conformational changes of lysozyme-based materials Techniques

Sample state

Available information

Merits

Demerits

SDS-PAGE

Solution

Primary structure

Intuitive

Identification of lysozyme ONLY but not quantitative analysis

Small amounts of sample were required (μg)

CD

Solution

Secondary structure (α-helix,

Fast, intuitive

Accurate sample concentrations and reference databases

β-sheet, turns and unordered

are essential Low protein concentration (∼1 mg/ml)

(random)

IR

Powder/ solution

Secondary structure

Marked characteristics

Not suitable for the analysis of samples containing water, as the hydroxyl can interfere with the determination

Information rich (functional group; spatial structure,

the

position and

amount

of

Unable to achieve quantitative analysis

substituent group) The analysis of data depends mainly on experience

UV-vis spectroscopy

Solution

Tertiary structure

Fast and easy

Unable to determine samples that are insoluble

Sensitive to conformational changes under

The spectra is dependent on the solvent

different conditions

Fluorescence

Solution

Tertiary structure

spectroscopy

Useful

for

in-depth

thermodynamic

properties and non-covalent acting forces

Not suitable for direct assay of lysozyme-based materials in the powder state

studies

NMR1H and NMR13C

Solution

Secondary structure

Convenient

High cost

Non-destructive to samples

The

preparation

of

samples is complicated

and

time-consuming Provide dynamic information

DSC

Powder/solution

Thermodynamic parameters

Ease and simplicity of operation

(Tm, △Hd, △G, △Cp, RI)

Sample preparation is very complicated if the sample is liquid Requires knowledge of database for operating the experiment

and

calculating

the

thermodynamic

parameters Errors in the measurement of result (i.e., ΔΗ) due to minor impurities or the baseline chosen in small enthalpy systems

36

Table 3 Recent advances of lysozyme-based biomaterials in food industry applications Lysozyme-based biomaterials

Food products

Results (as compared with the control)

Reference

Effective in reducing TVB-N and inhibiting the

Wang et al., (2017)

(a) Fish and meat-related products Lysozyme-collagen coating

Fresh salmon fillets

growth of bacteria Lysozyme-catechin incorporated

Minced pork

gelatin film

Inhibit lipid oxidation (TBARS) and microbial

Kaewprachu et al., (2015)

growth (total plate count) of the refrigerated minced pork

Thermochemically modified

Pork

lysozyme (60 °C for 10 min)

Reduce the counts of Pseudomonas and

Cegielska-Radziejewska &

Enterobacteriaceae in meat and increase the odour

Szablewski, (2013)

score of ground pork compared with the control Lysozyme-chitosan-organic

Pork

Control the growth of Escherichia coli and

rectorite/sodium alginate film

Staphylococcus aureus. Reduce the TVB-N value,

coating

increase the sensory quality, and extend the

Huang et al., (2012)

shelf-life of pork approximately 3 days Lysozyme incorporated into paper

Carpaccio

containing carboxymethyl cellulose

Effective in inhibiting E. coli and L. innocua.

Barbiroli et al., (2012)

Almost 1 log cycle reduction of thin meat slices in total aerobic count compared to the control

(b) Beverages Lysozyme

Unpasteurized beer

Efficient in preventing the growth of lactic acid

Silvetti et al, (2012)

bacteria (LAB) implicated in beer spoilage; have no impact on the flavour of beer; extend the shelf life of beer Lysozyme-chitosan beads

White wines

Could be regarded as an efficient antimicrobial

Liburdi et al., (2016)

agent during the winemaking process Lysozyme and submerged culture

Sherry wines

Effective against lactic acid bacteria and acetic acid

Roldán et al., (2017)

bacteria (c) Others Heat-denatured lysozyme

Salads and dressings

Reduce the infectivity of murine norovirus strain 1

Takahashi et al., (2016)

Lysozyme-gum arabic complex

Mayonnaise

Acts as a natural preservative and emulsifier

Hashemi et al., (2018)

Heat-denatured lysozyme

Berry fruit

May be an effective disinfectant for berry fruit, as it

Takahashi et al., (2018)

can inactivate hepatitis A virus (HAV) in addition to norovirus Lysozyme-chitosan films

Chicken eggs

Maintain the internal quality (especially Haugh unit and pH) of eggs for at least 3 weeks longer than uncoated ones

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Yuceer, & Caner, (2014)

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Highlights    

The bacteriostatic mechanism of lysozyme has been reviewed Techniques such as CD and IR could show the secondary structures (α-helix, β-sheet, turns and unordered) of lysozyme in detail Modifications via the Maillard reaction as well as other interactions have broadened applications of lysozyme in different orientations Lysozyme-based biomaterials are effective in maintaining the qualities of foods

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