Chemical and biological protection of food grade nisin through their partial intercalation in laminar hydroxide salts

Abstract

The use of antimicrobial agents within a matrix, specifically layered compounds, is of growing interest for reducing contamination due to food borne pathogens and deteriorative microorganisms, one of the main health problems worldwide. In this study, zinc layered hydroxide nanoparticles were synthesized as a matrix for nisin immobilization. Layered materials were characterized by X-ray diffraction, Fourier-Transform Infrared and Ultra Violet–Visible spectra, Scanning Electron Microscopy, and by Thermogravimetric Analysis. Thermal, chemical, enzymatic, and biological stabilities were assessed against Lactobacillus brevis as control strain. Free and immobilized nisin in solution were previously subjected to 25 and 121 °C, pH (7, 9) and inactivation with protease before antimicrobial tests that lasted 21 days. Immobilized nisin was found to maintain the activity levels after the protease action while the pure nisin solution lost its activity gradually. Furthermore, immobilized nisin treated at 121 °C and pH 7 showed higher activity than pure nisin after 21 days. These results may support that immobilizing nisin in zinc layered hydroxide salts promoted extended nisin inhibitory activity in solution after thermal, chemical or enzymatic treatments. This research provides an alternative to nisin application that could be used in processes where such operating conditions take place, as in dairy products.

Introduction

Antimicrobial activity is related with compounds that kill bacteria locally or stop their growth without provoking hazard to surrounding tissues. Most antimicrobial agents are molecules obtained from modified natural compounds as β-lactams, but their abuse has led to the development of bacterial resistance (Hajipour et al. 2012).

The use of natural antimicrobial agents, i.e. bacteriocins, has become more common. These molecules are peptides produced from the secondary metabolism of lactic-acid bacteria, and they are used in the food industry because of their ability to inhibit pathogens and deteriorative microorganisms. Nisin is a bacteriocin produced by Lactococcus lactis, and its antimicrobial activity is of selective spectra against several microorganisms. Nisin mode of action is by attaching to the cell membrane and producing pores, leading to leaking of intracellular compounds (Alves de Oliveira et al. 2015). Nisin is cataloged as GRAS (generally recognized as safe) by the Food and Agriculture Organization (FAO 2019), and it has been used for inhibition of Clostridium botulinum, Listeria monocytogenes, and other pathogens with a maximum dose of 250 ppm in the final product (Bouaziz et al. 2017).

Nevertheless, nisin bioavailability has been a restricting factor in medical and food industries because its efficiency reduces in time, probably due to interaction with other molecules found in food (Bouaziz et al. 2017), alkaline pH, high temperature, and/or interaction with proteases (Sanlibaba et al. 2009).

In order to protect bioactive molecules from degradation or rapid liberation, biocompatible nanomaterials that act as carriers have been developed, minimizing side effects in consumers (Ghamami et al. 2017; Barahuie et al. 2014). Novel nanocarriers that have been researched nowadays are laminar materials. These materials are compounds in which crystals are formed by stacking bi-dimensional units bound through weak interactions. Laminar compounds can be (a) Laminar hydroxide salts (LHS) with the formula M²⁺(OH)_2−x(A^m−)_x/m · nH₂O; or (b) Laminar double hydroxides (LDH), ${\text{M}}_{1-x}^{2+}{\text{M}}_x^{3+}{\left(\text{OH}\right)}_2{\left({\text{A}}^{m-}\right)}_{x/m}·{\text{nH}}_2\text{O}$ where M²⁺ represents a divalent metal, M³⁺ a trivalent metal, and A^m− is the intercalated ion (Nabipour et al. 2015; Barahuie et al. 2014; Carbajal Arizaga et al. 2007). These compounds are capable of retaining chemical species with compatible electric charges to those shown in the sheet (Carbajal Arizaga et al. 2007). Examples of intercalated compounds are ciprofloxacin, ibuprofen, dodecyl sulfate, low density polyethylene, para-amino salicylate, amino acids, and many others (Nabipour et al. 2015, 2017; Demel et al. 2014; Jaeger et al. 2014; Saifullah et al. 2013; Carbajal Arízaga 2012).

The aim of this study was to synthesize materials composed of Zn-LHS and nisin, characterize them, and test their chemical and biological stability through antimicrobial activity after different physical and enzymatic treatments.

Materials and methods

Synthesis of ZnHS and Nisin-ZnHS

Briefly, 200 mL of a solution containing ZnCl₂ (0.04 g/mL) was kept under stirring at room temperature for 20 min. Then, 200 mL of NaOH 0.1 M were added to obtain pH of 8, and after 20 min of stirring, precipitate (ZnHS) was collected by centrifugation (10,000 rpm, 24 °C for 10 min,), washed thoroughly with distilled water in equal synthesis volume, and dried at room temperature for 24 h. For nisin intercalation, 90 mL of a solution of 5 mg/mL nisin (Sigma Aldrich, St. Louis, MO, USA) was prepared, and 500 mg of dried ZnHS were added with constant stirring at room temperature for 4 h. Material (Nisin-ZnHS) was collected by centrifugation at 1600 rpm at 25 °C for 15 min and dried at room temperature for 24 h.

Characterization

Powder X-ray diffraction (XRD) patterns were collected with an Panalytical Empyrean X-ray diffractometer (Panalytical, Malvern, UK) using CuΚα radiation with a 2θ angle from 5 to 70° in a 0.02 step and collection time of 30 s. Fourier-transform infrared spectra (FTIR) was recorded in the range of 4000–400 cm⁻¹ in transmittance mode using a FTIR spectrophotometer (Thermo Scientific Nicolet iS5 iD5, Waltham, MA, USA). A thermogravimetric (TG) analysis was performed on a Discovery model from TA Instruments, (New Castle, DE, USA). Samples were weighed in the range from 5 to 10 mg. TG curves were recorded by heating samples from 25 to 900 °C at 10 °C min⁻¹ under nitrogen atmosphere. Scanning electron microscopy (SEM) was recorded in a FE-SEM (TESCAN, model MIRA 3 LMU, Brno, Kohoutovice, Czech Republic). Nisin concentration was measured by spectroscopy at 208 nm with a nanodrop 2000 (Thermo Scientific, Waltham, MA, USA) in supernatant during intercalation, and interfacial concentration was calculated as mentioned by Bouazis et al. (2017). For the liberation test, 17 mg of Nisin-ZnHS were dispersed in 50 mL of phosphate buffered saline (PBS) and kept in constant stirring for approximately 21 days (500 h); supernatant was measured with spectroscopy at 208 nm (nanodrop 2000, Thermo Scientific, Waltham, MA, USA).

Antimicrobial evaluation

Antimicrobial activity of zinc hydroxide salt (ZnHS) and intercalated ZnHS with nisin (Nisin-ZnHS) were evaluated by the plate count method and the agar diffusion assay. Lactobacillus brevis strain BTO7CC from the microorganism collection of the Laboratory of Molecular Biology (Guadalajara, MX) was used as reference strain during this study because of its capacity to be inhibited in a linear way by increasing nisin concentration as previously reported for L. sakei (Pongtharangkul and Demirci 2004). For the plate count method, L. brevis was harvested in Man, Rogosa, and Sharpe (MRS) broth at 37 °C for 24 h; then, optical density was adjusted to 1 × 10² cell/mL in MRS broth using an ultraviolet–visible (UV–Vis) spectrophotometer at 600 nm (Optizen POP, Seoul, Korea). Nisin-ZnHS and nisin were added to a final concentration of 2.5 mg of nisin/mL; ZnHS was evaluated at the same concentration of Nisin-ZnHS. Bacteria with and without materials were incubated at 37 °C for 24 h. Serial dilutions in sterile Phosphate Buffered Saline (PBS) pH 7.4 were carried out, and aliquots of 100 μL were spread on MRS agar. Plates were incubated at 37 °C for 24 h, and number of colony-forming units (CFU) was recorded. For agar diffusion assay, L. brevis was harvested to a concentration of 1 × 10⁵ cell/mL and spread on MRS agar plates, and four holes were bored; 100 μL of the solutions containing nisin (0, 100, 200, and 300 ppm) were added in the holes, and plates were incubated at 37 °C for 24 h. The diameter of the inhibition zone around each well was measured and plotted against nisin concentration to obtain a standard curve, and the concentration of nisin released from the hydroxide salt was correlated.

Chemical stability of immobilized nisin

Chemical, enzymatic and thermal stabilities of Nisin-ZnHS were tested. For stability with respect to time, solutions of pure and immobilized nisin were prepared to a final concentration of 3 mg/mL in PBS and kept in refrigeration (4 °C) throughout the experiment (21 days). The biological nisin activity was followed by the agar diffusion assay as previously described.

Chemical stability was evaluated with pure and immobilized nisin previously treated in solutions of pH 7 and 9 against L. brevis. The inhibition diameter was measured after incubation at 37 °C for 24 h. For thermal stability, solutions of pure and immobilized nisin were heated at 121 °C for 20 min and then, placed in contact with L. brevis. Plates were incubated at 37 °C for 24 h, and inhibition diameters were measured. The combination of pH (7 and 9) and heat treatments (25 and 121 °C) were also studied.

Being a peptide, nisin activity is reduced in contact with proteases, such as trypsin, actinase E, and proteinase K (Sanlibaba et al. 2009). For enzymatic stability, solutions of pure and immobilized nisin were added to a solution of proteinase K (Promega, V3021, UK) in 50 mM potassium phosphate at a final concentration of 1 mg/mL at 37 °C for 24 h. Enzyme was inactivated at 65 °C for 30 min; then, it was added to holes bored previously in MRS agar plates with L. brevis at a concentration of 1 × 10⁵ cell/mL. Inhibition diameter was measured after 24 h of incubation at 37 °C. Antimicrobial tests were performed in triplicate, and the mean ± standard deviation (SD) for each analysis was calculated. A one-way analysis of variance (ANOVA) was performed to determine the effect of immobilizing nisin in ZnHS using Statgraphics Centurion XVII software v17.

Results and discussion

X-ray diffraction, infrared spectra and thermogravimetric analysis

Figure 1 depicts XRD patterns, IR spectra and TG curves for Zinc layered hydroxide salt (ZnHS) and nisin-Zinc layered hydroxide salt (Nisin-ZnHS). The X-ray diffractogram (Fig. 1a) showed that the structures obtained, corresponded to the phase of Zn₅(OH)₈Cl₂ · 2H₂O (ICDD card 07-0155) for both materials (Sithole et al. 2012; Cousy et al. 2017). The layered structure remained with a displacement of the diffraction peak at 2θ angle from 11.6 to 8.3 after contact with the biomolecule, which corresponds to an opening of the interlayer space of 2.874 $\dot{\text{A}}$. This opening in the structure may suggest a nisin intercalation in the interlayer space, as reported by Bouaziz et al. (2017) in Zn/Mg and Zn/Al double layered hydroxides. Comparing both materials, LHS was observed to produce a bigger interlayer space, and thus, gave more cavity to the biomolecule than the LDH.

Fig. 1.

Zinc hydroxide salt (ZnHS) and Nisin-Zinc hydroxide salt (Nisin-ZnHS) characterization: a X-Ray Diffractogram; b Infrared spectra; c Thermogravimetric curves

In the IR spectra (Fig. 1b), a region around 3500–3000 cm⁻¹ was attributed to vibration of O–H bonds in both compounds, as well as O–H vibrations in the region of 1630–1250 cm⁻¹. Presence of Zn–O bonds can be elucidated with the signals centered at 500 cm⁻¹ (Carbajal Arízaga 2012). For hydroxide with immobilized nisin, a signal in the region of 1680–1610 cm⁻¹ was related to C=O tension (amide I), and a flexion of N–H (amide II) around 1545 cm⁻¹; both bands corresponded to peptide bonds (El-Jastimi and Lafleur 1997). A reduction in the band intensity located at 3500 cm⁻¹ of hydroxide bonds may suggest that the OH in the layered compound could be interacting with nisin residues (Jaeger et al. 2014).

Thermogravimetric curves are depicted in Fig. 1c. Zinc hydroxide salt curve resembles to those reported elsewhere (Lins et al. 2018; Cursino et al. 2015; Velazquez-Carriles et al. 2018). With respect to weight, Nisin showed thermal stability until reaching 600 °C, with a small weight loss of 10%; despite of this loss, peptide conformation could have been affected with temperature, and thus, its antimicrobial activity (Niaz et al. 2018). When intercalation took place, the composite showed a first event at 225 °C due to surrounding and interlayer water loss from hydroxide salt (20%). The next event at 450 °C, showed less weight reduction in the Nisin-ZnHS (10%) compared to the ZnHS alone, which could suggest that during intercalation, several Cl⁻ and OH⁻ ions inside the layers were substituted for nisin, thus, HCl liberation was reduced. Subsequently, ZnO was formed (Velazquez-Carriles et al. 2018).

Nisin adsorption and liberation test

Nisin adsorption in the zinc layered hydroxide salt, measured with UV–Vis, is depicted in Fig. 2. During the first 2 h of contact, the maximum amount of nisin that could be adsorbed in the hydroxide salt was achieved, with a concentration of 0.53 mg nisin/mg ZnHS, which corresponded to 530 UI/ZnHS mg, approximately; this value remained practically constant until 4 h later when the mixing was stopped. In the first 2 h, nisin was adsorbed with a ratio of 0.43 mg per hour, and after this time, adsorption reduced to 0.06 mg per hour (r = 0.98). Bouaziz et al. (2017) reported several nisin concentrations in different LDH in the range from 0.17 to 0.33 mg nisin/mg LDH in 4 h. The result obtained in this study using LHS was higher than LDH in lesser time, which could be achieved because the interlayer space was less uniform in the LDH structure containing two different metals than in the LHS matrix, providing a more suitable space for nisin to be intercalated.

Fig. 2.

Nisin adsorption in Zinc layered hydroxide salt

For the liberation test (Fig. 3), nisin in supernatant was measured with UV–Vis spectra at 208 nm. Liberation ratio was linear (r = 0.96) with a rate of 0.007 mg nisin/hour throughout the evaluation time. At 21 days (500 h), percentage of estimated nisin released from the hydroxide salt was of 50%. In a previous study that we conducted for liberation in water, nisin reached 20% of the estimated concentration in the first 50 h, while it was of 10% in PBS in the same time. The amount of different salts found in the solution that promote desorption–adsorption phenomena could support these data obtained. Nisin release from LDH synthetized by Bouaziz et al. (2017) showed a 70% liberation in 21 days. With these results, nisin might not only be intercalated in a higher dose but also capable of prevailing in the LHS matrix longer than in LDH, thus, prolonging the activity.

Fig. 3.

Nisin liberation from zinc layered hydroxide salt

Laminar hydroxide salts morphology

Scanning Electron Microscopy micrographs for morphological analysis are depicted in Fig. 4. A characteristic hexagonal structure for zinc layered hydroxide salts was obtained with an average longitude of 1 μm and height inferior to 0.1 μm (Fig. 4a). During synthesis, addition of NaOH plays an important role in defining crystallinity of LHS (Cousy et al. 2017), so in this test, an imperfect surface crystal was obtained, which could help in the adsorption of the biomolecule. This result is supported in a previous experiment in which a more crystalline material was placed in contact with nisin, and the amount of peptide retained in the structure was inferior to the imperfect crystal (data not shown). After nisin intercalation (Fig. 4b), the hexagonal morphology remained but roughness increased on the surface. Moreover, particle agglomeration, presumably nisin, could be observed; this structural property could allow a better interaction with microorganisms in antimicrobial tests (Bouaziz et al. 2017).

Fig. 4.

Scanning Electron Microscopy micrographs of a Zinc layered hydroxide salt; b Nisin-Zinc layered hydroxide salt

Antimicrobial activity evaluation

To verify antimicrobial activity of immobilized nisin, plate count was used. After 24 and 48 h of contact with immobilized nisin in layered hydroxide, growth of lactic-acid bacteria L. brevis was reduced in 0.5 and 1.0 Log colony forming units (CFU) per milliliter, respectively (Fig. 5a). This result proved that nisin, once adsorbed in the layered hydroxide, remained active.

Fig. 5.

Inhibition of Lactobacillus brevis in a 24 and 72 h. ZnHS: Zinc hydroxide salt; Nisin-ZnHS: Nisin-Zinc hydroxide salt; b inhibition halo of L. brevis (cm) with respect to nisin concentration (IU/mL)

Different tests were carried out to verify the stability of immobilized nisin. A curve was constructed to correlate the nisin concentration with the inhibition halo produced (Fig. 5b), which was used to determine the inhibition activity of immobilized nisin as previously described. Figure 6 shows activity of released and immobilized nisin as International Units per mL (UI/mL).

Fig. 6.

Activity of released and immobilized nisin. Horizontal axis: time (days); Vertical axis: Nisin activity (IU/mL)

Pure nisin activity, compared with neutral conditions (pH 7, 25 °C), was highly reduced with heat treatments for both pH conditions (30% for pH 7 and 40% for pH 9), as well as when placed in contact with proteinase K (more than 40%) by the end of day 23, while the activity of nisin in pH 9 and 25 °C was slightly reduced (less than 20%). Reduction in activity was almost linear, suggesting that the activity was constantly lost. Being a peptide, nisin may suffer denaturation with high temperatures, and alkaline conditions may also contribute to this degradation (Sanlibaba et al. 2009); these characteristics may be the reason of this activity loss. Proteinase K recognizes serine residues, and in the nisin structure, this residue can be found near the carboxylic end-group, and it helps the bacteriocin to attach and produce the pores in the bacterial cell membrane. (Alves de Oliveira et al. 2015).

On the other hand, nisin released from the zinc layered hydroxide salt showed difference in activity loss compared to pure nisin in all treatments. At neutral pH and 25 °C, an increment of activity was observed until day 15; until the end of the study, the activity remained practically constant but inferior to that of pure nisin. On the other hand, at 121 °C treatment, released nisin activity kept increasing, while pure nisin still diminished. Despite having slightly less activity than pure nisin, released nisin activity was less reduced than pure nisin. At this temperature, layered hydroxide salts tended to release water molecules from the inside and surface, and probably, part of the nisin molecule may occupy those empty spaces; thus, activity was less affected (Morales Borges et al. 2009). Nonetheless, the zinc layered hydroxide salt matrix was not able to protect the nisin in alkaline pH, probably due to hydroxide oxidation. When a layered hydroxide salt is placed in a basic solution, the relationship of M/OH changes, and when a critical value of this relationship is exceeded, oxidation takes place (Blaise et al. 2018). Finally, the activity of the immobilized nisin treated with the enzyme showed slightly higher values than the pure nisin by the end of day 20 and kept increasing, while the pure nisin continued losing activity. For this reason, part of the nisin active section may have been covered within the hydroxide salt, and thus, proteinase K was not able to reach and inactivate it.

These results suggested that nisin immobilization in zinc layered hydroxide salts allowed the biomolecule to keep its biological activity in solution longer than pure nisin when treated at neutral pH and high temperatures or in the presence of proteolytic enzymes. Further tests in some food products should be performed to support these results, especially in dairy foods that can be contaminated with foodborne pathogens as L. monocytogenes.

Conclusion

This study synthesized an inorganic matrix composed by zinc layered hydroxide salt for hosting the bacteriocin nisin. This matrix was able to maintain the activity of nisin higher after different treatments than when nisin was pure in solution. The zinc layered hydroxide nanoparticle can provide nisin protection, thus promoting peptide activity by controlled release in an aqueous system. According to these results, the ZnHS with bacteriocins can be considered as a strategy to extend food shelf-life.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflicts of interest.