ABSTRACT
The aim of this study was evaluated the effect of selected bioactive substances and nanoparticles on the immunoreactivity of edible packages based on chitosan using the ELISA. The analysed protein was the tropomyosin. The results confirmed the presence of the tropomyosin (3.77 ± 0.79–5.75 ± 0.01 µg/g) in control samples. This study demonstrated that the bioactive substances in the form of grape (0.61 ± 0.34–0.43 ± 0.16 µg/g), blueberry (0.58 ± 0.32–0.39 ± 0.27 µg/g), and parsley extracts (2.09 ± 1.28–0.79 ± 0.40 µg/g) reduces immunoreactivity (p < 0.05) of the tropomyosin. The elder pollen had no significant effect (p > 0.05) on immunoreactivity. ZnO and TiO2 nanoparticles also demonstrated immunoreactivity reduction (p < 0.05). The exception was silver nanoparticles, where the immunoreactivity increased with increasing concentration of grape extract (1.29 ± 0.01–5.47 ± 0.25 µg/g). The results confirmed the inhibitory effect of bioactive substances on the immunoreactivity of the used ELISA and also showed the need to consider immunoreactive substances when interpreting ELISA results.
Introduction
Crustaceans are one of the most important sources of food allergies and their main allergen is the tropomyosin protein (Shibahara et al., Citation2007). Tropomyosin is a heat-stable protein with a molecular weight ranging from 36 to 38 kDa and is associated with immunoglobulin E (IgE) antibody-mediated hypersensitivity (Werner et al., Citation2007). Although the tropomyosin is soluble in water, its extraction is facilitated by a strong salt solution (Baley, Citation1952). In sensitive individuals, allergic symptoms can appear within a few minutes after ingestion, in the form of nausea, vomiting, hives, asthma, and even anaphylactic shock (Davis et al., Citation2020; Poms et al., Citation2004). The group of crustaceans mainly includes shrimps, prawns, crabs, crayfish, and lobsters (Davis et al., Citation2020; Wild & Lehrer, Citation2005). Chitosan is obtained from the exoskeleton of crustaceans (Dutta, Citation2017) and it is a polysaccharide that can also act as a food allergen due to its structure and molecular weight (Kato Yasuko, Citation2005). Cases of allergy to chitosan are reported after oral ingestion, when systemic urticaria and breathing difficulties developed (Kato Yasuko, Citation2005).
Allergic individuals must avoid foods containing shellfish, which requires highly sensitive and accurate detection methods (Eischeid et al., Citation2021). A suitable method for the detection of shellfish allergens in food is the sandwich ELISA (Eischeid et al., Citation2021; Poms et al., Citation2004; Shibahara et al., Citation2007; Yu et al., Citation2019).
The use of chitosan in the food industry is driven by price, consumer acceptance as a natural bioactive polysaccharide and intrinsic antimicrobial activity. Thanks to its exceptional physicochemical properties, which are given by the polysaccharide skeleton, chitosan is a natural alternative to chemically synthesized antimicrobial polymers (van den Broek et al., Citation2015). Chitosan has unique properties such as biodegradability, biocompatibility, non-toxicity and renewability (Barikani et al., Citation2014), therefore it can be used as an alternative to the production of food packaging (Motelica et al., Citation2020).
Studies of materials used to make edible packaging also note additives that can increase their functionality. An active form of packaging can thus contribute to maintaining the nutritional profile of packaged foods. For example, antioxidants protect food from oxidation, which can result in a change in the colour, taste or smell of the food. Natural antioxidants can be, for example, plant extracts, essential oils, α-tocopherol, ascorbic acid and citric acid, bee pollen or propolis (Trajkovska Petkoska et al., Citation2021). Interactions between proteins and phenolics have been reported to alter protein immunoreactivity (Zhang et al., Citation2018). Immunoreaction is an immunologic reaction between antigen and antibody.
Nanotechnology has proven to be a promising method for the use of vitamins, antioxidants, bioactive or antimicrobial substances. Nanoparticles improve the mechanical, physical and barrier properties of edible packaging. Edible packaging can influence interactions with food and play an important role, for example, in releasing or trapping compounds (antimicrobials and antioxidants) and removing harmful gases (oxygen and water vapor) that affect product quality and shelf life. The most used nanoparticles include silver, zinc oxide, silica copper, gold, iron and others (Trajkovska Petkoska et al., Citation2021). For example Kumar et al. (Citation2019) dealt with the properties of a nanocomposite film based on chitosan, polyvinyl alcohol, and zinc oxide. And previously also titanium dioxide, which according to EFSA is not considered a safe substance for food since 2020.
The aim of this study was to determine the effect of the addition of selected bioactive substances and nanoparticles to edible chitosan-based packaging on immunoreactivity. Based on the results, the usability of these additives of bioactive substances and nanoparticles with the highest effect on immunoreactivity was assessed.
Material and methods
Production of packaging with the addition of plant extracts
1.5 g of low molecular weight chitosan was dissolved in 1% lactic acid. In the sample with no extract added, 135 mL of 1% lactic acid was used. For samples with some extract contained, the amount of 1% lactic acid varied depending on the addition of the extract. The samples were stirred for 15 min at 50 °C and 500 rpm. After that, plant extracts of blueberries (Vaccinium myrtillus L.), parsley (Petroselinum), and seedless red grapes (Crimson) were added at concentrations of 5, 10, and 20%, followed by mixing for 5 min and addition of glycerol. After 5 min, the solutions were poured into Petri dishes and allowed to dry for 48 h (Dordevic et al., Citation2021). The preparation of plant extracts is described by Tauferová et al. (Citation2021). Briefly, the aqueous extract (1:10 w/V) used to produce edible packaging was prepared from the pulp and skins obtained by juicing plant raw materials. The sample preparation scheme is shown below (Scheme 1).
Scheme 1. Sample preparation scheme.
Production of packaging with the addition of elder pollen
For packages with the addition of pollen, 1.5 g of low molecular weight chitosan and a given amount of elder pollen (0.075, 0.150, 0.300 g) were weighed. Subsequently, 135 mL of 1% lactic acid was added and the samples were transferred to a mixer, where they were mixed for 15 min at 50 °C and 500 rpm. Afterwards glycerol was added and after 5 min, the solutions were poured into Petri dishes and allowed to dry for 48 h.
Production of packaging with the addition of nanoparticles
135 mL of 1% lactic acid was added to the weighed amount of the respective nanoparticles (ZnO or TiO2) in concentrations of 0.05; 0.20 and 0.50%. Subsequently, 1.5 g of chitosan was added and the mixtures were stirred and heated until liquefied and then stirred in a mixer for 15 min at 50 °C and 750 rpm. In the next step, glycerol was added followed by stirring for 5 min. Afterwards, the solutions were poured into Petri dishes and allowed to dry for 48 h. In the case of the addition of red grape extract, this addition was added after 15 min of stirring in a volume of 13.5 mL, with 121.5 mL of lactic acid used initially (Tauferová et al., Citation2022).
Production of packaging with the addition of a colloidal solution of silver nanoparticles
135 mL of 1% lactic acid dissolved in a colloidal solution of silver nanoparticles with concentrations 1·10−3%, 3·10−3% and 5·10−3% a was added to 1.5 g of chitosan, the mixture was stirred and heated until liquefied, then the mixtures were stirred on a mixer for 15 min at 50 °C and 750 rpm. This was followed by the addition of glycerol and stirring for 5 min. After pouring into Petri dishes, the solutions were allowed to dry for 48 h. In the case of the addition of red grape extract, this addition was added after 15 min of stirring in a volume of 13.5 mL, with 121.5 mL of lactic acid solution used initially solved in the colloidal solution of silver nanoparticles (Tauferová et al., Citation2022).
Sample preparation
Sample extract preparation and quantification was based on the manual of Veratox® for Crustacea Allergen, No. 8520 (Neogen, GB). This is a sandwich enzyme-linked immunosorbent assay (S-ELISA) where the test antibodies react with shellfish proteins (polyclonal antibodies).
Briefly put: 1 g of the sample adjusted to the required size was weighed and the extraction additive and 25 mL of the extraction solution at a temperature of 30 °C were added to each sample. The extraction solution was prepared by adding extraction buffer (10 mM PBS) to 1 L distilled water according manual of Veratox® for Crustacea Allergen (Neogen, GB). The samples were then extracted in a water bath at 30 °C for 30 min with occasional shaking. After removing the tubes from the water bath, the samples were allowed to stand for 5 min and then centrifuged in a CF-10 centrifuge (Witeg, Germany) at 13,500 rpm for 10 min. The obtained supernatants were used for further determination.
The quantification was performed in compliance with the manual. Standards with a concentration of 0, 2.5, 5, 10, 25 µg/g of crustacea were used for quantification. Wash buffer solution was prepared by pouring all the wash buffer concentrate into 1-litre volumetric flask and topped to the mark. Measurements were performed at a wavelength of 650 nm on an Infinite M Nano spectrophotometer (Tecan, Austria). Results were expressed as µg/g of shellfish allergenic protein. The measurement was performed for each sample 3 times. The results expressed as protein were multiplied by a factor of 0.22 (on average crustaceans contain 22% protein), according to the manual of the Veratox® for Crustacea Allergen kit.
Quantification limit: 2.5 µg/g crustaceans
Quantification range: 2.5-25 µg/g crustaceans
Statistics
The results were statistically processed with the Kruskal–Wallis test (one-way ANOVA) at a significance level of p = 0.05 (Unistat ltd., Great Britain).
Results and discussion
The protein content with allergenic potential in chitosan-based edible packaging was determined using the sandwich enzyme-linked immunosorbent assay (ELISA) method.
In the analysed samples, the control (C) confirmed the higher presence of crustacean proteins (3.77 ± 0.79 a 5.75 ± 0.01 µg/g). Thus, a clean edible package of chitosan can be considered potentially allergenic. According to the legislation (Regulation (EU) No, Citation1169/Citation2011 of the European Parliament and of the Council, Citation2011), there is no minimum amount that can be considered allergenic. However, available studies mention the presence of 10 mg – 100 mg of crustaceans as sufficient to induce an allergic reaction (Miceli Sopo et al., Citation2015; Taylor et al., Citation2014).
The effect of the addition of extracts on the immunoreactivity of edible packaging
In the first phase of the research, the effect of the addition of selected bioactive substances on the immunoreactivity of edible packaging was investigated. The packaging was enriched with grape (GrE), blueberry (BlE), and parsley extracts (PaE) and elder pollen grains (PoE) in different concentrations. Extracts from blueberries, grapes, and parsley contain phenolic substances that have proven antioxidant properties (de Menezes Epifanio et al., Citation2020; Garcia-Lomillo & Luisa Gonzalez-SanJose, Citation2017; Kedage et al., Citation2007; Li et al., Citation2021).
The results shown in showed lower reactivity (p < 0.05) for samples with the addition of grape extract (0.61 ± 0.34 µg/g GrE 5%; 0.57 ± 0.20 µg/g GrE 10%; and 0.43 ± 0.16 µg/g GrE 20%) and blueberry extract (0.58 ± 0.32 µg/g BlE 5%; 0.57 ± 0.55 µg/g BlE 10%; and 0.39 ± 0.27 µg/g BlE 20%). A lower reactivity was found with the addition of parsley extract (p < 0.05) only when using 10% and 20% extract (0.77 ± 0.61 µg/g PaE 10% and 0.79 ± 0.40 µg/g PaE 20%), the addition of 5% extract had no effect (p > 0.05) on immunoreactivity (2.09 ± 1.28 µg/g PaE 5%). Addition of elder pollen (4.47 ± 0.46 µg/g PoE 0.05%; 3.59 ± 0.90 µg/g PoE 0.1%; and 2.45 ± 1.16 µg/g PoE 0.2%) had no effect on immunoreactivity (p > 0.05).
Table 1. Crustacean protein content in µg/g for individual packaging types with the addition of plant extracts and elder pollen.
Our results point to a trend of a decrease in the immunological response due to the addition of grape, blueberry, and parsley extracts (). The reason for the different change in reactivity with PoE is the lower concentration used and the form of the additive. Elder pollen was used in its native state, where the pollen exine represents a natural barrier against environmental influences. Even though the exchange of water (and therefore solutes) in dried pollen with the environment is confirmed (Elleman & Dickinson, Citation1990), this effect had no significant impact on the reactivity of the ELISA kit used, but a trend of decreasing immunoreactivity can be observed with increasing pollen concentration ().
Figure 1. Graphical representation of crustacean protein content as a function of the addition of bioactive substances.
The effect of the addition of nanoparticles on the immunoreactivity of edible packaging
The second phase of the research was focused on the addition of zinc nanoparticles (ZnO NPs), titanium nanoparticles (TiO2 NPs) and a colloidal solution of silver nanoparticles (col. AgNPs) in various concentrations into edible packaging. The selected nanoparticles have been shown to have antimicrobial properties (Kumar et al., Citation2021; Trajkovska Petkoska et al., Citation2021). The samples were monitored for changes in immunoreactivity not only depending on the nanoparticles used, but also with regard to the addition of grape extract, for which a decrease in immunoreactivity was recorded in the first phase of the research. TiO2 was used as the most common nanomaterial additive utilized in the past, however, since 2021, it is considered a genotoxic substance unsuitable for application in food (Younes et al., Citation2021). The ZnO and AgNPs were used as common nanoparticles used in food industry and in packaging materials. Both of them are characterized by no toxicity in low concentrations (Youn & Choi, Citation2022), nevertheless, according to EFSA recommendation, their application has to be evaluated for genotoxicity and migration in food packaging prior to actual use (More et al., Citation2021).
The results shown in demonstrate differences in crustacean protein content between samples fortified with grape extract and samples with no grape extract addition.
Table 2. Crustacean protein content in µg/g for individual packaging types with and without the addition of grape extract.
The crustacean protein content of the chitosan control sample without the addition of grape extract showed a higher (p < 0.05) immunoreactivity (5.75 ± 0.01 µg/g) than the control sample of chitosan with the addition of grape extract (2.64 ± 0.01 µg/g).
The addition of ZnO nanoparticles to experimentally produced edible packaging showed an increasing trend of immunoreactivity with increasing concentration of ZnO NPs in samples with the addition of grape extract (0.86 ± 0 µg/g ZnO NPs 0.05%; 0.95 ± 0.01 µg/g ZnO NPs 0.2%; and 1.35 ± 0.01 µg/g ZnO NPs 0.5%) as well as without the addition of grape extract (6.62 ± 0.10 µg/g ZnO NPs 0.05%; 8.47 ± 0.47 µg/g ZnO NPs 0.2%; and 8.48 ± 0.66 µg/g ZnO NPs 0.5%). Samples enriched with ZnO NPs and grape extract showed significantly lower (p < 0.05) immunoreactivity than samples with ZnO NPs without grape extract. Statistically significant differences were also confirmed between C and ZnO NPs in all concentrations used (p < 0.05).
With increasing concentration of titanium dioxide nanoparticles in packaging samples, antigen reactivity decreased in both, samples without grape extract (7.34 ± 0.32 µg/g TiO2 NPs 0.05%; 6.41 ± 0.06 µg/g TiO2 NPs 0.2%; and 4.33 ± 0.01 µg/g TiO2 NPs 0.5%), as well as in samples enriched with grape extract (1.22 ± 0.0 µg/g TiO2 NPs 0.05%; 1.00 ± 0.01 µg/g TiO2 NPs 0.2%; and 0.55 ± 0.0 µg/g TiO2 NPs 0.5%). Edible packaging containing TiO2 NPs and with the addition of grape extract showed statistical differences compared to the control (p < 0.05) except for the sample without the addition of grape extract TiO2 NPs. This result is due to the decreasing reactivity of tropomyosin with increasing TiO2 concentration, which is caused by the conformational change of the secondary structure of tropomyosin by the electrostatic forces of TiO2 (Chen et al., Citation1999).
A different situation was found for packaging samples containing a colloidal solution of silver nanoparticles and grape extract, when the immunoreactivity had an increasing tendency simultaneously with the increasing concentration of the AgNPs colloidal solution (1.29 ± 0.01 µg/g col. AgNPs 1·10−3%; 2.47 ± 0.01 μg/g col. AgNPs 3·10−3% to 5.47 ± 0 μg/g col. AgNPs 5·10−3%), while in samples without the addition of grape extract with increasing concentration of AgNPs colloidal solution there was a statistically significant (p < 0.05) decrease in immunoreactivity (5.45 ± 0.18 µg/g col. AgNPs 1·10−3%; 4.54 ± 0.06 µg/g col. AgNPs 3·10−3%; and 3.54 ± 0.02 µg/g col. AgNPs 5·10−3%) relative to the control sample. We believe this result is due to the oxidation of hydroxyl groups of flavonoids in grape flour by colloidal silver (Terenteva et al., Citation2015) and subsequent reduction of the reactivity of flavonoids with tropomyosin. We assume that the decrease in immunoreactivity is caused by electrostatic interactions of AgNPs with tropomyosin, similar to what was shown for the electrostatic interaction of AgNPs with lysozyme (Turbay et al., Citation2021). A different situation was found in samples with the addition of grape extract, where a statistically significant difference (p < 0.05) compared to the control sample was found only at concentrations of 1·10−3% and 5·10−3% col. AgNPs. Statistical significance (p < 0.05) was also noted between samples containing the same concentrations of AgNPs colloidal solution, differing by the addition of grape extract.
The differences between samples that differed by the addition of grape extract were statistically significant (p < 0.05) in all cases and are shown in for clarity.
Figure 2. Crustacean protein content depending on the addition of ZnO NPs, TiO2 NPs, col. AgNPs and grape extract.
Summary evaluation of the effect of nanoparticles and bioactive substances on immunoreactivity
Extracts from blueberries, grapes, and parsley contain phenolic compounds (de Menezes Epifanio et al., Citation2020; Ghafoor et al., Citation2009; Kedage et al., Citation2007; Li et al., Citation2021; Özkan et al., Citation2003; van den Broek et al., Citation2015; Xie et al., Citation2017), which can form soluble and insoluble complexes with proteins (Chung & Champagne, Citation2009). An interaction between proteins and phenolics has also been described, leading to a change in protein immunoreactivity (Zhang et al., Citation2018). In their study, Wai et al. (Citation2020) state that polyphenols can react with the mannose ends of proteins or with flagellin (Wai et al., Citation2020). This phenomenon is also used for the preparation of hypoallergenic foods (Plundrich et al., Citation2014).
Our results showing a reduction in immunoreactivity in packages with the addition of blueberries, grapes and parsley extracts are in line with the study by … Our results are consistent with the study by Xuan and Nguyen (Citation2012), who focused on the characterization of possible residual allergenic proteins in chitosan. Immunoreactivity of protein residues in chitosan samples, namely tropomyosin specific for crustaceans, was confirmed by the inhibition ELISA method. This is consistent with the observed allergic reactions that occur when exposed to products containing chitosan.
In their study, Ravindranathan et al. (Citation2016) point out that there is a number of inconsistencies related to immunoreactivity after chitosan application, because chitosans produced by different methods can induce different immune responses (Ravindranathan et al., Citation2016). For this reason, there is a need for warnings and information about the presence of tropomyosin in products containing chitosan to be available to consumers who may be sensitive to crustaceans (Xuan & Nguyen, Citation2012).
The obtained results confirm the influence of the food matrix on the reactivity of tropomyosin (Werner et al., Citation2007). In addition to the influence of the matrix itself, the influence of food processing by thermal heating was also confirmed (Kamath et al., Citation2013). In the case of our study, a lower temperature (40 °C) was used, so this effect cannot be assumed.
Our results confirmed the change in immunoreactivity due to the addition of a source of phenolic substances (grape extract). All samples supplemented with grape extract showed lower immunoreactivity (p < 0.05) than samples without grape extract, except for samples supplemented with of a colloidal solution of AgNPs 5·10−3%. For the colloidal solution of silver nanoparticles enriched with grape extract, the opposite dependence was observed than for the samples without the addition of grape extract. This relationship can be explained by the reaction of phenolics with colloidal silver. These results show that interaction between phenolic substances and proteins as well as between nanoparticles and proteins have an impact on the ELISA detection. Therefore, the interaction in immunoreactivity should be explored further and in more details in order to avoid misinterpretation of ELISA results.
The obtained results can also be used in applied sciences for the production of edible packaging based on chitosan with a lower potential effect on allergenicity. Nonetheless, this use requires further allergenicity testing on mathematical or biological models.
Conclusion
The results of this study confirmed the presence of allergenic proteins in chitosan-based edible packaging. The immunoreactivity of the proteins present was inhibited by the addition of grape, blueberry, and parsley extracts (p < 0.05), with the exception of the addition of 5% parsley extract, where no effect on the reactivity of the ELISA kit used was demonstrated (p > 0.05). Furthermore, there was no evidence of any effect on immunoreactivity with the addition of elder pollen (p > 0.05). All samples with the addition of nanoparticles (ZnO, TiO2) and grape extract showed lower immunoreactivity (p < 0.05) than samples without grape extract. The exception were samples with addition of colloidal solution of silver nanoparticles, where the opposite dependence was observed. In samples enriched with grape extract, an increasing trend of immunoreactivity was show, while in samples without the addition of grape extract, protein immunoreactivity decreased with increasing concentration of colloidal solution of silver nanoparticles. This relationship can be explained by the reaction of phenolic compounds with colloidal silver. Our results confirmed the change in immunoreactivity caused by adding a source of phenolic substances (grape extract) to packaging based on chitosan. The addition of grape extract affects the reactivity of the ELISA method used.
Acknowledgment
The research was supported by ITA VETUNI, from University of Veterinary Sciences, Brno, Czech Republic, grant number 2022ITA23.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Funding
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