Influence of packaging material on polyphenol content and antioxidant activity in some commercial beers

: Using two methods (ferric reducing antioxidant power and radical scavenging activity), the total polyphenol content (Folin–Ciocalteu reagent) and polyphenol patterns (HPLC) in 10 commercial lager beer brands produced in Romania was determined. Samples bottled in glass, plastic and aluminium packages were analysed for each brand when available. Results have indicated considerable variations in the total and individual phenolic contents as well as antioxidant activity across beer brands. A statistical analysis was performed to assess the influence of packaging type on the antioxidant activity and phenolic content of the beers. Statistical differences were found between the DPPH and FRAP methods in glass, aluminium and plastic material. Moreover, the antioxidant activity based on the DPPH method is influenced by the type of packaging material, while in the case of FRAP method, no statistical difference was reported. Furthermore, the same analysis has shown that the polyphenol concentration is invariant to the type of material.


Introduction
Beer is a fermented, low-alcohol beverage and one of the most consumed beverages around the world; it is rich in nutrients such as carbohydrates, amino acids, minerals, vitamins and other compounds such as polyphenols. Phenolic compounds may be a major factor in assuring the antioxidant potential of the diet and may contribute in maintaining the endogenous redox balance in humans (Arranz et al., 2012).
Since beer is inexpensive and preferred by many people, it is produced at industrial level worldwide under various brands. In terms of chemical contents, a series of scientific studies have shown its antioxidant activity (Zhao et al., 2010;Tafulo et al., 2010). Studies have also found that beer exhibits inhibitory effects on carcinogens and a positive influence on plasma lipid levels, plasma antioxidants and anticoagulant activities in hypercholesteraemic patients (Gerhauser, 2005;Nardiniet al., 2006;Gorinstein et al., 2007). Some very recent studies investigated in vitro the antioxidant effects using various methods for determining the correlation of commercial beer with polyphenolic profiles (Zhao et al., 2010;Tafulo et al., 2010).
The industrial production of beer also requires packaging containers that can preserve the chemical composition of the beer and antioxidant properties for as long as possible. However, the literature provides little information on the safety compliance of packaging materials for maintaining these properties. There have been some recommendations regarding the chemical nature of beer packaging, but there remains a lack of information about the influence of raw packaging materials on the chemical composition and properties of beer (Brunazzi et al., 2014).
Some studies quantified the amount of phthalates and adipates occurring in beer from plastic gaskets, lids and stoppers (Sendon et al., 2012) as well as tetra packs (Carnolet al., 2017). There have also been studies related to the safety of aluminium cans for beer packaging. Some authors demonstrated that there was virtually no difference in aluminium content between beverages in cans and beverages in glass bottles (Müller et al., 1993;Stahl et al., 2011).
According to our knowledge, there have been no studies on the correlation between the beer antioxidant activity and polyphenol content and the chemical nature of its packaging. The present study draws a comparison between the polyphenol content and antioxidant activity in ten commercial beer brands produced in Romania and packed in three different types of materials: glass bottles, plastic bottles, and aluminium cans. Testing the antioxidant activity (AA) of the beers facilitates the estimation of a protection degree after the AA ingestio, but the results may vary according to the method and/or packaging material. These factors were identified by qualitative characterisation of the samples. Therefore, the main objectives of this study were: (i) the evaluation and comparison of the phenolic profiles and antioxidant activities of commercial beers packaged in three different types of materials; (ii) the evaluation of the influence of packaging material on the antioxidant activity and polyphenols in beers, using a statistical analysis.

Beer samples
Ten of the most popular industrial lager beer brands produced in Romania were selected for this study and acquired from a local supermarket. According to the specifications of the producers, all brands come from the same batch, are bottled on the same day and have the following particularities: alcohol content (3.5-5.0 %, v/v); original gravity (8.0 -11.1 0 P) and raw material (malt, hop). For each brand, samples packed in glass bottles, plastic bottles and aluminium cans were purchased when available. Two beer brands lacked glass packaging and another two brands lacked plastic packaging. There were at least two different kinds of packaging for each brand. For each beer sample, beers from three bottles were combined, homogenised and degassed with intensive stirring for 30 min at room temperature. All experiments were performed during the marked shelf lives of the products.

Determination of total phenolic content
The total phenolic content was determined using the Folin-Ciocalteu reagent according to the method reported by Pereira da Silva (Pereira da Silva et al. 2011). Gallic acid (GA) was used as a calibration standard, and the results are expressed as GA equivalents (mg GA/100 mL beer).
For this experiment, an aliquot (1 mL) of beer was added to 1 mL of Folin-Ciocalteu reagent (diluted 1:1 with distilled water), and the mixture was added to 25 mL of distilled water (solution B). Then, 1 mL of solution B was mixed with a 20% sodium carbonate solution to a total of 5 mL. After 40 min of storage in the dark at room temperature, the absorbance was measured at 725 nm versus a prepared blank using a Jasco V-630 UV-VIS spectrophotometer (Japan). The correlation equation constructed with GA (1 to 10 mg L -1 ) was y = 0.1387x + 0.0204 (R 2 = 0.9990), and the limit of detection was 3.26 mg L -1 .

Determination of phenolic profile by HPLC
Chromatographic analyses of common phenolic compounds were performed on an Agilent

Antioxidant capacity 2.5.1. Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was performed according to previous reports by Benzie (Benzie & Szeto, 1999). The FRAP reagent was prepared by mixing 2.5 mL of 10 mM TPTZ in 40 mM HCl (1mL), 2.5 mL of 300 mM acetate buffer (pH = 3.6), and 2.5 mL of 20 mM FeCl3·6H2O. The FRAP reagent was added to 1 mL of each diluted beer sample, and the mixture was shaken. A reagent blank was prepared by adding 1 mL of water instead of beer samples. Absorbance readings of the samples and the reagent blank were taken after 4 min at 593 nm using a UVvisible spectrophotometer (Jasco V-630). The antioxidant activity was calculated from the calibration curve made with GA (y = 297.03x) with a range of 0.8-16.6 mM and good linearity (R 2 = 0.9953).

DPPH radical scavenging activity
The DPPH radical scavenging activity of the beer samples was determined according to the method reported by Brand-Williams (Brand-Williams et al. 1995) with minor changes. Each diluted beer sample (0.1 mL) was added to 2.9 mL of DPPH solution. The absorbance was measured at 517 nm after the solution had been allowed to stand in the dark for 60 min. The GA calibration curve was plotted as a function of the percentage of DPPH radical scavenging activity (y = 2.4024x + 0.8899) with a range of 0.16-3.32 mM and good linearity (R 2 = 0.9913). The results have been expressed as mg GA/L.

Statistical analysis
The antioxidant activity and polyphenol level of the beer obtained through the methods mentioned before and for different packaging materials was further analysed from a statistical point of view, using the statistical software R version 3.4.3 (R version 3.4.3 (2017-11-30) -"Kite-Eating Tree" 2017). Two-tailed ANOVA test was applied to verify the influence of the type of packaging material on the antioxidant activity and polyphenols. The samples considered were independent. The test requires that the samples are normally distributed and the variance of each group is homogeneous (homoskedasticity). The former condition was verified using the Shapiro test, while the latter uses Bartlett test. The impact of the DPPH and FRAP methods on the level of antioxidant activity of beer was an analysed based on the two-sample t-test. All data are reported as means ± standard deviation (SD) from triplicate determinations.

Total phenolic contents
Beer is considered one of the major sources of phenolic compound; the presence of the compounds contributes to colloidal, sensorial properties and to the flavour of beer. Flavour stability is one of the most important factors in determining the shelf life of packaged beer (Callemien & Collin, 2010). In this study, commercial beers were analysed, and the results are

Individual phenolic acids
Some studies established that the most abundant phenolic acids in beer are gallic acid, ferulic acid, and syringic acid (Zhao et al., 2010). Some of them are present in beer mainly in free forms, while others in conjugated forms (Nardini & Ghiselli, 2004). Hop and malt are the raw materials of beer which serve as an important source of phenolic compounds. Around 30% of polyphenols from beer comes from hops and 70%-80% originates from malt (Krofta et al., 2008).
In this study, the beer samples had relatively high levels of gallic, ferulic, caffeic, p-coumaic and vanillic acids, while the levels for cinnamic, p-hydoxibenzoic, syringic and chlorogenic acids were much lower (Table 1-other acids). Ferulic acid was among the five predominant phenolic acids detected in the beer samples, with a concentration of 2.04-7.89 mg/l. A number of research findings suggest that the main phenolic acid in barley, malted barley is ferulic acid (Szwajgier et al., 2005).
Gallic and ferulic acids were the dominant phenolic compounds identified in the tested beer samples and both of them accounted for > 60% of the total phenolic acids.
The results obtained in our experiment revealed that for the same type of packaging material, beer samples showed differences for some phenolic acids. For example, the glass packaging beers (bands 4 and 10) had both the highest and the lowest level of ferulic acid. Therefore, the type of packaging material is not the main influence on the content of this acid in beer. The original gravity (OG) value is very important to the execution of the brew and subsequent fermentation of the beer. One of the major disadvantages to the high gravity brewing process is the reduced foam stability in the final diluted beer (Stewart et al., 1998).
Oxygen in beer mostly enters process during filling. The amount of oxygen absorbed in the bottling stage depends on the method of bottling, primarily the use of inert gases, the type of machine used, and the capping system. It is a common practice for commercial brewers to measure the total package oxygen (TPO) and to use this value to evaluate the oxygen level in the final packaged beer. It represents the total amount of oxygen with the package, including both the headspace and the liquid. In beer industry, oxygen has a greater effect on the quality of the product, while TPO levels are kept below 100 ppm (Coetzee & Du Toit, 2015).
The same beer in different types of packaging material showed similar phenolic profiles, but significant variations appeared in the total and individual phenolic content, which have been determined through Folin-Ciocalteu and HPLC methods, respectively (Table 1). The difference between the two assays is that the FC method is not specific for phenolic compounds and suffers interference from other compounds (Dávalos et al., 2003). Therefore, the measurement of phenolic profile might provide more information about their antioxidant activity.

Antioxidant activity
The radical scavenging activity and reducing power were analysed to determine the antioxidant activity of beers. The DPPH assay measures a change in the stable radical DPPH by the electron donating ability of the sample (Rahmanet al., 2015). The FRAP value measures the reduction of the ferric ion (Fe 3+ ) to ferrous ion (Fe 2+ ) by donor electrons in the sample .
In the present study, all beer samples showed antiradical properties (Fig. 2). The highest antiradical activity was estimated in the glass packaging beer and the lowest one in plastic packaging beer. These differences may be due to the changes in the phenolic composition during storage. Slight changes in the structure or conformation of these compounds can cause significant changes in the antioxidant activity, which alters the overall oxidative capacity or flavour stability of the beer. Nowadays, flavour stability has become the most important factor in determining the shelf-life of the packaged beer. The flavour stability of the beer primarily depends on the oxygen content of the packaged beer (Bamforth. 2000).
Some studies show that the content of phenols decreases nearly 35% in the first two weeks of storage and it can be related to a higher level of phenol oxidation at the beginning of the storage (Vanderhaegen et al., 2006).

Effect of the packaging material
The influence of the packaging material on the antioxidant activity and the total phenolic content was analysed from statistical point of view. For each type of beer, whenever one of the packaging material was not available, the corresponding value is regarded as unavailable. For the methods considered to determine the antioxidant activity of the examined beers -DPPH and FRAP -the differences between glass, aluminium and plastic materials were studied. The relative order of DPPH assays was plastic< Al< glass and for FRAP assays was glass<plastic<Al.
Comparing the methods, AA was about two times higher for DPPH assays, as resulting from the mean plotted in Fig. 4. This observation may result from the different mechanisms of analysing the antioxidant activity; these mechanisms are described by the singlet electron transfer (SET) based activity for FRAP values and the mixed mode with SET and hydrogen atom transfer based activity for DPPH radical scavenging activities (Kareem et al.,, 2015).
In the case of the FRAP method, a similar analysis was performed. The box plot in Fig. 4 suggests that the means of the observations within each group are similar, fact also proved by the ANOVA test. The resulting p-value = 0.8747 is greater than the significance level, and therefore the null hypothesis is accepted. Fig. 5 shows the box plot of total polyphenols detected in glass, plastic and aluminium. The tests for normality and homoskedasticity of data were performed before applying the two-tailed ANOVA test with the same significance level as in the previous analysis. The resulting p-value is 0.808, implying that the difference between the means of the groups is not significant. was performed to compare whether the mean of the antioxidant activity changes when using the two methods mentioned above. For example, in the case of glass material, the null hypothesis is , while the alternative hypothesis states that the used method significantly influences the level of antioxidant activity of the beer. The reported p-value of is notably smaller than the significant level , and hence, the null hypothesis is rejected. Thus, in the case of the glass material, the antioxidant activity of the beer is influenced by the type of method applied. We repeated the analysis for aluminium and plastic packaging materials, considering the impact of the DPPH and FRAP again. The reported pvalues are of order , suggesting that regardless of the type of material that is used, the method has a significant effect on the level of detected antioxidant activity.

Conclusions
There were considerable variations in phenolic profiles (total and individual phenolic contents) and antioxidant activities in commercial beer across different brands. These differences may be due to the original gravity, the natural aging process and possible changes in the phenolic composition during storage.
The statistical analysis highlighted the impact of the DPPH and FRAP methods on the levels of antioxidant activity in beer when the type of material might play a significant role. The results indicate that the DPPH method is influenced by the type of material, while the FRAP remains invariable. Moreover, the analysis indicates a significant difference between the levels of antioxidant activity detected using DPPH and FRAP, regardless of the type of material. The statistical test that we have applied has revealed that the polyphenol concentration is similar across all types of material.