Effect of dissolved oxygen on the oxidative and structural characteristics of protein in beer during forced ageing

Abstract

Oxygen is contributing to the deterioration of the beer and shortening the shelf-life of the packaged product. The effect of dissolved oxygen (DO) on oxidative and structural characteristics of protein in beer during forced ageing was examined. Results showed that increased DO decreased obviously protein content in beer, and further reduced antioxidant activities of beer and lipid transfer protein 1 (LTP1). Meanwhile, the increase of DO decreased significantly the free thiol groups content and enhanced the disulfide bonds level in beer and LTP1. Results from circular dichroism, surface hydrophobicity and zeta-potential illustrated that the increase of DO dramatically changed the secondary and tertiary structure of LTP1 with the decrease in the surface hydrophobicity, α-helix and β-turn contents, and the increase in the random coil and negative zeta potential. These results indicated that increased DO could damage the structure of LTP1 and had a negative impact on oxidative stability of beer.

Graphical Abstract

Beer, dissolved oxygen, LTP1, oxidative stability, protein structure

Introduction

Beer, one of the world’s most widely consumed alcoholic drink, is an integral part of diet in many countries. Fresh beers with unique fragrance and refreshing taste are attractive to consumers, whereas inevitable occurrence of some off-flavours during storage results in a significant decrease in potability of beer (Li et al., 2016). Oxygen is considered the most adverse factor on beer quality, and it is contributing to the deterioration of the beer and shortening the shelf-life of the packaged product (Barth, 2013). Dissolved oxygen (DO) has a dual function (Hempel et al., 2013), since it is beneficial for yeast cells to grow at the initial period of fermentation, but during storage it can generate undesirable residues, damage the colour, flavour and quality of beer and even shorten its shelf-life. DO in beer promoted the production of carbonyl compounds, such as trans-2-nonenal and 2-heptenal, which had a great impact on beer characteristics due to their low flavour thresholds and sensitivity to ageing (Baert et al., 2012). Thus, it is of importance to improve beer flavour stability by controlling the DO in beer during brewing, distribution and storage.

It has been found that certain protein in beer exhibited free radical scavenging property and played a positive role in oxidative stability of beer (Abrahamsson et al., 2012). The antioxidant activity of protein in beer was mainly due to the high reactivity and free radical scavenging activity of thiol groups it contained (Zheng et al., 2017). Previous studies indicated that thiol-containing protein could reduce the accumulation of H₂O₂ and inhibit the formation of acetaldehyde during beer storage, which had a significant role in maintaining the flavour stability of beer (Wu et al., 2012; Vichi et al., 2015). It is worth mentioning that lipid transfer protein 1 (LTP1) in beer contained a large number of thiol groups, which were closely associated with the oxidative stability of beer (Wu et al., 2011). In the scientific literature, there are few studies investigating the effect of DO on the oxidative and structural characteristics of beer protein during forced ageing. The interrelationships between protein and oxidative stability of beer are not clarified thoroughly.

Therefore, the objective of this study is to investigate the effects of DO on oxidative and structural characteristics of protein during forced ageing of beer, and to clarify the contribution of protein, especially LTP1, to oxidative stability of beer.

Materials and methods

Materials and chemicals

A total of 96 bottles of lager beers with 11.0 °P of original gravity and 4.3% (v/v) of ethanol content were purchased from local supermarket (Guangzhou, China). All chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Oxygenation and forced ageing treatment of beer samples

Beer samples (24 bottles per oxygen content condition) were filled with high-purity oxygen under aseptic conditions to be 2, 4 and 6 mg L⁻¹, respectively. The beer samples (twenty four bottles) without addition of high-purity oxygen was used as a control with a DO of 0.5 mg L⁻¹. Then, all beer samples were aged at 60 °C for 7 days according to the method of Yao et al. (2018) and Cai et al. (2019).

Total protein content assay

Total protein content of beer was determined according to Bradford method using bovine serum albumin as a standard (Bradford, 1976).

LTP1 isolation and purification

Beer samples were concentrated 50 times under vacuum, and lyophilised to dryness in a Scientz-18N freeze dryer (Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China), and then fully dissolved in 30 mL Tris-HCl buffer. Subsequently, the samples were centrifuged at 10 000 g for 30 min at 4 °C. The LTP1 was isolated and purified using the previous method proposed by de Almeida et al. (2013). The solution containing LTP1 was collected and further purified through dialysis with molecular weight cut-off 1000 Da overnight, then lyophilised for further use. In addition, the purification effects were identified by SDS-PAGE (Figure S1). Forty μL of sample was mixed with 10 μL 5-fold dilution of loading buffer, then placed in a boiling water bath and allowed to heat for 10 min. SDS-PAGE was carried out with the 5% stacking gel and 12% separation gel. The loading volume of sample was 10 µL. Finally, the gel was stained for 30 min with 0.25% Coomassie blue R-250 and then destained with destaining solution.

The molecular weight (Mw) distributions of protein in beer

Protein was precipitated by ammonium sulphate with final 80% saturation, dissolved in phosphate buffer at pH 7.0. The solution was dialysed via dialysis membrane with a molecular weight cut-off 1000 Da overnight. Then, samples were centrifuged for 30 min at 5000 g and 4 °C, and the supernatants were collected. The molecular weight distributions of protein were measured by HPLC (Agilent 1260, USA) as described by Zhou et al. (2018). The size distribution of samples was determined based on the elution time of the following molecular markers: Tyr-Tyr (362 Da), bacitracin (1423 Da), aprotinin (6511 Da), cytochrome C (12 327 Da) and bovine serum albumin (66 430 Da).

Antioxidant activity assay

ABTS radical cation scavenging activity and DPPH radical scavenging activity of beer with 4-fold of dilution and the purified LTP1 (1 mg mL⁻¹) were detected according to the method previously reported by Zhao & Zhao (2012), and Zhao et al. (2010), respectively.

Quantification of free thiol groups (SH) and disulfide bonds (S-S)

The number of SH and S-S in beer and LTP1 were determined using Ellman’s reagent as described by Winther & Thorpe (2014). The contents of SH and S-S were presented as µmol g⁻¹ protein.

Surface hydrophobicity assay

The surface hydrophobicity assay was carried out as previously described (Jin et al., 2019). ANS was the fluorescence probe used to determine the surface hydrophobicity values of LTP1. The LTP1 solutions (0.005–0.5 mg mL⁻¹) were prepared in 0.01 m pH 7.0 phosphate buffer. Fluorescence was measured at an excitation wavelength of 365 nm and an emission wavelength of 484 nm. The index of surface hydrophobicity was obtained from a plot of initial slope of the fluorescence intensity vs. protein concentration.

Circular dichroism and zeta-potential assay

The sample was prepared by directly dissolving lyophilised LTP1 into 0.01 m pH 7.0 phosphate buffer and the final concentration was 1 mg mL⁻¹. Circular dichroism spectra were acquired using a Chirascan circular dichroism spectrophotometer (Applied Photophysics, UK) over the wavelength range from 180 to 260 nm. The resulting data was analysed with the CDNN software. The zeta-potential of LTP1 was measured using a Malvern Zetasizer Nano ZS potential analyser (Malvern Instruments Ltd., Malvern, Worcestershire, UK).

Statistical analysis

All the results are reported as means ± standard deviation. Statistical calculation of experimental data was performed using SPSS 23 software (SPSS Inc., Chicago, IL, USA) for one-way ANOVA and Duncan’s multiple-range test.

Results and discussion

Effect of DO on beer proteins during forced ageing

The effect of DO on protein content in beer during forced ageing was examined, and the results are shown in Fig. 1. After forced ageing for 7 days, the protein content in beer samples with DO of 0.5, 2, 4 and 6 mg L⁻¹ decreased by 15.42%, 16.93%, 20.59% and 22.42%, respectively. The results indicated that the increase of DO might accelerate protein degradation or precipitation, and cause more serious damage to beer when compared with thermal ageing (Cai et al, 2019). Previous studies have shown that the initial DO concentration had a decisive effect on the level of reactive oxygen species (ROS) in beer. The higher the DO content, the larger is the number of ROS and hence the more oxidation consumption of beer protein (Kunz et al., 2014). Furthermore, ROS could damage the structure and physical-chemical properties of protein, thereby destroy its functional activity (Weids et al., 2016; O'Brien et al., 2019).

Figure 1

Effect of DO on total protein concentration in beer during forced ageing. DO, Dissolved oxygen; DO of 0.5 mg L⁻¹ (■), 2 mg L⁻¹ (●), 4 mg L⁻¹ (▲) and 6 mg L⁻¹ (◆).

SDS-PAGE analysis of LTP1 in beer affected by DO during forced ageing is shown in Figure S2. With increasing DO, the intensity of the beer protein bands decreased significantly (P < 0.05) during forced ageing. By 7 days of forced ageing, all protein bands with a DO of 6 mg L⁻¹ almost disappeared. Among them, the protein Z had a molecular weight range of 35–55 kDa, and the LTP1 with the molecular weight around 10 kDa (Cai et al., 2019). The increase in DO in beer caused the disappearance of the LTP1 band under the same ageing condition, which clearly demonstrated that the DO could damage the protein subunit of LTP1.

The Mw of beer protein with different DO during forced ageing was measured by HPLC, and the results are shown in Figure S3. Protein in beer was mainly distributed in molecular weight range of 10–50 kDa. There were similar distributions in the molecular weight of beer protein with different DO during forced ageing. After 7 days of ageing, as for beers with different DO, the proportion of proteins with Mw < 3 kDa increased by 6.63%–7.44%, and proteins with Mw 5–10 kDa increased by 2.59%−3.97%, whereas the proportion of proteins with Mw > 10 kDa decreased significantly. All these results suggested that the macromolecular protein in beer were degraded with prolonged time of forced ageing. DO caused oxidative damage in proteins and also exacerbated protein degradation to a certain extent (Wu et al., 2012).

Effect of DO on antioxidant capacity of beer and LTP1 during forced ageing

Figure 2 shows the antioxidant capacity of beer and LTP1 evaluated by ABTS and DPPH radical scavenging assay. By 7 days of forced ageing, the antioxidant capacity of beer and LTP1 decreased significantly (P < 0.05). ABTS free radical scavenging activity reduced by 2.25%, 11.20%, 11.28% and 11.86% (Fig. 2a), and DPPH radical scavenging activity decreased by 8.67%, 37.00%, 37.24% and 37.24% (Fig. 2b), respectively, for the beers with DO of 0.5, 2, 4 and 6 mg L⁻¹. However, ABTS radical scavenging capacity of LTP1 decreased by 36.76%, 53.11%, 53.42% and 54.99% (Fig. 2a), and DPPH radical scavenging capacity of LTP1 reduced by 5.18%, 32.87%, 33.18% and 35.55% (Fig. 2b), respectively. All these results showed that the antioxidant capacity of beer and LTP1 decreased with the increase of the DO. Although LTP1 exhibited a greater radical scavenging ability than beer, the antioxidant capacity of LTP1 was significantly reduced during forced ageing. This might be due to the free thiol groups in LTP1 was oxidised by DO, which caused a significant decrease in antioxidant activity of LTP1 (Lund et al., 2015). These results further demonstrated that DO resulted in the decrease in antioxidant activity of beer and LTP1.

Figure 2

Effect of DO on ABTS radical cation (a) and DPPH radical (b) scavenging activity of beer (solid line) and LTP1 (dashed line) during forced ageing. DO, Dissolved oxygen; DO of 0.5 mg L⁻¹ (■), 2 mg L⁻¹ (●), 4 mg L⁻¹ (▲) and 6 mg L⁻¹ (◆). [Colour figure can be viewed at wileyonlinelibrary.com]

Effect of DO on SH and S-S in beer and LTP1 during forced ageing

As presented in Fig. 3, the change of SH and S-S content in beer and LTP1 followed the same trend at different DO during forced ageing. As the DO increased, the SH contents in beer and LTP1 decreased by 91.98%, 90.73%, 82.87% and 95.01%, and by 47.95%, 73.73%, 75.42% and 81.23% (Fig. 3a), respectively, which was consistent with antioxidant activities of beer and LTP1 (Fig. 2). In contrast, the S-S content in beer and LTP1 increased by 6.79-, 10.55-, 11.81-, and 13.89-fold, and by 3.78-, 6.08-, 6.86-, and 8.13-fold (Fig. 3b), respectively. These results further demonstrated that the presence of DO accelerated the free thiol groups-oxidation reaction and the disulfide bond was formed (de Almeida et al., 2013). Therefore, the degree of oxidation of beer and LTP1 was increased.

Figure 3

Effect of DO on free thiol groups (SH, solid line) and disulfide bonds (S-S, dashed line) in beer (a) and LTP1 (b) during forced ageing. DO, Dissolved oxygen; DO of 0.5 mg L⁻¹ (■), 2 mg L⁻¹ (●), 4 mg L⁻¹ (▲) and 6 mg L⁻¹ (◆).

Effect of DO on secondary structure of LTP1 during forced ageing

The effects of different concentrations of DO on the secondary structure of LTP1 in beer during forced ageing is characterised by circular dichroism. As shown in Fig. 4, only LTP1 from fresh beer with a negative peak near 220 nm corresponding to the α-helical in the n−π* region. Secondary structure of the LTP1 in untreated beer displayed a negative peak around 208 nm, and its intensity decreased with forced ageing time extended. It also should be noted that the LTP1 in beer with high DO after forced ageing showed almost no characteristic absorption peaks of α-helix. These indicated that the level of α-helical in LTP1 decreased and the α-helical hydrogen bonds were disrupted by DO during forced ageing. Furthermore, the LTP1 in beer with oxygen addition had a characteristic absorption peak near 195 nm due to the presence of β-sheet structure, which was closely related to the protein aggregation (Du et al., 2015).

Figure 4

Circular dihedral spectrum of LTP1 in beer affected by DO during forced ageing. DO, Dissolved oxygen; A-D, forced ageing on 1^st, 3^rd, 5^th and 7^th day, respectively. [Colour figure can be viewed at wileyonlinelibrary.com]

Table 1 shows the secondary structure contents of LTP1 affected by DO during forced ageing. After treatment with oxygen addition, the α-helix and β-turn contents of LTP1 in beer were significantly reduced compared with control, but increased in the irregular curl content. These results demonstrated that DO could increase the misfolding of LTP1 molecule and its structure was changed from order to disorder during forced ageing. Further analysis revealed that the secondary structure conformation of LTP1 was changed by DO, which might result in the losing its original biological activity, such as the ability to scavenge free radicals.

Table 1

Secondary structural contents of LPT1 affected by DO during forced ageing

Time of beer forced ageing (days)	DO (mg L−1)	β-sheet (%)	Random coil (%)	Helix (%)	β-turn (%)
0	0.5	43.79a	15.73a	17.77a	22.71a
1	0.5	41.19b	16.80a	17.76a	24.24a
	2	41.35b	31.69c	11.57b	15.38b
	4	41.41b	31.76c	11.48b	15.35b
	6	41.46b	31.76c	11.45b	15.32b
3	0.5	40.75b	18.57ab	17.58a	23.10a
	2	41.41b	31.76c	11.48b	15.35b
	4	41.40b	31.77c	11.48b	15.35b
	6	41.42b	31.79c	11.46b	15.33b
5	0.5	39.92bc	19.69b	17.55a	22.83a
	2	41.31b	31.88c	11.47b	15.34b
	4	41.36b	31.88c	11.45b	15.31b
	6	41.36b	31.88c	11.45b	15.31b
7	0.5	39.12c	20.61b	17.50a	22.78a
	2	41.30b	31.85c	11.49b	15.36b
	4	41.41b	31.88c	11.38b	15.33b
	6	41.42b	31.90c	11.38b	15.30b

Results with different letters in the column for each index are significantly different (P < 0.05).

Effect of DO on surface hydrophobicity and zeta-potential of LTP1 during forced ageing

As shown in Fig. 5, the change of surface hydrophobicity and zeta-potential in LTP1 showed the same trend during forced ageing. With the increase in DO, after 7 days of forced ageing, the surface hydrophobicity decreased by 83.54 − 85.89% (Fig. 5a), and zeta-potential of LTP1 decreased by 12.24–13.94 mV (Fig. 5b). The decrease in zeta-potential implied that DO would promote more negatively charged residues exposed in LTP1, the binding amount of ANS^– was greatly decreased and then the surface hydrophobicity was reduced. In addition, other studies have shown that protein-aggregates were formed in part by intermolecular disulfide bonds, which contributed to inhibit the increase of surface hydrophobicity (Elahi & Mu, 2017; Ma et al., 2018). The increase of DO would promote the formation of disulfide bonds by oxidation of SH and lead to the reduction of surface hydrophobicity, which corresponds to the results of SH and S-S content (Fig. 3). Furthermore, ROS formed by DO would lead to protein aggregation, and enable the hydrophobic group to bury inside (Weids et al., 2016; O'Brien et al., 2019), which resulted in reducing the surface hydrophobicity of LTP1. Therefore, these results further suggested that the tertiary structure and biological activity of LTP1 changed with the increase of DO and the oxidative stability of beer was reduced.

Figure 5

Effect of DO on surface hydrophobicity (solid line) and zeta-potential (dashed line) of LTP1 during forced ageing. DO, Dissolved oxygen; DO of 0.5 mg L⁻¹ (■), 2 mg L⁻¹ (●), 4 mg L⁻¹ (▲) and 6 mg L⁻¹ (◆).

Conclusion

The increase of DO significantly changed the structure and antioxidant activities of LTP1 during beer forced ageing. Meanwhile, the effect of DO on LTP1 during beer forced ageing was related to the reduction of free thiol groups content, the decrease of antioxidant capacity and the disruption of the secondary and tertiary structure in LTP1. All these results demonstrated that the DO was an unfavourable factor for the structure of protein, particularly LTP1 and beer quality during forced ageing.

Acknowledgments

The authors would like to acknowledge the Science and Technology Project of Guangdong Province (Nos. 2018A050506008 and 2016A010105003), the National Natural Science Foundation of China (31972062), the Science and Technology Project of Guangzhou (201903010056), the Science and Technology Project of Sichuan Province (2018JZ0039) and the 111 Project (B17018) for their financial support.

Author contribution

Xuyan Zong: Investigation (lead); Writing-original draft (lead). Huirong Yang: Methodology (equal). jinxiaofan jinxiaofan: Data curation (equal); Software (equal); Visualization (equal). Charles Stephen Brennan: Supervision (equal). Teodora Emilia Coldea: Writing-review & editing (equal). Linfei Cai: Formal analysis (equal); Validation (equal). Haifeng Zhao: Conceptualization (lead); Funding acquisition (lead); Project administration (lead); Supervision (lead).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Ethics approval was not required for this research.

Peer review

The peer review history for this article is available at https://publons.com/publon/10.1111/ijfs.14894.

Data availability statement

Research data are not shared.