Effects of Wheat Malt-Derived Endo-Β -1, 4-Xylanase Supplementation on The Quality of Wheat Beer

: Endo-β -1, 4-xylanase (EC 3.2.1.8) is a key enzyme in the degradation of Arabinoxylan (AX). AX and its degradation products are the key substances that affect the viscosity, turbidity, filtration speed, alcohol, foam performance, and health care function of wheat beer. A wheat malt-derived endo-β -1, 4-xylanase was isolated and purified by our group in the previous stage. The aim of this study was to investigate the effects of the wheat malt-derived endo-β -1, 4-xylanase addition on the quality and composition of AX in hopped wort and wheat beer. The results showed that the content and the average degree of polymerization (avDP) of water-soluble arabinoxylan (WEAX) in hopped wort and wheat beer decreased with the increase of the addition level of the wheat malt-derived endo-β -1,4-xylanase. When the addition of wheat malt-derived endo-β -1,4-xylanase was 2.5 times of the activity of endo-β -1,4-xylanase in raw malt, the degradation rate of WEAX in hopped wort was the maximum (33.71%), and the avDP of WEAX was the minimum (29.74) at the addition level of the endo-β -1,4-xylanase was 2 times. The viscosity and turbidity of hopped wort and wheat beer decreased continuously with the increase of the addition level of the endo-β -1,4-xylanase, while the foam stability of wheat beer increased continuously. Wheat malt-derived endo-β -1,4-xylanase could decrease the content of 1320-6200 Da polysaccharide in wheat beer, but increase the content of<1320 Da polysaccharide.


INTRODUCTION
Wheat beer is a top-fermented beer with a prominent flavor and distinctive foam and which is made up of more than 40 percent wheat malt (Jie, 2019). In Germany, one out of every ten beers sold is a wheat beer (Faltermeier, 2014). In recent years, Chinese wheat beers, including cloudy beers as well as craft beers, have also become popular with consumers.
Arabinoxylan (AX) is the main non-starch polysaccharide in wheat cell walls (Rakszegi, 2014), accounting for 70% of endosperm cell wall composition (Ravn, 2016). AX in cereal is linked by (1, 4)-β-d-xylopyranose residues to form the main chain and α-L-arabinfuranose can be mono-substituted or double-substituted at the C(O)-3 or/and C(O)-2 positions of xylose residues (Fadel, 2017). Arabinoxylan will be degraded in the process of wheat malt preparation and wort mashing, a high concentration of WEAX and high molecular weight will improve the viscosity and turbidity of the wort and beer, medium viscosity WEAX and molecular weight are conducive to foam stability, and small molecular weight WEAX has certain prebiotic functions (Han, 2000;Evans, 1999;Mendis, 2014). Arabinoxylans degradation requires a series of enzyme, among which endo-β-1,4-xylanase is the key enzyme. Endo-β-1, 4-xylanase can be derived from microorganisms or plants. Studies have found that some endo-β-1, 4-xylanase derived from Bacillus subtilis (Moteshafia, 2022) and Paenibacillus terrae (Kim, 2016) contain inhibitory proteins, which can reduce the degradation effect of the enzyme. In the study of plant-derived endo-β-1,4-xylanase, there were more research on barley, oat, and rye, but fewer research on wheat. Our research group has isolated and purified a wheat malt-derived endo-β-1, 4-xylanase, which molecular weight is about 58000 Da (Peng, 2020) and confirmed that the wheat malt-derived endo-β-1, 4-xylanase can not only degrade WUAX into WEAX, but also degrade WEAX into smaller molecules (Peng, 2020), but the application of the enzyme in beer production has not been carried out yet. The aim of this study was to investigate the effects of the wheat malt-derived endo-β-1, 4-xylanase addition on the quality of wheat beer. The results would provide a theoretical basis for the application of wheat malt-derived endo-β-1, 4-xylanase in wheat beer brewing.

The Brewing Process of Wheat Beer
The brewing process of wheat beer was as shown in Figure 1. The control and five experimental groups were made in three batches in parallel.
(1) Mashing: malt was combined with water at the ratio of 1:3.5.
(2) Boiling: hop was added at 1.0 g/L, and the wort was boiled to adjust the concentration to 12%.
(3) Fermentation: beer yeast was added at a ratio of 1 g/L.

Determination of Crude Polysaccharide Content, The Average Degree of Polymerization Of WEAX, and Monosaccharide Content in Wort and Beer
Hopped wort and wheat beer were filtered using 0.45 μm membranes and then alcoholized with 95% ethanol. The mixture was mixed evenly and left to stand overnight at 4℃, followed by centrifugation at 5000 r/min for 15 min. The precipitate obtained was crude polysaccharides. Contents were determined by gas chromatography, referring to methods by He et al (He, 2017) and Liu et al (Liu, 2020).

Determination of Molecular Weight of Crude Polysaccharides
The molecular weight determination methods of crude polysaccharides in hopped wort and wheat beer were referred to Peng (Peng, 2019).

Electron Microscope Analysis
The microstructure of wheat beer polysaccharides was observed by scanning electron microscopy (SEM) following methods described by Li (Li, 2020) and Deng (Deng, 2018). Hopped wort and wheat beer were filtered using 0.45 μm membranes and alcoholized with 95% ethanol. Precipitation was collected and freeze-dried.

Statistical Analysis
All data used in analyses were the means of at least three replicates. Microsoft Excel 2016, DPS, and SPSS were used for data processing. OriginPro 2020 software was used for data analysis and graphs. IBM SPSS Statistics 25 software was used for one-way analysis of variance (ANOVA) and multiple comparisons (DUNCAN test). A difference analysis was completed in SPSS at a 95% confidence level. Wheat malt-derived endo-β-1, 4-xylanase was added at 0.5, 1.0, 1.5, 2.0 and 2.5 times of endo-β-1, 4-xylanase activity in raw malt. Wheat malt-derived endo-β-1, 4-xylanase was not added as control.

Changes of Polysaccharides Components in Hopped Wort and Wheat Beer
With the increase of wheat malt-derived endo-β-1,4-xylanase addition level, the contents of WEAX in hopped wort continued to decrease, that is, the degradation rate of WEAX continued to increase. The maximum degradation rate of WEAX was 33.71% at 2.5 times enzyme addition level, and the maximum degradation rate of WEAX was found at 2 times enzyme addition level compared with other addition levels. It was 3.37%. With the increase of wheat malt-derived endo-β-1,4-xylanase addition level, the avDP of WEAX in hopped wort decreased, and the avDP was the minimum when the wheat malt-derived endo-β-1,4-xylanase was added with 2 times activity. With the increase of wheat malt-derived endo-β-1,4-xylanase addition level, the content of arabinogalactan (AG) in the hopped wort decreased, that is, the AG degradation rate increased. The contents of AG reached the minimum (no significant difference) when 2 times and 2.5 times enzyme activities were added, and the maximum degradation rate of AG was 23.39%. The contents of mannose-polymer (MP) and glucose polymer (GP) in hopped wort increased with the increase of wheat malt-derived endo-β-1,4-xylanase addition level, and the contents did not change beyond 1.5 times addition level.
With the increase of wheat malt-derived endo-β-1,4-xylanase addition, the contents of WEAX in wheat beer decreased, and the contents of WEAX reached the minimum value (no significant difference) at 2 and 2.5 times addition level, and the degradation rate was about 11.4%. The avDP of WEAX decreased with the increase of wheat malt-derived endo-β-1,4-xylanase addition level, indicating that WEAX molecule became smaller, and the avDP was the minimum when the wheat malt-derived endo-β-1,4-xylanase addition level was 2 times. The contents of AG and MP in wheat beer increased with the increase of wheat malt-derived endo-β-1,4-xylanase addition level. The contents of AG and MP at 0.5 and 1 times addition had maximum values (no significant difference), and had a significant difference compared with the control group. The contents of AG and MP were 0.04 mg/mL and 0.03 mg/mL, respectively. There was no significant difference in the contents of AG and MP between 2 and 2.5 times addition. The increase of wheat malt-derived endo-β-1,4-xylanase addition not only promoted the degradation of arabinoxylan, but also promoted the degradation of other polysaccharides. Among them, the content of WEAX and avDP had the greatest changes when the addition level is 1.5 and 2 times. : hopped wort without the addition of wheat malt-derived endo-β-1, 4-xylanase, WE0.5-WE2.5: hopped wort with 0.5-2.5 times wheat malt-derived endo-β-1, 4-xylanase added, BE0: wheat beer without the addition of wheat malt-derived endo-β-1, 4-xylanase, BE0.5-2.5: wheat beer with 0.5-2.5 times wheat malt-derived endo-β-1, 4-xylanase added.
Different letters in each line indicate significant difference (P <0.05).

Changes in Molecular Weight of Polysaccharides in Hopped Wort and Wheat Beer
The changes of molecular weight and composition of crude polysaccharides in hopped wort and wheat beer with different addition levels of wheat malt-derived endo-β-1, 4-xylanase are shown in Figure 2. The molecular weight of polysaccharide was divided into 5 components according to the peak shape of the chromatographic. The molecular weight, relative content and absolute content of the 5 polysaccharide components are shown in Table 2.
In the hopped wort (Fig. 2a), the peak shape and peak molecular weight of the five polysaccharides components is constant, but the peak height and width is different. The content increase of the polysaccharide which MW is less than 242 Da was the most obvious, the polysaccharide content increased by 90% (WE0.5) to 93.75% (WE1.5). The content of polysaccharide in this part increased first and then decreased, and the maximum increase rate was 93.75% when the enzyme activity addition level is 1.5 times. The second, content of 242-1320 Da (Region IV) polysaccharide also increased significantly, which content increased by 64.60% (WE2) -75.46% (WE1), but the content increased first and then decreased. The content of polysaccharide (Region Ⅱ) with a molecular weight of 6200-21700 Da decreased the most significantly, and the content reduction rate was 12.5%-40.90%, the polysaccharide content with molecular weight 1320 to 6200 Da (region Ⅲ) also decreased by 20.8%-39.37%.
In wheat beer (Fig. 2b), with the increase of the addition level of wheat malt-derived endo-β-1, 4-xylanase, the four polysaccharide chromatographic peaks (Q1, Q2, Q3, Q4) remained unchanged in terms of peak shape and peak molecular weight, and the difference in peak height and peak width also indicated the difference in the content of polysaccharide components corresponding to the molecular weight. The content of polysaccharide region Ⅰ with a molecular weight of 21700 Da to 48000 Da decreased obviously, which may be due to the condensation of macromolecular polysaccharide. Secondly, the content of polysaccharide 1320-6200 Da (region Ⅲ) was also decreased by 7.21%-15.32%. The content of polysaccharide which MW is less than 242 Da increase the most obvious, and the content of polysaccharide increased by 45.45% (WE1.5) to 72.73% (WE0.5). The increase rate of the content of polysaccharide decreased first and then increased the content of the polysaccharide with a molecular weight of 242-1320 Da (Region IV) also increased by 8.82% (WE2.5) -18.42% (WE1).

Changes of Basic Indexes of Hopped Wort and Wheat Beer
The turbidity and viscosity of hopped wort and the turbidity, viscosity and foam stability of wheat beer of different addition levels of wheat malt-derived endo-β-1, 4-xylanase are shown in Figure 3.
The viscosity and turbidity of hopped wort and wheat beer decreased continuously with the addition level increase of wheat malt-derived endo-β-1, 4-xylanase, while the foam stability of wheat beer was increased and when the enzyme addition level was 2.5 times the increase was up to 14 s. The correlation between wheat beer basic indexes and the content of polysaccharide in hopped wort and wheat beer is shown in Table 3, the viscosity was significantly positively correlated with the content of WEAX, the avDP of WEAX, the content of AG and MP, and the content of polysaccharides with molecular weights of 6200-21700 Da and 1320-6200 Da. The foam stability was positively correlated with the content of WEAX, GP, MP, and polysaccharides with molecular weights of 6200-21700 Da, 1320-6200 Da and 342-1320 Da. The addition of endo-β-1,4-xylanase significantly increased the content of polysaccharides with molecular weights of 6200-2100 Da and 342-1320 Da in the beer by 33.33% and 8.8%-18.3% respectively, which may be the main reason for the increased foam stability of wheat beer.

Electron Microscopic Analysis of Polysaccharide in Hopped Wort and Wheat Beer
Scanning electron microscopy can be used to detect the morphological characteristics of biopolymers, including polysaccharides, proteins, and peptides (Rozi, 2019). Fig.  4 shows the SEM microscopic images of polysaccharide in six hopped wort and six wheat beer with the different addition of wheat malt-derived endo-β-1, 4-xylanase. It can be seen from the SEM that the degree of loosening, fragmentation, and surface roughness of the polysaccharides increased with the increase of the addition level of wheat malt-derived endo-β-1, 4-xylanase, but the degree of fragmentation of the polysaccharides did not change significantly beyond the 1.5 times addition level. The above phenomenon may berelated to the increasing degradation degree of polysaccharides such as AX with the increase of the addition level of wheat malt-derived endo-β-1, 4-xylanase.

CONCLUSION
The content of water-soluble arabinoxylan (WEAX) and average degree of polymerization (avDP) in hopped wort and wheat beer decreased with the increase of the addition level of wheat malt-derived endo-β-1,4-xylanase. When the addition of wheat malt-derived endo-β-1,4-xylanase was 2.5 times of the activity of endo-β-1,4-xylanase in raw malt, the degradation rate of WEAX in hopped wort was the maximum (33.71%), and the avDP of WEAX was the minimum (29.74) at the enzyme addition level is 2 times. With the increase of the addition level of wheat malt-derived endo-β-1,4-xylanase, the polysaccharide with a molecular weight of 1320-21700 Da was mainly degraded into polysaccharide with a molecular weight of less than 242 Da and 242-1320 Da. With the addition of wheat malt-derived endo-β-1,4-xylanase, the turbidity and viscosity of the hopped wort and wheat beer decreased and the foam stability of the wheat beer increased.