Cloudy wheat beer enriched with okra [Abelmoschus esculentus (L.) Moench]: Effects on volatile compound and sensorial attributes

ABSTRACT In order to explore the effect of okra on the cloudy and flavor characteristics of wheat beer, fresh okra and dried okra were added separately into boiling wort. The beer without okra was used as control sample. Aroma characteristics were analyzed using gas chromatography (GC), high-performance liquid chromatography, headspace–solid-phase microextraction–GC–mass spectrometry (HS-SPME-GC-MS), and electronic nose. The results showed that okra addition significantly increased the turbidity and viscosity. Principal components analysis reflected that three beers were apart from each other. After adding okra, the higher alcohols decreased, while the esters increased. In conclusion, okra can be used to improve stability and aroma characteristics.


Introduction
Okra [Abelmoschus esculentus (L.) Moench], an annual herb plant, belongs to the malvaceae family. Okra is well known as "green ginseng," because it contains abundant protein, polysaccharides, vitamins, and unsaturated fatty acid. [1,2] Okra polysaccharides include galactose, galactan, araban, and pectin, [3,4] which has been widely used as food additive and nutrient supplement. [5] Water-extractable okra polysaccharide is shown having very good viscoelasticity, [6] which makes it a good thickening and suspension agent. In addition, okra polysaccharide can provide special flavor and nutrition to beverage. Previous researches indicated that okra fruit exhibited potential immune-stimulating effects, attributing to its thick and slimy polysaccharides. [1] Furthermore, okra is beneficial to resist gastric irritative and inflammative diseases. [7] The oligomeric proanthocyanidins extracted from okra seeds exhibited a great inhibitory activity of starch hydrolases, contributing to controlling hyperglycemia as a functional ingredient. [8] Okra is extremely perishable with a short shelf life because of the high water contents and fast respiration rate. [9] Recent years, cloudy wheat beer has been a potential drink relying on its thick, soft, and durable foam, outstanding wheat malts aroma, faint hop flavor, and sweet taste. [10,11] Nowadays, 4-vinylguaiacol (4-VG), by a clove-like, slightly phenolic odor note, is widely researched as the typical aroma of wheat beer differing from lager beer. [12] The appearance of cloudy wheat beer is turbid due to the retained active ale yeasts. Specifically, it gets rid of the filtration treatment and high-temperature pasteurization, so reserves active yeasts, amino acid, phenolic compounds, microelements including potassium, calcium, zinc, magnesium, etc. [13] The retained active yeasts are suggested to be a nutritional ingredient. However, cloudy wheat beer has its own disadvantages as well. High contents of pentosan and protein may pose a threat to the nonbiological stability in the saccharification and fermentation due to the compositional significance of wheat malt and barley malt. [14,15] It has a great significance to research how to maintain the stability of cloudy wheat beer. Some works attempted to increase the physicochemical or sensorial properties of the beer by adding some food products during brewing. For example, goji berries have been added during the fermentation process, bringing beer desirable sensory characteristics and high antioxidant capacity. [16] The addition of green tea, oolong tea, and black tea during brewing process was found having a profound influence on beer flavor character due to the volatiles changes connected with the behavior of yeast fermentation.- [17] To our best knowledge, okra has not been applied in the production of alcoholic beverage. In this present work, okra was added during brewing in order to increase the viscosity and turbidity of cloudy wheat beer, improve the yeasts suspending power, bring a new flavor, and finally enhance the sensory quality of cloudy wheat beer. Since fresh okra was more expensive than dried okra as a result of the planting seasonal limitation and short storage time, both fresh and dried okra were used in this study. Okra was added into boiling wort, and the obtained okra cloudy wheat beer was compared with ordinary beer in terms of cloudy characteristics and volatile compounds.

Materials and reagents
Okra was purchased from local supermarket in Tai

Okra preparation
Fresh okra was rinsed carefully and drained off. Okra pods were crosscut into 0.5 cm thick slices, and stalks were removed. The okra slices were divided into two parts. One part was transferred into a mixer (Kenwood Electronic, England) to make fresh okra pulp (FOB), and the other one was dried in an oven (Shanghai Shuli Instrument Co., Ltd., Shanghai, China). The drying process included two steps: (1) enzyme deactivation at 100°C for 15 min; (2) drying at 50°C for 20 h to reduce the moisture content below 5.0%. The dried slices were grounded and passed a 0.25 mm miller sieve (ZKA-WERKE, Germany).

Brewing process
Three kinds of cloudy wheat beers were prepared, namely beer with FOB, beer with dried okra powder (DOB), and control beer (CB). The flowchart of cloudy wheat beer production process was presented in Fig. 1. The brewing process started with the saccharification after mixing 55% barley malt and 45% wheat malt. Spent gains were removed by filtration, and the filtered wort was divided into three groups to produce CB, FOB, and DOB. The okra was added with hops during boiling process, with the dosage as 1 g dry weight okra per 1 L wort.

Physicochemical indices
Alcohol, real extract, real fermentation degree (RFD), diacetyl, total acid, FAN, TN, pH, chromaticity, and turbidity were determined according to Analytica EBC methods. Transmittance was read at 680 nm using UV-2600 spectrophotometer (Unico, Shanghai, China). Viscosity was measured by a falling-ball viscometer (Thermo Electron GmbH, Karlsruhe, Germany) at 20°C. Determination of pectin was performed according to M-hydroxydiphenyl method with galacturonic acid (GalA) as the standard. [6] High performance liquid chromatography analysis Ferulic acid and 4-VG were measured using high performance liquid chromatography (HPLC) equipped with an ultraviolet detector at 230 nm. Samples were degassed, passed through a 0.45 μm filter, and analyzed using Atlantis C18 column (250 mm × 4.6 mm, 5 μm, Waters., USA). The mobile phase was deionized water:acetonitrile:phosphoric acid = 599:400:1; and flow rate was 1 mL/ min. Column temperature was maintained at 30°C and the injection volume was 20 μL.
Organic acid was measured by HPLC equipped with a Aminex HPX-87H column (300 mm × 7.8 mm, 9 μm, Bio-Rad., USA) and an ultraviolet detector at 215 nm. After degassing treatment, 20 μL sample was injected into the column and the column was eluted using 0.0033 mol/L sulfuric acid at a flow rate of 0.6 mL/ min at 42°C. The organic acid content was determined by external standard method using a SHIMADZU CLSS-VP system.

Monosaccharide compositions
The monosaccharides compositions of the okra polysaccharides were measured by GC method after hydrolysis, reduction, derivatization, and extraction. [18]

Volatile compounds
The absolute contents of volatile compounds were measured by a distillation-GC method according to the previous study. [19] The relative contents of volatile compounds were analyzed using headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) according to the method of Molina-Calle et al. with some modifications. [20] A volume of 25 mL sample was poured into a 60 mL glass vial with 5 g of sodium chloride. The above glass vial was sealed and preincubated at 40°C for 10 min to equilibrate, using a hotplate stirrer (VWR Co., USA). Volatiles were extracted using an SPME fiber coated with divinylbenzene/carbonex/polydimethylsiloxane (DVB/CAR/PDMS 50/30 μm) (Supelco, Bellefonte, PA, USA). The SPME fiber was exposed to the sample headspace for 40 min at 40°C. Afterward, it was transferred to the injection port (230°C ) and stayed for 5 min for desorption. GC instrument was equipped with a VF-Wax MS column (60 m × 0.25 mm × 0.5 μm, Agilent J & W, USA). The oven temperature was maintained at 30°C for 4 min, then increased to 60°C at 3°C/min and held for 5 min, then increased to 220°C at 3°C/min and to 230°C at 2°C/min, finally maintained at 230°C for 25 min. The MS spectra fragments were compared with those obtained from a database (NIST11 & NIST11s).

Electronic nose
A volume of 5 mL sample was transferred into a 150-mL triangular flask and held at 25°C for 60 min for equilibration. The headspace was analyzed with the electronic nose (E-nose) PEN3 (Airsense Analytics, Germany), composed of an array of ten temperature-moderated metal-oxide sensors. [21] The signal output was sustained for 6 min.

Sensory evaluation
The sensory evaluation of the beers was done by a well-trained panel made of five females and five males, 30-50 years old. Quantitative descriptive sensory analysis was applied by a 10-point interval scale (1 = none, 10 = extremely strong). Four aspects including appearance, odor, taste, and in mouth sensation were deemed to measure the quality of the beers. The specific method was complied by European Brewery Convention Analytica, Section 13, sensory analysis, and Ducruet et al. [16] Statistical analysis All the data were statistically analyzed by SPSS Statistics 22 software (IBM, United States). Significant differences between various treatments were examined by least significant difference method. p < 0.05 was considered as statistically significant difference. The volatiles of E-nose analysis were compared with the principal components analysis (PCA) with WinMuster software (Airsense, Germany). All analyses were performed in triplicate.

Quality analysis
The live yeast in cloudy wheat beer is helpful to maintain body intestinal balance. However, these yeasts tend to precipitate during storage, which makes turbidity a crucial parameter to evaluate the stability of cloudy wheat beer. As seen in Table 1, the turbidity increased and transmittance decreased for samples of FOB and DOB in comparison with the control, which can be attributed to the addition of okra extract. Adding okra extract increased the chromaticity of the beer. The chromaticity of DOB was higher than FOB, which can be explained by the browning reactions during okra drying for the former. [22] The pH of CB was 4.43, while the value of FOB and DOB was 4.48, which was corresponding to the okra extract in wort.
The alcohol content of FOB and DOB is higher than that of CB, suggesting that the sugar from okra could be transformed into alcohol by beer yeasts. [23] The increasing FAN in FOB and DOB indicated that okra addition was in favor of yeast growth and reproduction. RFD showed the similar tendency. Diacetyl, the main crude taste substance, is regarded as the symbol for beer maturity. When the diacetyl content is below 0.10 mg/L, the quality of wheat beer is considered to be eligible; once overweighs the threshold value of 0.15 mg/L, it would cause impure taste. The diacetyl contents of all the samples are below 0.10 mg/L, and no significant difference was found between them. Pectin was the mainly extracted polysaccharide from okra and greatly increased in FOB and DOB. Sticky pectin served the purpose of improving the yeast suspending ability, thus affected the turbidity and viscosity of cloudy wheat beer.
4-VG is one of the typical flavors of ale beer and contributes to clove-like flavor. 4-VG was suggested to be a degradation product of ferulic acid by a thermal effect such as malt kilning, wort boiling, and sterilization. [12] In addition, feruloyl esterases produced by brewing yeasts could accomplish the decarboxylation of ferulic acid to form 4-VG. [24] Consequently, 4-VG gradually increased as ferulic acid decreased during brewing process. The contents of ferulic acid and 4-VG in CB were both slightly higher than those in FOB and DOB, implying okra addition depressed the 4-VG formation.
The correlations among transmittance, chromaticity, turbidity, viscosity, pectin, and FAN were investigated and summarized in Table 2. Transmittance had negative correlations with turbidity (p < 0.01) and FAN (p < 0.01). Turbidity had positive correlations with viscosity (p < 0.05) and FAN (p < 0.05). Okra addition directly increased pectin and FAN content in cloudy wheat beer, which was beneficial to the viscosity improvement and cloudy characteristic. The result indicated that the increasing viscosity and FAN played a vital role in the turbidity stability.

Organic acid and monosaccharide analysis
Organic acid was an essential part of flavor compounds as its contents directly decided the harmony of beer taste. It was observed that malic acid and fumaic acid increased and citric acid, pyruvic acid, and succinic acid decreased after adding okra in cloudy wheat beer. In addition, for oxalic and lactic acid, no significant difference was found among CB, FOB, and DOB (Table 3). Succinic acid, a dicarboxylic acid, is the main nonvolatile organic acid affecting beer taste and has the largest content in fermentation. This acid has special salty and bitter taste. [25] FOB and DOB tended to be more delicate and harmonious as the succinic acid decreased. Okra polysaccharides are mainly composed of glucose, followed by xylose and arabinose, with mannose and galactose content as the lowest (Fig. 2). Ale yeasts made use of glucose and free mannose to metabolize sugars to alcohol by Embden-Meyerhof-Parnas pathway, while non-fermented sugars, arabinose, xylose, and galactose could not participate in yeasts metabolic activity. [11] No difference was found in glucose and mannose content among the beer samples. FOB has the highest arabinose content, while CB has the highest xylose content.

Distillation-GC analysis
The volatiles, playing a vital role in the taste and aroma, can be divided into crude taste substances (diacetyl, aldehyde, and sulfide) and aromatic substances (higher alcohol and ester). GC analysis indicated that the addition of okra had a significant influence on the aroma of beers; the ester  content increased whereas the alcohol content decreased (Table 4). Commonly, the main ester and higher alcohol in beers are ethyl acetate (fruity) and isoamyl alcohol (pungent), respectively. The contents of ester and higher alcohol were related to malt type and yeast strain. Moreover, overexpression of the genes encoding branched chain amino acid amino transferases and alcohol dehydrogenases leads to an increase in higher alcohol formation. [26] Therefore, the decreasing of higher alcohol content in both okra beers may be relative to the enzyme overexpression. As one of the main volatile acids influencing the flavor of cloudy wheat beer, acetic acid's content in CB was much higher than that of FOB and DOB. Acetaldehyde is the most important aldehyde and emerging as the primary fermentation product by yeast metabolism. Acetaldehyde content in green beer is about 20-40 mg/L and in mature beer, the content is below 10 mg/L. The acetaldehyde content in CB, FOB, and DOB had no difference and met the requirements of mature beer.

HS-SPME-GC-MS analysis
As seen in Table 5, 57 volatile compounds were detected in the cloudy wheat beers, including 19 alcohols, 18 esters, 10 acids, 3 aldehydes, 3 terpenes, 3 ketones, and 1 phenolic compound. The results were in accordance with the analysis of distillation-GC method ( Table 4). The relative content of total alcohol in CB was more than that of FOB and DOB, whereas the total esters in CB were less than FOB and DOB. The relative contents of volatile compounds are different among the three beers. For instance, the contents of 1-octen-3-ol, 1, 10-decanediol, 1-decanol, n-tridecan-1-ol, and 1-tetradecanol in FOB and DOB were higher than those of CB. In addition, there were no significant difference in the contents of 1propanol, 1-heptanol, 1-octanol, citronellol, geraniol, and cedrol. From the perspective of the esters, the contents of hexyl acetate, ethyl oenanthate, ethyl octanoate, octyl acetate, phenylethyl acetate, ethyl laurate, and ethyl palmitate had no significant difference, which means the above esters were more likely derived from the malts, hops, and fermentation process rather than additive okra. The relative content of  isoamyl alcohol was the highest in alcohol, isoamyl acetate and phenylethyl acetate were the highest in ester, and octanoic acid was the highest in acid. To sum up, adding okra increased the total ester content, and the beer tastes mellowness and freshness, and no off-flavor was found.
Although the percentages of aldehydes, terpenes, ketones, and phenolic compounds were lower than those of alcohols, esters, and acids, they contribute greatly to the beer flavor. Benzaldehyde content in CB (0.12%) was found much higher than that in FOB (0.04%) and DOB (0.02%). The relative content of terpenes in FOB was higher than that of DOB and CB (e.g., D-limonene and styrene). As a typical flavor compound of cloudy wheat beer, styrene is formed via two pathways. One is the thermal decarboxylation of cinnamic acid during wort boiling process; the other is enzymatic decarboxylation during fermentation. [27] Styrene in FOB and DOB was higher than that in CB. Caryophyllene was only detected in FOB, which suggested okra addition brought new aroma component. The relative contents of ketones, including 3-octanone, 6-methyl-5-hepten-2-one, and dihydro-5-pentyl-2(3H)-furanone, had little difference in three beers. 4-Ethenyl-1,2-dimethoxy-benzene with aroma like vanilla beans extract was the only phenol compound detected from GC-MS. Its relative contents in CB, FOB, DOB were 0.05%, 0.28%, 0.25%, respectively.

Electronic nose
PCA is a two-dimensional map containing the first and second principal components (PCs). PCA can be used to analyze the volatile components to highlight the differences among various samples. The higher the contribution rate, the better the PCs exactly reflect the original multi-index information. Generally, the total accumulative contribution rate over 85% showed the feasibility of the methods. [28] As shown in Fig. 3, the contribution rates of the first and second PCs were 99.01% and 0.96%, respectively, indicating that these two PCs contained most of the volatile compounds information. The points of each beer were concentrated in the circle and they could be clearly separated from each other. Moreover, they all had a positive correlation with PC1 and PC2. Specifically, the DOB had the strongest positive relevance with PC2, and the weakest positive relevance with PC1. The FOB had the weakest positive relevance with PC2. As seen in Fig. 3, it is noteworthy that the absolute linear distance between FOB and DOB was far more close to each other than to CB, which indicated that okra addition had obvious influences on the aroma of cloudy wheat beer.
E-nose could recognize the flavor differences via data analysis. Ten types of metal-oxide sensors could reflect response signals for the variety of odor from multi-angle. The capabilities of ten sensors and their correlations with PC1 and PC2 were listed in Table 6. It was observed that all sensors contributed greatly to PC1. W1C, W3C, and W5C had a negative correlation with PC1, while others exhibited positive correlation. Furthermore, W5C was the sensor which had the strongest positive correlation with PC2.
The sensors position in radar fingerprint chart can represent the contribution rates (Fig. 4). The further the distance from original points, the greater the impact the sensor made. For instance, the value of W1C, W3C, W6S, W5C, and W3S sensors was close to the original points, indicating that they were not sensitive to their corresponding aroma. W5S, W1S, W1W, W2S, and W2W sensors, sensitive to three cloudy wheat beers, stood for broadrange, broad-methane, sulfur-organic, broadalcohol, and sulph-chlor, respectively. The response value of CB was higher than that of FOB and DOB, which depended on not only what odor molecules are composed of, but also the concentration. [29] Furthermore, radar fingerprint chart of FOB and DOB was almost overlapped, which implied that they were more likely to have similar volatiles.

Sensory evaluation
The results of sensory evaluation were shown in Table 7. From the perspective of beer appearance, the color intensity and turbidity of FOB and DOB were higher than CB. The difference between FOB and DOB was that FOB had higher turbidity and DOB had darker color. Grainy-like aroma of three beers took the highest score was more outstanding. 4-VG, the typical flavor of ale wheat beer, behaved as phenolic-like aroma. Okra addition increased the grassy-like aroma as the value of FOB and DOB was higher than CB. It is found that CB got better evaluation in terms of caramel-like aroma. The bitterness and sourness value of CB was higher than FOB and DOB because succinic acid was highest in CB, which was in accordance with the result   Table 3. Foam of FOB and DOB was smooth, thick, and abundant than CB. Besides, the bubble size and quantity of FOB exhibited better sensory value than DOB.

Conclusion
In terms of the stability of cloudy wheat beer, it was reasonable that adding okra into boiling wort could enhance the turbidity, viscosity and foam of beers, and the suspending capability of active yeasts. As for the flavor and taste of cloudy wheat beer, okra addition decreased the ratio of higher alcohols and esters; the esters increased, whereas the higher alcohols decreased significantly. It was noteworthy that the contents of volatile compounds and aroma characteristics between FOB and DOB were similar. Fresh okra addition in boiling wort could enrich the varieties of volatiles. For example, caryophyllene was detected only in FOB. On contrast, dried okra has the advantages from the perspective of material cost.  beer with fresh okra pulp; DOB: beer with dried okra powder. *Significant at p < 0.05; **significant at p < 0.01; and ***significant at p < 0.005. Various letters in the same line indicate significant difference. Each item evaluated by an expert panel (n = 10), minimum score = 1 and maximum score = 10.