Aroma Perceptual Interactions among Ester Compounds and Their Effects on Sensory Attributes of Huangjiu

YU Haiyan, XIE Jingru, XIE Tong, CHEN Chen, AI Lianzhong, TIAN Huaixiang,

(1.School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai 201418, China;2.School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China)

Abstract: Gas chromatography-mass spectrometry (GC-MS) coupled with an external standard method was used for the quantification of 20 esters in four typical types of Huangjiu.Gas chromatography-olfactometry (GC-O) was used to determine the odor traits of 14 odor-active esters.Further, four important ester compounds were selected for perceptual interaction investigation by sensory analysis and the S-curve method.Synergistic effects were observed in binary mixtures of ethyl lactate (a) with ethyl acetate (b), and ethyl phenylacetate (c) with ethyl 2-methylbutanoate (d).At 50% correct detection probability, the overall odor thresholds of Huangjiu aromatic reconstitution were reduced from 8.04% (V/V) to 4.32% (V/V), and 11.56% (V/V) to 2.28% (V/V) by these two mixtures (a/b, c/d), respectively.Besides, the a/b (90:10, m/m)and c/d (93:7, m/m) mixtures could obviously enhance the intensity of fruity aroma compared with the single compounds.It was indicated that there was a synergistic aroma enhancing effect between these ester compounds, which affected the fruity aroma of Huangjiu.These results will provide a theoretical foundation for Huangjiu flavor regulation.

Keywords: Huangjiu; ester compounds; odor threshold; S-curve; aroma perceptual interaction

Huangjiu, a kind of traditional fermented rice wine, has been produced for over 5 000 years in China.It is popular among consumers for its ester aroma and bittersweet taste[1].Esters are the most abundant group of aroma compounds in Huangjiu[2].They exert great function in constituting the aroma characteristics of Huangjiu due to their low odor threshold and desirable fruity odor[3].The ester compounds in Huangjiu mainly derived from microbial metabolism during fermentation, and chemical reactions during aging.Gas chromatography-mass spectrometry (GC-MS) becomes the most frequently-used technology for the analysis of Huangjiu complex odorants in recent years[4-5].Both qualitative and quantitative analysis of ester compounds can be fulfilled by this technology[6-7].Besides, only a small fraction of volatiles (key aroma compounds) are associated with aroma perceptions of Huangjiu[8], and esters tend to be involved.In combination with odor activity value (OAV) determination, analysis of these key aroma components by gas chromatography-olfactometry (GC-O)identified that ethyl lactate, ethyl acetate[3,5], ethyl hexanoate[9],and ethyl phenylacetate[10]made a great contribution to the special flavor of Huangjiu.

It is recognized that the wine aroma relates to enhancing and limiting interactions occurred between aroma components, instead of equalling to the total number of aroma components[11].Nevertheless, the complexity and diversity of the interactions between different aroma components make it difficult to verdict specific perceptual interactions between components[12].In the last few years,the perceptual interactions in liquor and wine have been founded by sophisticated sensory analysis methods.For example, due to ethyl phenylacetate's fruity-odor, different levels (100, 2 500 and 58 000 μg/kg) of ethyl phenylacetate was, added to a recombinant system, respectively.It is found that the fruity odor was suppressed in the recombinant system, which contained no ethyl phenylacetate, but other redissolved substances according to tested concentration.The synergistic effects of 2-methylbutyl acetate[13], ethyl 2-methylbutanoate, ethyl 3-hydroxybutanoate and ethyl 2-hydroxy-4-methylpentanoate on fruity aroma perception was researched by Lytra et al[14-17].Perceived interaction does not only exist in wine, but also in food.According to the study of the interaction between the 24 representative aroma components in oolong tea, Zhu Jiancai et al[11]drew the conclusion that the masking effect was produced by the combination of ethyl phenylacetate with certain structural and aroma compounds in the recombinant system.In contrast, the addition of a low (1 400 μg/kg) or high level (11 500 μg/kg)of phenylacetate was found to have an enhancing effect on the floral aroma.Moreover, the addition of phenylethyl acetate at a threshold concentration (3 200 μg/kg) resulted in a significant increase in perceived sweetness[18].The synergistic effect can make these compounds modify the fruity aroma perception in the matrices at subthreshold concentrations.Exploring perceptual interactions is an important step in understanding the complex aroma characteristics of Huangjiu.However, the interactions between the specific aroma components in Huangjiu have not yet been found.

The primary purposes of this study were to 1)quantify the ester compounds in Huangjiu by GC-MS and external standard method; 2) determine the important ester compounds by GC-O odor trait analysis; 3) evaluate the perceptual interaction of four important ester compounds(ethyl lactate, ethyl acetate, ethyl phenylacetate and ethyl 2-methylbutanoate) by S-curves; 4) explore the effects of these important esters on sensory attributes via compound addition.Based on these results, this work can offer a theoretical reference for the adjustment and control of Huangjiu flavor by evaluating the perceptual interactions between these ester compounds.

1 Materials and Methods

1.1 Materials and reagents

Four types of typical Huangjiu (“Jia Fan” (S1), “Xiang Xue” (S2), “Yuan Hong” (S3), and “Shan Niang” (S4) were purchased from Zhejiang Pagoda Brand Shaoxing Rice Wine Co.Ltd., Shaoxing, China.These classifications refer to the processing technique in producing Huangjiu[3].All four types of Huangjiu, which were brewed in 2018 with the same rice materials and then stored in pottery jars, kept under 4 ℃and light-avoiding conditions, which were identical with the samples in the work of Yu Haiyan et al[2].

Ethanol (≥99.7%), ultrapure water was supplied by.Milli-Q water system.Ethyl acetate, ethyl isobutyrate, ethyl 2-methylbutanoate, ethyl isovalerate, isoamyl acetate, ethyl pentanoate, ethyl hexanoate, ethyl heptanoate, ethyl lactate,ethyl octanoate, ethyl nonanoate, ethyl 2-furoate, ethyl propanoate, ethyl caprate, ethyl butanoate, ethyl benzoate,diethyl succinate, ethyl phenylacetate, 2-phenylethyl acetate, ethyl 3-phenylpropionate, 2-octanol (internal standard) andn-alkane standards (C6-C30) were of chromatographic purity.All of these reagents were provided by Sigma-Aldrich (USA).

1.2 Instruments and equipments

GZX-9070MBE electronic balance (Shanghai Boxun Industrial Co.Ltd., Shanghai); HHS thermostat water bath(Antou-guorui Experimental Instrument Factory, Jintan,Zhejiang); DVB-CAR-PDMS/100 μm Headspace solid phase microextraction (HS-SPME) equipment, 7890 GC connected with 5973C MS and equipped with DB-Wax and DB-5 column (60 m × 0.25 mm, 0.25 μm), 7890 GC connected with olfactory detector (Agilent technologies, New York, USA);Milli-Q water system with 18.2 MΩ·cm resistivity (Millipore,Saint-Quentin-en-Yvelines, France).

1.3 Methods

1.3.1 HS-SPME of volatile compounds

A 100 μm DVB-CAR-PDMS fiber was inlet in the head space of a glass vial for 50 min under 50 ℃, then after 5 min of keeping the sample still under room temperature with no other operations to set the equilibration, the water bath liquid would be continuously stirred at 250 r/min during room temperature for 15 min.At the GC inlet, desorption of aroma components proceeded at 250 ℃ for 5 min[19].

1.3.2 GC-MS determination of volatile compounds in Huangjiu

The tested molecular mass of conditions for GCMS analysis were as previously reported[2].According to Yu Haiyan et al[2], silica capillary column used were DBWax and DB-5 column, and the quantitative method was an external standard method.The analysis was conducted in triplicate.

1.3.3 External standard curves

A model solution containing mass volumetric proportion of 14% ethanol was prepared (ethanol and ultrapure water).The model solution was diluted to gradient concentrations of 1:2, 1:5, 1:10, 1:20, 1:50 and 1:100.Each solution with 5 g model and 20 μL internal standard (2-octanol, 410 mg/L)were immitted in 20 mL vial for producing the calibration curves.The calibration curves were established as Zhu Jiancai et al[20].A calibration curve of aroma components were established, along with the validation range and determination factor (R2).The experiments were conducted in triplicate.The formula for aroma component concentrations calculation were enumerated in equation (1):

WhereCx,Cx,AxandAirespectively refered to volatile compound concentration, internal standard concentration,volatile compound peak and internal standard peak;aandbrespectively refered to the standard curve slope factor and intercept of each compound standard.

1.3.4 OAV and GC-O analysis of odor-active compounds in Huangjiu

The OAV of each compound was calculated as the ratio concentration and referential olfactory threshold[21-23].GC-O analysis was conducted by referring to previous research[2].After the training of distinguishing each odor trait and estimating its aroma intensity correctly, ten panelists were designated to participate in this study.Each panelist should sniff and record the odor information in triplicate.

1.3.5 Descriptive sensory analysis

Sensory analysis was implemented according the report of Lytra et al[16].Samples were evaluated in individual compartment at the controllable environment temperature of 20 ℃ using covered black ISO glass[24]containing approximately 50 mL liquid and encoded with a random three-digit number, lasts about 5 min.The esters should be remained stable for the first 10 min according to literature description[16].Therefore, the solutions provided to the panelists ought to be updated at 10-minute intervals.

Sixteen panelists (8 males and 8 females, aged(24.5 ± 2.2) a) formed the sensory panel.All were professionally trained and selected on the basis of their ability to assess the aroma of Huangjiu.They attended five meetings a week for 8 weeks to cultivate expertise for odor testing.

The dilute alcohol solution (14% ethanol) was employed as matrix for the olfactory threshold examination of ethyl lactate (a), ethyl acetate (b), ethyl phenylacetate (c), ethyl 2-methylbutanoate (d), a/b mixture and c/d mixture via three-alternative forced-choice (3-AFC) test[25].The initial concentrations of a, b, c, d, a/b mixture and c/d mixture for olfactory threshold examination were respectively 1.6, 0.1,0.1, 0.1, 0.1 and 0.1 mg/kg, each set of threshold examination comprised 10 tests with increasing concentrations differentiated by a factor of 2.0.The concentration that acquired 50% correct detection probability was stated as the olfactory threshold, whose assessment referred to the work of Cometto-muniz[26]and Cameleyre[13]et al.As a psychometric function, the concentration/response function accorded with the S-curve (y= (1 + e(-λx))-1) withλreferred as fitting coefficients given by Sigmaplot 14.0 software(Systat Software Inc., Germany).The probability of detection was revised by the chance factor (P= (3p- 1)/2), wherepreferred to the correct response proportion for each concentration,Preferred to the proportion corrected for accidental impacts,which was one thirds as per the 3-AFC method.

In order to determine whether the esters a and b, c and d had aroma perceptual interactions, the above panelundertook the examination of specific mixtures’ olfactory thresholds.Eight different combination systems (14% ethanol solution and 14% ethanol solution containing 95 000 μg/kg a, 9 500 μg/kg b, or 104 500 μg/kg of an a/b mixture (90:10,m/m); 14% ethanol solution and 14% ethanol solution containing 4 500 μg/kg c, 350 μg/kg d, or 4 850 μg/kg of the c/d mixture (93:7,m/m) were applied as matrix to determine the olfactory threshold of aromatic reconstitution(AR, which contained all the volatile compounds with actual concentrations detected by GC-MS).All the ratios were the actual mass ratios of the compounds in the sample.Besides,the mass gradients of aromatic reconstitution in each set of threshold examination were 0.1, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3,12.5, 25, 50 mL diluted in 50 mL combination system.

Besides, Feller’s additive model was employed for certain mixtures to evaluate their interaction effects[27].The mixture calculation probabilityp(AB) was defined asp(AB) =p(A) +p(B) –p(A)p(B), and the measurement probabilityp(AB) was identified through the 3-AFC method.Likewise, the statistical analysis results were presented by S-curves.If the panel’s detection performance for the mixture was inferior to the probabilities summation, inhibition would be manifested.Results that performed above the probabilities summation manifests some form of mutual enhancement or synergy (hyper-addition).Performance matched the probabilities summation, no mixture interactions (simple additive effect) were manifested.

Compound supplementation experiments were employed to investigate the effects of Huangjiu aroma compounds on given sensory attributes.Based on previous research[2,19], eight sensory attributes (“esteric”, “alcoholic”,“sauce”, “fruity”, “caramel”, “Qu”, “sweet” and “sour”) were designated to quantify the sensory characteristics.The first sample contained AR, while the other six ones contained both AR and respectively supplemented with 95 000 μg/kg a,9 500 μg/kg b, 104 500 μg/kg of the a/b mixture (90:10),4 500 μg/kg c, 350 μg/kg d, or 4 850 μg/kg of the c/d mixture(93:7).Each sample should be evaluated with a 9-point scale evaluation (0, none; 5, medium; and 9, strong).Each panelist was demanded to perform the experiments in triplicate.

1.4 Statistical analysis

Statistical analysis of volatile compound concentrations and AIs was executed by analysis of variance (ANOVA);Duncan’s test was used to assess the differences between samples with significant changes indicated byP< 0.05.XLSTAT, version 7.5 (Addinsoft, New York, USA)undertook all statistical analysis.Sigma Plot 12.0 (Systat Software Inc., Germany) software and Origin 9.0 (OriginLab Corporation, Northampton, USA) were employed to fit S-curves and draw column chart respectively.

2 Results and Analysis

2.1 Qualitative and quantitative analysis of ester compounds in Huangjiu

As shown in Table 1, all the ester components were qualitatively identified by retention index, authentic standardsand mass spectrum comparison with NIST11.L library.A total of 20 ester components were identified in the four types of Huangjiu.The sugar content disparity of four Huangjiu samples resulted in the disparities of ester concentrations (138 000-211 000 μg/kg), even categories.Ester compounds formed by esterification of fatty acids were fundamental to the olfactory profile of Huangjiu, they provided fruity and floral odor, imparting a mellow aroma[28].These compounds with OAV ≥ 1 made a relatively strong impression on the aroma of Huangjiu.

Table 1 Ester concentrations determined in Huangjiu with standard curves, coefficients of determination and validation ranges

Table 2Aroma intensities of odor-active ester compounds identified by GC-O in Huangjiu

As Table 2 listed, GC-O analysis detected 14 odoractive ester compounds.According to the qualitative and quantitative results and AIs, ethyl propionate (AI of 3.01-4.35), ethyl phenylacetate (AI of 3.00-4.23), ethyl 2-methylbutyrate (AI of 3.02-4.13), ethyl hexanoate (AI of 3.18-3.67), ethyl acetate (AI of 2.54-3.57), ethyl butanoate(AI of 1.54-3.35), and ethyl lactate (AI of 2.05-2.85) were determined as the esters with greatest contribution to the aroma profile of Huangjiu.This is consistent with the results of Chen Shuang et al[19].Ethyl acetate, ethyl hexanoate,and ethyl lactate contributed with fruity aroma, ethyl 2-methylbutyrate and ethyl phenylacetate contributed with fruity and floral aromas, ethyl propionate and ethyl butanoate contributed with pineapple aroma[19].

Table 3 Composition of AR used in olfactory tests

2.2 Synergistic effects of ester compounds combinations assessed by S-curves

Previous studies proved ester compounds’ vital role in determining the odor profiles of alcoholic beverages,especially Huangjiu[19].As most ester compounds had similar detection thresholds, which inclined to produce perceptual interactions.Such interactions were probable factors in producing the complex Huangjiu aroma.This phenomenon has already been confirmed to be occuring in red wine[29].

Among the seven ester compounds with a strong aroma intensity, sensory interactions were found only in a/b mixture and ethyl phenylacetate c/d mixture.Specific results were as follow: the olfactory thresholds of a, b, c, d, a/b (90:10,m/m),and c/d (93:7,m/m) in AR (Table 3) assessed by the panel,were 159, 22.4, 12.5, 9.71, 17.1 and 8.29 mg/L, respectively.The contrast between values of these olfactory threshold and those identified in the dilute ethanol solution revealed that there was no distinct matrix effect (95% confidence interval on the detection probabilities;P< 0.01).These threshold measurements were almost identical with those reported in the literature except for 2-methylbutyrate[13].The reason for the obvious difference in the threshold within the matrix of ethyl 2-methylbutyrate could be resulting from the difference in raw materials and fermentation principles between Madeira wines and Huangjiu in reported literature[31].The results of olfactory threshold determination obtained in the two matrix (dilute ethanol solution and AR) indicated that four ester compounds were presented at the concentrations well inferior to their olfactory threshold, i.e.present at sub-threshold concentrations.This result confirmed that these compounds hardly exerted olfactive effects on the studied matrix.

At 50% correct detection probability, the threshold value 4.32% (V/V), obtained from experiment was lower than 8.04% (V/V) which was calculated by Feller’s additivemodel (Fig.1A), disclosing a hyper-additive effect (synergy)(P< 0.05).It is manifested that at subthreshold concentrations,an ethyl lactate/ethyl acetate mixture contributed to a significant reduction for the olfactory threshold in Huangjiu aromatic reconstitution.A possible reason for this phenomenon was synergy between the esters.As a result, the pineapple fragrance imparted by these esters was enhanced.A similar observation has been made in liquor[13].

Fig.1 S-curve for olfactory threshold determination based on Feller’s model

Similarly, in the ethyl phenylacetate/ethyl 2-methylbutanoate mixture, the threshold value 2.28% (V/V)obtained from experiment was inferior to 11.56% (V/V) ,which was calculated by Feller’s additive model (Fig.1B) at 50% detection probability, disclosing a hyper-additive effect(synergy) (P< 0.05).The threshold was reduced by a factor of 5.1.It also indicated that at subthreshold concentrations,the ethyl phenylacetate/ethyl 2-methylbutanoate mixture contributed to a significant reduction for Huangjiu olfactory threshold, meaning that ethyl phenylacetate and ethyl 2-methylbutanoate would exert a greater influence on the fruity scent when combined in specific ratio.The above result signified that these two compounds has made considerable contribution on the overall aroma by interacting with each other while reducing the fruity odor threshold.A similar observation was made by Lytra et al[29], who found that some volatile components had synergistic or additive effects at specific threshold concentrations.According to S-curves,even at subthreshold concentrations, these four esters exerted an important function on enhancing the overall aroma of Huangjiu through perceptual interactions.

2.3 The effects of compound supplement on descriptor intensities

As shown in Fig.2, the effects of four vital aroma compounds (ethyl lactate, ethyl acetate, ethyl phenylacetate and ethyl 2-methylbutanoate) on AR sensory attributes.The four ester compounds addition increased the intensities of the ester and fruity descriptors to different extents.Compared with the AR, the ester and fruity descriptor intensity ratios increased from 1.22 to 1.25 and 1.15 to 1.21, respectively.Among them, the effects of ethyl phenylacetate and ethyl 2-methylbutanoate on the fruity descriptor intensity were insignificant, and the intensity of the sauce descriptor was weakened.Maybe their own fruity odor resulted in this phenomenon, so that the increase of fruity aroma made the sauce aroma harder to be perceived.When they were supplemented to the AR in a specific proportion (90:10,m/m),the increase of ester and fruity descriptor intensities turned extremely significant and the enhancement was higher than that of their single summation.This might be caused by the synergy between them.The influence of the sauce descriptor intensity became insignificant, which might root from the masking effect of the sauce descriptor offsetting the elicited enhancements when adding separately.The addition of ethyl 2-methylbutanoate affected the overall aroma balance of AR,increasing the intensity of fruity descriptor while weakening that of caramel descriptor.When ethyl 2-methylbutanoate was presented with ethyl phenylacetate at a certain ratio(93:7,m/m), the effect of the fruity descriptor intensity was enhanced, potentially due to synergistic effects between ethyl phenylacetate and ethyl 2-methylbutanoate.Meanwhile, the weakening effect of the caramel descriptors intensity became insignificant, perhaps due to their simultaneous presence.

These results indicated that these four ester compounds even altered the holistic threshold and perception when presented at subthreshold concentrations, which was coincident with the investigation of Cameleyre et al[14].To some extent, the aroma synergistic effect of ethyl lactate and ethyl acetate were confirmed, along with that of ethylphenylacetate and ethyl 2-methylbutanoate.These results revealed the important aroma contributions of these four ester compounds as natural enhancers of fruity aromas, as all these compounds were produced during the fermentation process of Huangjiu, additionally confirmed the results of Lytra et al[15].To regulate the fruity aroma of Huangjiu, it is necessary to control the aroma-producing flora during the fermentation process.

Fig.2 Impacts of ethyl lactate, ethyl acetate, ethyl phenylacetate, and ethyl 2-methylbutanoate on aroma intensity of AR

3 Conclusion

In this study, the comprehensive qualitative and quantitative analysis of ester components in Huangjiu was more accurate than our previous research[2], additionally involving the interaction of four important ester compounds that effected Huangjiu aroma profile.The results of sensory analysis and S-curves revealed that, at 50% correct detection probability, the dualistic mixtures of ethyl lactate/ethyl acetate and ethyl phenylacetate/ethyl 2-methylbutyrate respectively made the overall threshold of Huangjiu reduced from 8.04%to 4.32% and 11.56% to 2.28%.This demonstrated the existence of synergistic effects between the components of these mixtures.Furthermore, when ethyl lactate/ethyl acetate and ethyl phenyl acetate/ethyl 2-methylbutyrate were simultaneously presented in a specific ratio (90:10; 93:7),more significant enhancing effects on the ester and fruity aromas arose, which indirectly verified a synergistic effect between them.S-curves provided both theoretical basis and practical guidance of some key aromatic esters’ properties and interactions in Huangjiu.The ester mixture in Huangjiu impacts the overall aroma significantly when presented in the certain concentration ratio.