HAN Xiaoyun , LIU Ying , TAN Wenxian , WANG Yanan ,SONG Yong , ZHAO Qiuhong , MAO Zhiwei ,DU Baochang , SUN Qingshen
(1.Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University,Harbin 150500, China; 2.Key Laboratory of Microbiology, College of Heilongjiang Province, School of Life Sciences,Heilongjiang University, Harbin 150080, China; 3.Key Laboratory of Molecular Biology, College of Heilongjiang Province,School of Life Sciences, Heilongjiang University, Harbin 150080, China; 4.Gannan Forest Farm, Qiqihar 162100, China)
Abstract: Mulberry pulp is tender and juicy, which is a kind of food homologous to medicine and food.Mulberry has such medical applications as anti-aging, lowering blood sugar and preventing cardiovascular diseases.However, fresh mulberry fruits are perishable and intolerant to storage.While the mulberry beverages have a tedious taste and high production cost.In order to solve the above problems, the cold mulberry “Longsang 1” (Morus abla L.cv.Longsang 1) was used as the raw material, which was mixed with the local mountain grape (Vitis amurensis Rupr.) to obtain a new type of compound beverage.Longsang 1 mulberry pulp was mixed with different proportions of mountain grape juice and white sugar.The single factor and response surface analysis were used to optimize the best formula of the beverage through fuzzy mathematics sensory evaluation method.After that, electronic nose, electronic tongue and gas phase-ion mobility spectroscopy (GC-IMS) technology were used to evaluate the sensory characteristics and the flavor components of the beverages before and after compounding.The results showed that the optimal conditions for obtaining the compound beverage were mulberry juice content of 14.18%, mountain grape juice content of 10.60%, and sugar of 8.00% when the sensory scores were used as the evaluation index.Under these conditions, the optimal flavor and taste were obtained, and the total solubility solid content was (14.38±0.19) Brix.The principal component analysis (PCA) results of the electronic nose showed that the flavors of the two beverages were significantly different.The electronic tongue analysis showed that the compound beverage had better sourness and umami taste.A total of 33 compounds were detected from the two beverages by GC-IMS.Compared with the mulberry beverage, there were 17 compounds in the composite beverage with higher content, giving the composite beverage a pleasant flavor, which was consistent with the results of the electronic tongue analysis.This research expands the application range of wild grapes, reduces the production cost of mulberry beverages, and has an excellent market prospect.
Keywords: mulberry; Vitis amurensis Rupr.; flavor substance; fuzzy mathematics model
Mulberry is a member ofMorusofMoraceaegenus[1].The main mulberry varieties distribute in Asia, especially widely grow in China[2].The ripening time of mulberries varies from July to September depending on the location, and the ripe fruits are dark purple to black[3].Mulberries are mostly reddish purple or black, poly-flower fruit, and contain essential nutrients necessary for health[4], as well as various active substances such as resveratrol, anthocyanin, etc, which can be used for beauty and anti-aging, anti-tumor, as well as lowering blood pressure, lowering blood sugar, and preventing cardiovascular disease[5-7], with application prospects in human nutrition and food industry[8-9].However, mulberry fruit is a highly perishable berry due to its high water content, fragile structure, and elevated respiration rate[10-11].An effective method to extend the shelf life of this berry is to process the products in a short period of time.The processed products include mulberry jam, mulberry fruit wine and mulberry drink.However, mulberry-based beverages are not very popular because of their tedious taste.At present, mulberry compound beverages are showing a diversified trend in the market, including bitter gourd mulberry fruit juice drink, red bean wolfberry mulberry fruit juice drink, blueberry mulberry compound drink, red date mulberry compound drink, mulberry and orange mixed drink, and mulberry wolfberry cassia health drink, which can integrate the functional characteristics of two or more kinds of raw materials, and develop a kind of new fortified drink.
VitisamurensisRupr., a member of Vitaceae family, is a common wild grape in Asia[12].In China, this berry grows mainly in Heilongjiang and Jilin provinces[13].This fruit is a dark purple grape which is extremely cold-resistant[14], with thickened peel, low sugar content, high acidity and strong pigment.Its juice is rich in a variety of biologically active substances such as anthocyanins and resveratrol, with antioxidant, anti-inflammatory, analgesic and neuroprotective effect[15].For many years, it has been used as a traditional Chinese herb[12].
Mulberry andVitisamurensisRupr.are berry fruits that are mainly used in their fresh forms, which limits their applications and long term consumption.Both of the berries grow in Heilongjiang province, where the climate and soil might endow the fruits special features.In our previous study, we have characterized and proved the hypoglycemic effects ofMorusablaL.cv.Longsang 1 leaf-derived water extracts[16].
In this experiment, mulberry,VitisamurensisRupr.and sugar were used as raw materials to obtain a kind of compound beverage, of which the physical and chemical properties and flavor substances were analyzed.This study might extend the use of these berries in Heilongjiang province.
1.1 Materials and reagents
Mulberries (MorusablaL.cv.Longsang 1) andVitisamurensisRupr.were harvested in the mulberry base in Gannan County (Qiqihar, China) in 2018 and 2019; citric acid (food grade) and potassium sorbate (food grade) were purchased from Shandong Kunda Biotechnology Co., Ltd.(Linyi, China); copper sulfate (AR CAS: 7758-98-7), potassium sulfate (AR CAS: 7778-80-5), methyl red (AR CAS: 493-52-7), anhydrous ether (AR CAS: 60-29-7) and sodium hydroxide (AR CAS: 1310-73-2) were purchased from Tianjin Comeo Chemical Reagent Co., Ltd.(Tianjin, China); sulfuric acid (AR CAS: 7664-93-9) and hydrochloric acid (AR CAS: 7674- 01- 0) were purchased from Beijing Chemical Plant (Beijing, China); anhydrous ethanol (AR CAS: 64-17-5) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.(Tianjin, China); methanol (CR CAS: 67-56-1) was purchased from Dima Technology (Beijing, China).
Fig.1 Preparation schematic of mulberry-Vitis amurensis Rupr.compound beverage
1.2 Factors affecting the preparation of mulberry-Vitis amurensis Rupr.beverage
The flowchart of the beverage preparation was firstly determined as shown in Fig.1, then different amount of mulberry andVitisamurensisRupr.ratios, i.e.1∶0.5，1∶0.75，1∶1，1∶1.25 and 1∶1.5 were prepared.Different amount of sucrose (W/V) and material/liquid were also considered.The fuzzy mathematics-based model was established for sensory evaluation.
1.2.1 Pretreatment and processing flowchart of the beverage preparation
Mature mulberries andVitisamurensisRupr.were selected and washed thoroughly.Both the fruits were made juice for use.
1.2.2 Establishment of fuzzy mathematics-based model
A panel of 10 subjects (5 males and 5 females) without any sensory abnormalities were selected to score different samples using the sheet delivered with different criteria as shown in Table 1.All the participants must meet all the following requirements: the sensory assessors were required to refrain from eating spicy food for 12 h before the assessment.The mouth should be rinsed with water between two samples, and the next sample should be assessed at an interval of 10 minutes, and discussion should be avoided during the assessment.
Table 1 Sensory evaluation criteria of the compound beverage
Fuzzy mathematics model establishment.The evaluation factor set was a collection of sensory evaluation factors for the mulberry-VitisamurensisRupr.compound beverage, and the target factor setU={color (U1), smell (U2), state (U3), taste (U4)}; weight setA={color weight (Al), smell weight (A2), state weight (A3), taste (A4)}; comment setV={very good (V1), good (V2), fair (V3), poor (V4), very poor (V5)}, based on 100 points,V1is 100 points,V2is 80 points,V3is 60 points,V4is 40 points, andV5is 20 points.
The sensory evaluation standards corresponding to each level of comment set were given in Table 1.According to the sensory evaluation results, three single-factor evaluation matrices were established and analyzed by fuzzy mathematics evaluation methods.
1.2.3 Effect of mulberry andVitisamurensisRupr.juice ratios (V/V)
Different mulberry andVitisamurensisRupr.juice ratios, i.e.1∶0.5, 1∶0.75, 1∶1, 1∶1.25 and 1∶1.5, were prepared.The amount of sucrose was 8% and the juice was diluted with twice the volume of water.The score from fuzzy mathematics model was used to determine the optimal ratio.
1.2.4 Effect of sucrose amounts
Different amounts of sucrose (W/V), i.e.4%, 6%, 8%, 10% and 12%, were added.The ratio of mulberry andVitisamurensisRupr.juice was 1∶0.75 and the juice was diluted with 2 volumes of water.The score from fuzzy mathematics model was used to determine the optimal amount of sucrose used.
1.2.5 Effect of juice and water ratios
The ratio of mulberry andVitisamurensisRupr.juice was fixed at 1∶0.75 and the sucrose was fixed at 8% (W/V).Different juice and water ratios, i.e.1∶1, 1∶2, 1∶3, 1∶4 and 1∶5, were prepared using water.The score from fuzzy mathematics model was used to determine the optimal ratio.
1.2.6 Response surface optimization
According to the single factor experiments, the main factors affecting beverage flavor including mulberry andVitisamurensisRupr.juice ratios, amounts of sucrose used and dilution factors were used as independent variables, the corresponding parameters were recorded using Design Expert V8.0.6.1 software as shown in Table 2.
Table 2 Independent variables used for beverage optimization
1.3 Determination of physical-chemical properties of the beverage
The protein and fat contents in the beverage were analyzed based on the national standard Kjeldahl method and Soxhlet extraction method, respectively.The soluble solids in the beverage were measured using a hand-held refractometer, and the pH change of the beverage was measured using a digital pH meter.
1.4 Flavor substance analysis of the beverage
1.4.1 Electronic nose analysis
The electronic nose is usually composed of a gas sampling unit and a sensor array.Before the experiment, the prepared beverage samples were accurately pipetted into 50 mL clean glass bottles with disposable 10 mL syringes, then the samples were equilibrated for 30 min at 25 ℃ to develop headspace for detection.Parameters for electronic nose (iNose system, US ISENS) were: sample preparation time for 5 s; detection time for 60 s; measurement count for 1 s; automatic zero adjustment time for 10 s; cleaning time for 240 s; internal flow at 400 mL·min-1; injection flow rate at 400 mL·min-1.Each parallel was collected once, and 3 stable detection data were finally selected for PCA.
1.4.2 Electronic tongue analysis
40 mL of different beverage sample was poured into an electronic tongue cup and measured with the SA-402B taste analysis system (Insert, Japan).The working electrodes were: gold electrode, palladium electrode, silver electrode, platinum electrode, titanium electrode, tungsten electrode, and the auxiliary electrode was a platinum electrode with a diameter of 2 mm, and silver/chlorine silver (Ag/AgCl) was used as a reference electrode.Distilled water was used as the cleaning solvent.The sampling time was 180 s (n=5).To ensure that the data was stable and reliable, the measurement started after the sensor reached equilibrium, data were collected 5 times for each sample through 5 sensors, and the average values were calculated.The data were analyzed using the laboratory analytical viewer (LAV) analysis software of the equipment configuration.
1.5 Volatile substance analysis of the beverage
The beverage volatile substances were analyzed in Shandong Jinan Haineng Instrument Co., Ltd.After homogenization of the beverage samples, 1 mL of sample was placed in a 20 mL headspace bottle, and the sample was injected after incubation at 60 ℃ for 10 min, using FlavourSpec© gas phase-ion mobility spectrometer (GAS, Dortmund, Germany) for analysis.Gas-ion mobility spectrometry unit: analysis time 20 min, column type FS-SE-54 15m ID: 0.53 mm, column temperature 60 ℃, carrier gas/drift gas N2, IMS temperature 45 ℃; automatic top empty sampling unit: injection volume 500 μL, incubation time 10 min, incubation temperature 60 ℃, injection needle temperature 65 ℃, incubation speed 500 r·min-1.Gas chromatography conditions: 0.00 min, E1 flow rate 150 mL·min-1, E2 (carrier gas) 2 mL·min-1; 2.00 min, E1 flow rate 150 mL·min-1, E2 (carrier gas) 2 mL·min-1; 20.00 min, E1 flow rate 150 mL·min-1, E2 (carrier gas) 100 mL·min-1.
The analysis software supporting the instrument includes LAV, three plug-ins and GC × IMS Library Search, which can analyze samples from different angles.LAV: view the analysis spectrum, each point in the figure represents a volatile organic compound.Reporter plug-in: directly compare the spectral differences between samples (two-dimensional top view and three-dimensional spectrum).Gallery Plot plug-in: fingerprint comparison, intuitively and quantitatively compare the differences of volatile organic compounds between different samples.Dynamic PCA plug-in: dynamic principal component analysis, used for cluster analysis of samples, and quickly determine the type of unknown samples.GC × IMS Library Search: the NIST database and the IMS database built in the application software can perform qualitative analysis of the substances, and the database can be expanded by using standard products.
1.6 Data analysis
All the experiments were done in triplicate, and Origin 9.0 was used for statistical analysis.The results were expressed as (mean±std), and the significance test was performed through ANOVA.
Mulberry juice has been studied extensively because of its possible functions.Studies have shown that the anthocyanins of mulberry juice had anti-fatigue effects[9].VitisamurensisRupr.contains such active substances as oligostilbenes, flavonoids, anthocyanins and phytochemicals, which accounts for its potential use in developing new products such as compound beverages in this study.Another study by Xuan et al showed that the active substance, Vam3, isolated fromVitisamurensisRupr., could alleviate colitis-related tumorigenesis through inhibiting NF-Kappa B signaling pathway, suggesting its anti-inflammatory effects[20].Therefore, combination of the two raw materials may find new use in drink industry.Our study firstly optimized the compound beverage based on hedonic analysis using fuzzy mathematics model, and then characterized the beverage.
2.1 Establishment of fuzzy matrix mode and single factor test results
2.1.1 Establishment of fuzzy matrix model
A panel of 10 people evaluated the beverage based on the color, aroma, tissue state and taste.The number of votes for each quality factor in different grades was recorded, and the fuzzy relational matrix was obtained after normalization of sensory evaluation scores for the compound beverage.
whereRj(j= 1,2,3,…,t) is the sample number, and each row represents the evaluation result of a quality factor.According to the principle of fuzzy matrix transformation,H=K×R, the comprehensive evaluation result of samplejis calculated,Hj=K×Rj.
In this study, the user survey method was used to determine the weight of each factor, based on the importance of different factors in the compound beverage sensory evaluation.The weight set of the four quality factors wasK= 0.17, 0.33, 0.21, 0.29, that is, aroma was the most important quality index, followed by taste, while color and tissue state had relatively little influence on the sensory quality of the complex beverage.Weight refers to the proportion of each factor in all evaluated factors from a factor set, which can be expressed ask=k1,k2,k3,…,Km.The weight sum of elements in the weight set is 1.0 according to the normalization principle.
2.1.2 Single factor test results
Fig.2(a) revealed that the sensory scores of the compound beverage increased firstly, and then decreased with the increased proportion of mulberry juice, with the best flavor and the highest comprehensive sensory score when the ratio was 1∶0.75, scoring (78.98±0.75).It can be seen from Fig.2(b) that as the amounts of sucrose added increased, the sensory score of the compound beverage increased firstly and then decreased, with the optimal taste when 8% sugar (W/V) was added, scoring (76.66±2.37).The optimal ratio of juice to water was 2.0 as shown in Fig.2(c), scoring (81.68±1.42).
Fig.2 Single factor test results
2.2 Response surface test results
2.2.1 Response surface test design and results
The results of the response surface analysis to optimize the mulberry mountain grape compound beverage were shown in Table 3, and a quadratic multinomial regression model was obtained of sensory scoreYversus mulberry juice toVitisamuresisRupr.juice ratio (A), surcose content (B), dilution factor (C):
Y=+88.08+3.76A-1.59B+0.27C+0.93AB-2.88AC-1.04BC-4.05A2-4.43B2-4.91C2
Table 3 Response surface design and results
Fig.3 showed the interactive effects between the ratios of mulberry juice toVitisamurensisRupr.juice, the amount of sucrose added and the dilution factor, the ratio of mulberry juice toVitisamurensisRupr.juice, and the dilution factor.All these factors showed significant effects on the sensory evaluation of the compound drink, which were in consistence with the ANOVA results in Table 4.
Fig.3 Response surface diagram of the influence of the interaction of various factors on the comprehensive score of compound beverage
2.2.2 Verification test
In order to further verify the accuracy and feasibility of the model, the content of mulberry juice,VitisamurensisRupr.juice and surcose were set as 14.18%, 10.63% and 8.0%, respectively.The sensory score of the compound beverage was (83.38±0.03) after 3 verification tests, indicating the parameters obtained by surface optimization were reasonable and credible, and had practical value.
In this experiment, mulberry andVitisamurensisRupr.provided by the mulberry base in Gannan County, Qiqihar were selected as the raw materials of the compound beverage.When the mulberry juice content was 14.18%, theVitisamurensisRupr.juice content was 10.63%, and the surcose content was 8.0%, the mulberry-VitisamurensisRupr.compound beverage tasted best with a unique flavor, which was the most acceptable.Compared with pure mulberry juice beverage, the sensory quality of mulberry-VitisamurensisRupr.compound beverage increased significantly, which proved that the addition ofVitisamurensisRupr.increased the acidity of the beverage significantly and was more acceptable.Therefore, this work aimed to compare the sensory and flavor features of the two drinks.
2.3 Determination of physical and chemical indicators of the beverage
Table 5 showed that the total soluble solid (TSS) content was (10.98±0.14) and (14.38±0.19) Brix for mulberry beverage and mulberry-VitisamurensisRupr.beverage, respectively.In our study, the mulberry beverage with TSS content at 10.98 Brix was enough, which was lower than that reported[21].It could be explained by the differences in species or growth location.After mixed withVitisamurensisRupr., the TSS content increased, but with more pleasant flavor.Except for the TSS content, all the other parameters (pH, protein and fat content) showed decreased tendency, which could be explained by the low protein and fat content inVitisamurensisRupr..
Table 5 Changes of physical and chemical parameters in mulberry beverage
2.4 Sensory analysis
The purpose of PCA in E-nose or E-tongue detection is to use a few new variables to represent the original information as much as possible after the dimensionality reduction processing[22].The scatter plots for main component were obtained after the PCA of the original data obtained by E-nose evaluation as shown in Fig.4(a), where each point represented one sample, and the distance between points represented the characteristic difference among different detection points.The contribution rates of principal components 1 and 2 were 93.2% and 6.2%, with the total contribution rate of 99.8%, indicating that the principal components 1 and 2 already contained a large amount of information which could reflect the sample’s overall information.The main component analysis results showed that the aroma components of the two beverages were obviously different, which laid a foundation for the subsequent analysis of flavor substances.
Fig.4 Flavor analysis of the mulberry beverage (sample 1) and mulberry-Vitis amurensis Rupr.beverage (sample 2)
Electronic tongue technology uses sensor arrays, some of which show different responses to various gases, and combines signal processing methods based on pattern recognition or artificial neural networks[23].The electronic tongue response radar chart was shown in Fig.4(b).The dots represented eight representative flavors.Both drinks responded on 8 flavors.The sour, salty and umami taste of the compound beverage were much higher than that of mulberry drinks which might be explained by the interaction of many components after addingVitisamurensisRupr.to produce acid, alcohol and lipid.The compound beverage had higher sour, salty, and umami values, and tasted better.It should be due to the addition ofVitisamurensisRupr..There was no significant difference in other tastes between the two drinks.
2.5 Analysis of beverage volatile substances
GC-IMS is a new separation and detection technology that has emerged in recent years[24].In this study, the volatile organic compounds were identified by GC-IMS as shown in Fig.5.The abscissa in Fig.5(a)～Fig.5(d) was the ion migration time (ms), the ordinate was the retention time (s); red represented the substance composition; the abscissa 1.0 red vertical line was the RIP peak (reactive ion peak).The red dot indicated that the substance was higher in the compound beverage, and the blue dot indicated that the substance was lower in the compound beverage.Fig.5(a) and Fig.5(b) were the gas phase ion mobility spectra of mulberry beverage and compound beverage, respectively.The mulberry beverage was used as a reference in Fig.5(d).The compound beverage spectrum deducted the peak in the mulberry beverage.The red dots indicated that the substance was higher in the compound beverage, and the blue dots indicated that the substance was lower in the compound beverage.It could be seen that the content of more substances in the compound beverage was higher than that in the mulberry beverage, and there were also some substances with lower content than the mulberry beverage.From the above results, it could be seen that the flavor difference between the compound beverage and the mulberry beverage was significant.
The odor fingerprint analysis technology can obtain not only corresponding response signals according to different flavor substances, but also the overall information of the volatile organic components in the sample.In Fig.5(e), sample 1 was a mulberry beverage, and sample 2 was a composite beverage.Some substances were followed by -M and -D, indicating that they were monomer and dimer of the same substance.The complete volatile organic compound information of each sample and the difference of volatile organic compounds between the samples could be seen from the figure.
Table 6 is a list of meteorological ion mobility spectrum of compounds.A total of 30 compounds were detected, among which aldehydes accounted for approximately 51.5%, followed by ketones 18.1%.The most abundant aldehyde compounds in beverages were nonanal, hexanal, 3-methylbutanal and furfural, among which 15 compounds showed higher ion intensities in mulberry-VitisamurensisRupr.beverage than those in mulberry beverage alone.
Table 6 List of compounds and their ion intensities in the gas phase ion mobility spectrum
VitisamurensisRupr.and mulberry were used to develop the compound beverage with pleasant flavor by optimizing the formula.Electronic nose, electronic tongue and GC-IMS techniques were used to characterize mulberry beverage complexed withVitisamurensisRupr.It could be concluded that the addition ofVitisamurensisRupr.improved the sensory and flavor characteristics of mulberry beverage.