The protective effect of cyclodextrin on the color quality and stability of Cabernet Sauvignon red wine

Caiyun Liu ,Lulu Wu ,Shuyue Fan ,Yongsheng Tao, ,Yunkui Li,

1 College of Enology,Northwest A&F University,Yangling 712100,China

2 Ningxia Helan Mountain’s East Foothill Wine Experiment and Demonstration Station of Northwest A&F University,Minning 750104,China

Abstract The impact of cyclodextrins (CDs) on wine quality and stability remains largely unknown.This study systematically assessed the protective effect of the post-fermentation addition of CDs on color stability of red wine from the viewpoints of color characteristics,copigmentation and phenolic profiles.The grey relational analysis (GRA) and principal component analysis (PCA) methods were employed to dissect the key effective determinants related to color quality.The addition of CDs induced a significant hyperchromic effect of 8.19-25.40%,a significant bathochromic effect and an enhancement of the color intensity.Furthermore,the evolution of anthocyanin forms and the content of monomeric anthocyanins revealed that β-CD is a superior favorable cofactor during wine aging,but for long-term aging,2-HP-β-CD and 2-HP-γ-CD are more beneficial in promoting the formation of polymerized anthocyanins and color stability.This work provides an important reference for the use of CDs to enhance the color quality and stability of red wines.

Keywords: cyclodextrins,color properties,copigmentation,Helan Mountain’s East Foothill,red wine aging

1.lntroduction

Wine has grown in popularity among Chinese consumers due to its long history,unique vinification,and graceful quality,and higher wine quality is being demanded.Color is one of the most influential sensory indicators used by consumers to evaluate wine quality.However,red wine is prone to color deterioration because of several factors that negatively affect its stability in storage (Fanetal.2023a).This has become a widespread problem for wine production in Chinese continental arid and semi-arid environment regions,such as Ningxia Helan Mountain’s East Foothill.Some local wines show significant chromatic quality declines even after a few months of aging (Zhang Betal.2021).

Red wine color is mostly a result of the amount and variety of anthocyanins present,which are typically found in monomeric,combined,and polymeric forms.In general,monomeric anthocyanins account for the vivid red color during the initial stages of fermentation,but the wine color is predominantly determined by the amount of anthocyanin polymerization during the latter phases of maturation (Prat-Garcíaetal.2020).Furthermore,copigmentation,which primarily consists of anthocyaninanthocyanin and anthocyanin-cofactor combinations,is one of the essential elements affecting wine color (Lietal.2019).According to Lietal.(2017),copigmentation in young red wine may contribute between 30% and 50% of the color.Researchers have explored numerous strategies to overcome this long-standing problem for the wine industry,such as adjusting the pH and storage temperature,as well as the addition of phenolic compounds (Scrimgeouretal.2015;Lietal.2018;Gambutietal.2022).However,many of these methods fall short of improving quality,and some even have detrimental effects on the organoleptic characteristics.There is growing interest in the copigmentation issue,including attempts to control copigmentation by adding copigments that enrich the color palette in direct correlation with consumer perception (Lietal.2023a).

Cyclodextrins (CDs) have developed into the most promising exogenous stabilizers in food.CDs are natural,tasteless,colorless,odorless,non-caloric,noncarcinogenic,biodegradable,and non-toxic compounds.CDs are cyclic compounds made up ofα-D-glucopyranose units connected byα-1,4-glycosidic bonds,resulting in a cavity that is hydrophobic within a hydrophilic exterior(Liuetal.2022).Among them,α-CD andβ-CD are cyclic oligosaccharides containing six and seven glucose molecules,respectively,that have been employed by the food industry in order to improve sensory properties,shelf life,and component sequestration.

Through non-covalent host-guest interactions,hydrophobic cavities can form inclusion complexes with various organic guest molecules,giving them the unique capacity to trap a wide variety of molecules.When guest molecules are encapsulated in CD cavities,they are protected from evaporation,degradation,and oxidation,and their release is controlled or delayed (Liuetal.2022).Because of their abundance and affordable cost,β-CD are the most frequently employed CDs in the food industry.However,the water solubility (1.85%)and encapsulation ability ofβ-CD are insufficient for many commercial applications.Chemical modification is frequently adopted to alter functional performance.Hydroxypropyl-β-CD (HP-β-CD) is the most commonly used derivative because of its improved solubility and encapsulation properties (Santanaetal.2021).Researchers have widely discussed and evaluated the utility of natural and modified CDs.For example,CDs can prevent color fading by enhancing the chemical stability of the colorant (Gonzalez Pereiraetal.2021).Lopez-Nicolasetal.(2007) employed the CIELAB color space approach to evaluate the efficacy of natural and modified CDs as browning inhibitors by adding them to apple and pear juices.They discovered that maltosyl-β-CD could act as a “secondary antioxidant” that reduced browning and improved the juice’s natural antioxidant activity.Furthermore,the addition of chlorogenic acid (CGA) with the HP-β-CD to grape juice demonstrated their significant contribution to the protection of color (Shaoetal.2014).Recent studies have investigated the mechanism of color protection further by evaluating the impact of CDs on the thermodynamic and kinetic characteristics of anthocyanin-3-O-glucoside (Fernandesetal.2013).According to these findings,the substitution of specific hydroxyl groups onβ-CD with other functional groups plays a crucial role in the host-guest recognition process.

CDs,as the host molecule,can form inclusion complexes with various guest molecules.The formation and stability of inclusion complex mainly depend on the suitability of the size and shape of the host and guest and the strength of their interaction.Therefore,different types of CDs have different encapsulation ability for the same guest molecule.The novelty of this study lies in exploring the effect of post-fermentation addition of CDs on the color quality and stability during wine aging,which is not attempted so far.So this study achieved the following three key goals: (i) to evaluate the protective effect of post-fermentation additions ofα-CD,β-CD,2-HPβ-CD and 2-HP-γ-CD on the color quality and stability of wines aged for 1,3,5,and 7 months;(ii) to compare the copigmentation effect of four CDs and seek the most optimal one to suit the different storage periods of the wines;and (iii) to forecast the primary determinants influencing color evolution based on the relationship between the varying trends of phenolic compounds and color characteristics.The results of this study will provide details on the application of CDs to solve color stability problems of the wine industry in Helan Mountain’s East Foothill Wine Region in Ningxia Hui Autonomous Region of China.

2.Materials and methods

2.1.Materials

Grapes: Cabernet Sauvignon grapes were harvested in October 2021 from the Helan Mountain’s East Foothill region of Ningxia,China (106°27´E,38°47´N).The pH,reducing sugar (calculated by glucose) and titratable acidity (calculated by tartaric acid) of the collected berries were 3.83,262.83 and 6.85 g L-1,respectively.

Yeast:SaccharomycescerevisiaeBV818 was purchased from Angel Yeast Co.,Ltd.(Hubei,China).

Main reagents: The reagents of hydrochloric acid,ethanol,methanol,sulfur dioxide,vanillin,and acetaldehyde were purchased from GHTECH (Guangzhou,China);gallic acid monohydrate,caffeic acid,malvidin-3-O-glucoside and (+)-catechin were purchased from Shanghai Yuanye Biotechnology Co.,Ltd.(Shanghai,China);α-CD,2-HP-β-CD and 2-HP-γ-CD were produced by Shanghai Aladdin Biochemical Technology Co.,Ltd.;andβ-CD was sourced from Shanghai Bohr Chemical Reagent Co.,Ltd.(Shanghai,China).

2.2.Winemaking experiment

The green and moldy berries were manually removed,while the remaining bunches were de-stemmed and slightly crushed before they were put into five individual containers (5 L each) with a 60 mg L-1SO2supplement.Then,the must was supplemented with 20 mg L-1Gestown pectinase (Shanghai,China) and immersed at 4°C for 48 h.Alcoholic fermentation was initiated by the introduction of 200 mg L-1S.cerevisiaeBV818yeast when the must was warmed up to 21°C.The fermentation temperature was maintained at (25±1)°C.The fermentation process was monitored by determining the must density and temperature.When the residual sugar was less than 2 g L-1,60 mg L-1of SO2was added to stop the alcoholic fermentation.The fermented wines were mixed and evenly divided into five aliquots,to which 1.5 g L-1α-CD,1.5 g L-1β-CD,2 g L-12-HP-β-CD and 2 g L-12-HP-γ-CD were added separately,and the control wine (CK) had no additive.The samples were stored in 750 mL brown bottles in the underground cellar of the College of Enology (Northwest A&F University,Yangling,China) at a constant temperature (15 to 16°C) and relative humidity (75%),and subjected to analysis after 1,3,5,and 7 months of storage.

2.3.Determination of color characteristics

The CIELAB parameters for the CK and CD-treated groups were measured using D65 illumination and a 10°observer.Using deionized water as a reference,all the wine samples were filtered through 0.45 μm filters.A glass cuvette with a path length of 2 mm was selected,and a Cary 60 ultraviolet-visible spectrophotometer from Agilent Technologies (Santa Clara,USA) was used to scan the visible light absorption spectrum of the samples from 400 to 780 nm,with a 1 nm scanning interval.The color parametersL＊,a＊,b＊,C＊abandhabof the wine were determined by calculating the absorbances at 450,520,570,and 630 nm.L＊ represents lightness,ranging from 0(black) to 100 (white).Thea＊ andb＊ parameters indicate the green-red color channel and the yellow-blue color channel,respectively.The chroma value (C＊ab) reflects the vividness of the color as determined by the chroma and is related toa＊ andb＊.The hue angle (hab) more precisely represents a specific color.The closerhabis to 0 degree,the closer the color is to the red hue (Lietal.2017).

The color difference values (ΔE＊ab),which describe how the wine color changes over time,were calculated.The Euclidean distance between two points in threedimensional space,defined byL＊,a＊,andb＊,was used to calculate ΔE＊ab(Zhaoetal.2021).

Color parametersL＊,a＊ andb＊ were calculated according to eqs.(1)-(8):

whereAis the absorbance;Tis the transmittance;X,YandZstand for the tristimulus values of the sample;andXn,YnandZndenote the tristimulus values of the standard D65 illuminant,assigned as 94.825,100.000 and 107.381,respectively.

ChromaC＊aband huehabwere calculated according toL＊,a＊ andb＊:

Then the color difference ΔE＊abwas calculated according to the CIEDE2000 color-difference formula:

Furthermore,the hyperchromic effect (ΔA) was measured at 520 nm absorbance,and the shift in the maximum absorption wavelength (Δλmax) was used to measure the bathochromic effect (Zhaoetal.2022).ΔAand Δλmaxwere calculated according to eqs.(13)-(14):

where ΔAis the hyperchromic effect expressed in %,andAandA0represent sample absorbance at 520 nm with and without copigments,respectively.

where Δλmaxrepresents the difference between the maximum absorbance with copigment (λmax) and without copigment (λmax0).

2.4.Determination of monomeric anthocyanins

The LC-20AT high-performance liquid chromatography(HPLC) system (Shimadzu,Kyoto,Japan) was employed to detect the monomeric anthocyanins.The device comprised a column oven,an autosampler,an array photodiode detector,and a Synergi Hydro-RP C18 column (250 mm×4.6 mm,4 μm,80 Å).Before analysis,the wine samples were filtered using a 0.22 μm organic filter membrane.The mobile phase was composed of solvent A (purified water:acetonitrile:formic acid=32:4:1(v/v)) and solvent B (purified water:acetonitrile:formic acid=16:20:1 (v/v)),and was administered at a flow rate of 1.0 mL min-1.The gradient program was carried out as follows: 0 to 35% B for 45 min;35 to 100% B for 1 min;100% B isocratic for 4 min;and 100% to 0% B for 1 min.The temperature of the column was 35°C.The volume of injection was 20 μL,and the wavelength of detection was 520 nm.According to the retention time and maximum absorption wavelength,the anthocyanin species represented by each peak were determined based on the HPLC data (Lanetal.2021).

2.5.Determination of free anthocyanin ratio (FA%),copigmented anthocyanin ratio (CA%) and polymeric anthocyanin ratio (PA%)

The wine was treated with 20 μL of 10% (v/v)acetaldehyde and allowed to stand for 45 min at 25°C,then the absorbance (Aacet) was determined.In addition,2 mL of wine was mixed with 160 μL of 5% (w/v) SO2,and the absorbance (ASO2) was recorded after 10 min.Finally,model wine (5 g L-1tartaric acid,12% ethanol,and 0.2 mol L-1NaCl,pH adjusted to 3.6 with NaOH) was used to dilute the wine samples by a factor of 20 in order to assess the copigmented pigment (Awine).The absorbance was measured using a glass cuvette with a 2 mm light path at a wavelength of 520 nm.The value of Awinewas multiplied by 20 to account for the dilution factor.FA%,CA%,and PA% were determined using the equations from Lanetal.(2021) as expressed in eqs.(15)-(17):

2.6.Determination of phenolic compounds

The total phenols,total flavonoids,and total anthocyanins in the samples were determined.Samples (0.25 mL) diluted with 10% ethanol (v/v) were transferred into test tubes,followed by the addition of 0.25 mL of 0.1% HCl in 95% ethanol (v/v) and 4.55 mL of 2% HCl (v/v).The solution was mixed well and left to stand for 15 min.A Cary 60 UV-Vis spectrophotometer(Agilent Technologies Inc.,Santa Clara,USA) was used to determine the absorbance of samples in a 2 mm quartz cuvette at 280,360,and 520 nm,respectively.The total phenol (A280),total flavonoid (A360),and total anthocyanin (A520) contents were assessed from standard curves made using dilutions of gallic acid(in 10% ethanol),caffeic acid (in 10% ethanol),and malvidin-3-O-glucoside (in 10% ethanol) at 280,360,and 520 nm,respectively (Liuetal.2023).Lanetal.(2021) reported thep-DMACA method for determining total flavanols at 640 nm.

The total tannin content of the samples was measured using the vanillin assay (Herderich and Smith 2005).A 0.5 mL methanol-diluted sample (red wine diluted 10-fold)was pipetted and poured into the centrifuge tube,then 3 mL of the 4% vanillin solution (made in methanol) and 1.5 mL of the 36% hydrochloric acid were added.After thoroughly mixing,the samples were placed in darkness for approximately 40 min.At a wavelength of 510 nm,the absorbance of the samples was recorded,and the content was determined using the standard curve that was developed using diluted (+)-catechin.

2.7.Determination of phenol monomers

The method of Lietal.(2021) was modified for the measurement of phenol monomers.Aliquots of 1 mL of the samples with 1 mL of ethyl acetate and 0.5 mL of acetonitrile were mixed and vortexed for 10 s,then the mixture was centrifuged at 7,500×g for 15 min (HC-30182;Anhui Zhongke Zhongjia Scientifc Co.,Ltd.,Tianjin,China),and the supernatant was removed.The extraction was repeated twice.The supernatant was evaporated to dryness with a 9901S centrifugal vacuum concentrator (Automatic Science Instrument Co.,Ltd.,Tianjin,China).The residue was dissolved in methanol for HPLC analysis.The phenolic acids were analyzed using a Shimadzu HPLC that was fitted with a Synergi Hydro-RP C18 column (250 mm×4.6 mm,4 μm,Phenomenex,Tianjin,China) at a flow rate of 1 mL min-1.Solvents (A) 0.1% acetic acid (v/v)in water:acetonitrile (8:1) and (B) 0.1% acetic acid (v/v)in water:acetonitrile (4:5) were prepared for the mobile phase.The gradient was 0-35% B from 0 to 45 min,35-100% B from 45 to 50 min,100% B isocratic from 50 to 55 min,100-0% B from 56 to 62 min,and 0% B isocratic from 56 to 62 min.The oven temperature was 30°C,and the detection wavelength range was 210-400 nm.

2.8.Statistical analysis

The main components influencing color were predicted using GRA.In this study,GRA was employed to determine the relationships between phenolic compound variation trends and color characteristics.Based on the GRA results,the compounds that were most related to the color characteristics were screened out.The results of each experiment are given here as the mean and standard deviation.The results were compared using one-way analysis of variance (ANOVA) and the Duncan test (α=0.05),andP＜0.05 indicated statistical significance.Origin 2021 was selected for data visualization and PCA.

3.Results and discussion

3.1.Effect of CD addition on color quality

Table 1 depicts the influences of CK,α-CD,β-CD,2-HPβ-CD and 2-HP-γ-CD on the red wine CIELAB color parameters.The color characteristics of the CK changed considerably from 1 to 7 months of aging,with values ofL＊,b＊,C＊ab,andhabgradually increasing while the value ofa＊ gradually decreased.These changes are consistent with the observations of the evolution of color swatches(Fig.1) in red wine,i.e.,the brick red hues took the place of the ruby,chroma and vibrancy.

Fig.1 Comparison and evolution of color swatches for CK,α-CD,β-CD,2-HP-β-CD and 2-HP-γ-CD wines during aging.CK,control check;CD,cyclodextrin;HP,hydroxypropyl.

Although these color changes are predictable,the wine samples with the addition of CDs exhibited excellent color characteristics at all the various stages of aging.In comparison to CK,theα-CD andβ-CD groups had lowerL＊ values and highera＊ andC＊abvalues at 1 month of storage (P＜0.05),as seen in Fig.1 and Table 1.After 3 months of storage,the addition ofβ-CD to the wine resulted in the best color performance,with a decline inL＊values of 7.12 and an improvement ina＊ values of 4.31.After 5 months of storage,the addition of 2-HP-β-CD drastically increased the color intensity (lowerL＊ values),reddish tonality (highera＊ values),and color saturation(C＊abvalues) compared to the CK and other groups(P＜0.05).At 7 months of storage,four CDs exhibited color preservation properties.These results indicate that it is critical to consider the effects of different structural CDs over the long term rather than just the short term or immediate effects,especially for red wines with aging times of several years or more.

Table 1 Comparison and evolution of the CIELAB parameters (L＊,a＊,b＊,C＊ab,and hab values) for CK,α-CD,β-CD,2-HP-β-CD,and 2-HP-γ-CD wines during aging1)

Additionally,the bathochromic shifts and hyperchromic effects of the wines containing various CDs are shown in Table 2.In comparison to the other samples,the ΔAofβ-CD addition was 34.93%,with Δλmaxdecreasing by 6 nm after 3 months of storage.After 5 months of storage,the hyperchromic effect and bathochromic shift were more prominent in wines containing 2-HP-β-CD.The CD groups showed a bathochromic shift and hyperchromic impact after 7 months of storage,withβ-CD having a weaker effect.These changes primarily came about as a result of the physicochemical interactions between hydrophobic compounds in a polar solution,which involved intramolecular and intermolecular forces brought on by hydrophobicity,hydrogen bonding,and Van der Waals’ forces and altered the maximum absorption magnitude and wavelength (Sunetal.2022).

Studies have reported that ΔE＊abvalue changes of ＞2.7 CIELAB units could be visibly perceptible (Martínezetal.2001).Similar to the discussion above,the effects of CD addition after fermentation on the ΔE＊abvalues seen in Table 2 were also apparent.Of these,β-CD (ΔE＊ab=12.77)was most effective at 3 months of storage,2-HP-β-CD(ΔE＊ab=14.62) was surprisingly effective at 5 months of storage,and except forβ-CD,the remaining three CDs were effective at 7 months of storage (P＜0.05).These modifications make the red wine richer by enhancing the color tendency and intensity (Fanetal.2023b).

3.2.Effect of CD addition on anthocyanins

In this study,five predominant monomeric anthocyanins were detected: delphinidin-3-O-glucoside (Dnd-G),cyanidin-3-O-glucoside (Cnd-G),petunidin-3-O-glucoside(Ptnd-G),peonidin-3-O-glucoside (Pnd-G),and malvidin-3-O-glucoside (Mvd-G).Their corresponding acylated anthocyanins were detected as well,including peonidin-3-O-Acetly-Glucoside (Pnd-AG),malvidin-3-O-Acetly-Glucoside (Mvd-AG),peonidin-3-O-Coumayl-Glucoside(Pnd-CG),and malvidin-3-O-Coumayl-Glucoside (Mvd-CG).Anthocyanins gradually decreased as the wine aged,as can be seen in Table 3,indicating they were degrading and losing color.This is consistent with the fact that they are phenolic substances that degrade quickly.They can also be combined with other substances,either directly or indirectly,to form polymeric anthocyanins,such as vitisins,pinotins,flavanyl-pyranoanthocyanins,portisins,oxovitisins,xanthylium,and other polymeric pigments (Lietal.2023b).In the later stages of storage(5 and 7 months),the addition ofα-CD,2-HP-β-CD and 2-HP-γ-CD led to a dramatic reduction in the monomeric anthocyanin content,with the total detectable anthocyanin content being half of the control group or even less than that,which is consistent with the formation of a substantial amount of polymeric anthocyanins.

Table 2 Comparison and evolution of bathochromic (Δλmax),hyperchromic (ΔA) and color difference values (ΔE＊ab) for α-CD,β-CD,2-HP-β-CD and 2-HP-γ-CD wines during aging

To further explore the major color contributors among the anthocyanins,the GRA was used to investigate the link between the anthocyanin fluctuation trends and the color parameters.The closer the sequence of two linked elements,the higher the grey relational grade(GRG) score between them (Linetal.2019).The GRG of anthocyanins and the CIELAB color parameters are shown in Table 4.The GRG values ranged from 0.514 to 0.700,suggesting that the monomeric anthocyanins had a certain degree of influence on the color characteristics.Pnd-CG,Pnd-AG,Mvd-CG,and Ptnd-G were considered to be the primary factors influencing color with high GRG values over 0.63,according to a ranking of the GRG values from high to low.Note thatβ-CD consistently protects Pnd-CG,Pnd-AG,Mvd-CG,and Ptnd-G throughout storage.The protection ofβ-CD against anthocyanin degradation could possibly be due to a combination of partial inclusion of the anthocyanin and several external associations such as hydrogen bonding,hydrophobic interactions,and steric phenomena(Kalantarietal.2021).In the case of copigmentation,the accumulation of pyran ring acyls reduces the sensitivity of anthocyanins to the nucleophilic attack of water,preventing chalcone formation and likely color fading.From this perspective,the acylated anthocyanins seem more stable (Giusti and Wrolstad 2003).Based on our discussion of anthocyanins and color,we noticed that adding CDs to wine can enhance and improve the color qualities,but at the same time the monomeric anthocyanin content was dramatically reduced,which meant the various anthocyanin forms may have changed.Thus,the changes in the FA%,CA%,and PA% over time were determined and graphed (Fig.2).Throughout the whole aging process,the CA% decreased while the FA% and PA% increased gradually in the CK.Some other studies have also reported this phenomenon(Brouillard and Dangles 1994;Gutiérrezetal.2005).This was most likely caused by cofactor degradation,which would shift the equilibrium of copigmentation toward the reactants (Zhang Xetal.2021).Moreover,copigmented anthocyanins would be less likely to form if polymeric pigments were generated,as copigmentation is considered the first step in the production of polymeric pigments (Xueetal.2022).

Fig.2 Comparison and evolution of the copigmented anthocyanin ratios (CA%),free anthocyanin ratios (FA%) and polymeric anthocyanin ratios (PA%) for CK,α-CD,β-CD,2-HPβ-CD and 2-HP-γ-CD wines during aging.CK,control check;CD,cyclodextrin;HP,hydroxypropyl.Different letters indicate signifcant differences (P＜0.05) among wines in the same time point.Bars are mean±SD (n=3).

In combination with the GRG values (Table 4),PA%was found to have the maximum color potency,with a GRG of 0.778.During the aging process,the proportion of polymeric pigments increased,and their significance in color is gaining more attention.In comparison to the control group,four CDs aged 5 to 7 months had significant increases in PA% and correspondingly significant favorable color features.In particular,the effect of 2-HPβ-CD was more pronounced,leading to the proposition that 2-HP-β-CD is more advantageous in promoting the formation of polymeric anthocyanins,thereby protecting the color.Based on the color changes during storage and the results for the monomeric anthocyanins,the protection of color byβ-CD is most likely caused by the formation of copigmented anthocyanins and this effect is very strong during short-term storage but much less pronounced than that caused by polymeric anthocyanins during long-term storage.

3.3.Effect of CD addition on non-anthocyanin profiles

Fig.3 shows the evolution of the total phenols (Fig.3-A),total flavonoids (Fig.3-B),total tannins (Fig.3-C),and total flavanols (Fig.3-D) during the aging process.The higher contents of total phenols in theα-CD,β-CD,2-HPβ-CD and 2-HP-γ-CD wines (P＜0.05) as compared to the CK demonstrates that the addition of CDs had a crucial protective effect.Therefore,CDs can be considered to protect phenolics from oxidation,and the improved retention of phenolic compounds can be attributed primarily to the unique structures of the CDs.When compared to the control group,β-CD significantly reduced the total tannins and total flavanols after 3 months of storage,while it significantly increased the total flavonoids.At the same time,the addition of 2-HP-β-CD and 2-HP-γ-CD increased the total flavonoid content.The protective effect of CDs is primarily due to their ability to encapsulate phenolics.In general,flavonoids withβ-CD have higher inclusion stability constants than phenolic acids (Zhuetal.2016).According to Zhangetal.(2017),β-CD possesses significant selective inclusion characteristics for the flavonoid compounds in the complex system of chimonanthus praecox extract.When wine was stored for 5 months,the addition of 2-HP-β-CD significantly reduced the contents of total tannins and total flavanols,whereasβ-CD,2-HP-β-CD and 2-HP-γ-CD significantly increased the content of total flavonoids compared with the control group.At 7 months of storage,the addition of the four CDs increased the total flavonoid content while reducing total tannin and total flavanol contents compared with the control group.The protective effect of CDs is primarily due to their ability to encapsulate phenolics,and both the physical protection barrier and the complexation effect may be protective mechanisms for phenolic compounds(Mangolimetal.2014).

Although non-anthocyanidin phenolics are not typically red in color,they may affect the expression and enrichment of pigments in wine (Chengetal.2019).To investigate the impacts of CDs on key phenolic monomers,a total of 12 phenolic monomers,including 7 non-flavonoids (gallic acid,chlorogenic acid,vanillic acid,caffeic acid,syringic acid,p-coumaric acid,and trans-ferulic acid) and 5 flavonoids (protocatechuic acid,cianidanol,L-epicatechin,quercetin,and kaempferol),were detected and quantified (Table 5).For the CK,the contents of the remaining 6 non-flavonoids,excluding caffeic acid,reached their maximum values with aging at 3 months and then gradually declined.In terms of flavonoids,protocatechuic acid reaches its peak in the 3rd month and then declines,while cianidanol andL-epicatechin gradually go down,and quercetin and kaempferol go up with aging.

Previous studies have shown that phenolic substances have varying degrees of influence on color (Castro-Lópezetal.2016a).Based on this,the GRG in this study indicated that chlorogenic acid,vanillic acid,protocatechin and syringic acid were eventually found to possess the higherL＊,a＊ andC＊abpotency with the GRG above 0.80 (Table 4).Four CDs were protective against chlorogenic acid,vanillic acid,syringic acid,protocatechin and cianidanol,and the effects of 2-HP-β-CD andα-CD became quite obvious with the prolongation of aging time.The diverse protective effects of CDs on flavonoids could possibly be due to their distinct reaction modes.This hypothesis was similar to that of Chengetal.(2019),who postulated that variations in the hydrophilicity of flavonoids due to structural variations in those compounds lead to diverse interactions between flavonoids and CDs,which affect their stability.According to this viewpoint,Castro-Lópezetal.(2016b) asserted that geometric isomerization and oxidation of polyene chains were the main contributors to the instability and subsequent degradation of flavonoids.The encouraging results of this study strongly indicate that CDs improve wine color deterioration by protecting the phenolics.

3.4.Multivariate statistical analysis

In order to assess the overall effects of the CD additions,PCA was employed to analyze sample differences and generate the graphical relationship of the wines based on their chemical compositions.The PCA of analytical variables,as seen in Fig.4,explained 82.1% of the variation in the first two dimensions of the data.PC1 was the most representative factor,accounting for 55.6% of the variability,with PC2 accounting for the remaining 26.5%.Fig.4-A presents thea＊ and phenolic substances in the right correlation loadings on PC1,which decreased with aging,and in the left on PC1,the color parameters (L＊,b＊,habandC＊ab) and non-anthocyanin compounds increased with aging.In line with this,Fig.4-B shows the pattern of changes in the various samples and the matching score plot that was used to ascertain the correlation between PC components and wines.The samples were clearly divided along the first axis according to their aging time,which can be seen as a “maturation” axis because ageing increases from right to left.This indicated that PC1 had a stronger advantage for differentiating the samples that were aged for 1,3,5,and 7 months from the others.

Fig.4 Principal component analysis (A,loading plot;B,score plot) of parameters.CK,control check;CD,cyclodextrin;HP,hydroxypropyl.CA%,copigmented anthocyanin ratios;FA%,free anthocyanin ratios;PA%,polymeric anthocyanin ratios.1M,3M,5M,and 7M indicate 1,3,5 and 7 months of storage,respectively.

The PCA scores can be used to quantify the differences in the distributions of the samples at different aging stages.The 1 month-aged samples are located on the PCA plot in the right quadrant,and were mainly characterized by FA% and the respective monomeric anthocyanins,such as Ptnd-G,Pnd-G,Mvd-G,Mvd-AG,Pnd-CG,and Mvd-CG.Moreover,there was no statistically significant difference between the CDs and the CK group.During 3 months of aging,the samples were greatly separated from one another.In particular,the wine withβ-CD addition showed amazing results.This was anticipated because the amounts of CA%,anthocyanins,and other color-related variables (suchL＊,a＊,andC＊ab)were modified by the addition of theβ-CD,indicating that theβ-CD group would have higher chromatic intensities and more vivid hues.However,the compositions of samples after 5 months of aging were very different from those after 3 months.Non-anthocyanin compounds have a strong influence on sample color.Additionally,there was a significant difference between the CD treatmentsand the control wines.Among them,2-HP-β-CD addition displayed superior chromatic intensity and color stability.However,the modes of action are completely different fromβ-CD addition,as 2-HP-β-CD could promote the polymerization of anthocyanins to protect the color by affecting the phenolic substances and ensuring sufficient pigment polymerization during wine aging.After 7 months of storage,the CK wine clearly lost its color and was distributed in quadrant III of the PC1-PC2 plane,which was relatively low and displayed a degenerated color property that was associated with a greater negative correlation withL＊.However,the CD treatments could significantly improve the color properties.

Table 5 Comparison and evolution of the non-anthocyanin contents (mg L-1) for CK,α-CD,β-CD,2-HP-β-CD and 2-HP-γ-CD wines during aging

The data obtained with PCA highlighted the fact that the use of the CDs has a strong influence on the wine color quality,and confirmed the relevant impact of different CDs on the color stability of wines at different stages of aging.Color durability and intensity are widely known to be affected by the kinds and concentrations of the ingredients used in copigmentation during the winemaking process.When we compared the color traits of all the samples,we discovered that the wines with CDs added had a clear improvement in color.Furthermore,the results showed that the copigmentation effects of CD treatments on anthocyanins result in diverse modes of action for various CDs.Regarding theβ-CD treatment,the results showed thatβ-CD is a superior favorable cofactor during wine aging.Howardetal.(2013) found that chokeberry anthocyanins were stable in the cavity of theβ-CD inclusion complex for a long time,and demonstrated thatβ-CD is a suitable capsule matrix for anthocyanins.But for long-term aging,2-HP-β-CD is more beneficial in promoting the formation of polymerized anthocyanins and color stability.The 2-HP-β-CD cavity can provide superior microenvironmental protection due to the hydroxypropyl substitution,which allows guest molecules to easily enter the 2-HP-β-CD cavity and disrupt the intramolecular network of strong hydrogen bonds by opening and expanding the nativeβ-CD.Additionally,these compounds and the associated host-guest CD complexes are more water-soluble due to the presence of external primary and secondary hydroxyl groups,which enhances their activities (Shaoetal.2014).

4.Conclusion

The effects of post-fermentative additions of CDs on color quality and stability were investigated in this study.At 3 and 5 months of storage,wines with addedβ-CD and 2-HP-β-CD had improved color characteristics,as evidenced by lowerL＊ and highera＊ andC＊abvalues.Furthermore,this improvement was accompanied by significant changes in ΔAandλmax,and these color enhancements were observable (ΔE＊abvalue＞2.7 CIELAB units).Additionally,the compounds that were most strongly related to color were screened out based on the GRA results.The results clearly showed that the effect of non-anthocyanins on color is not negligible during storage.Furthermore,by contrasting the copigmentation effects of several CDs,different CDs were found to have various modes of action.Therefore,the most suitable type of CDs should be selected according to the specific storage needs.However,the working mechanism of these effects may be attributed to the interactions between the wine components and the additive molecules.This complexity calls for more evidence,especially the exploration at the chemical molecular level.Most importantly,this study established new knowledge about the influence of CDs on red wine quality and provides a basis for the application of CDs in wine production.

Acknowledgements

This work has been supported by the Regional Collaborative Innovation Project in Xinjiang Autonomous Region of China (2022E02011),the National Key R&D Program of China (2019YFD1002500) and the Key Project of Research and Development Plan in Ningxia Hui Autonomous Region of China (2018BBF02001).We thank Computing Center in Xi’an,China for data calculations and analysis.

Declaration of competing interest

The authors declare that they have no conflict of interest.