Effects of different pretreatments on physicochemical properties and phenolic compounds of hawthorn wine

ABSTRACT

The physicochemical properties, organic acid, and phenolic profiles of hawthorn wines prepared by different pretreatments were analyzed. It was found that fermentation together with smashed seeds was favorable for ethanol production, while thermal maceration, addition of pectinase and amyloglucosidase significantly increased the levels of sugar-free extract, residual sugar, total phenolics, flavonoids, and most individual phenolic comounds, showing beneficial effects on improving nutritive value of hawthorn wine. Addition of pectinase and amyloglucosidase caused an increase of methanol content by 34.88% and 26.97%, respectively, while thermal maceration decrease the methanol content by 36.20%. Addition of amyloglucosidase yielded the highest contents of procyanidins as well as flavanols, while thermal maceration and addition of pectinase were favorable for improving phenolic acid level. All the pretreatments significantly increased the contents of succinic acid and malic acid in the final wines, among which thermal maceration and addition of pectinase yielded the highest content of succinic acid and malic acid respectively.

Resumen

En este estudio se analizaron las propiedades fisicoquímicas, así como los perfiles de ácidos orgánicos y fenólicos, de los vinos de espino preparados mediante diferentes pretratamientos. Se constató que la fermentación, junto con las semillas trituradas, favorece la producción de etanol, mientras que la maceración térmica, la adición de pectinasa y de amiloglucosidasa aumentaron significativamente los niveles de extracto sin azúcar, azúcar residual, fenólicos totales, flavonoides y la mayoría de los compuestos fenólicos individuales, mostrando efectos beneficiosos para mejorar el valor nutritivo del vino de espino. La adición de pectinasa y amiloglucosidasa provocó un incremento del contenido de metanol en 34.88% y 26.97% respectivamente, en tanto que la maceración térmica disminuyó el contenido de metanol en 36.20%. La adición de amiloglucosidasa produjo los mayores contenidos de procianidinas y de flavanoles; la maceración térmica y la adición de pectinasa, por su parte, fueron favorables para mejorar el nivel de ácido fenólico. Todos los tratamientos previos aumentaron significativamente el contenido de ácido succínico y de ácido málico en los vinos finales; entre los tratamientos aplicados, la maceración térmica y la adición de pectinasa produjeron el mayor contenido de ácido succínico y de ácido málico, respectivamente.

KEYWORDS:

• Hawthorn
• wine
• phenolic compound
• organic acid
• smashed seed
• thermal maceration
• pectinase
• amyloglucosidase

PALABRAS CLAVE:

• espino
• vino
• compuesto fenólico
• ácido orgánico
• semilla triturada
• maceración térmica
• pectinasa
• amiloglucosidasa

1. Introduction

Earth is a plant-oriented planet and horticulture plants presented in earth with a wide number of species, cultivars, accessions, genotypes (Guney et al., 2019; Zia-Ul-Haq et al., 2014). Among horticulture plants, fruits has special importance because they include high content of non-nutritive, nutritive, and bioactive compounds such as flavonoids, phenolics, anthocyanins, phenolic acids, and as well as nutritive compounds such as sugars, essential oils, carotenoids, vitamins, and minerals (Senica et al., 2019; Vijayan et al., 2008). Hawthorn (Crataegus spp.) belongs to the Rosaceae family and is widely distributed in northern temperate zones, including those of Asia, North America, and Europe (Edwards et al., 2012). The hawthorn fruit is rich in phenolics, terpenes, steroids, organic acids, and has long been used in natural health products and traditional medicine with the purpose to stimulate digestion, improve circulation, regulate gastrointestinal function, and treat cardiovascular diseases such as chronic heart failure, hyperlipidemia, hypertension, and arteriosclerosis (Chang et al., 2002; Edwards et al., 2012; Wu et al., 2014; Yang & Liu, 2012). Recently, the hawthorn fruit extracts had been reported to exhibit potent antioxidant, anticancer, antihypertensive, antidiabetic, neuroprotective activities, and most of them were ascribed to the phenolic compounds in hawthorn fruit (Chang et al., 2013; Li et al., 2013; Miao et al., 2016; Wen et al., 2015; Zheng et al., 2019).

Due to its health promoting properties and medicinal effects, hawthorn tree has long been cultivated widely in China and the hawthorn fruits are usually used for eating, health diets and making various commercial products such as wine, drink, jam, jelly, and candy (Chang et al., 2002). Among the processing products, hawthorn wine seems to be more promising and popular with consumers due to its delicate flavor and nutritive health functions (He et al., 2013; Liu et al., 2018). However, the quality and the content of bioactive compounds as well as health benefits of fruit wine could be influenced by maceration pretreatment, pressing condition, yeast strain, fermentation process and storage (Akalin et al., 2018; Behrends & Weber, 2017; Ferreira-Lima et al., 2016; Lachowicz et al., 2017; Segade et al., 2014). As compared with grape wine, knowledge on hawthorn wine making is still limited. He et al. (2013) investigated the typical properties and antioxidant capacities of hawthorn wines fermented by different wine yeasts and found that the general composition, content of total anthocyanins, polyphenols, flavonoids, and antioxidant capacity showed expected variations. Microwave and heating pretreatments of hawthorn fruits were reported to facilitate the extraction of phenolics, anthocyanins, ascorbic acid, and procyanidins (Liu et al., 2018). Pulp contact during fermentation could intensify and diversify the aroma profile of hawthorn wine, while pectinase treatment was favorable for wine clarification and filtration (Zhang et al., 2017). Many other factors which could affect the quality and nutritive characteristics of hawthorn wine remain unknown up to date.

Hawthorn fruit is not so juicy as compared with other fruits such as grape, cherry, blueberry, and apple. So the pretreatment process is crucial for hawthorn wine making. With the purpose of optimizing the pretreatment process for improving the quality and nutritive value of hawthorn wine, this work evaluates the effects of different pretreatments on general physicochemical properties, organic acid composition, and phenolic profile of hawthorn wine.

2. Materials and methods

2.1. Materials

2.1.1. Hawthorn fruits

The Chinese hawthorn (Crataegus pinnatifida Bge.) fruits were obtained from Jiyuan, Henan Province, China. Fresh harvested hawthorn fruits without insect damage or mechanical injury were washed with distilled water and used for pretreatment and wine making after the surface water was removed by flow air.

2.1.2. Yeast and inoculum

The yeast of Saccharomyces cerevisiae was isolated from naturally fermented grape must and maintained on potato dextrose agar. The inoculum was prepared by inoculating the yeast in a sucrose solution (5% w/v) at 28°C for 24 h.

2.1.3. Standards

Standards of citric acid, succinic acid, malic acid, quinic acid, lactic acid, tartaric acid, oxalic acid, shikimic acid, gallic acid, protocatechic acid, coumalic acid, vanilllic acid, p-coumaric acid, sinapic acid, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, ferulic acid, caffeic acid, catechin, epicatechin, procyanidin B1, procyanidin B2, procyanidin C1, vitexin, apigenin, quercetin, rutin, hyperoside, isoquercitrin, luteolin-7-O-β-D-glucuronide, kaempferol, kaempferol-3-O-glucoside were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A).

2.2. Pretreatment and fermentation

Hawthorn fruits were divided into five groups and subjected to different pretreatments as described in Table 1. After pretreatment, the resulting must was added with 60 mg/kg of SO₂ (in form of K₂S₂O₅) and 160 g/kg of sugar respectively and then inoculated with the yeast inoculum (5% v/v) and fermented at 22–24°C for 14 days. After fermentation, the wine was separated by filtration with silk cloth of 200 meshes and then stored at 15–20°C for another 30 days before further analysis.

Table 1. Description of different pretreatments

Tabla 1. Descripción de los diferentes tratamientos previos.

Pretreatment	Description
Control (C)	2000 g of fresh hawthorn fruits were mixed with 1600 mL of distilled water, and then crushed without breaking of seeds
Thermal maceration (T)	2000 g of fresh hawthorn fruits were macerated with 1600 mL of boiling water for 15 min and then crushed without breaking of seeds
Fermentation together with smashed seeds (S)	2000 g of fresh hawthorn fruits were mixed with 1600 mL of distilled water and then smashed with a beater (JYL-G12, Joyoung, China)
Addition of pectinase (P)	2000 g of fresh hawthorn fruits were crushed together with 1600 mL of distilled water without breaking of seed, and then 0.2% of pectinase (>40 PA/mg, DSM, Germany) was added into the must
Addition of amyloglucosidase (A)	2000 g of fresh hawthorn fruits were crushed together with 1600 mL of distilled water without breaking of seed, and then 0.35‰ of amyloglucosidase (from Aspergillus niger, 1 × 10^5 U/g, Shanghai Yuanye Bio-Technology Co., Ltd, China) was added into the must

2.3. Clarity and general chemical analysis

Hawthorn wine was centrifuged at relative centrifugal force of 7260 g for 10 min and the resulting supernatant was used for further determination. The clarity of hawthorn wine was evaluated by recording the transmittance at 700 nm with a UV/Vis spectrophotometer (Specord 50, Analytik Jena AG, Germany) by using distilled water as blank control. Chemical analysis of total sugar, reducing sugar, titratable acidity, sugar-free extract, methanol, and alcoholic content was performed according to the methods described in the National Standards of the People’s Republic of China of GB/T 15038–2006.

2.4. High-performance liquid chromatography (HPLC) analysis of organic acids

HPLC analysis of organic acids in hawthorn wine was performed by using a 1525 binary HPLC system coupled with a 2998 Photodiode Array Detector (Waters Corp., Wilford, MA, USA) according to the method described by Li et al. (2017). A Welch Ultimate AQ-C18 column (4.6 × 250 mm, 5 µm, Welch Science &Technology Co., Ltd, Shanghai, China) was used to separate the organic acids with (NH₄)₂HPO₄ solution (0.02 mol/L, pH 2.4) as elution solvent at a flow rate of 1.0 mL/min. Chromatograms were recorded at 210 nm and compared with those of standards. Contents of organic acids were calculated according to the external calibration curves prepared with organic acid standards, and the results were reported as milligram per 100 mL of hawthorn wine.

2.5. Phenolic determinations

2.5.1. Total phenolic content

The total phenolic content (TPC) in hawthorn wine was determined with Folin-Ciocalteu phenol reagent (F9252, Sigma-Aldrich, USA) according to the method described by Singleton et al. (1999) with minor modifications. The reaction system consisted of 0.5 mL of diluted hawthorn wine, 2.5 mL of Folin-Ciocalteu phenol reagent, and 2.0 mL of sodium carbonate solution (7.5% w/v). The absorbance of the resulting reaction mixture was recorded at 760 nm and chlorogenic acid was used as standard. The result was reported as milligram of chlorogenic acid equivalents per liter of hawthorn wine.

2.5.2. Total flavonoid content

The total flavonoid content (TFC) in hawthorn wine was determined by using a colorimetric method described in previous study (J. C. Liu et al., 2018). The reaction system consisted of 0.2 mL of diluted hawthorn wine, 0.3 mL of sodium nitrite (5% w/v), 0.3 mL of aluminum nitrate (10% w/v), and 4.0 mL of sodium hydroxide (4% w/v). The absorbance of the resulting reaction mixture was recorded at 510 nm and rutin was used as standard. The result was reported as milligram of rutin equivalents per liter of hawthorn wine.

2.5.3. Total monomeric anthocyanin content

The total monomeric anthocyanin content (TMAC) in hawthorn wine was determined with the pH-differential method described in previous study (J. C. Liu et al., 2018). Cyanidin-3-glucoside with a molecular weight of 449.2 and molar absorptivity of 26900 was used to calculate the total monomeric anthocyanin content in howthron wine, and the result was reported as milligram of cyanidin-3-glucoside equivalents per liter of hawthorn wine.

2.5.4. Total proanthocyanidin content

The total proanthocyanidin content (TPAC) in hawthorn wine was measured by means of vanillin assay according to the method described in previous study (J. C. Liu et al., 2018) with minor modifications. The reaction system consisted of 0.5 mL of wine sample, 3.0 mL of vanillin/methanol (4% w/v) and 1.5 mL of HCl (37% w/v). The absorbance of the resulting reaction mixture was recorded at 500 nm and procyanidin B2 was used as standard. The result was reported as milligrams of procyanidin B2 equivalents per liter of hawthorn wine.

2.5.5. Ultrahigh-performance liquid chromatography (UPLC) analysis of phenolic compounds

For the determination of individual phenolic compounds, the centrifuged wine sample of each treatment was extracted with ethyl acetate (1:2 v/v) twice, and the combined organic phases were evaporated under reduced pressure with a rotary evaporator at 37°C. Then the remaining residue was redissolved with methanol solution (80% v/v) and subjected to determination after filtration by using an ACQUITY UPLC H-Class System (Waters Corp., Wilford, MA, USA). A Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) was used to separate the phenolic compounds at 30°C with 0.1% of formic acid solution (solvent A) and methanol (solvent B) as elution solvents. The gradient elution was carried out at a constant flow rate of 0.2 mL/min and programmed as follows: 0 min, 95% A; 2 min, 95% A; 4 min, 92% A; 6 min, 90% A; 13 min, 90% A; 16 min, 87% A; 23 min, 85% A; 25 min, 82% A; 32 min, 82% A; 42 min, 70% A; 43 min, 0% A;. Chromatograms were monitored at 280 nm and 360 nm with an Acquity UPLC Tunable UV Detector and compared with those of authentic standards. Contents of phenolic compounds were calculated according to the external calibration curves prepared with phenolic standards, and the results were reported as milligram per liter of hawthorn wine (mg/L).

2.6. Statistical analysis

Statistical analysis was performed with IBM SPSS Statistics 22.0 software and the results were reported as mean ± standard deviation for triplicate. One-way analysis of variance (ANOVA) and Duncan’s tests were used to discern the significant differences between different pretreatments.

3. Results and discussion

3.1. General physicochemical properties

As shown in Table 2, all the treatments yielded an alcoholic content of 11.49–13.54% (v/v), which is generally satisfactory for fruit wine. Among them, fermentation together with smashed seeds yielded the highest alcoholic content, which indicates pulping with smashed seeds might be favorable for ethanol production. This might be due to the release of chemical components from hawthorn seeds when they were smashed together with the pulps. Hawthorn seed contains various fatty acids, sterols and vitamin E (Wang et al., 2018), which could improve the ethanol tolerance in yeast and enhance the alcohol production (Mishra & Kaur, 1991; Xiao et al., 2010; Zhang et al., 2016). The lower residual sugar content in the final wine prepared by smashed seed treatment also suggests a high fermentation ability of yeast in the must of pulping with smashed seeds. The contents of sugar-free extract in wines prepared by thermal maceration, addition of pectinase and amyloglucosidase were higher than others, suggesting an enhancement of extraction of soluble substances from hawthorn fruit. Maceration with boiling water is a typical process in hawthorn wine or juice production. The thermal process may cause degradation of cell wall polysaccharide and breakdown of tissue structure, facilitating extraction of soluble solids, polysaccharides, phenolics from fruits and vegetables (L. L. Zhang et al., 2019; Liu et al., 2018; De Roeck et al., 2008). Enzymatic maceration with pectinase or amyloglucosidase may also cause rupture of cell structure and promote release of cell components by degradation of polysaccharides and starch in hawthorn fruit. Addition of pectinase and amyloglucosidase also resulted in an increase of residual sugar and methanol content in hawthorn wine. This may be explained by the increase of initial sugar content in the must due to the enzymatic degradation of polysaccharides and starch as well as promotion of release of cell components. Typically, commercial pectinase preparation includes pectinesterase, polygalacturonase and pectinlyase. During treatment with pectinase in wine-making process, methanol is produced as a result of the hydrolysis of methyl ester groups in pectin by pectinesterase, leading to an increase of methanol content in the final wines (Cabaroglu, 2005; Revilla & González-SanJosé, 1998). The increase of methanol content in hawthorn wines with treatment of amyloglucosidase might be ascribed to the existence of small amount of pectinesterase in commercial amyloglucosidase preparation which is produced from Aspergillus niger. Thermal maceration pretreatment gave the lowest methanol content in the final wine due to the heat inactivation of endogenous pectinesterase, which occurs naturally in fruits and is responsible for the release of methanol in fruit juices and wines without addition of pectinase (Hou et al., 2008; Miljic et al., 2016). However, thermal maceration resulted in a hawthorn wine with poor clarity, so the wine must be clarified by some other methods. Whereas the wines of other four treatments all showed a satisfactory clarity after centrifugation and did not need any further clarification. There was no significant difference in the titratable acidity among the control, thermal maceration pretreated and smashed seed treated wines, whereas addition of pectinase and amyloglucosidase yielded a higher titratable acidity in the final wines. This might be ascribed to the enzymatic deesterification and degradation of pectin, leading to increase of total acidity. Treatment with pectinase also caused a significant increase of titratable acidity in plum juice, apple juice and mash (Chang et al., 1995; Zhang et al., 2011).

Table 2. General physicochemical properties of hawthorn wine prepared by different pretreatments.

Tabla 2. Propiedades fisicoquímicas generales del vino de espino preparado mediante diferentes pretratamientos.

Item	Treatment
	C	T	S	P	A
Clarity (%)	97.56 ± 1.54^a	67.54 ± 2.66^b	96.37 ± 1.77^a	98.56 ± 0.90^a	98.44 ± 1.15^a
Alcoholic content (% v/v)	11.96 ± 0.46^b	11.90 ± 0.34^b	13.54 ± 0.43^a	11.49 ± 0.31^b	11.53 ± 0.40^b
Sugar-free extract (g/L)	41.73 ± 1.63 ^c	55.66 ± 1.32^a	39.62 ± 1.47 ^c	50.24 ± 0.79^b	48.79 ± 1.42^b
Total sugar (g/L)	2.17 ± 0.04^d	3.85 ± 0.11 ^c	1.76 ± 0.11^d	9.41 ± 0.62^a	4.99 ± 0.38^b
Reducing sugar (g/L)	1.67 ± 0.05^d	2.89 ± 0.08 ^c	1.53 ± 0.07^d	8.73 ± 0.78^a	4.26 ± 0.30^b
Titratable acidity (g/L)	17.03 ± 0.71 ^c	17.47 ± 0.81^bc	16.72 ± 0.56 ^c	19.60 ± 1.05^a	18.69 ± 0.90^ab
Methanol (mg/L)	86.10 ± 2.46^b	54.93 ± 3.81 ^c	84.43 ± 3.32^b	116.13 ± 9.36^a	109.32 ± 6.08^a

Different letters for each sample indicate the significant differences at p < 0.05

Las distintas letras para cada muestra indican la presencia de diferencias significativas a p < 0.05.

3.2. Organic acids

Eight organic acids, including citric, succinic, malic, quinic, lactic, tartaric, oxalic, and shikimic acid were determined in hawthorn wines. As shown in Table 3, citric acid was the predominant organic acid in hawthorn wines, accounting for 48.73–58.07% of the total organic acids. This is similar to the results of Han et al. (2019), who reported that citric acid contributed to more than 70% of the total organic acids. Other research also indicated citric acid was the predominant organic acid in hawthorn fruit (Liu et al., 2010).

Table 3. Contents of organic acid of hawthorn wines prepared by different pretreatments.

Tabla 3. Contenido de ácido orgánico de los vinos de espino preparado mediante diferentes pretratamientos.

Organic acid	Treatment
	C	T	S	P	A
Oxalic acid	1.73 ± 0.02^b	2.11 ± 0.06^a	1.67 ± 0.05^b	0.76 ± 0.03^d	1.17 ± 0.04 ^c
Tartaric acid	0.85 ± 0.03 ^c	5.66 ± 0.13^b	0.98 ± 0.05 ^c	7.38 ± 0.12^a	7.40 ± 0./09^a
Quinic acid	313.93 ± 7.72 ^c	292.94 ± 8.82^d	300.40 ± 6.40 ^cd	374.46 ± 9.89^a	354.49 ± 7.76^b
Malic acid	158.56 ± 5.41^e	210.99 ± 4.61^b	180.49 ± 3.98^d	232.28 ± 6.63^a	197.33 ± 6.11 ^c
Shikimic acid	3.47 ± 0.06^b	2.79 ± 0.04^e	3.37 ± 0.06 ^c	3.77 ± 0.04^a	3.09 ± 0.06^d
Lactic acid	23.59 ± 1.46^d	70.08 ± 4.10^a	27.00 ± 1.02 ^cd	65.44 ± 2.12^b	29.99 ± 1.12 ^c
Citric acid	900.49 ± 16.77^a	898.23 ± 22.94^a	900.40 ± 25.25^a	927.50 ± 17.20^a	902.71 ± 39.08^a
Succinic acid	148.01 ± 8.15 ^c	360.46 ± 1134^a	208.23 ± 6.06^b	211.63 ± 8.32^b	221.94 ± 8.59^b
Total	1550.62 ± 20.45^d	1843.25 ± 34.42^a	1622.54 ± 21.39 ^c	1823.21 ± 30.56^a	1718.13 ± 17.75^b

Different letters for each sample indicate the significant differences at p < 0.05

Las distintas letras para cada muestra indican la presencia de diferencias significativas a p < 0.05.

No significant difference was observed in the content of citric acid among all the treatments. However, thermal maceration increased the content of succinic acid by 143.54% as compared with the control, while other three treatments showed an increase of 40.23–49.95%. The highest content of malic acid was observed in the hawthorn wine prepared by pectinase treatment with an increase of 46.49% as compared with the control, while thermal maceration, fermentation together with smashed seed, and addition of amyloglucosidase showed an increase of 33.07%, 13.83%, and 24.45% respectively. Addition of pectinase and amyloglucosidase also resulted in higher contents of quinic acid and tartaric acid in the final wines, but showed lower oxalic acid level. Thermal maceration and addition of pectinase caused a significant increase of lactic acid level in the final wine.

The organic acid profile of fruit wine can be influenced by the fruit, yeast strain, and fermentation conditions, which had been investigated intensively (Chidi et al., 2018). However, the knowledge about the effects of pretreatments on the release or production of organic acids during wine making is still limited. M. M. Liu et al. (2018) reported a significant increase of malic acid and succinic acid in persimmon wine by pretreatment with addition of pectinase, which is similar to the present result.

3.3. Total phenolics, flavonoids, anthocyanins, and proanthocyanidins

As compared with the control treatment in which the fresh hawthorn fruits were crushed and then fermented directly without any other treatment, thermal maceration, addition of pectinase and amyloglucosidase increased the TPC in the final wines by 16.74–34.39% (Table 4), indicating an enhancement of release of phenolic compounds by thermal maceration and enzymatic treatment. Pre-fermentation treatment of crushed grapes at 60°C for 60 min or 80°C for 15 min caused an increase of TPC of 2.97–105.41% in the fresh wines, depending on the variety and temperature (Atanackovic et al., 2012). Premaceration of persimmon pulp at 60°C for 24 hours and addition of pectinase also resulted in higher TPC and TFC in persimmon wine (M. M. Liu et al., 2018). It is suggested that the pectinase could degrade the pectin substances in cell walls and cause rupture of cell structure, promoting liberation of bound phenolics and enhancing the extraction of free phenolic substances (Ducasse et al., 2010). Interestingly, fermentation together with smashed seeds also caused a significant increase of TPC in the final wine. This might be due to the extraction of phenolic compounds from smashed seeds. As for TFC, thermal maceration, addition of pectinase and amyloglucosidase also resulted in an increase of 28.41%, 67.27%, and 26.57%, respectively, while fermentation together with smashed seeds showed no significant effect on TFC as compared with the control. The highest level of TMAC in the final wine was achieved by addition of amyloglucosidase, which is comparable to the control. However, thermal maceration, addition of pectinase and fermentation together with smashed seeds led to a decrease of TMAC of 51.65%, 71.94%, and 52.47% respectively as compared with the control. Normally, thermal and pectinase pretreatment both can enhance the extraction of anthocyanins by destroying the cell structure (El Darra et al., 2016; Ducasse et al., 2010; Liu et al., 2016). However, the degradation of anthocyanins can also be accelerated by thermal treatment (Danisman et al., 2015), which could counteract the promoting effect and thereby decrease the anthocyanin content in the final wine. The decrease of TMAC by pectinase treatment may be the results of degradation of anthocyanin by enzymatic deglycosylation with glucosidase activity and various esterases activity existed in the commercial pectinase preparation (Arnous & Meyer, 2010; Versari et al., 1997). Interestingly, addition of amyloglucosidase yielded a quite high TMAC in the final wine which was comparable to the control, indicating that amyloglucosidase are more suitable for the release of anthocyanin from hawthorn pulp. The commercial amyloglucosidase preparation may also contain some esterases activity, which might lead to degradation of anthocyanin. But the amyloglucosidase may cause more other polysaccharide degradation than pectinase, which might affect the release of anthocyanins from hawthorn pulp and improve the yield of anthocyanins. Several researches had documented the enhancement of extraction of bioactive compounds from plant materials by treatment with amyloglucosidase and ascribed it to the degradation of the cell wall polysaccharides (Alrahmany & Tsopmo, 2012; Sahne et al., 2017). Further research should be done to elucidate the mechanism involved in the enhancement of anthocyanin extraction by amyloglucosidase treatment. Thermal maceration pretreatment also caused a significant decrease of TPAC in the final wine, indicating the proanthocyanidins and anthocyanins in hawthorn fruits are more susceptible to thermal degradation as compared with other phenolic compounds. However, addition of pectinase and amyloglucosidase led to an increase of TPAC of 15.29% and 31.49% in the final wine, which is similar to the results of TPC and TFC.

Table 4. TPC, TFC, TMAC, and TPAC of hawthorn wines prepared by different pretreatments.

Tabla 4. TPC, TFC, TMAC y TPAC de los vinos de espino preparados mediante diferentes pretratamientos.

Item	Treatment
	C	T	S	P	A
TPC	1545.74 ± 75.66 ^c	1804.43 ± 24.51^b	2134.50 ± 68.12^a	2077.41 ± 87.41^a	1808.80 ± 21.18^b
TFC	526.41 ± 16.64 ^c	675.97 ± 10.81^b	558.98 ± 24.84 ^c	880.51 ± 27.39^a	666.28 ± 12.51^b
TMAC	3.65 ± 0.38^a	1.76 ± 0.18^b	1.73 ± 0.10^b	1.02 ± 0.09 ^c	3.66 ± 0.13^a
TPAC	1040.35 ± 87.44 ^c	815.74 ± 28.42^d	955.90 ± 54.86 ^c	1199.52 ± 41.62^b	1367.93 ± 51.57^a

Different letters for each sample indicate the significant differences at p < 0.05

Las distintas letras para cada muestra indican la presencia de diferencias significativas a p < 0.05.

3.4. Individual phenolic compounds

Based on available phenolic standards, 11 individual phenolic acids and 9 individual flavonoids in hawthorn wine were identified and quantified by using the UPLC method. As shown in Table 5, chlorogenic acid, epicatechin, procyanidin B2 and C1 were the main phenolic compounds in the hawthorn wines, accounting for 64.99–70.10% of the total amount of detected phenolic compounds. Previous researches also indicated the high content of chlorogenic acid, epicatechin, procyanidin B2 and C1 in hawthorn fruit (Cui et al., 2006; Liu et al., 2011; Wen et al., 2015). However, no hyperoside and isoquercitrin was found in the hawthorn wines of the present research. This might be ascribed to the difference among various raw materials of hawthorn fruit and degradation or transformation of these two compounds during wine making process, since their initial content in hawthorn fruit is rather low. Otherwise, this work revealed the existence of neochlorogenic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, ferulic acid, procyanidin B1, rutin, and luteolin-7-O-β-D-glucuronide with a relatively high content in hawthorn wine, which had not been noticed in above reference works.

Table 5. Contents of phenolic compounds of hawthorn wines prepared by different pretreatments.

Tabla 5. Contenido de los compuestos fenólicos de los vinos de espino preparados por diferentes pretratamientos.

	Treatment
Phenolic compounds	C	T	S	P	A
Gallic acid	4.90 ± 0.45 ^c	3.71 ± 0.41^d	5.91 ± 0.32^b	5.18 ± 0.27^bc	10.36 ± 0.61^a
Protocatechic acid	1.66 ± 0.13^d	2.51 ± 0.19 ^c	3.05 ± 0.23^b	4.67 ± 0.39^a	1.71 ± 0.16^d
Coumalic acid	0.51 ± 0.05^b	0.97 ± 0.13^a	0.51 ± 0.07^b	0.54 ± 0.03^b	0.47 ± 0.04^b
Neochlorogenic acid	30.62 ± 1.65^b	29.20 ± 2.24^b	27.62 ± 0.87^b	39.91 ± 2.62^a	39.09 ± 1.96^a
Chlorogenic acid	111.57 ± 7.98^d	152.99 ± 11.18^ab	123.89 ± 12.90 ^cd	170.45 ± 7.49^a	139.47 ± 10.13^bc
Vanillic acid	5.27 ± 0.37^b	14.73 ± 1.11^a	3.84 ± 0.36^b	13.57 ± 1.14^a	14.13 ± 0.98^a
Cryptochlorogenic acid	21.42 ± 1.03 ^c	27.51 ± 1.67^a	19.62 ± 0.78 ^c	27.74 ± 1.65^a	24.27 ± 1.06^b
p-Coumaric acid	11.14 ± 0.92^b	17.97 ± 1.85^a	9.07 ± 0.56^b	18.89 ± 10.88^a	10.41 ± 0.86^b
1,3-Dicaffeoylquinic acid	16.20 ± 1.15^b	23.08 ± 2.58^a	11.97 ± 0.79 ^c	20.29 ± 1.26^a	21.88 ± 1.35^a
Ferulic acid	19.37 ± 1.51^d	26.49 ± 1.30^b	21.12 ± 1.43 ^cd	31.95 ± 2.44^a	23.65 ± 2.67^bc
Sinapic acid	14.38 ± 1.32^d	21.54 ± 1.58^b	10.30 ± 0.50^e	30.88 ± 2.04^a	17.19 ± 1.55 ^c
Procyanidin B1	59.86 ± 4.06 ^c	68.87 ± 2.85^b	75.70 ± 2.67^a	76.79 ± 3.70^a	64.99 ± 2.97^bc
Procyanidin B2	673.55 ± 36.08 ^c	743.14 ± 46.25^bc	539.85 ± 36.96^d	792.04 ± 35.87^b	897.12 ± 37.61^a
Procyanidin C1	249.24 ± 21.37^b	300.06 ± 22.28^a	195.88 ± 15.60 ^c	328.68 ± 31.41^a	323.68 ± 19.78^a
Epicatechin	246.41 ± 20.77^b	304.47 ± 17.24^a	164.82 ± 8.22 ^c	296.74 ± 18.72^a	321.50 ± 17.51^a
Rutin	48.34 ± 4.58 ^c	104.22 ± 11.14^a	67.45 ± 5.07^b	65.85 ± 4.79^b	71.81 ± 4.47^b
Vitexin	7.16 ± 0.78^d	28.39 ± 1.86^b	16.22 ± 1.44 ^c	37.54 ± 3.46^a	13.98 ± 1.44 ^c
Luteolin-7-O-β-D-glucuronide	56.58 ± 4.31^b	104.45 ± 6.46^a	63.25 ± 3.49^b	100.93 ± 7.86^a	67.40 ± 6.96^b
Apigenin	4.01 ± 0.32 ^c	12.43 ± 1.59^a	8.81 ± 0.77^b	8.34 ± 0.79^b	4.99 ± 0.54 ^c
Kaempferol	5.25 ± 0.38^b	1.01 ± 0.06 ^c	4.52 ± 0.46^b	8.32 ± 0.68^a	4.54 ± 0.38^b

Different letters for each sample indicate the significant differences at p < 0.05

Las distintas letras para cada muestra indican la presencia de diferencias significativas a p < 0.05.

The hawthorn wines prepared by different pretreatments showed different profiles of phenolic composition. As shown in Table 5, most of the detected phenolic compounds, including protocatechic acid, neochlorogenic acid, chlorogenic acid, vanillic acid, cryptochlorogenic acid, p-coumaric acid, 1,3-dicaffeoylquinic acid, ferulic acid, sinapic acid, procyanidin B1, procyanidin C1, epicatechin, vitexin, luteolin-7-O-β-D-glucuronide, and kaempferol, showed the highest level in the final wine prepared by pectinase treatment, and thereby the total phenols also was the highest. This indicates pectinase treatment is favorable for production of hawthorn wine with high content of phenolic compounds, which is in agreement with the results of TPC and TFC. The final wine prepared by amyloglucosidase treatment also contained high content of neochlorogenic acid, 1,3-dicaffeoylquinic acid, procyanidin C1, epicatechin as well as total phenols, which were comparable to those of pentinase treatment. In addition, the content of gallic acid and procyanidin B2 in the final wine prepared by amyloglucosidase treatment were the highest among all treatments. As a traditionally used process in hawthorn wine production, maceration with boiling water before fermentation also yielded a hawthorn wine with high content of phenolic compounds, in which the contents of coumalic acid, rutin and apigenin were the highest among all treatments, and the contents of chlorogenic acid, vanillic acid, p-coumaric acid, 1,3-dicaffeoylquinic acid, procyanidin B2, procyanidin C1, epicatechin, luteolin-7-O-β-D-glucuronide as well as total phenols were comparable to those of pentinase treatment. This indicates that this traditional process is still a useful method for producing hawthorn wine with health benefits. Unexpectedly, fermentation together with smashed seeds resulted in a final wine with quite lower contents of most phenolic comounds, which is inconsistent with the result of TPC. This might be explained by the chemistry of Folin-Ciocalteu assay used in the experiment. The determination of TPC by Folin-Ciocalteu reagent relies on the reducing capacity of phenolic compounds, but the reaction is not specific for phenolic compounds and many other oxidation substrate can interfere in an inhibitory, additive, or enhancing manner (Huang et al., 2005; Singleton et al., 1999). The release of other antioxidants from smashed seeds, such as neolignans, squalene, tocopherols, and unanticipated phenolic compounds which were not identified in the present research (Peng et al., 2016; Salmanian et al., 2014; Wang et al., 2018), might be responsible for the higher TPC value than the total phenols estimated by UPLC.

Figure 1 summarized the profiles of phenolic compounds of different pretreatments according to the classes they belong to. Procyanidins (including procyanidin B1, B2, and C1) were the predominant phenolic compounds in hawthorn wine, contributing to 55.95–62.04% of the total phenols and 66.71–72.64% of the total flavonoids determined by UPLC. Addition of amyloglucosidase yielded the highest contents of procyanidins as well as flavanols (including procyanidins and epicatechin), which were higher than those of thermal maceration and addition of pectinase. However, the contents of total phenolic acids in the final wines of thermal maceration pretreatment and pectinase treatment were higher than that of amyloglucosidase treatment. This indicates that thermal maceration and addition of pectinase are favorable for the release of phenolic acid, while amyloglucosidase treatment is helpful for improving the level of flavanols. The phenolic acids in the hawthorn wine can be classified to two groups as hydroxycinnamic acids and hydroxybenzoic acids, which contributed to 91.34–95.01% and 4.99–8.66% of the total phenolic acids determined in the present research, respectively. The content of total hydroxycinnamic acids in the final wine prepared by pectinase treatment was higher than those of thermal maceration pretreatment and amyloglucosidase treatment, but the content of total hydroxybenzoic acids showed the opposite trend. Fermentation together with smashed seeds resulted in a significantly decrease of total amount of procyanidins, flavanols as well as flavonoids, but the contents of hydroxycinnamic acids, hydroxybenzoic acids as well as total phenolic acids were comparable to those of the control treatment. Pectinase treatment yielded a comparable promoting effect to thermal maceration pretreatment for release of procyanidins, flavanols, and flavonoids, but it was superior for phenolic acids as well as hydroxycinnamic acids. These suggest that the mechanism involved in the change of different groups of phenolic compounds in hawthorn fruit related to different pretreatments during wine making are differentiated, which may be associated with the tissue structure, linkage between phenolic compound and other components, and chemical nature of the phenolic compound itself. Further research should be performed to elucidate it.

Figure 1. Contents of different groups of phenolic compounds in hawthorn wines prepared by different pretreatments.

Figura 1. Contenido de los diferentes grupos de compuestos fenólicos en los vinos de espino preparados por diferentes pretratamientos.

4. Conclusion

Pretreatment could significantly influence the physicochemical properties, organic acid composition, and phenolic profile of hawthorn wine. Fermentation together with smashed seeds was favorable for ethanol production, but resulted in relatively lower TFC, TMAC, TPAC, and most individual phenolic comounds. Thermal maceration pretreatment, addition of pectinase and amyloglucosidase could significantly increase the sugar-free extract, residual sugar content, TPC, TFC, and most individual phenolic comounds, showing beneficial effects on improving nutritive value of hawthorn wine. However, addition of pectinase and amyloglucosidase also caused a significant increase of methanol content, while thermal pretreatment yielded the lowest methanol content. Chlorogenic acid, epicatechin, procyanidin B2 and C1 were the main phenolic compounds in hawthorn wine. Addition of amyloglucosidase yielded the highest contents of procyanidins as well as flavanols, while thermal maceration pretreatment and addition of pectinase were favorable for the improvement of phenolic acid level. All the treatments significantly increased the contents of succinic acid and malic acid in the final wine, among which thermal pretreatment yielded the highest content of succinic acid and addition of pectinase resulted in the highest level of malic acid. It is suggested that the effects of different pretreatment on the organic acids and phenolic compounds depend on not only the pretreatment methods, but also the chemical nature of the compounds themselves. In conclusion, thermal maceration pretreatment was still an promising method for producing hawthorn wine with low methanol content and relative high content of phenolics, while addition of pectinase and amyloglucosidase were more favourable for producing hawthorn wine with improved health benefits due to the higher content of phenolics and organic acids. Further research should be performed to reveal the mechanism involved in the change of organic acids and phenolic compounds related to different pretreatments during hawthorn wine making process.

Disclosure statement

The authors declare that they have no conflicts of interest.

Additional information

Funding

This work was funded by the Agricultural Science and Technology Innovation Program (ASTIP) of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2016-ZFRI)