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Article

Effects of Cynanchum bungei Decne Addition on the Physicochemical Properties and Antioxidant Activity of Rice Wine

by
Gonglin Cai
1,
Hangmeng Dong
1,2,
Shoulong Liu
1,
Huabin Zhou
1,2,* and
Hailong Yang
1,2,*
1
College of Life & Environmental Science, Wenzhou University, Wenzhou 325035, China
2
Zhejiang Provincial Key Laboratory for Water Environment and Marine Biological Resources Protection, Wenzhou University, Wenzhou 325035, China
*
Authors to whom correspondence should be addressed.
Fermentation 2023, 9(8), 700; https://doi.org/10.3390/fermentation9080700
Submission received: 15 June 2023 / Revised: 17 July 2023 / Accepted: 20 July 2023 / Published: 26 July 2023
(This article belongs to the Section Fermentation for Food and Beverages)
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Abstract

:
Cynanchum bungei Decne is an agricultural crop with a high starch content and contains bioactive compounds with anti-tumor, anti-depressant, anti-oxidant, and other activities. In this work, three concentrations of C. bungei Decne, namely, 5%, 15%, and 25%, were added to media to brew C. bungei Decne rice wine. The basic physical and chemical properties, antioxidant activities, sensory characteristics, and volatile components of C. bungei Decne rice wine were determined. Furthermore, the effects of C. bungei Decne extract on the cell viability, alcohol dehydrogenase activity, and glucose absorption capacity of Saccharomyces cerevisiae were analyzed. The results showed that the main active compound contents and antioxidant activity of the rice wine were increased with the increase in the C. bungei Decne added. However, the vitality of Saccharomyces cerevisiae was inhibited by C. bungei Decne to some extent. Combined with a sensory evaluation, 15% C. bungei Decne was found to be the optimal additive concentration with which to brew C. bungei Decne rice wine. These data provide a theoretical basis for the development of C. bungei Decne rice wine.

1. Introduction

Chinese rice wine has a long history and is rich in nutrients such as protein, polysaccharide, phenolics, minerals, amino acids, etc. [1]. Modern research has shown that Chinese rice wine has anti-atherosclerosis, anti-fatigue, anti-aging, and other effects [2,3]. Traditionally, rice wine is an amber-coloured wine made from glutinous rice through the fermentation of multiple microorganisms, representing the early use of microbes. However, traditional rice wine is facing a crisis of weak market prospects. A potential solution to this crisis is to develop health products [1] by incorporating medicinal and edible plants into rice wine, such as bamboo leaves [4], Eucommia ulmoides [5], and ginkgo [6].
C. bungei Decne is an agricultural crop with a high starch content of 40–60% and has a lengthy record of cultivation, mainly in China, Korea, Japan, and India [7]. C. bungei Decne contains not only a large amount of starch but also steroid glycosides and acetophenones, which have anti-tumor, anti-depressant, anti-oxidant, and other effects [8,9]. In ancient times, people believed C. bungei Decne to have the effects of calming the mind and nourishing the blood, liver, and kidneys, etc., and it has been used as a medicinal and edible plant since the late Tang Dynasty. At present, C. bungei Decne is typically used as a feedstock for the manufacturing of starch, which results in the excessive loss of active ingredients [10]. C. bungei Decne and other starchy plants can be used to produce wine. During the brewing process, the active components of the materials are released into the wine under the action of microorganisms. Therefore, brewing wine is a feasible way to efficiently utilize the resources of C. bungei Decne.
To utilize C. bungei Decne more effectively and produce rice wine with greater health functions, different concentrations of C. bungei Decne were added to glutinous rice to prepare wine, and the physicochemical properties, antioxidant capacity, and consumer acceptance of the brewed wines were analysed. In addition, the effects of C. bungei Decne extract on the cell viability, alcohol dehydrogenase activity, and glucose absorption capacity of the yeast were also analysed in this study. The results of these experiments provide the necessary data for the production of C. bungei Decne rice wine.

2. Materials and Methods

2.1. Materials

Fresh C. bungei Decne tubers of commercial maturity (diameter over 0.8 cm) were obtained from a planting garden in Binhai County at a longitude 34.01 and latitude 119.84 (Jiangsu Province, China) in November 2021.

2.2. Brewing Method

Fresh C. bungei Decne tubers were thoroughly cleaned and ground in a juicer to produce a homogenate. The brewing process of the rice wine was performed as described by Zhou et al. [11]. Briefly, the glutinous rice was soaked, washed, drained, steamed, and allowed to cool to approximately 30 °C. Then, the cooked glutinous rice (0.625 kg) was weighed and placed in a 2 L container. Next, 96 g of Monascus koji (self-produced from Monascus purpureus CGMCC 16790 in the laboratory), 0.4 g of rice wine yeast (Saccharomyces cerevisiae; Angel Company, Yichang, China), 1.6 g of saccharifying enzyme (Angel Company, China), and 1.0 L of distilled water were added in turn. After that, 50 g, 150 g, and 250 g C. bungei Decne homogenate were added, i.e., 0% (YW), 5% (AYW), 15% (BYW), and 25% (CYW) ratios of homogenate weight to distilled water, respectively. After mixing, fermentation was performed in an incubator under conditions of 28 °C for 10 days. Afterwards, the lees were removed via filtration. The filtrate was placed into a 500 mL bottle, and the wine was boiled at 100 °C for 30 min. The wine sample was then stored in a cool and dark place to mature for three months.

2.3. General Component Measurements

The wine sample was centrifuged (8000 rpm, 15 min), and the supernatant was used for the determination. The reducing sugar content was determined through the 3,5-dinitrosalicylic acid method using glucose as a standard [12]. For the protein content determination, the supernatant of the wine sample (1 mL) was completely mixed with 5 mL of Coomassie bright blue reagent, and the absorbance at 595 nm was recorded after 6 min of reaction. The protein content was calculated using bovine serum protein as a standard. Alcohol, amino nitrogen, and total acid were determined according to the national standard of the People’s Republic of China (GB/T 13662-2018, yellow wine) [13], where the total acid was calculated based on the acetic acid.

2.4. Total Phenolic and Total Flavonoid Measurements

The total phenolics was assayed according to the method of Gasinski et al. [14] using gallic acid as a standard. Briefly, the supernatant of the wine sample (0.5 mL) was mixed with 2.5 mL of Folin–Ciocalteau reagent and left to stand for 6 min, and then 2 mL of saturated sodium carbonate solution was added into the system. The mixture was reacted in the dark for 90 min, and after that, the absorbance at 765 nm was recorded. The results were expressed as the gallic acid equivalent per millilitre of wine sample (mgGAE mL−1).
The total flavonoid content analysis was performed according to the approach of Chu et al. [15] using rutin as a standard. Briefly, the supernatant of the wine sample (1 mL) was mixed with 0.3 mL NaNO2 (5%) and left to stand for 6 min, and then 0.3 mL Al(NO3)3 (10%) was added, sufficiently mixed, and reacted for an additional 8 min. Finally, 3 mL NaOH (4%) was added and left to stand for another 15 min, and the absorbance at 500 nm was recorded. The results were expressed as rutin equivalents per millilitre of wine sample (μgRE mL−1).

2.5. γ-Aminobutyric Acid (GABA) Content Measurements

A double volume of sulfosalicylic acid (4%) was added into the supernatant of the wine sample and left to stand for 30 min at 37 °C. For the pre-column derivation, 1 mL of the sample was thoroughly mixed with 2 mL of OPA solution (containing 1 mL methanol, 20 mg phthalaldehyde, 4 mL 0.4 mol L−1 boric acid buffer at pH 10.2, and 60 µL mercaptoethanol) and allowed to stand for 2 min. The analytical sample was filtered through a 0.22 μm microporous membrane and analysed using the HPLC method with an Agilent AavanceBio AAA column, column temperature of 40 °C, injection volume of 10 μL, gradient elution with a 0.25 mol L−1 sodium acetate solution (pH 5.9, A) and acetonitrile (B), a flow speed of 1.0 mL min−1, and a detection wavelength of 332 nm. The gradient elution was 0 min, A:B = 90%:10%; 30–35 min, A:B = 40%:60%; 40–45 min, A:B = 90%:10%. The γ-aminobutyric acid in the sample was quantitatively calculated based on the standard curve [16].

2.6. Acetophenone Content Measurements

The supernatant of the wine sample was filtered through a microporous filter membrane (0.22 μm), and the acetophenone content was analysed via HPLC (Agilent 1206). The chromatographic conditions were based on the method of Sun et al. [17], with slight modifications: an Agilent ZORBAX SBC18 column, column temperature of 25 °C, flow speed of 1.0 mL min−1, binary mobile phase of acetonitrile/0.1% formic acid (20/80, v/v), and injection volume of 5 μL. The contents of 4-hydroxyacetophenone and 2,4-dihydroxyacetophenone in the rice wine sample were calculated based on their standard curves, respectively.

2.7. Antioxidant Capacity Measurement

The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity was measured as described by Pinteus et al. [18]. Briefly, the supernatant of the wine sample (0.2 mL) was thoroughly mixed with 2 mL of DPPH ethanol solution (0.2 mmol L−1) and then allowed to react in the dark for 30 min, and the absorbance at 517 nm was recorded. The results were expressed in terms of the vitamin C equivalent per mL of rice wine (μg VCE mL−1). The 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging ability was measured following the method of Tian et al. [19]. Briefly, the ABTS radical was generated by mixing ABTS (7 mmol L−1) and potassium persulfate (2.45 mmol L−1) and reacting them for 12 h in the dark. The absorbance at 734 nm was adjusted to 0.7 ± 0.02 to obtain the ABTS working solution. The supernatant of the wine sample (0.1 mL) was thoroughly mixed with 3 mL of the ABTS working solution and reacted for 6 min, and the absorbance at 734 nm was recorded. The results were expressed as the vitamin C equivalent per mL of rice wine (μgVCE mL−1). In addition, the ferric-ion-reducing antioxidant power (FRAP) was analysed as described by Tian et al. [19]. In brief, the supernatant of the wine sample (0.15 mL) was mixed with 2.85 mL of freshly prepared FRAP reagent, which was obtained by mixing 10 mmol L−1 of TPTZ solution, 20 mmol L−1 of ferric chloride, and 0.3 mol L−1 of acetate buffer (pH 3.6) in a ratio of 1:1:10. After reaction in the dark for 30 min, the absorbance at 593 nm was recorded. The results were expressed as Trolox equivalents per millilitre of rice wine (mgTRE mL−1).

2.8. Sensory Assessment

A sensory evaluation was performed following the national standard of the People’s Republic of China (GB/T 13662-2018, yellow wine) [13] and Yu et al. [20]. In brief, the samples were served in random coded cups, and ten trained members (five men and five women) evaluated their flavour characteristics (alcohol, ester, sour, sweet), visual characteristics (clarity, amber colour), and overall properties. The quantification of the above indicators was expressed using an intensity scale from 0 to 9 (0: none; 1–2: very weak; 3–4: ordinary; 5–6: medium; 7–8: strong; 9: high intensity).

2.9. Volatile Compound Identification

The volatile compounds were analysed using the solid-phase microextraction–gas chromatography–mass spectrometry method (SPME-GC-MS). A total of 6 mL of wine sample, 10 μL of the internal standard (2-octanol, 10 mg L−1), and 2 g of NaCl were mixed in a 15 mL SPME bottle. The SPME bottle was kept at 70 °C for 15 min to maintain equilibrium. At the same temperature, the SPEM extractor was inserted into the top of the bottle mouth for headspace extraction for 40 min. Afterwards, the SPEM extractor was then quickly withdrawn and inserted into the GC-MS injection port. The chromatographic column was an HP-INNOWAX (60.0 m × 250 μm, 0.25 μm). The temperature of the column was initially maintained at 40 °C for 5 min, then increased at 5 °C per minute to 120 °C and at 10 °C per minute to 240 °C, and finally held for 10 min at 240 °C. In the MS system, the electron energy was 70 eV, and the temperatures of the quadrupole, transmission line, and ionisation source were 150 °C, 240 °C, and 230 °C, respectively. Scanning was performed in the full-scan mode, and the scan range was 20 to 500 m/z. The results were retrieved from the NIST database, the impurity peaks were removed, and the volatile components in the wine samples were determined qualitatively and quantitatively [14]. The results of the compound identification were used to generate an R language heat map.

2.10. Measurement of the Glucose Uptake of Yeast Cells

Fresh C. bungei Decne was dried at 50 °C, pulverized, and extracted with 70% ethanol. The extract was obtained via centrifugation, concentrated to dryness under a vacuum, and redissolved in dimethyl sulfoxide (DMSO) [21]. Extract contents equivalent to 5%, 15%, and 25% of fresh C. bungei Decne, respectively, were added to the YPD media to test the effects of C. bungei Decne on the growth and metabolism of yeast. DMSO was used as a blank control. All the groups were inoculated with activated rice wine yeast (Angel Company, China) and cultured on a shaker at 28 °C for one week. Samples were taken every 24 h.
For the glucose uptake experiment, the yeast was collected via centrifugation (4000 rpm, 4 °C) for 5 min, and the precipitate was re-suspended with normal saline. After that, 0.5 mL of the suspended yeast solution was added into 5 mL of preheated glucose solution (8%). The mixture was incubated at 30 °C for 30 min, and then the supernatant was collected via centrifugation. The glucose content in the supernatant was determined with the DNS method [22]. The glucose absorption capacity was expressed as milligrams of glucose consumed per mL of yeast suspension per minute (mg mL−1 min−1).

2.11. Measurement of the Viability of Yeast Cells

The vitality of the yeast was determined using the 2,3,5-triphenyltetrazolium chloride (TTC) method, as described by Tanaka et al. [23], with slight modifications. Firstly, 0.2 mL of yeast sample was added into 2.0 mL of 1% TTC solution and homogenized in the dark for 5 min. The pellet was then collected via centrifugation, mixed with 2 mL of dimethyl sulfoxide, and centrifuged. After that, the absorbance of the supernatants was determined at 485 nm.
The yeast’s viability was also visually analysed through fluorescence microscopy, as described by Kwolek-Mirek et al. [24], with slight modifications. Briefly, on day 7, 1.5 mL culture solutions of all the wine samples, respectively, were taken and centrifuged at 4000 rpm for 10 min at 4 °C, and the supernatant was removed. After washing twice with phosphate-buffered saline (PBS) buffer, the precipitate was subsequently mixed with 0.4 mL of normal saline. Then, 0.1 mL of the resulting solution was added into 2 mL of PBS buffer and 0.01 mL of fluorescein diacetate (FDA, 0.5 mg mL−1) and stained for 10 min in the dark. The slides were prepared and observed under an inverted fluorescence microscope with 525 nm emission and 488 nm excitation, respectively.

2.12. Determination of Alcohol Dehydrogenase (ADH) Activity

The yeast cells were collected via centrifugation, washed twice with physiological saline, and ultrasonically disrupted for 10 min with 100 mmol L−1 of Tris-HCl buffer (containing 1 mmol L−1 dithiothreitol, 10 mmol L−1 MgCl2, and 1 mmol L−1 EDTA, pH 7.0) in an ice bath. After that, the mixture was centrifuged, and the supernatant was collected for enzyme detection [25].
The ADH activity was detected according to the method of Li et al. [26], and the protein content of the supernatant was determined using the Coomassie bright blue method. For ADH activity determination, 1.5 mL of Tris-HCl buffer (pH 7.0, 50 mmol L−1), 0.5 mL of 40% ethanol, and 1.0 mL of NAD (4 mg mL−1) were mixed and incubated at 37 °C for 20 min, and then the reaction was initiated by adding 0.1 mL of preheated (37 °C) crude enzyme solution. The absorbance at 340 nm was monitored for 5 min, and the maximum value was recorded [26]. One unit of ADH activity was defined as the amount of enzyme required to create a 0.01 increase in OD340 nm under the determining conditions, and the result was expressed as units per milligram of protein.

2.13. Data Analysis

All data are displayed as the mean ± standard deviation. After testing using the Shapiro–Wilk method for a normal distribution, the data were analysed using one-way analysis of variance (ANOVA), followed by Duncan’ multiple ranges using SPSS 23.0 software, and statistical significance was set at p < 0.05. Pearson’s method was used for the correlation analysis.

3. Results and Discussion

3.1. Basic Physical and Chemical Characteristics of C. bungei Decne Rice Wine

The basic components of the brewed rice wine were analysed, and the specific results are shown in Table 1. The alcohol content of AYW, BYW, and CYW was approximately 14% (v/v), and there was no significant difference between them (p > 0.05). However, the alcohol content of YW was 16.35% (v/v), significantly (p < 0.05) higher than the contents of the C. bungei Decne addition groups, indicating the inhibition effect of C. bungei Decne on alcohol production. Similar results were also shown in the case of rice wines brewed from a substrate containing Pyracantha fortuneana and bamboo leaves [4,27]. Nevertheless, in terms of the total acid, amino acid nitrogen, protein, and reducing sugar, their contents in YW, AYW, BYW, and CYW were increased successively and showed significant differences, which may be related to the high starch and protein contents in C. bungei Decne itself.

3.2. Total Phenolic and Flavonoid Contents of Rice Wine

Phenolic compounds are the secondary metabolites of plants that can combat oxidative damage and have other healthy functions [28]. In the process of rice wine brewing, the hydrolase derived from various microorganisms is beneficial for the release of phenolic compounds from the plant matrix [29]. Table 2 shows that there was no significant difference in the total phenolic content between YW and AYW, whereas the total phenolic content was significantly increased in BYW and CYW (p < 0.05). These results indicated that the addition of C. bungei Decne homogenate had no significant influence at a low concentration but had a significant effect at a high concentration in terms of the increase in the total phenolic content in rice wine. Similar results were also obtained by Lu et al. [30], who concluded that the total phenolics in rice wine could be improved by adding exotic plants.
Similar to polyphenols, the flavonoids in rice wine are mainly derived from the plant raw materials. Microbial metabolism can improve the release of flavonoids during the brewing process [31]. As shown in Table 2, the flavonoid concentration in YW was the lowest, at50.76 μg mL−1, while the concentrations in AYW, BYW, and CYW were 94.09 μg mL−1, 125 μg mL−1, and 197.42 μg mL−1, respectively, indicating that the concentration of flavonoids in the rice wine was also increased with the increase in the added amount of C. bungei Decne homogenate. Similar results were previously obtained in a study of Acanthopanax senticosus rice wine [32].

3.3. GABA Content in Rice Wine

GABA is a non-protein amino acid and can act as a neurotransmitter in the human body. Current research suggests that high levels of GABA may play a role in the treatment of depression, blood pressure, and the immune response, which is related to the digestive system [33]. Recently, many GABA-containing foods have been developed to satisfy the demand for healthy foods [34]. There are many GABA-producing strains involved in the fermentation process of rice wine; therefore, rice wine has a high GABA content and is considered as an alternative food with a high concentration of GABA [35]. As shown in Table 2, the content of GABA in the wine sample was increased through the addition of C. bungei Decne, and a much higher content was determined in AYW, BYW, and CYW than in the blank group (YW). These results showed that the addition of C. bungei Decne homogenate could also increase the GABA content in rice wine.

3.4. Acetophenone Content in Rice Wine

It has been proven that through the quantitative analysis of main functional components, one can evaluate the involvement of additives in the brewed wines [36]. The main bioactive components in C. bungei Decne are acetophenones [37]. Among them, 4-hydroxyacetophenone and 2,4-dihydroxyacetophenone are representative compounds. Therefore, they were selected as indicative substances in the wine samples to be analysed. As shown in Figure 1, no acetophenone compounds were detected in YW, whereas the characteristic peaks of two indicative substances appeared in AYW, BYW, and CYW. Table 2 shows that the content of acetophenone compounds in the wine sample increased significantly (p < 0.05) with the increasing addition of C. bungei Decne. This indicates that the active substances of C. bungei Decne can be released into the liquor during brewing, thus improving the functions of rice wine.

3.5. Antioxidant Capacity of Rice Wine

The antioxidant capacity of rice wine is mainly derived from the rich polyphenols and flavonoids in the wine [38]. The ABTS and DPPH free radical scavenging and FRAP of all the wine samples are shown in Table 2. There was a significant (p < 0.05) difference in the scavenging capacity against DPPH between the YW and C. bungei Decne addition groups (AYW, BYW, and CYW), and the activity of CYW was significant (p < 0.05). The ABTS radical scavenging capacity of the C. bungei Decne addition groups was also higher than that of YW, but there was no statistical significance between YW and AYW. This indicated that a low concentration of C. bungei Decne had no significant effect on the improvement of ABTS scavenging ability, while a high concentration of C. bungei Decne (>15%) could increase this activity of the rice wine, in accordance with the change trend of the total flavonoid content in the wine samples. For FRAP, we found that the Trolox equivalent data of YW, AYW, BYW, and CYW were sequentially increased, with significant differences (p < 0.05). Collectively, CYW had the strongest antioxidant capacity among all the groups, with a DPPH scavenging capacity of 23.45 μg VCE mL−1, ABTS scavenging capacity of 171.98 μg VCE mL−1, and FRAP of 0.14 mg TRE mL−1, respectively. The results of the antioxidant power analysis of the four wine samples showed that the addition of C. bungei Decne could significantly enhance the antioxidant power of the wine up to a certain addition level. Similar results were also obtained in studies of Ganoderma lucidum beer and bamboo rice wine [4,39], owing to the rich polyphenols and flavonoids in the plant matrices. Our correlation analysis (Table 3) also showed that the antioxidant capacity of C. bungei Decne rice wine was closely related to the contents of total phenolics and total flavonoids.

3.6. Volatile Compound Identification

To compare the changes in the flavour of the rice wine with the addition of C. bungei Decne, the volatile compounds of YW and CYW were determined, and the results are shown in Figure 2. In total, 46 volatile compounds were detected in YW and CYW. In YW, the volatile compounds included seven esters, eight alcohols, four aldehydes, one acid, five alkanes, six aromatic compounds, one nitrogenous compound, and two other compounds. In CYW, the volatile compounds included 10 ester compounds, 9 alcohols, 7 aldehydes, 2 acids, 3 alkanes, 5 aromatic compounds, 4 nitrogen compounds, and 3 other compounds. The esters, alcohols, and aldehydes of CYW were 1.4 times, 1.1 times, and 2 times higher than those of YW, respectively, indicating that the addition of C. bungei Decne homogenates can change the volatile components and improve the major volatile flavour components of rice wine.
Esters are the main source of flavour in rice wine. A high concentration of esters can significantly improve the quality and taste of rice wine. In the fermentation process, yeast can produce medium-chain fatty acid esters. Ethyl acetate (with a fruity aroma), hexanoic acid ethyl ester (a strong fruity and wine aroma), ethyl phenylacetate (a honey aroma), and octanoic acid ethyl ester (a typical fruit and brandy aroma) exhibit different flavours [40] and were detected in YW and CYW. In addition, 1-butanol, 3-methyl acetate (with a banana and pear scent), and propanoic acid, 2-hydroxy ethyl ester (S) (a strong wine scent) were detected in CYW [41]. Acetate is the main ester in rice wine, and it accounted for 86.55% and 82.80% of the ester contents of YW and CYW, respectively. The level of acetate depends on the concentration of its substrates and the activity of the yeast’s own alcohol acetyltransferase [42]. The alcohols and acids in rice wine are another key factor determining the quality of rice wine. They are mainly produced by yeasts, lactic acid bacteria, and other microorganisms during the brewing process and are the key substances that determine the quality of rice wine [40].
Small amounts of aldehydes and ketones, aromatic compounds, alkanes, nitrogen compounds, and other compounds were also detected in the wine samples. They accounted for 12.85% and 12.62% of the volatile compounds detected in YW and CYW, respectively. Many of these trace volatiles are prerequisites for the formation of other components and have their own unique aromas.

3.7. Effects of C. bungei Decne Extract on Yeast Vitality

In the fermentation process of rice wine, the degree of alcohol is closely related to the vitality and quantity of the yeast. In order to further explore the reason for the alcohol decrease in the C. bungei Decne addition groups, the influence of the C. bungei Decne extracts on yeast vitality was analysed. As shown in Table 4, the addition of C. bungei Decne could promote the cell viability and glucose uptake capacity of the yeast in the first 3 days. On day 4, there was no significant difference in glucose uptake capacity between the YW and C. bungei Decne addition groups, while the cell viability of YW was significantly (p < 0.05) higher than that of the C. bungei Decne addition groups. After that, the cell viability and glucose uptake capacity of YW were consistently higher than those of the C. bungei Decne addition groups. Under the fluorescence microscope (Figure 3), we could see that the fluorescence intensity of the yeast cells on day 7 decreased with the addition of C. bungei Decne extract. These results showed that the addition of C. bungei Decne extract had a promoting effect on the cell vitality of the yeast in the early growth stage but had an inhibitory effect in the later growth stage. Similar results were also observed by Sa et al. in their study on the inhibition effect of Myrcia tomentosa extract on the growth of Candida albicans [43]. Interestingly, the analysis of the ADH enzyme activity of yeast cells showed that the highest ADH enzyme activity in all the groups was determined on the second day, and the ADH enzyme activity of the control group (YW) was significantly higher than that in the C. bungei Decne extract addition groups. These results indicated that C. bungei Decne extract could reduce the ability of yeast to produce ethanol to some extent. This may be due to the fact that small-molecule active substances such as phenolics, terpenoids, and acetophenones in C. bungei Decne can inhibit the metabolism of yeast, which has been reported previously [44].

3.8. Sensory Evaluation

Sensory evaluation is the most intuitive expression of the consumer acceptance of foods, and it is also the key to the marketing of new alcoholic beverages. As shown in Figure 4, the highest clarity was obtained for YW, and the highest sweetness was recorded for CYW, because it contained a higher content of reducing sugar (Table 1). In terms of colour, the amber colour of the wine body was increased with the increase in C. bungei Decne. However, the lowest overall evaluation was recorded for CYW, while most of the evaluators assigned BYW relatively high balance in terms of its flavour characteristics (sweet, alcoholic, sour, and ester) and the highest overall evaluation. Overall, YW and AYW had simple tastes, obvious aromas, and clear appearances. BYW had good overall balance in terms of taste, an obvious aroma, and an excellent fragrance, which were highly favoured by consumers. On the other hand, the taste of CYW was too strong, which led to low acceptance by consumers. This was due to the fact that C. bungei Decne contains a variety of volatile compounds and has a characteristic odour [45], which can negatively affect the flavour of rice wine with excessive addition. In summary, BYW was the wine sample with the best acceptability.

4. Conclusions

Based on the research results, it is possible to improve the health function of rice wine by adding C. bungei Decne. The levels of polyphenols, flavonoids, acetophenones, and γ-aminobutyric acid in rice wine were increased significantly. Furthermore, the antioxidant capacity of rice wine was also increased significantly with the increase in the added amount of C. bungei Decne homogenate. In addition, C. bungei Decne could also improve the volatile flavour compounds, and 15% was the suitable concentration according to the consumer sensory evaluation.

Author Contributions

Conceptualization, H.Y.; investigation, G.C. and H.D.; data curation, G.C. and S.L.; methodology, G.C. and H.Y.; resources, H.Z. and H.Y.; supervision, H.Z. and H.Y.; validation, H.Z. and H.Y.; writing—original draft, G.C. and H.D.; writing—review and editing, H.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research and Development Project of Zhejiang Province (2020C02038), China.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00700 g001 550
Figure 1. HPLC chromatogram of 4-hydroxyacetophenone and 2,4-dihydroxyacetophenone. ST, standards. YW, AYW, BYW, and CYW are the groups to which we added C. bungei Decne at 0%, 5%, 15%, and 25%, respectively.
Figure 1. HPLC chromatogram of 4-hydroxyacetophenone and 2,4-dihydroxyacetophenone. ST, standards. YW, AYW, BYW, and CYW are the groups to which we added C. bungei Decne at 0%, 5%, 15%, and 25%, respectively.
Fermentation 09 00700 g001
Fermentation 09 00700 g002 550
Figure 2. Heat maps of volatiles in CYW and YW. CYW1–CYW3 represent three repeats of the samples with 25% C. bungei Decne, and YW1–YW3 represent three repeats of the control group. The unit of the data bar is μg mL−1.
Figure 2. Heat maps of volatiles in CYW and YW. CYW1–CYW3 represent three repeats of the samples with 25% C. bungei Decne, and YW1–YW3 represent three repeats of the control group. The unit of the data bar is μg mL−1.
Fermentation 09 00700 g002
Fermentation 09 00700 g003 550
Figure 3. Fluorescent images of cell viability of yeast on day 7. (A–D) represent the groups to which we added C. bungei Decne at 0%, 5%, 15%, and 25%, respectively.
Figure 3. Fluorescent images of cell viability of yeast on day 7. (A–D) represent the groups to which we added C. bungei Decne at 0%, 5%, 15%, and 25%, respectively.
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Fermentation 09 00700 g004 550
Figure 4. Radar chart of a single index score of rice wine. YW, AYW, BYW, and CYW are groups to which we added C. bungei Decne at 0%, 5%, 15%, and 25%, respectively.
Figure 4. Radar chart of a single index score of rice wine. YW, AYW, BYW, and CYW are groups to which we added C. bungei Decne at 0%, 5%, 15%, and 25%, respectively.
Fermentation 09 00700 g004
Table
Table 1. Basic physical and chemical properties of rice wines.
Table 1. Basic physical and chemical properties of rice wines.
CharacteristicYWAYWBYWCYW
Reducing sugar (mg mL−1)7.92 ± 0.66 d11.36 ± 1.02 c13.19 ± 0.35 b17.70 ± 0.12 a
Alcohol content (%, v/v)16.35 ± 0.24 a13.9 ± 0.43 b14.49 ± 0.60 b14.69 ± 1.09 b
Total acid (mg mL−1)4.83 ± 0.26 d7.29 ± 0.09 c7.50 ± 0.09 b8.53 ± 0.05 a
Amino acid nitrogen (mg mL−1)0.72 ± 0.00 d0.96 ± 0.02 c1.25 ± 0.03 b1.32 ± 0.01 a
Protein content (μgBSE mL−1)61.50 ± 1.07 d74.19 ± 3.38 c88.83 ± 0.54 b98.14 ± 0.45 a
Significant differences at p < 0.05 within the index (each row) are indicated by different letters. YW, AYW, BYW, and CYW are the groups to which we added C. bungei Decne at 0%, 5%, 15%, and 25%, respectively.
Table
Table 2. Contents of the main active compounds and antioxidant capacity of rice wine brewed from C. bungei Decne with different levels of fortification.
Table 2. Contents of the main active compounds and antioxidant capacity of rice wine brewed from C. bungei Decne with different levels of fortification.
AssaysYWAYWBYWCYW
Total phenolic content (mg GAE mL−1)0.65 ± 0.00 b0.63 ± 0.03 b0.66 ± 0.02 b0.74 ± 0.03 a
Total flavonoid content (μg RE mL−1)50.76 ± 1.36 d94.09 ± 2.41 c125.00 ± 4.17 b197.42 ± 1.05 a
GABA content (μg mL−1)29.97 ± 0.53 d44.87 ± 0.98 c56.09 ± 1.22 b73.28 ± 2.87 a
2′,4′-Dihydroxyacetophenone (μg mL−1)ND8.64 ± 0.11 c20.14 ± 0.14 b38.39 ± 0.21 a
4-Hydroxyacetophenone (μg mL−1)ND2.88 ± 0.09 c6.15 ± 0.09 b9.75 ± 0.03 a
DPPH radical scavenging activity (μg VCE mL−1)6.80 ± 0.23 c8.98 ± 0.30 b9.82 ± 1.11 b23.45 ± 0.77 a
Fe3+-reducing antioxidant power (FRAP) (mg TRE mL−1)0.03 ± 0.00 d0.07 ± 0.00 c0.08 ± 0.01 b0.14 ± 0.01 a
ABTS radical scavenging activity (μg VCE mL−1)139.61 ± 3.99 b143.10 ± 3.21 b166.78 ± 2.05 a171.98 ± 0.48 a
Significant differences at p < 0.05 within the index (each row) are indicated by different letters. YW, AYW, BYW, and CYW are the groups to which we added C. bungei Decne at 0%, 5%, 15%, and 25%, respectively.
Table
Table 3. Correlation coefficients (R2) between the contents of total phenolics and total flavonoids and scavenging activities against DPPH•, ABTS•, and FRAP of the brewed rice wines.
Table 3. Correlation coefficients (R2) between the contents of total phenolics and total flavonoids and scavenging activities against DPPH•, ABTS•, and FRAP of the brewed rice wines.
Total PhenolicsTotal FlavonoidsDPPH• Scavenging ActivityFRAPABTS• Scavenging Activity
Total phenolics10.87040.95590.85000.7813
Total flavonoids 10.93960.99320.9067
DPPH• scavenging activity 10.94700.7647
FRAP 10.8537
ABTS• scavenging activity 1
Table
Table 4. Effect of C. bungei Decne extract on the vitality of yeast.
Table 4. Effect of C. bungei Decne extract on the vitality of yeast.
Time (d) 1234567
Content
Glucose uptake (mg mL−1 min−1)YW0.70 ± 0.01 b0.69 ± 0.02 b0.70 ± 0.01 c0.78 ± 0.05 a0.77 ± 0.02 a0.90 ± 0.03 a0.92 ± 0.04 a
AYW0.75 ± 0.01 a0.75 ± 0.03 a0.74 ± 0.02 b0.75 ± 0.00 a0.77 ± 0.00 ab0.81 ± 0.03 bc0.80 ± 0.01 b
BYW0.71 ± 0.01 ab0.70 ± 0.03 b0.78 ± 0.02 a0.78 ± 0.03 a0.76 ± 0.01 b0.82 ± 0.03 b0.76 ± 0.02 bc
CYW0.75 ± 0.04 a0.73 ± 0.01 ab0.79 ± 0.00 a0.77 ± 0.02 a0.80 ± 0.04 a0.77 ± 0.02 c0.74 ± 0.01 c
Cell viability (A485 nm)YW0.32 ± 0.00 c0.41 ± 0.01 c0.41 ± 0.02 c0.68 ± 0.02 a0.75 ± 0.02 a0.80 ± 0.00 a0.89 ± 0.02 a
AYW0.36 ± 0.00 b0.44 ± 0.00 b0.63 ± 0.01 b0.58 ± 0.01 c0.69 ± 0.03 b0.77 ± 0.03 ab0.73 ± 0.01 b
BYW0.35 ± 0.00 b0.44 ± 0.01 b0.65 ± 0.00 b0.57 ± 0.01 c0.71 ± 0.01 b0.74 ± 0.04 b0.74 ± 0.03 b
CYW0.44 ± 0.02 a0.46 ± 0.00 a0.73 ± 0.03 a0.61 ± 0.02 b0.68 ± 0.02 b0.73 ± 0.01 b0.74 ± 0.04 b
ADH enzyme activity
(U mg−1)		YW4317.36 ± 58.34 a4599.01 ± 32.92 a4326.09 ± 78.98 a3998.69 ± 102.34 a3760.45 ± 46.43 a3627.53 ± 65.45 a3366.24 ± 21.92 a
AYW3858 ± 252.62 b4417.46 ± 80.65 b4294.26 ± 30.48 a3744.78 ± 27.84 b3385.19 ± 132.02 b3411.96 ± 42.12 b3380.69 ± 60.28 a
BYW3913.20 ± 50.81 b4281.82 ± 128.79 bc4262.79 ± 50.15 a3772.68 ± 82.71 b3486.03 ± 89.96 b3422.98 ± 40.75 b3368.65 ± 102.55 a
CYW4069.12 ± 55.74 ab4209.22 ± 26.80 c4131.19 ± 27.31 b3804 ± 44.76 b3536.62 ± 63.81 b3290.31 ± 57.99 c3262.30 ± 60.28 a
Significant differences at p < 0.05 within the index on each day are indicated by different letters. YW, AYW, BYW, and CYW are the groups to which we added C. bungei Decne at 0%, 5%, 15%, and 25%, respectively.
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Cai, G.; Dong, H.; Liu, S.; Zhou, H.; Yang, H. Effects of Cynanchum bungei Decne Addition on the Physicochemical Properties and Antioxidant Activity of Rice Wine. Fermentation 2023, 9, 700. https://doi.org/10.3390/fermentation9080700

AMA Style

Cai G, Dong H, Liu S, Zhou H, Yang H. Effects of Cynanchum bungei Decne Addition on the Physicochemical Properties and Antioxidant Activity of Rice Wine. Fermentation. 2023; 9(8):700. https://doi.org/10.3390/fermentation9080700

Chicago/Turabian Style

Cai, Gonglin, Hangmeng Dong, Shoulong Liu, Huabin Zhou, and Hailong Yang. 2023. "Effects of Cynanchum bungei Decne Addition on the Physicochemical Properties and Antioxidant Activity of Rice Wine" Fermentation 9, no. 8: 700. https://doi.org/10.3390/fermentation9080700

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