Effects of red wine supplementation on characteristic modulations during fermentation of green onion kimchi

Green onion (pa) kimchi represents a traditional lactic acid fermented dish originating from Korea. In parallel, red wine stands as a yeast-fermented alcoholic beverage derived from grape juice. This investigation systematically assessed the impact of red wine supplementation on the microbial, antioxidant, and sensory attributes of green onion kimchi throughout a 48-hour fermentation process at 35°C. Notably, the addition of red wine expedited the decrease in pH within the green onion kimchi. Furthermore, a substantially elevated antioxidant activity, total viable count, and lactic acid bacteria (LAB) population were observed during the fermentation of red wine-supplemented green onion kimchi in comparison to its conventional counterpart. Importantly, hedonic sensory acceptance demonstrated no significant disparities between the regular and red wine-supplemented green onion kimchi. The outcomes gleaned from this inquiry suggest that the incorporation of red wine could be contemplated as a means to enhance the health-promoting attributes of green onion kimchi, endowing it with antioxidant and probiotic qualities.


Introduction
The term "kimchi" is a comprehensive descriptor for a class of lactic acid fermented vegetable dishes originating from Korea [1].The diversity of kimchi is evident in the existence of approximately 200 distinct types in Korea, distinguished primarily by the principal ingredients employed in their preparation, such as cabbage (baechu) kimchi, diced radish (kkakdugi) kimchi, ponytail radish (chonggak) kimchi, mustard leaf (gat) kimchi, and green onion (pa) kimchi [2].Broadly, the kimchimaking process involves salting vegetables to diminish water activity, incorporating additional elements (such as red chili powder or gochugaru, garlic, ginger, fish sauce, fermented seafood, and/or other vegetables), and subjecting the mixture to fermentation to foster the proliferation of lactic acid bacteria (LAB) and the development of distinctive flavors [3].Internationally recognized as a functional food, kimchi earns acclaim for its well-balanced nutritional composition and comparatively low energy content [4].The habitual consumption of kimchi is correlated with the robust health and extended life expectancy of the Korean population, surpassing 83 years [5].Beyond its nutritional value, kimchi has been acknowledged for its diverse health benefits, including antimicrobial, antioxidative, antimutagenic, anticarcinogenic, anti-obesity, and anti-diabetic properties [6].Red wine constitutes an alcoholic beverage typically produced through the yeast fermentation of dark-colored grape juice, inclusive of grape skin, imparting the characteristic red hue.The natural pigments within the grape skin contribute to the wine's distinctive coloration.The standard wine production process involves the crushing of grapes with their skin, the introduction of a yeast starter, and subsequent fermentation, facilitating the conversion of sugar into ethanol by yeast organisms [7].Modest consumption of red wine has been associated with various health benefits, encompassing the regulation of blood sugar, the maintenance of cardiovascular health, a reduced risk of cancer, and protection against aging-related diseases [8].The role of red wine in promoting cardiovascular health is underscored by the phenomenon known as the French paradox, wherein France exhibits a low incidence of coronary heart disease despite the populace's high daily intake of foods rich in saturated fats and cholesterol.This paradox is attributed to the routine consumption of red wine, replete with antioxidants that contribute to cardiac well-being [9].Predominantly, red wine is rich in polyphenols, which serve as potent antioxidants.Key polyphenols in red wine, such as resveratrol, anthocyanins, catechins, and tannins, have been scientifically demonstrated to confer positive effects on human health [10].
The objective of this research was to assess the microbial characteristics, antioxidant activity, and sensory acceptance of green onion kimchi supplemented with red wine during a fermentation process at 35°C.The incorporation of red wine was anticipated to introduce additional microorganisms integral to the kimchi fermentation process, thereby enhancing the flavor profile of the kimchi.Moreover, the potential health advantages associated with red wine consumption were expected to complement the existing benefits of kimchi.Notably, there is a dearth of studies investigating kimchi production involving red wine, making this research particularly novel.Consequently, the outcomes of this study are poised to offer unique insights that can contribute to advancing the status of kimchi as a functional food.

Red wine and kimchi preparation
Red wine was produced using conventional methods employed by Domaine de Candie, a winery in Toulouse, France.In brief, Cabernet Sauvignon grapes were harvested in October 2016, and the resulting must, obtained from the crushed grapes, was blended with commercial yeasts (0.25 g/L, SafOEno NDA 21, Fermentis by Lessafre, France) and diammonium phosphate (0.25 g/L, Fermaid K by Laffort, France).The red wine underwent extraction after fermenting for 20 days at 25°C, resulting in an alcohol content of 12-14%.
Green onion (pa) kimchi was prepared following a published recipe [11] with slight modifications.Green onions sourced from a local farmer in Toulouse, France, underwent preparatory steps that involved removing roots and tough outer layers before being uniformly cut into 8-10 cm lengths and salted with Korean anchovy sauce (20 mL/ 100 g green onions, Chung Jung One brand).A seasoning paste was concocted by cooking 10 g glutinous rice flour in 100 mL water until achieving translucency.Red chili powder (10 g, Shinsumi brand), garlic powder (5 g, Ducros brand), ginger powder (5g, Ducros brand), and caster sugar (3 g, Béghin Say brand) were incorporated into the paste.Post-cooling, the paste was amalgamated with the pre-salted green onions.Red wine (10 mL/ 100 g green onions) was introduced into the mixture and thoroughly mixed.Subsequently, the composite blend was placed in a sealed glass jar and subjected to a 48-hour fermentation at 35°C in an incubator.

pH and microbial analyses
Kimchi samples (10 g) underwent homogenization with distilled water (100 mL) using a handheld blender and were subsequently filtered through cheesecloth to yield kimchi extracts.The pH of the kimchi was ascertained using a pH-meter (MP120, Mettler Toledo, Greifensee, Switzerland) following the manufacturer's instructions.Microbial analysis encompassed assessments of the total viable count (TVC) and lactic acid bacteria (LAB) count, following established methodologies [12].For TVC, kimchi samples underwent serial dilution in saline solution (0.85% NaCl), were spread over platecount agar (Merck, Darmstadt, Germany), and were then incubated at 37°C for 24 hours.LAB were cultivated on De Man, Rogosa, and Sharpe (MRS) agar supplemented with 10% NaCl (Difco, Detroit, MI, USA) for 24 hours at 30°C.Microbial counts were computed as the average of duplicate measurements for each group and expressed as log colony-forming units (CFU)/g.

Antioxidant activity (DPPH free radical scavenging) analysis
The DPPH (2,2-diphenyl-1-pycrylhydrazyl) free radical scavenging activity was assessed following a previously established protocol [13] with some modifications.Concisely, kimchi samples were macerated in 95% ethanol (1:1 w/v) for 1 h before subjecting them to DPPH analysis.The resulting solution underwent filtration using a nylon syringe filter (porosity 0.22 μm).Subsequently, 0.1 mL of the filtered solution was combined with 1.4 mL of DPPH solution (6 M in methanol, Sigma Aldrich), stirred, and allowed to stand for 30 minutes in the absence of light before spectrophotometric measurement at 520 nm using a Genesys 30 Vis Spectrophotometer (Thermo Fisher Scientific, MA, USA).Ascorbic acid (Sigma Aldrich) served as the standard solution.The outcomes were expressed as mg ascorbic acid equivalent (AAE)/g of the sample.

Sensory analysis
Kimchi samples obtained on the second day of fermentation underwent a hedonic rating analysis and hedonic ranking analysis [14] employing a cohort of 67 untrained panelists in Toulouse, France.The panel comprised individuals of Franco-Korean descent and others accustomed to kimchi consumption, primarily international university students aged 21-28 years.In the hedonic rating analysis, the kimchi samples were assessed for their appearance, aroma, texture, and flavor using a 9-point Likert scale (ranging from 1 denoting extreme dislike to 9 indicating extreme liking).In the hedonic ranking analysis, each panelist was tasked with selecting their preferred sample from two kimchi variants presented to them: green onion kimchi with or without red wine supplementation.Subsequently, each panelist ranked their chosen sample as the first preference, with the alternative sample assigned a second-ranking position.

Statistical analysis
All data, expressed as the mean ± standard deviation (SD), were analysed using IBM SPSS Statistics v26 software.The data of pH, microbial, and sensory analyses were evaluated using two-way analysis of variance (Anova) followed by Tukey's HSD test (p<0.05).The data of hedonic rating and ranking analysis were evaluated using student's t-test and Friedmann test respectively (p<0.05).

Results and discussion
In the initial phases of kimchi fermentation, there is a dynamic and interconnected fluctuation in both acidity and microbial composition.Notably, there is a marked elevation in microbial load, particularly lactic acid bacteria (LAB), whose enzymatic activities contribute to the generation of organic acids, subsequently influencing a reduction in pH.The acidity levels and the specific composition of organic acids in kimchi play pivotal roles as determinants of the flavor and sensory attributes of the final product [15].
As illustrated in Figure 1, there was a pronounced decline in pH observed within the initial 24 hours of green onion kimchi fermentation, plummeting from an initial value of 6.68 to 4.35.Over the subsequent 24 hours, the pH continues to decrease, ultimately stabilizing within the range of 3.88-3.95.Intriguingly, the introduction of red wine as a supplement expedites the pH reduction in green onion kimchi during the initial 24 hours of fermentation, transitioning from 6.52 to 3.91.It is noteworthy that the optimal pH range associated with the most desirable flavor of kimchi has been documented to fall between 4.0 and 4.5 [16].Concerning microbial proliferation during the fermentation of green onion kimchi, Figure 2 illustrates a notable escalation in viable microorganisms and lactic acid bacteria (LAB).This increase is particularly pronounced in red wine-supplemented kimchi as compared to the conventional counterpart.Notably, the red wine itself harbors microorganisms that are introduced into the initial mixture of green onions at the onset of kimchi fermentation.These microorganisms predominantly include LAB, specifically Lactobacillus, Leuconostoc, Oenococcus, and Pediococcus, which play a pivotal role in the malolactic fermentation process in wine [17].Consequently, the heightened presence of LAB in red wine-supplemented green onion kimchi may be correlated with an expedited pH reduction when compared to regular green onion kimchi.The LAB in kimchi exhibit a diverse array of extracellular enzymes that facilitate the breakdown of cellulose in plant tissues and the conversion of simple sugars into lactic acid.In the initial stages of fermentation, Leuconostoc emerges as the dominant LAB due to its lower acid tolerance and microaerophilic characteristics.As the fermentation progresses, the acidic conditions favor the proliferation of LAB with heightened acid tolerance, predominantly Lactobacillus and Weissella [18].Moreover, the acidic milieu acts as a deterrent against the growth of spoilage and pathogenic microorganisms in kimchi [19].Green onions and red wine are recognized reservoirs of antioxidants.Green onions, renowned for their antioxidative potential, contain chlorophyll, quercetin, vitamin A, vitamin C, and sulfur-rich compounds [20].Similarly, red wine boasts polyphenols, contributing to its antioxidant properties [9].As depicted in Figure 3, the supplementation of red wine enhances the antioxidant activity of green onion kimchi, possibly stemming from synergistic interactions among the antioxidants inherent in green onions and red wine.A comparable augmentation in antioxidant activity was noted in a prior investigation where black raspberry supplemented cabbage kimchi [21].The observed increase in antioxidant activity during fermentation aligns with findings from other studies [22,23], likely attributed to the softening texture of green onions during fermentation, facilitating the release of antioxidants and the formation of organic acids endowed with antioxidant properties.Notably, the established link between the antioxidant activity of kimchi and its potential health benefits, particularly in cancer prevention, has been substantiated by previous scientific studies [24][25][26][27]  Figure 4 illustrates the sensory acceptance of green onion kimchi, both regular and red winesupplemented, following a 48-hour fermentation period.Hedonic scores, evaluating appearance, aroma, texture, and flavor, exhibited no significant disparities between the two kimchi variants.Furthermore, the hedonic ranking analysis corroborated this finding, indicating a lack of noteworthy distinction between regular and red wine-supplemented green onion kimchi (p > 0.05).It is imperative to note that pH, a pivotal factor governing the sensory acceptance of kimchi [15], exhibited no discernible difference between the two variants at the 48-hour mark (Figure 1).However, by the third day of fermentation, both regular and red wine-supplemented green onion kimchi developed an excessively sour taste coupled with a pungent aroma, rendering them unpalatable.
Our findings would propose a novel application of agricultural commodities for health, which will in turn support agricultural activities and sustainability.In particular, this study has been directed towards the third point presented in the United Nation's Sustainable Development Goals (SDGs), which is ensure healthy lives and promote well-being [28].Furthermore, our findings also support the development of a novel functional food by integrating two different traditional foods originating from different cultures.This would provide a creative insight and broaden the scientific view for future development of functional foods.

Conclusion
In conclusion, our results indicate that incorporating red wine into green onion (pa) kimchi could be a viable strategy to enhance its functional attributes without compromising its hedonic sensory acceptance.The introduction of red wine demonstrated notable improvements in antioxidant activity, viable microorganism count, and lactic acid bacteria levels, alongside expediting the pH reduction in green onion kimchi.Future investigations could focus on characterizing the probiotics present in red wine-supplemented green onion kimchi and conducting in vitro analyses using viable cells to delve deeper into the antioxidant properties of this enhanced kimchi variant.This study supports the future agriculture and sustainability by providing a novel insight for functional food development that promotes health and well-being.

Figure 1 .
Figure 1.Changes in pH during fermentation of green onion kimchi with or without red wine supplementation at 35°C.Data (n=4) are expressed as mean±SD.Different letters indicate significant differences among samples (p<0.05).

Figure 3 .
Figure 3. Changes in antioxidant activity during fermentation of green onion kimchi with or without red wine supplementation at 35°C.Data (n=3) are expressed as mean±SD.Different letters indicate significant differences among samples (p<0.05).AAE: ascorbic acid equivalent.

Figure 4 .
Figure 4. Hedonic sensory acceptance (scale 1-9) of green onion kimchi with or without red wine supplementation fermented at 35°C for 48 hours.Data (n=67) are expressed as mean±SD.No significant difference was observed among samples (p>0.05).