Formulation of a Functional Probiotic Beverage Using Maesil (Prunus mume) Syrup By-Product Fermented by Lactiplantibacillus plantarum KFOM 0042

Abstract

Maesil (Prunus mume) syrup is the most common form of maesil consumption in Korea; however, its production generates large quantities of by-products. This study aimed to develop a functional probiotic beverage through the lactic acid fermentation of maesil syrup by-products (MSBs). To optimize fermentation, eight strains of Lactiplantibacillus plantarum were tested, and KFOM 0042 was selected based on its superior acid production in MSBs. The effects of MSB concentration (5%, 10%, 15%, or 20%), pH levels (3, 4, or 5), and sugar type (none, glucose, fructose, or sucrose) were evaluated. The optimal conditions were found to be 20% MSB at pH 4 or 5, either without added sugar or with sucrose. After fermenting under these conditions for 4 days, the probiotic beverages were stored at 4 °C for 30 days to assess stability. All formulations maintained LAB counts above 7 log CFU/mL for 18 days, but only the beverage with sucrose sustained these levels for 25 days. Additionally, antioxidant activity, total polyphenol, and flavonoid content increased post-fermentation, with the highest lactic acid levels observed at pH 5. Overall, this study presents a novel strategy for upcycling MSB into a probiotic beverage with enhanced functional and microbial stability.

1. Introduction

Prunus mume, belonging to the Rosaceae family, is an important staple fruit crop in East Asian countries [1]. The Korean name for P. mume is maesil, which is also commonly known as Japanese apricot or Chinese plum [2]. Maesil is rich in volatile compounds and essential nutrients, including organic acids, amino acids, minerals, dietary fiber, and phenolic compounds, and it exhibits various biological activities such as antidiabetic, liver-protective, antitumor, antimicrobial, antioxidant, and anti-inflammatory effects [3,4,5]. It is consumed in diverse forms, including pickles, flavoring agents, alcoholic beverages, and medicinal extracts, with maesil syrup (Maesil chung) being the most popular in Korea [6,7]. Maesil syrup, a fruit-juice concentrate produced by fermenting maesil with sugar, has recently gained popularity as a seasoning that imparts sweetness and a distinctive flavor to foods [8]. However, as maesil syrup consumption has grown, the amount of solid fruit residues, typically discarded as by-products after syrup extraction, has also steadily increased. Current food waste management strategies, such as landfilling, incineration, and composting, are considered inefficient in terms of resource utilization and contribute to greenhouse gas emissions that adversely affect the environment, highlighting the need for more eco-friendly and sustainable alternatives [9].

To address these issues, upcycling technologies have recently gained attention. This process involves transforming discarded materials, by-products, or waste into new products of higher quality or added value compared to the original [10]. Unlike traditional waste treatment methods, upcycling minimizes environmental impact, extends resource lifespan, and contributes to the realization of a circular economy [11]. The global adoption of upcycling is expanding, and it is increasingly recognized as a key strategy for sustainable development [12]. In particular, its importance in the food industry has grown significantly in recent years, and related research is being actively pursued [13,14]. However, upcycled food products still face consumer concerns due to their waste-based origins, necessitating additional efforts to improve consumer acceptance [15].

Lactic acid bacteria (LAB) are widely used in the production of fermented foods worldwide due to their ability to convert sugars into lactic acid. This process lowers the pH and enhances the preservation of food products [16]. Lactic acid fermentation not only extends shelf life but also facilitates the production of bioactive compounds with health benefits, including antimicrobial, anticancer, anti-inflammatory, antioxidant, and antiviral effects [17,18]. Moreover, LAB fermentation supports gastrointestinal health and modulates the host’s immune response [19]. Among LAB, Lactiplantibacillus plantarum is a Gram-positive probiotic species renowned for its exceptional metabolic versatility and ecological adaptability. It is found in diverse environments, including vegetables, dairy products, meat, and grass silage [20,21]. L. plantarum is commonly used in food fermentation due to its nonpathogenic nature and its ability to improve the quality of fermented products [20]. Recently, there has been growing interest in utilizing L. plantarum to produce probiotic beverages from various plant-based matrices [22,23,24]. These plant-based probiotic beverages offer an alternative to fermented dairy products, as they are free from lactose and animal-derived fats, thus reducing associated health concerns [25].

With the growing demand for plant-based probiotics and the increasing emphasis on sustainable food waste utilization, this study aimed to investigate the potential of converting by-products generated during maesil syrup production into a functional, plant-based probiotic beverage through lactic acid fermentation using L. plantarum. In addition, this study focused on optimizing fermentation parameters to enhance microbial viability and metabolic activity in the upcycled beverage. Fermentation trials were conducted under various conditions, including strain selection, substrate concentration, sugar supplementation, and pH adjustment. Changes in physicochemical properties, microbial activity, and organic acid profiles were analyzed during fermentation and subsequent storage. This study presents an LAB fermentation-based upcycling strategy that supports the valorization of food by-products while offering a sustainable and health-promoting alternative to commercial probiotic beverages.

2. Materials and Methods

2.1. Preparation of Maesil Syrup By-Product

Maesil syrup by-product (MSB) refers to the maesil fruits and flesh pulps that were discharged after separating maesil syrup following a sugaring process of 100 days. MSB was extracted with hot water, then concentrated to achieve a total soluble solid content of 71.00 °Bx, supplied by Slow Food (Hadong, Gyeongsangnam, Republic of Korea). For the proximate composition of the MSB extract, the total carbohydrate (determined by the sulfuric acid-phenol method) content was 46.87% (w/w), while the crude ash content was 0.19% (w/w). Both crude protein and crude lipid were not detected. The carbohydrates in the MSB extract consisted of glucose, fructose, and sucrose in a ratio of 1.3:1.1:1.0 (w/w). The extract contained malic acid and citric acid at concentrations of 10,960.35 mg/L and 17,300.15 mg/L, respectively. In addition, the total polyphenol content was 2042.10 mg GAE/L. To assess its suitability as a fermentation substrate for L. plantarum, the MSB extract was diluted to 20% (v/v).

2.2. Screening of Lactic Acid Bacteria for Fermentation

Eight strains of L. plantarum, isolated from Baechu kimchi, Pa kimchi, and Dongchimi, were selected based on their ability to thrive in low pH conditions to determine the most suitable strain for MSB fermentation. These strains were preserved at −80 °C in de Man, Rogosa, and Sharpe (MRS) broth (KisanBio, Seoul, Republic of Korea) supplemented with 30% (v/v) glycerol. For activation, L. plantarum strains were streaked onto MRS agar plates and incubated at 30 °C for 48 h to obtain single colonies. One colony was then selected and subcultured in MRS broth at 30 °C for 18 h. The cultures were harvested by centrifugation at 4000 rpm for 10 min at 4 °C, washed twice with distilled water, and adjusted to an optical density of 0.8 at 600 nm using a Genesys 180 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The suspension contained approximately 4.7 × 10⁸ CFU/mL, as confirmed by plate counting on MRS agar. Each strain was inoculated at 1% (v/v) into the 20% (v/v) MSB extract, preadjusted to pH 5 with food-grade sodium carbonate (Na₂CO₃, 10 g/100 mL) [26], and fermented at 30 °C for 4 days. Fermentation performance was assessed by monitoring pH changes using a pH meter (Orion Star A211, Thermo Scientific, Waltham, MA, USA).

2.3. Fermentation Optimization and Storage Stability

To determine the optimal fermentation conditions for the MSB probiotic beverage, the effects of MSB extract concentration (5%, 10%, 15%, or 20% (v/v)), pH (3, 4, or 5), and sugar type (none, glucose, fructose, or sucrose) were evaluated. The pH of the MSB beverage was adjusted using food-grade Na₂CO₃ (10 g/100 mL), and glucose, fructose, or sucrose was added at 15% (w/v) based on a previous study [16]. Fermentation was conducted at 30 °C for 4 days, and characteristics were assessed by measuring pH, acidity, and viable counts of LAB. The pH and acidity of the beverages were measured using a pH meter. To estimate LAB counts, the samples were serially diluted 10-fold in sterilized 0.85% (w/v) saline, and appropriate dilutions were spread onto MRS agar plates. The plates were incubated at 30 °C for 48 h using AnaeroPack-Anaero (MGC, Tokyo, Japan), and results were reported as log CFU/mL. Subsequently, to assess the storage stability of MSB probiotic beverages fermented under optimized conditions, samples were stored at 4 °C for 30 days. During storage, pH, acidity, and LAB counts were measured using the same methods applied in the fermentation optimization experiments.

2.4. Antioxidant Capacity of the MSB Probiotic Beverage

2.4.1. DPPH Radical Scavenging Activity

The antioxidant capacity of the MSB probiotic beverages was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, with slight modifications based on a previous study [27]. Briefly, 0.1 mM DPPH (Thermo Scientific, Waltham, MA, USA) in 80% (v/v) methanol was prepared and allowed to stand for 30 min, after which the absorbance was adjusted to 0.650 ± 0.020 at 517 nm. Then, 2.95 mL of the DPPH solution was mixed with 0.05 mL of the MSB beverages, and the mixture was incubated at room temperature for 30 min. The absorbance was subsequently measured at 517 nm using a spectrophotometer.

2.4.2. ABTS Radical Scavenging Activity

Additionally, the antioxidant activity of the MSB beverages was assessed using the ABTS radical scavenging assay, with minor modifications based on a previous study [28]. A solution containing 1 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH, Sigma-Aldrich, St. Louis, MO, USA) and 2.5 mM 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma-Aldrich, St. Louis, MO, USA) in PBS (pH 7.2) was incubated in a water bath at 70 °C for 30 min and then cooled to room temperature. The absorbance of the resulting ABTS radical solution was adjusted to 0.650 ± 0.020 at 734 nm. Subsequently, 0.98 mL of the ABTS solution was combined with 0.02 mL of the MSB beverage, and the mixture was incubated at 37 °C for 10 min. Absorbance was then measured at 734 nm using a spectrophotometer.

2.4.3. Ferric Reducing Antioxidant Power

The ferric reducing antioxidant power (FRAP) assay was performed based on a previous study [29]. The FRAP reagent was prepared by mixing 0.3 M sodium acetate buffer (pH 3.6), 10 mM 2,4,6-Tri(2-pyridyl)-1,3,5-triazine (TPTZ, Sigma-Aldrich, St. Louis, MO, USA) in 40 mM HCl, and 20 mM FeCl₃·6H₂O in a 10:1:1 (v/v/v) ratio. A total of 0.95 mL of the FRAP reagent was combined with 0.05 mL of the MSB beverage, and the mixture was incubated at room temperature for 30 min. After incubation, absorbance was immediately measured at 593 nm using a spectrophotometer. Antioxidant activity was expressed as mg ascorbic acid equivalents (VCE)/L.

2.5. Phytochemical Content Analysis of the MSB Probiotic Beverage

2.5.1. Total Polyphenol Content

Total polyphenol content (TPC) of the MSB beverage was determined using the Folin–Ciocalteu method [30]. Briefly, 0.2 mL of the MSB beverage was mixed with 2.6 mL of distilled water, followed by the addition of 0.2 mL of Folin–Ciocalteu’s reagent (Sigma-Aldrich, St. Louis, MO, USA). The mixture was incubated at room temperature for 6 min, followed by the addition of 2.0 mL of 7% (w/v) Na₂CO₃ solution. The reaction was continued at room temperature for 90 min, and the absorbance was measured at 750 nm using a spectrophotometer. TPC was expressed as mg gallic acid equivalents (GAE)/L.

2.5.2. Total Flavonoids Content

Total flavonoids content (TFC) of the MSB beverage was determined according to a previous study [31]. Briefly, 0.5 mL of the MSB beverage was mixed with 3.2 mL of distilled water, followed by the addition of 0.15 mL of 5% sodium nitrate solution. After 5 min, 0.15 mL of 10% (w/v) aluminum chloride was added, followed by the addition of 1.0 mL of 1 M sodium hydroxide. The absorbance was immediately measured at 510 nm using a spectrophotometer. TFC was expressed as mg catechin equivalents (CE)/L.

2.6. Organic Acids Analysis of the MSB Probiotic Beverage Using an LC System

Quantitative analysis of oxalic, malic, lactic, and citric acids in the MSB probiotic beverage was performed using a liquid chromatography (LC) system (Nexera XR; Shimadzu, Kyoto, Japan) equipped with a photodiode array detector. Organic acids were separated using a Zorbax Eclipse C18 column (250 mm × 4.6 mm, 5 μm; Agilent Technologies, Santa Clara, CA, USA) under isocratic conditions with 10 mM potassium dihydrogen phosphate buffer as the mobile phase. Chromatographic conditions included a flow rate of 0.5 mL/min, a column temperature of 40 °C, and an injection volume of 10 μL. Detection of organic acids was performed at 210 nm.

3. Results & Discussion

3.1. Selection of LAB for MSB Probiotic Beverage

To identify the optimal strain for MSB probiotic beverage fermentation, eight L. plantarum strains were evaluated. The L. plantarum used in this study is one of the nineteen probiotic LAB strains approved by the Ministry of Food and Drug Safety (MFDS) in Korea [32], and it is extensively used in the fermentation of plant-based foods [22,23,24]. The pH of the 20% (v/v) MSB extract was adjusted to approximately 5 using food-grade Na₂CO₃ to allow clearer observation of pH reduction during fermentation. The MSB beverage was inoculated with each strain and fermented at 30 °C for 4 days. Changes in pH during fermentation are shown in Figure 1. In the control sample without LAB inoculation, the pH remained stable, whereas all LAB-inoculated samples exhibited a gradual decrease in pH. Among them, the MSB beverage inoculated with L. plantarum KFOM 0042 showed the greatest pH reduction of 1.13, resulting in a final pH of 3.85. This pH decrease, primarily due to lactic acid production, is a defining feature of LAB fermentation [33,34]. Therefore, L. plantarum KFOM 0042, which demonstrated the most significant acidification effect, was selected as the optimal strain for MSB fermentation, and all subsequent experiments were carried out using this strain.

3.2. Optimization of Fermentation Conditions for MSB Probiotic Beverages

3.2.1. MSB Extract Concentration

To determine the optimal MSB extract concentration for the probiotic beverage using L. plantarum KFOM 0042, MSB extract was prepared at concentrations of 5%, 10%, 20%, and 30%. The results are shown in Figure 2. After 4 days of fermentation at 30 °C, all tested concentrations exhibited a decrease in pH, accompanied by increases in acidity and LAB counts (Figure 2). Although the 5% MSB extract showed the greatest pH reduction (1.48 ± 0.08), its corresponding increases in acidity and LAB counts were lower than those observed in the 10% and 20% extracts. Notably, the 20% MSB extract showed the highest acidity (1.14 ± 0.01) and LAB count (8.80 ± 0.01 log CFU/mL) compared to those of the other concentrations after 4 days. Based on these findings, the 20% MSB extract was selected as the optimal concentration for fermentation with L. plantarum KFOM 0042.

3.2.2. pH and Carbon Source

We also evaluated the effects of pH and carbon source on the fermentation of MSB extract to determine the optimal conditions for developing an MSB probiotic beverage. Previous studies have shown that pH-controlled cultures yield higher microbial growth, and sugar utilization patterns vary depending on the LAB strain [35,36]. Accordingly, the pH of the 20% MSB extract was adjusted to 3, 4, or 5. To assess the impact of different carbon sources on the fermentation process, physicochemical changes were analyzed in samples supplemented with glucose, fructose, or sucrose. All carbon sources were added at a concentration of 15% (w/v), based on a previous study that evaluated optimal sugar concentrations [16].

As shown in Figure 3, during the 4-day fermentation, MSB beverages with an initial pH of 3 exhibited no significant changes in pH or acidity, and LAB counts declined to below 5 log CFU/mL. In contrast, MSB beverages adjusted to pH 4 and 5 showed average pH reductions of 0.38 ± 0.04 and 0.98 ± 0.05, respectively, along with corresponding increases in acidity of 0.53 ± 0.09 and 0.69 ± 0.10 (Figure 3A,B). LAB counts in beverages at pH 4 and 5 remained above 7 log CFU/mL throughout fermentation (Figure 3C). Notably, unsupplemented samples and those with added sugars maintained LAB counts near 8 log CFU/mL, suggesting these conditions are suitable for probiotic beverage development. Carbon sources are essential for the growth of LAB [37], and the total carbohydrate content of MSB extract was 46.87%, indicating that the 20% the MSB beverage likely provided sufficient carbohydrates to support LAB growth. However, high concentrations of sugars can induce osmotic stress, which may reduce probiotic viability [38]. For this reason, it is thought that the beverages supplemented with glucose and fructose showed slightly lower viable cell counts compared to those without sugar addition. In contrast, sucrose is known to impose only a transient osmotic stress because external and internal sugars equilibrate after some time [39]. Accordingly, it is considered that sucrose exerted less osmotic stress on L. plantarum KFOM 0042 in the MSB beverage compared to glucose or fructose, resulting in higher viable LAB counts. In addition, consistent with previous reports identifying sucrose as the optimal carbon source for the growth of L. plantarum [40], the present study confirmed that sucrose was the most effective substrate for promoting the growth of L. plantarum KFOM 0042, compared to other carbon sources. Therefore, the optimal fermentation conditions for the MSB probiotic beverage using L. plantarum KFOM 0042 were determined to be a 20% MSB extract adjusted to pH 4 or 5, under both non-supplemented and sucrose-supplemented conditions.

3.3. Evaluation of the Storage Stability of the MSB Probiotic Beverage

According to the criteria established by the FAO/WHO [41], probiotic beverages must contain a minimum of 6 to 7 log CFU/mL of viable microorganisms throughout their shelf life to be considered effective [42]. Therefore, maintaining microbial stability under refrigerated conditions is crucial for the development of MSB probiotic beverages. To assess this, the storage stability of MSB probiotic beverages fermented under four optimal conditions, pH 4 with no added carbon source (pH 4 + N), pH 4 with sucrose (pH 4 + S), pH 5 with no added carbon source (pH 5 + N), and pH 5 with sucrose (pH 5 + S), was evaluated during refrigerated storage (Figure 4).

Compared to the results observed at the end of fermentation (day 4), the pH and acidity of all MSB probiotic beverages remained stable during 30 days of storage at 4 °C (Figure 4A,B). However, LAB counts, which initially exceeded 8 log CFU/mL, declined slightly after 8 days of storage (Figure 4C). Notably, in the MSB probiotic beverage adjusted to pH 4 with no added carbon source (pH 4 + N), LAB counts dropped to 2.00 ± 0.17 log CFU/mL by day 21, and no viable cells were detected by day 30. Similarly, for the pH 5 + N condition, LAB counts remained at 6.18 ± 0.05 log CFU/mL on day 21 but became undetectable by day 30. In contrast, MSB probiotic beverages fermented under sucrose-supplemented conditions (pH 4 + S and pH 5 + S) retained LAB counts above 7 log CFU/mL on day 21, with viable counts still at 5.31 ± 0.19 and 5.44 ± 0.02 log CFU/mL, respectively, by day 30. Carbon sources are critical for the growth and survival of probiotics [37], and sucrose was found to positively influence the viability of L. plantarum KFOM 0042 in the MSB probiotic beverage. This result is consistent with previous findings that sucrose supplementation improves the storage stability of probiotic beverages during refrigeration [43,44]. Therefore, sucrose supplementation effectively extended the refrigerated shelf life of the MSB beverage by approximately one week, highlighting its potential as a probiotic product with enhanced storage stability.

3.4. Measurement of Antioxidant Activity

To evaluate the functionality of MSB probiotic beverages fermented under the four conditions described above, antioxidant activity was assessed (Figure 5). On day 4 of fermentation, the DPPH radical scavenging activity of MSB probiotic beverages significantly increased under all conditions, with an average rise of 81.06 ± 15.19 mg VCE/L compared to pre-fermentation values (Figure 5A). Similarly, ABTS radical scavenging activity also showed a significant increase following fermentation (Figure 5B). These results are consistent with previous studies reporting enhanced antioxidant activity after LAB fermentation of fruits and vegetables [45,46,47]. In addition, several studies have shown that fermenting fruit matrices similar to maesil with L. plantarum enhances antioxidant activity. For example, peach (Prunus persica) and plum (Prunus domestica L.) juices fermented with L. plantarum showed significant improvements in antioxidant capacity [48,49]. These results support the widespread application of L. plantarum in fruit-based probiotic beverages and suggest that the enhanced antioxidant activity observed in MSB beverages is consistent with previously reported findings. However, no significant change in FRAP was observed before and after fermentation (Figure 5C). Since DPPH, ABTS, and FRAP assays operate through different mechanisms, discrepancies among their results are common. It is widely accepted that employing multiple assay methods is necessary for a comprehensive evaluation of antioxidant capacity [50,51]. Overall, all MSB probiotic beverages under the tested conditions demonstrated significant improvements in DPPH and ABTS radical scavenging activities, supporting their potential as functional probiotic beverages with enhanced antioxidant properties. These findings suggest that LAB fermentation of by-products like MSB extract can facilitate the development of value-added functional beverages.

3.5. Measurement of Phytochemical Content

Changes in TPC and TFC were also evaluated in the MSB probiotic beverages (Figure 6). After 4 days of fermentation, all samples showed significant increases in both TPC and TFC, and these elevated levels remained stable during 30 days of refrigerated storage. Lactic acid fermentation has been reported to enhance antioxidant activity and increase TPC and TFC, and similar trends were observed in the MSB probiotic beverages fermented with LAB in this study [52]. Furthermore, the increase in polyphenols and flavonoids is known to contribute directly to enhanced antioxidant activity [53], suggesting that the elevated antioxidant capacity observed in the MSB probiotic beverages is closely linked to the increased TPC and TFC levels [54]. In addition, polyphenols and flavonoids exhibit various biological activities, including antitumor, cardioprotective, antibacterial, antifungal, anti-allergic, and anti-inflammatory effects. Their bioavailability is reported to be improved through LAB fermentation, which may further enhance their functional efficacy [52,55,56,57]. Therefore, the increased phytochemical content following fermentation, along with its stability during storage, supports the potential of the MSB beverage as a probiotic product with enhanced functional properties.

3.6. Quantification of Organic Acid

Changes in the contents of organic acids, including lactic acid, oxalic acid, citric acid, and malic acid in the MSB probiotic beverage, were measured during storage using HPLC, as shown in

Table 1. Changes in organic acids in MSB probiotic beverages.

Organic Acid (mg/L)	MSB Probiotic Beverage
	Day	pH 4 + N	pH 4 + S	pH 5 + X	pH 5 + S
Lactic acid	0	ND	ND	ND	ND
	4	1452.21 ± 123.50 aB	1251.05 ± 22.93 cC	1814.32 ± 4.70 bA	1667.26 ± 28.10 bA
	6	1475.99 ± 82.80 aB	1275.04 ± 3.87 bcC	1853.79 ± 20.7 aA	1746.91 ± 104.99 abA
	18	1462.64 ± 23.11 aB	1301.44 ± 9.65 abC	1859.36 ± 19.49 aA	1876.43 ± 45.04 aA
	34	1508.38 ± 8.84 aB	1339.93 ± 21.04 aC	1695.04 ± 9.19 cA	1691.99 ± 23.99 bA
Oxalic acid	0	83.52 ± 6.02 aB	81.83 ± 1.09 aB	95.64 ± 5.41 aA	81.79 ± 1.32 abB
	4	83.68 ± 7.73 aA	79.03 ± 0.79 aA	82.85 ± 0.96 bcA	76.38 ± 1.74 bA
	6	81.54 ± 4.57 aA	77.87 ± 1.39 aA	84.08 ± 2.54 bA	78.93 ± 3.96 abA
	18	78.48 ± 1.00 aA	80.42 ± 2.99 aA	82.89 ± 1.71 bcA	84.76 ± 3.39 aA
	34	84.83 ± 1.22 aA	79.31 ± 2.23 aB	76.33 ± 0.61 cB	78.01 ± 0.99 abB
Citric acid	0	724.47 ± 66.33 aA	710.84 ± 14.90 aA	801.21 ± 53.75 aA	668.98 ± 5.29 aB
	4	678.87 ± 41.01 abA	595.80 ± 17.06 bB	513.66 ± 32.89 bC	479.05 ± 13.39 bC
	6	629.94 ± 46.16 abA	578.23 ± 8.74 bcAB	517.22 ± 5.75 bB	476.46 ± 34.12 bB
	18	575.11 ± 15.04 bA	550.81 ± 11.71 cdA	474.98 ± 1.35 bcB	447.93 ± 11.01 bB
	34	584.13 ± 11.13 abA	538.30 ± 18.16 dB	422.77 ± 5.33 cC	384.03 ± 8.69 cD
Malic acid	0	230.31 ± 19.22 aA	224.15 ± 7.81 aA	234.71 ± 5.14 aA	210.96 ± 2.86 aA
	4	ND	ND	ND	ND
	6	ND	ND	ND	ND
	18	ND	ND	ND	ND
	34	ND	ND	ND	ND

Results are expressed as mean ± standard deviation; n = 3. ND: not detected. Mean values with different uppercase letters within the same row are significantly different (p < 0.05). Mean values with different lowercase letters within the same column are significantly different (p < 0.05).

and Figure 7. Lactic acid levels increased markedly in all MSB probiotic beverages following fermentation, with significantly higher concentrations observed in samples adjusted to pH 5 compared to pH 4 (Figure 7A). Lactic acid, a primary organic acid produced during lactic acid fermentation, indicates enhanced fermentation activity under the pH 5 condition [58,59]. It is known to improve shelf life, flavor, and nutritional value, thereby positively influencing the quality and functionality of probiotic beverages [60]. Oxalic acid, citric acid, and malic acid have been reported as major organic acids present in maesil [61,62]. Oxalic acid was detected at an average concentration of 85.70 ± 6.68 mg/L in all day 0 samples, and its levels remained unchanged during fermentation and storage (Figure 7B). Citric acid content gradually declined throughout fermentation and storage; after 30 days, samples adjusted to pH 5 exhibited significantly lower citric acid levels than those adjusted to pH 4 (Figure 7C). Citric acid serves as a metabolic substrate for LAB, promoting lactic acid production directly and indirectly by preserving fermentable substrates [63]. This mechanism explains the significantly higher lactic acid levels observed in the pH 5-adjusted beverages compared to those at pH 4. Additionally, malic acid was present at an average concentration of 225.03 ± 7.26 mg/L in day 0 samples but was not detected in any samples post-fermentation (Figure 7D). This disappearance is likely due to malolactic fermentation, in which LAB convert malic acid into lactic acid and CO₂ [64,65]. These findings suggest that pH adjustment significantly influences organic acid metabolism during fermentation and storage. Moreover, the substantial increase in lactic acid, which was not originally present in the MSB extract, confirms the metabolic activity of LAB utilizing sugars and organic acids from MSB, thereby supporting the beverage’s potential as a functional probiotic product.

4. Conclusions

This study demonstrated the potential to convert MSB into a plant-based probiotic beverage through lactic acid fermentation. To our knowledge, there are no studies that have enhanced functionality using MSB. In this study, MSB beverages fermented under optimal conditions showed improved probiotic viability, storage stability, antioxidant activity, and phytochemical content. Fermentation at pH 5 more effectively promoted lactic acid production and enhanced microbial activity and fermentation efficiency. This study highlights the potential of using MSB as a sustainable food ingredient and supports a practical upcycling strategy for converting food by-products into value-added, plant-based probiotic beverages. Further study will be needed to evaluate the probiotic characteristics of L. plantarum KFOM 0042 through safety and stability analysis.