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

Comments 1: The authors wrote in the purpose of the paper that the study also aims to improve consumer acceptance of recycled foods. They did not carry out such an acceptance assessment.

Response 1: Thank you for pointing this out. We agree with your comment. This study aimed to increase the value of by-products by enhancing their functionality through the fermentation of wasted maesil with lactic acid bacteria. As you recommended, we revised the paragraph to focus on this primary objective as follows:

[Lines 72-79]: 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 micro-bial 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.

Comments 2: The authors should complete the information about the probiotic bacterial strains used. Where can one find information that these are tested bacteria and proven to be probiotic strains? Just indicating that they have been isolated from fermented foods does not entitle them to be called probiotics, one can only assume that they are potential probiotics. Nor can the name probiotic beverage be used. As L. plantarum originates from different habitats, it evolves in different ways, resulting in a high intra-species genetic diversity in this species. Usually, probiotic bacterial cells are stored in the Culture Collection of Agricultural Microbiology of a country. What were the criteria for selecting these 8 strains of Lactiplantibacillus plantarum? Has there been any previous research taking into account the technological characteristics of potential probiotics?

Response 2: Thank you for your comment. The L. plantarum used in this study is one of the 19 probiotic lactic acid bacteria strains approved by the Ministry of Food and Drug Safety (MFDS) in Korea and is widely used in the development of plant-based fermented beverages. Therefore, among the strains we possess, the 8 L. plantarum strains tested in this experiment were selected based on their ability to grow at low pH. The finally selected L. plantarum KFOM 0042 strain for fermentation was confirmed for its safety and stability as a probiotic through genomic analysis (data not shown). Whole genome sequencing results using the Oxford Nanopore platform showed multiple probiotic marker genes, and no toxic genes were detected. These findings suggest that L. plantarum KFOM 0042 has the potential to be defined as a probiotic strain. We plan to submit the related results soon.

As you recommended, we added the related sentences and references as follows:

[Lines 99-101]: 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.

[Lines 187-189]: The L. plantarum used in this study is one of the 19 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 [22-24].

[Lines 382-383]: Further study will be needed to evaluate the probiotic characteristics of L. plantarum KFOM 0042 through safety and stability analysis.

Comments 3: Lines 177-188. In my opinion, the authors made the wrong assumption to first raise the pH value of the drink with sodium carbonate and then lower it with fermentation. This is a long process and completely unnecessary, because you can add enough cells of a potential probiotic and keep the natural low pH (without carbonate) for the shelf life of the beverage. Therefore, the choice of Lactiplantibacillus plantarum KFOM 0042 due to its acidification effect is of no practical importance. In this case, survival in an acidic environment should be the selection criterion.

Response 3: Thank you for your comment. In general, lactic acid bacteria are known to survive at a pH range of 4 to 7 (Hakkinen et al., 2024; Popova-Krumova et al., 2024). The initial pH of MSB extract used in this study was around 2.8-2.9, which is too acidic for any lactic acid bacteria to survive. Initially, we aimed to use lactic acid bacteria isolated from MSB extract for fermentation in probiotic beverages, but no colonies were detected. This indicated that lactic acid bacteria are not present in MSB extract and have difficulty surviving under these conditions. A previous study reported that the pH of blueberry and blackberry juice (initial pH 3) was adjusted with sodium carbonate prior to probiotic fermentation (Wu et al., 2021). Consequently, we adjusted the pH of MSB extract from 3 to 5 to determine the optimal pH conditions. Although it might seem unnecessary to lower the pH again through lactic acid fermentation, this process can produce beneficial substances like lactic acid, thereby enhancing the functionality of the product.

Comments 4: Lines 215-218 The authors cite their previous studies as the basis for selecting the best carbon source. And here there are contradictions with the reviewed manuscript. They write in Article 16: ‘When evaluating the effect of different sugar sources and concentrations on lactic acid fermentation, 15% fructose was shown to give a higher acid yield than observed with other sugar sources.’ Such a discrepancy is puzzling.

Response 4: Thank you for your comment. We did not select the type of sugar based on previous studies, but we applied the 15% concentration, which was assessed as optimal, to various sugar sources for fermenting MSB extract. To enhance clarity, we revised the sentences as follows:

[Lines 224-228]: 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].

Comments 5: Lines 228- 229. Previous studies have reported that L. plantarum efficiently metabolises glucose, fructose, and sucrose [21]. Article 21 is written based on literature, not research. I have read it, it does not include this information about sugars.

Response 5: As you recommended, we deleted the original sentence and added the sentence  as follows:

[Line 237]: Carbon sources are essential for the growth of LAB [37],

Comments 6: The conclusions should be supplemented by the information/responses contained in the purpose of the study.

Response 6: As you recommended, we revised the conclusion in the manuscript as follows:

[Lines 374-383]: 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.

Comments 1: Line 101, 155, 178 - Na2CO3 must be written correctly, numbers 2 and 3 must be written as a subscript. Line 183, 185 - plantarum must be written in italics

Response 1: Thank you for your comment. We corrected the formatting of Na₂CO₃ by converting the numbers into subscripts and italicizing the species names in lines 110, 118, 165, 186, 187, 195, and 198.

Comments 2: What is the percentage level of soluble solid contents of MSB extract? What else does it contain (total solid, protein, sugars, etc.)?  A correct and complete description of the product used in the study is required.

Response 2: As you recommended, we added the sentences related to the composition of the MSB extract as follows:

[Lines 86-96]: Maesil syrup by-product (MSB) referred 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, and supplied by Slow Food (Hadong, Gyeongsangnam, 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 2,042.10 mg GAE/L.

Comments 3: Line 97-98:” The cultures were harvested by centrifugation at 4,000 rpm for 10 min at 97 4°C, washed twice with distilled water, and adjusted to an optical density of 0.8 at 600 nm”. Why did the authors not standardize the inoculum to an optical density of a 0.5 McFarland standard which corresponds to 1.5 × 108 CFU.mL-1 (McFarland, 1907) but adjusted to an optical density of 0.8? The microbial load for the microorganism is not mentioned. What was the initial microbial load for the fermentation, as the inoculum used certainly influenced the results obtained in this study?

Response 3: Thank you for your comment. As many recent studies have used optical density for bacteria inoculum preparation (Valdiviezo-Marcelo et al., 2023; Lu et al., 2024), we adjusted to an optical density of 0.8 and counted the initial microbial load. As you recommended, we added the initial microbial load for fermentation as follows:

[Lines 108-109]: The suspension contained approximately 4.7 × 10⁸ CFU/mL, as confirmed by plate counting on MRS agar.

Comments 4: Line 112-114:” 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),….ˮ L. plantarum is an aerobic microorganism. Why did the authors incubate the plates in anaerobiosis? In fact, have more types of LAB grown in the plates? Does this mean that a pure culture was not used?

Response 4: Thank you for your comment. L. plantarum is known to be a facultative anaerobe, and grows well under both anaerobic and aerobic conditions. Thus, in this study, we cultured L. plantarum under anaerobic condition. Additionally, MSB extract was plated on MRS agar without LAB inoculation under both conditions, and no colonies were observed, indicating that only the inoculated strain was purely cultured.

Comments 5: The graphs in figure 2 are not correct and must be redone. The described data must be put in line graphs (OXY) and not bar graphs, to be able to correctly observe the changes in the analyzed parameters during fermentation.

Response 5: As you recommended, we revised Figure 2 by replacing the bar graphs with line graphs and added the related sentence as follows:

[Lines 212-214]: 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 the other concentrations after 4 days.

Comments 6: Line 213-217: ˮ Previous studies have shown that pH-controlled cultures yield higher microbial growth, and sugar utilization patterns vary depending on the LAB strain [34,35]. Accordingly, the pH of the 20% MSB extract was adjusted to 3, 4, or 5. To assess the impact of various carbon sources on the fermentation process, physicochemical changes were analyzed in samples supplemented with 15% (w/v) glucose, fructose, or sucrose, based on a previous study…..ˮ but they did not report the initial concentration of sugars to be able to see the influence of the microorganism on the final product. Please rephrase.

Response 6: As you recommended, we added the total carbohydrate content and sugar composition of the MSB extract and discussed the influence of the microorganism on the probiotic beverage as follows:

[Lines 89-92]: 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).

[Lines 92-93]: The carbohydrates in the MSB extract consisted of glucose, fructose, and sucrose in a ratio of 1.3:1.1:1.0 (w/w).

[Lines 237-239]: 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.

Comments 7: The discussion section contains only a few comparisons to previously published papers. For this reason, comparisons should be made with studies that used the same matrices and microorganisms. Please rephrase.

Response 7: Thank you for your valuable feedback. As you recommended, we have revised the Results and Discussion sections of the manuscript.

[Lines 239-249]: 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.

[Lines 278-280]: This result is consistent with previous findings that sucrose supplementation improves the storage stability of probiotic beverages during refrigeration [43,44].

[Lines 296-302]: 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.