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Effects of Different Yeasts on the Physicochemical Properties and Aroma Compounds of Fermented Sea Buckthorn Juice

Fermentation 2025, 11(4), 195; https://doi.org/10.3390/fermentation11040195

by Bo Peng^1,2,3,†, Liyue Fei^1,2,3,†, Ziyi Lu^1,2,3, Yiwen Mao^1,2,3, Qin Zhang^1,2,3, Xinxin Zhao^1,2,3, Fengxian Tang^1,2,3, Chunhui Shan^1,2,3, Dongsheng Zhang^1,2,3,* and Wenchao Cai^1,2,3,*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3: Anonymous

Fermentation 2025, 11(4), 195; https://doi.org/10.3390/fermentation11040195

Submission received: 24 February 2025 / Revised: 27 March 2025 / Accepted: 2 April 2025 / Published: 7 April 2025

(This article belongs to the Special Issue Alcoholic Fermentation)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigated the fermentation of Sea buckthorn juice (SBJ) with two commercial Saccharomyces cerevisiae yeast strains (RW and RV171). The single and mixed fermentations were compared with the physicochemical properties. The volatile compounds were identified using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS).

The following suggestions are offered for revision.

The methods of bioinformatics should be presented in the manuscript.

In the manuscript, many abbreviations are not consistent.

What are SJ and FSJ?

The resolution of Figure (1~7) are not enough. The authors should provide higher resolution figures. The figure legends should be rewritten.

The representation of significant figures should be consistent.

For example, 1.86 ± 0.03 g/L (Line 140).

Line 159 : (50.53 ± 0.03 and 173.63 ± 0.33)

The chemical names need to be standardized, as the same compound is referred to by several different names. The names of compounds in the Abstract, the main text, and the tables are inconsistent. Please revise them

Line 18

Saccharomyces cerevisiae should be presented.

Line 26

RW, RV and RW, RV and RWRV

Line 54

The full name of SBJ should be presented.

Line 63

(1)The full name of FSBJ should be presented.

(2) The reason for choosing the two yeast strains (RW and RV171) should be presented.

Line 75

(1) What is “Shen Qiuhong”? Please cite the reference.

(2) What is SJ?

Line 85

“0.03% of pre-activated yeast strains”

(1) Why is the added amount of bacteria less than the usual experimental quantity?

(2)What are the concentrations of the yeasts (CFU/mL)?

Line 87

“When the alcohol content reached 0.45 ± 0.05% by volume”

(1) The method for determination of ethanol should be presented.

(2) What is the reason for completing the fermentation with 0.45% ethanol?

(3) How many days were required to complete the fermentation?

Line 92

(1)The full name of DNS should be presented.

(2) Dinitrosalicylic acid (DNS) method is primarily used to estimate the concentration of reducing sugars. Sucrose is a non-reducing sugar. Is the sucrose fully consumed by the end of fermentation?

Line 106

(1) •OH radical scavenging activity (•OH-SA)

(2) reducing power capacity (RP-CA)

Line 137

It shall be Figure 1. Please check the correctness.

Line 150

What is FSJ? It shall be FSBJ.

Line 160

“it’s a* was significantly lower.”

However, the value of a* is higher than that of RV group.

Line 166

What is FSJ? It shall be FSBJ.

Line 205~206

“its ability to scavenge the three free radicals was significantly higher than that of the other two SBJs.”

Please check the correctness.

Line 207

greengage

Line 231

7 alcohols (A1 to A7),

Line 233

7 hydrocarbons (F1 to F7),

Line 281

7 types

Line 285

64.16 mg/L is not correct. Please check the correctness.

Line 290~297

One compound (A2, cas 1960-12-8) is described in the manuscript with three names.

Phenylethyl Alcohol, (S)-1-phenylethanol (Table 1)

Phenylethyl Alcohol (Line 294, 296)

phenyl alcohol (Line 290)

Line 308

7 hydrocarbons

Line 338, 432 and abstract (Line 25)

“15, 14, and 11” should be corrected to “14, 13, and 10”.

Line 350

One compound (C1: cas 160-32-1) is described in the manuscript with three names.

ethyl caprylate (line 350)

ethyl octanoate (Table 3)

Octanoic acid, ethyl ester (Table 2)

Line 360

“Among them, ethyl caproate had the highest OAV”

It is not correct. Please check the correctness.

Line 361

One compound (C4: cas 123-66-0) is described in the manuscript with two names.

Butanoic acid, 3-methyl-ethyl ester (Table 2,3)

ethyl isovalerate (Line 361)

Comments on the Quality of English Language

No comment

Author Response

This manuscript investigated the fermentation of Sea buckthorn juice (SBJ) with two commercial Saccharomyces cerevisiae yeast strains (RW and RV171). The single and mixed fermentations were compared with the physicochemical properties. The volatile compounds were identified using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS).

The following suggestions are offered for revision.

Reply: Thank you very much for the positive comments and constructive suggestions. Please find the following detailed responses to your comments and suggestions.

The methods of bioinformatics should be presented in the manuscript.

Reply: Thank you for your thoughtful and rigorous work. We have added an introduction to bioinformatics methods to the MS and all revisions have been highlighted by using red colored text.

Content added to the instruction:

In the production of yeast fermented beverages, the quality and volatile compound content are closely related to the yeast used, so the selection of yeast is one of the key factors in determining the quality of fermented beverages. Han et al. (2020) used three different yeasts to ferment greengage alcoholic beverage (GAB) and found that although fermented GAB from RV171 and BV818 showed greater antioxidant capacity, GAB fermented by Lalvin 71B showed the highest efficiency, suitable sugar-acid ratio, and the richest volatile flavor compounds, and so Lalvin 71B was considered to be a more suitable strain for producing fermented GAB. Therefore, different yeasts fermenting SBJ may exhibit different physicochemical qualities and produce different flavor substances. In order to investigate the effect of yeast strains on the physicochemical qualities of fermentation of sea buckthorn juice (FSBJ), a redundancy analysis (RDA) was carried out with different yeast strains as “explanatory variables” and physicochemical indexes as “response variables” to evaluate the “ex-planatory variables” and “response variables” components. Redundancy analysis (RDA) was conducted to evaluate the linear relationship between the components of “explan-atory variables” and “response variables”. In addition, to understand the effect of dif-ferent yeasts on the volatile compounds in SBJ, principal component analysis (PCA) was used to understand the overall separation trend of volatile compounds among the sample groups, which revealed whether there were any differences in volatile compounds among the sample groups

In the manuscript, many abbreviations are not consistent.

What are SJ and FSJ?

Reply: Thank you for your thoughtful and careful work. I'm very sorry that I didn't standardize the abbreviations throughout the text, it was a mistake in my writing. SJ stands for sea buckthorn juice and FSJ stands for fermented sea buckthorn juice, I have now standardized the abbreviations throughout the text to SBJ for sea buckthorn juice and FSBJ for fermented sea buckthorn juice. All revisions have been highlighted by using red colored text.

Thank you once again for your constructive comments.

The resolution of Figure (1~7) are not enough. The authors should provide higher resolution figures. The figure legends should be rewritten.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

The representation of significant figures should be consistent.

For example, 1.86 ± 0.03 g/L (Line 140).

Line 159 : (50.53 ± 0.03 and 173.63 ± 0.33)

Reply: Thank you for your thoughtful and constructive work. We have standardized valid numbers to 2 decimal places throughout the text and all revisions have been highlighted by using red colored text.

The chemical names need to be standardized, as the same compound is referred to by several different names. The names of compounds in the Abstract, the main text, and the tables are inconsistent. Please revise them

Reply: Thank you for your rigorous and careful work. We have standardized the names of the compounds in the abstract, text and tables throughout the text, and all revisions have been highlighted by using red colored text.

Line 18

Saccharomyces cerevisiae should be presented.

Reply: Thank you for your thoughtful and valuable comment. We have added an introduction to Saccharomyces cerevisiae to the text and all revisions have been highlighted by using red colored text.

Content added to the Abstract: Flavor components are important factors that affect the quality of fermented beverages. Yeast is one of the most important factors affecting the flavor of beverages during the fermentation process, where yeast converts sugars into alcohol and produces flavor substances.

Content added to the instruction: In the present study, two commercial yeast strains were selected to prepare FSBJ. RV171 and RW are both commonly used commercial yeasts. RV171 is a low-alcohol sweet brewing active dry yeast, suitable for fermentation of high acidity fruits, with certain conversion ability to malic acid, and at the same time, it can generate more esters and glycerol substances, adding alcohol and ester flavor to the fermentation broth; RW has excellent tannin and pigment extraction power, and it can retain the original color of the fruits, highlighting the fruity aroma of the fermentation broth. Therefore RW and RV171 are suitable for fermenting most fruits, providing ester and alcohol flavor to the fer-mentation broth, and fermentation is stable and durable.

Line 26

RW, RV and RW, RV and RWRV

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 54

The full name of SBJ should be presented.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 63

The full name of FSBJ should be presented.
The reason for choosing the two yeast strains (RW and RV171) should be presented.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to the instruction: RV171 and RW are both commonly used commercial yeasts. RV171 is a low-alcohol sweet brewing active dry yeast, suitable for fermentation of high acidity fruits, with certain conversion ability to malic acid, and at the same time, it can generate more esters and glycerol substances, adding alcohol and ester flavor to the fermentation broth; RW has excellent tannin and pigment extraction power, and it can retain the original color of the fruits, highlighting the fruity aroma of the fermentation broth. Therefore RW and RV171 are suitable for fermenting most fruits, providing ester and alcohol flavor to the fer-mentation broth, and fermentation is stable and durable.

Line 75

What is “Shen Qiuhong”? Please cite the reference.
What is SJ?

Reply: Thank you for your thoughtful and constructive work. Hippophae rhamnoides L. subsp. mongolica ‘Shenqiuhong’ is an excellent large-fruited sea buckthorn variety which was introduced from Russia and Finland in 1990, and has been cultivated in northeast China, Daxinganling and northern Xinjiang. Second, SJ stands for sea buckthorn juice, and we have now standardized the acronym sea buckthorn juice throughout the text to SBJ. All revisions have been highlighted by using red colored text.

Content added to section 2.1: The variety of sea buckthorn berries called Hippophae rhamnoides L. subsp. mongolica ‘Shenqiuhong’ (Wang et al., 2024).The berries were stored at -20℃ until use.

Line 85

“0.03% of pre-activated yeast strains”

Why is the added amount of bacteria less than the usual experimental quantity?
What are the concentrations of the yeasts (CFU/mL)?

Reply: Thank you for your thoughtful and valuable comment. Because we used commercially brewed active dry yeast and used sterilized 5% dextrose solution to activate the powder for 30 min before using it for sea buckthorn juice fermentation. In addition, our main objective was not to make sea buckthorn wine, but rather to make yeast-fermented sea buckthorn beverage (<0.5% abv), so the amount of yeast powder added in this study was less than that in some yeast-fermented fruit wine articles. In addition, in both articles (Cai et al., 2018) and (Han et al., 2020), the amount of yeast powder added is ≦0.03%, so 0.03% (0.3 g/L) powder addition can meet the experimental requirements of this study. Finally, yeast powder was added to sea buckthorn juice at 0.03% (0.3 g/L), after activation, and the number of live bacteria in the juice was greater than 10⁶ CFU/mL.

Line 87

“When the alcohol content reached 0.45 ± 0.05% by volume”

The method for determination of ethanol should be presented.
What is the reason for completing the fermentation with 0.45% ethanol?
How many days were required to complete the fermentation?

Reply: Thank you for your rigorous and careful comment. Referring to the method of Zhao et al. (2022), according to the first method in GB 5009.225–2016 ″Determination of Ethanol Concentration in National Standard Liquor for Food Safety”, the sample density at 20°C was measured by the density bottle method, and the comparison table was used to calculate the alcohol content based on the density of the alcohol aqueous solution.

Because we made a yeast-fermented beverage rather than a fermented wine, the national standard (GB 7101-2022) stipulates that the ethanol content of the beverage should not be more than 0.5% by mass fraction, so we used an ethanol content of 0.45 ± 0.05% as the end point of fermentation of sea buckthorn juice.

After many trials, we found that the fermentation time of about 20 h can achieve the required alcohol content.

Content added to section 2.3: The SBJ was then sterilized and inoculated with 0.3 g/L of pre-activated yeast strains (RW, RV171 [RV], and RW&RV171 (1:1) [RWRV]) to prepare three separate fermentation mixtures (SBJ viable bacteria count > 10⁶ CFU/mL). The mixtures were placed in an incubator at 20℃ and fermented for 20 h. When the alcohol content reached 0.45 ± 0.05% by volume, fermentation was stopped. According to the first method in GB 5009.225–2016 ″Determination of Ethanol Concentration in National Standard Liquor for Food Safety”, the sample density at 20℃ was measured by the density bottle method, and the comparison table was used to calculate the alcohol content based on the density of the alcohol aqueous solution.

Line 92

The full name of DNS should be presented.

3,5-dinitrosalicylic acid (DNS)

Dinitrosalicylic acid (DNS) method is primarily used to estimate the concentration of reducing sugars. Sucrose is a non-reducing sugar. Is the sucrose fully consumed by the end of fermentation?

Reply: Thank you for your careful comment. We have added the full DNS name in the text. Secondly, as you have commented, we also think that using reducing sugar content to describe the data of DNS assays would be more rigorous. All revisions have been highlighted by using red colored text.

Line 106

(1) •OH radical scavenging activity (•OH-SA)

(2) reducing power capacity (RP-CA)

Reply: Thank you for your rigorous and careful work. •OH-SA and RP-CA, like DPPH and ABTS, are chemical antioxidant assays.

Line 137

It shall be Figure 1. Please check the correctness.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 150

What is FSJ? It shall be FSBJ.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 160

“it’s a* was significantly lower.”

However, the value of a* is higher than that of RV group.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to section 3.1.2: The L* and b* of the FSBJ obtained using RWRV were significantly higher than those of the FSBJs obtained using RW and RV (50.53 ± 0.03 and 173.63 ± 0.33, respectively), whereas its a* is significantly lower but higher than that of RV.

Line 166

What is FSJ? It shall be FSBJ.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 205~206

“its ability to scavenge the three free radicals was significantly higher than that of the other two SBJs.”

Please check the correctness.

Reply: Thank you for your thoughtful and careful comment. We did find that the FRAP of RV was greater than that of RW but less than that of RWRV. in addition, the DPPH-SA, ABTS-SA, and •OH-SA of RV were higher than those of RW and RWRV.

Line 207

Greengage

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 231

7 alcohols (A1 to A7),

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 233

7 hydrocarbons (F1 to F7),

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 281

7 types

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 285

64.16 mg/L is not correct. Please check the correctness.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 290~297

One compound (A2, cas 1960-12-8) is described in the manuscript with three names.

Phenylethyl Alcohol, (S)-1-phenylethanol (Table 1)

Phenylethyl Alcohol (Line 294, 296)

phenyl alcohol (Line 290)

Reply: Thank you for your valuable comment. We have standardized the expression of A2 compounds where they occur throughout the text to phenylethyl alcohol, and all revisions have been highlighted by using red colored text.

Line 308

7 hydrocarbons

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Line 338, 432 and abstract (Line 25)

“15, 14, and 11” should be corrected to “14, 13, and 10”.

Reply: Thank you for your rigorous and careful comment. However, in accordance with Table 3, we think that it is indeed the 15, 14, and 11 volatile compounds that have OAVs > 1 in the RW, RV, and RWRV.

Line 350

One compound (C1: cas 160-32-1) is described in the manuscript with three names.

ethyl caprylate (line 350)

ethyl octanoate (Table 3)

Octanoic acid, ethyl ester (Table 2)

Reply: Thank you for your valuable comment. We have standardized the expression of C1 compounds where they occur throughout the text to ethyl caprylate, and all revisions have been highlighted by using red colored text.

Line 360

“Among them, ethyl caproate had the highest OAV”

It is not correct. Please check the correctness.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to section 3.2.6: Among them, ethyl hexanoate had the highest OAV, followed by ethyl isovalerate, phenylethyl alcohol and 3-Methylbutyl 3-methylbutanoate ranked 3 and 4.

Line 361

One compound (C4: cas 123-66-0) is described in the manuscript with two names.

Butanoic acid, 3-methyl-ethyl ester (Table 2,3)

ethyl isovalerate (Line 361)

Reply: Thank you for your valuable comment. We have standardized the expression of C4 compounds where they occur throughout the text to ethyl hexanoate, and all revisions have been highlighted by using red colored text.

Cai, W., Tang, F., Shan, C., Yang, L., Zhan, Q., Zhao, X., & Ning, M. (2018). Changes in volatile compounds of fermented mixed drink using commercial yeast strain. Przemysl Chemiczny, 97(8), 1398-1405.

Han, X. Y., Zhao, Y. Y., Hu, B. W., Yang, H. Y., Peng, Q., & Tian, R. G. (2020). Influence of different yeast strains on the quality of fermented greengage (Prunus mume) alcoholic beverage and the optimization of fermentation conditions. Lwt-Food Science and Technology, 126, Article 109292. https://doi.org/10.1016/j.lwt.2020.109292

Wang, X. J., Liu, B. C., & Xie, Q. (2024). Analysis of genetic diversity in the free-pollinated F₁population of Hippophae rhamnoides L. subsp. mongolica 'Shenqiuhong' via simple sequence repeat markers. Genetic Resources and Crop Evolution. https://doi.org/10.1007/s10722-024-02284-w

Zhao, X. X., Xue, Y., Tang, F. X., Cai, W. C., Hao, G. F., & Shan, C. H. (2022). Quality improvement of jujube wine through mixed fermentation with Saccharomyces cerevisiae and Bacillus licheniformis. Lwt-Food Science and Technology, 164, Article 113444. https://doi.org/10.1016/j.lwt.2022.113444

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Effects of different yeasts on the physicochemical properties and aroma compounds of fermented sea buckthorn juice” discusses the fermentation of sea buckthorn juice using different yeast strains (Angel RW and RV171), which improves its taste, reduces acidity, enhances antioxidant activity, and produces characteristic volatile compounds, with RV171-FSBJ showing the highest fermentation efficiency and free radical scavenging ability. Increasing its antioxidant activity through fermentation could also provide health benefits, considering the role of antioxidants in protecting against chronic diseases. Article requires revisions. Comments below.

Comments:

Introduction

- It is necessary to add information about the medicinal properties of sea buckthorn.

Methods

- A subsection of reagents should be added.

- In spectrophotometric methods, more details should be provided about the equipment used in the study and the concentrations employed

Results and Discussion

- The results have been presented appropriately, however, there is a lack of discussion regarding the results obtained in the context of the scientific literature.

Conclusion

- What are its industrial implementations?

- Where can this be applied?

- Does it have an impact on human/animal health?

References

- The references require thorough revision. The citation style used is incorrect. The format for authors and journals should be standardized according to the journal's guidelines.

Author Response

The manuscript entitled “Effects of different yeasts on the physicochemical properties and aroma compounds of fermented sea buckthorn juice” discusses the fermentation of sea buckthorn juice using different yeast strains (Angel RW and RV171), which improves its taste, reduces acidity, enhances antioxidant activity, and produces characteristic volatile compounds, with RV171-FSBJ showing the highest fermentation efficiency and free radical scavenging ability. Increasing its antioxidant activity through fermentation could also provide health benefits, considering the role of antioxidants in protecting against chronic diseases. Article requires revisions. Comments below.

Reply: Thank you very much for the positive comments and constructive suggestions. Please find the following detailed responses to your comments and suggestions.

Comments:

Introduction

- It is necessary to add information about the medicinal properties of sea buckthorn.

Reply: Thank you for your thoughtful and valuable comment. We have added information about the medicinal properties of sea buckthorn in MS and all revisions have been highlighted by using red colored text.

Content added to introduction: Sea buckthorn berries are rich in nutrients and contain a variety of functional components and bioactive substances such as phenols, flavonoids, sterols, ascorbic acid, tocopherol, fatty acids, carotenoids, and organic acids. Yadav et al. showed that sea buckthorn berries have a high content of vitamin C (VC), almost 4–100 times that of other fruits. In addition, sea buckthorn is not only rich in VC, but also contains a large amount of oils and is rich in polyunsaturated fatty acids n-3, n-6 and n-9. Therefore, as a plant with both medicinal and culinary values, sea buckthorn has sig-nificant health benefits and has been traditionally used in traditional oriental medicine systems for the treatment of liver damage, cardiovascular disease and stomach disorders, among others. For example, sea buckthorn ex-tracts can exert antiplatelet effects by inhibiting thrombin-activated platelets, as well as antimicrobial effects by inhibiting adhesion and biofilm formation.

Methods

- A subsection of reagents should be added.

- In spectrophotometric methods, more details should be provided about the equipment used in the study and the concentrations employed

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to section 2.2:

2.2. Chemical and reagents

1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2'-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid (ABTS⁺) were supplied by Shanghai Macklin Biochemical Technology Co., Ltd; Folin-phenol, gallic acid standard, rutin standard were supplied by Shanghai Yuanye Biotechnology Co., Ltd; other chemical reagents (analytical purity) were provided by Tianjin Beifang Tianyi Chemical Reagent Factory.

Content added to section 2.6: The method of Zhao et al. (2023) was referred to for ABTS radical scavenging activity (ABTS-SA) and DPPH radical scavenging activity (DPPH-SA) detection with slight modification. The ABTS stock solution was prepared by taking 0.20 mL of ABTS and adding 0.20 mL of potassium persulfate and reacting under dark conditions for 12 h. The solution was diluted with anhydrous ethanol (approx. 40-50 times) to A₇₃₄=0.70±0.02. Take 0.10 mL of sample solution and add 3.00 mL of ABTS reserve solution and shake well, let it stand at 35 ± 2℃ for 10 min, and then measure the absorbance value (A_s) at 734 nm. Anhydrous ethanol was used to replace the ABTS reserve solution in the control group (A_d), and distilled water was used to replace the sample in the blank group (A₀). 20 μL of sample solution was added to 80 μL of deionized water and 100 μL of 0.20 mM DPPH solution, and placed at 37℃ for 45 min, and the absorbance value (A_s) was measured at 517 nm. Methanol was used as a control in-stead of DPPH solution (A_d), and distilled water was used as a blank instead of sample (A₀). Both ABTS+ and DPPH were calculated using the formula (1):

% clearance =(1-(A₀-(A_s-A_d)/A₀)) *100% (1)

The reducing power capacity (RP-CA) assay was performed by referring to the method of (Kwaw et al., 2018) with slight modification. The sample solution was diluted 100-fold to 1 mL, and 0.05 mL of HCl (0.01 M), 0.4 mL of potassium ferricyanide (0.02 M), 0.4 mL of 0.02 M FeCl3 and 0.7 mL of distilled water were added to the sample solution, and the mixture was incubated in an incubator at 37℃ in darkness for 30 min, and the absorbance value was measured at 720 nm. The RP-CA of FSBJs is expressed in millimoles of ascorbic acid.

The hydroxyl free radical scavenging capacity (•OH-SA) assay was performed by referring to the method of Wen et al. (2025) with slight modification. Take 1 mL of the sample solution, add 1.5 mL of 1.8 mM ethanol-salicylic acid solution, 2 mL of 1.8 mM ferrous sulfate solution and 0.1 mL of 0.3% hydrogen peroxide, mix well, and then take a water bath at 37℃ for 30 min, and then determine the absorbance value (A_s) at 510 nm. The control group was made by replacing the three reagent solutions with distilled water (A_d), and the blank group was made by replacing the sample with distilled water (A₀). •OH-SA were calculated using the formula (2):

% clearance =(1-(A_s-A_d)/A₀) *100% (2)

The above antioxidant tests were carried out using TAS-964 enzyme marker (Taian Technology Co., Ltd.) for absorbance value detection, and each sample was repeated three times.

Results and Discussion

- The results have been presented appropriately, however, there is a lack of discussion regarding the results obtained in the context of the scientific literature.

Reply: Thank you for your thoughtful and careful work. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to section 3.1.1: Therefore, different yeast strains showed different sugar metabolizing abilities, among which the RW strain had higher sugar metabolizing ability and showed higher bioconversion efficiency. Zhao et al. (2022) selected 10 commercial yeasts to ferment jujube wine and found that date wine fermented by different yeast strains presented different sugar contents, which was consistent with the results of this paper.

Content added to section 3.1.3: The reason for the RW-FSBJ being higher than the RV-FSBJ may be that the RW is widely used in the fermentation of fruit wines such as grapes, blueberries, and strawberries, and therefore it has better fermentation properties. The differences in TFC, TPC, and TAC among the three FSBJs may result from differences in the individual adaptability of yeasts and their abilities to produce hydrolase.

Content added to section 3.2: A large number of research papers and reviews have shown that yeast fermentation for aroma production is closely related to certain key yeast genes in the fermentation process. In addition many fruit and vegetable juices often do not have the ability to convert volatile compounds precursors into aroma compounds on their own, but certain yeast strains possess the necessary enzymes to facilitate the conversion of precursors to release aroma. This may therefore be the reason why two different yeasts of the same strain produce different volatile compounds.

Conclusion

- What are its industrial implementations?

- Where can this be applied?

- Does it have an impact on human/animal health?

Reply: Thank you for your thoughtful and constructive comment. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to introduction: With the natural antioxidant properties, flavor advantages and high nutritional value of sea buckthorn resources, this product has a broad prospect in the field of functional beverages, nutritional supplements, etc. It is especially suitable for the health beverage market, and the development of regional characteristic agricultural products. In the future, we can focus on the optimization of fermentation parameters, flavor stability control and the synergistic fermentation effect with other probiotics, and further carry out animal experiments such as cell, nematode and mouse to evaluate the safety and functionality of the product, so as to accelerate the industrialization process in the food industry, and to contribute to the high-value utilization of sea buckthorn resources and the development of the whole industrial chain.

References

- The references require thorough revision. The citation style used is incorrect. The format for authors and journals should be standardized according to the journal's guidelines.

Reply: Thank you for your thoughtful and constructive comment. We have made modifications in the text and the revision have been highlighted by using red colored text.

Kwaw, E., Ma, Y. K., Tchabo, W., Apaliya, M. T., Wu, M., Sackey, A. S., Xiao, L. L., & Tahir, H. E. (2018). Effect of lactobacillus strains on phenolic profile, color attributes and antioxidant activities of lactic-acid-fermented mulberry juice. Food Chemistry, 250, 148-154. https://doi.org/10.1016/j.foodchem.2018.01.009

Wen, J., Huang, R. J., Li, S., Jiang, L., Shao, L. H., Zhang, Q., & Shan, C. H. (2025). Polysaccharides from sea buckthorn - Ultrasound-assisted enzymatic extraction, purification, structural characterization, and antioxidant activity analysis. Food Chemistry-X, 26, Article 102265. https://doi.org/10.1016/j.fochx.2025.102265

Zhao, X. X., Tang, F. X., Cai, W. C., Peng, B., Zhang, P. L., & Shan, C. H. (2023). Effect of fermentation by lactic acid bacteria on the phenolic composition, antioxidant activity, and flavor substances of jujube-wolfberry composite juice. Lwt-Food Science and Technology, 184, Article 114884. https://doi.org/10.1016/j.lwt.2023.114884

Zhao, X. X., Xue, Y., Tang, F. X., Cai, W. C., Hao, G. F., & Shan, C. H. (2022). Quality improvement of jujube wine through mixed fermentation with Saccharomyces cerevisiae and Bacillus licheniformis. Lwt-Food Science and Technology, 164, Article 113444. https://doi.org/10.1016/j.lwt.2022.113444

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

This manuscript is dealing with the investigation of different yeasts usage in the fermentation of sea buckthorn juice its physicochemical properties and aromatic compounds.

In the abstract explain all abbreviations when you introduce them for the first time.

In the abstract are missing numerical values of the most important results. Insert it.

Explain in the introduction section is alcohol present in the fermented sea buckthorn juice.

In the subsection 2.1. are missing information regarding sea buckthorn berries.

What is geographical origin? Insert it.

What was agro-technical approach during the plant growing? Insert it.

Did you use any fertilizers? Inert it.

You have to be precise when you giving information regarding cultivar of fruit which was used. It is not sound scientific when you said according to the producer . . . Variety have to be confirmed by the expert. Correct it.

In the subsection 2.2. explain following.

What is the reason to use two different yeast with same strain together in one fermentation?

Express amount of added yeast in g per hL .

Did you have gelation process during the fermentation? You made a paste from fruit in blender and release pectin. Explain.

Did you add any substances to prevent growth of unwanted microorganisams? Explain.

What was the final content of alcohol expressed in vol%?

How did you monitored alcohol content to know when to stop fermentation? Explain

How long fermentation did take? Explain.

In the subsection 2.5. give detailed explanation of methods which were used.

In which units were expressed results in methods in subsection 2.5.

Line 137 Did you meant on the figure 1

Explain in the discussion what is the reason for different sugar content when you use two different yeasts of same strain.

Explain in the discussion why first yeast showed better results in the table 1.

According to which phenolic compounds were expressed TPC, TFC and TAC. Insert it, it is very important.

Highlight in the discussion that different yeasts of same strain were responsible for various antioxidant activity and phenolic content in wine. Kindly consider to cite Food and Feed Research, 51 (1), 119-129, 2024

In the subtitle of subsection 3.1.4. change antioxidant to antiradical

In the subsection 3.2. explain what is the reason for different aromatic profile obtained from two different yeasts from same strain.

Make figure 5 in better resolution.

Highlight what are unwanted volatile compound which could influence on the sensory properties.

Make figure 6 in better resolution.

Author Response

Dear Authors,

This manuscript is dealing with the investigation of different yeasts usage in the fermentation of sea buckthorn juice its physicochemical properties and aromatic compounds.

Reply: Thank you very much for the positive comments and constructive suggestions. Please find the following detailed responses to your comments and suggestions.

In the abstract explain all abbreviations when you introduce them for the first time.

Reply: Thank you for your thoughtful and constructive comment. We have made modifications in the text and the revision have been highlighted by using red colored text.

In the abstract are missing numerical values of the most important results. Insert it.

Explain in the introduction section is alcohol present in the fermented sea buckthorn juice.

Reply: Thank you for your thoughtful and constructive comment. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to abstract: Physicochemical analyses showed that RV171-FSBJ had the highest total reducing sugar (0.069 ± 0.02 g/L) and total acid content (1.86 ± 0.03 g/L), as well as the highest fermentation efficiency and free radical scavenging capacity (1, 1-diphenyl-2-picrylhydrazyl (DPPH) 98.54±0.03%, 2, 2'-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 88.35±0.14%, ·OH 48.61±0.4%). RWRV-FSBJ had the highest content of functional compounds (total flavonoid content (TFC) 176.09±0.44 μg/mL, Total phenolic content (TPC) 157.9 ±1.35 μg/mL, Total anthocyanin concentration (TAC) 0.04±0.004 μg/mL) and good color (L^* 50.53±0.04, a^*27.98±0.04, b ^*173.64±0.34).

In the subsection 2.1. are missing information regarding sea buckthorn berries.

What is geographical origin? Insert it.

What was agro-technical approach during the plant growing? Insert it.

Did you use any fertilizers? Inert it.

In the subsection 2.2. explain following.

Reply: Thank you for your valuable and careful comment. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to section 2.1: Frozen sea buckthorn berries were purchased in November 2022 from a farmer's market (Shihezi City, Xinjiang, China). The berries were grown in Korla, Xinjiang, China (without pesticide spraying), and workers harvested the fruits in October 2022 when they were ripe and similar in shape and size, with intact skins, no mechanical damage, no pests or diseases, and no residues on the stalks. Immediately after harvest, the berries were placed in -20℃ cold storage for freezing and preservation. The variety of sea buckthorn berries called Hippophae rhamnoides L. subsp. mongolica ‘Shenqiuhong’ (Wang et al., 2024). The berries were stored at -20℃ until use.

What is the reason to use two different yeast with same strain together in one fermentation?

Reply: Thank you for your thoughtful and constructive comment. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to the introduction: In the present study, two commercial yeast strains were selected to prepare FSBJ. RV171 and RW are both commonly used commercial yeasts. RV171 is a low-alcohol sweet brewing active dry yeast, suitable for fermentation of high acidity fruits, with certain conversion ability to malic acid, and at the same time, it can generate more esters and glycerol substances, adding alcohol and ester flavor to the fermentation broth; RW has excellent tannin and pigment extraction power, and it can retain the original color of the fruits, highlighting the fruity aroma of the fermentation broth. Therefore RW and RV171 are suitable for fermenting most fruits, providing ester and alcohol flavor to the fer-mentation broth, and fermentation is stable and durable.

Express amount of added yeast in g per hL .

Reply: Thank you for your thoughtful and constructive comment. We have made modifications in the text and the revision have been highlighted by using red colored text.

Did you have gelation process during the fermentation? You made a paste from fruit in blender and release pectin. Explain.

Reply: Thank you for your careful comments. As you say some fruits will come out gelatinized first after pulping, such as jujubes. However, sea buckthorn did not gelatinize when making sea buckthorn fruit into sea buckthorn juice using a wallbreaker due to its high oil and water content, and it did not gelatinize during the yeast fermentation process.

Did you add any substances to prevent growth of unwanted microorganisams? Explain.

Reply: Thank you for your careful comments. As you say some fermented drinks are made to ensure that they are not contaminated by stray bacteria during the fermentation process, so preservatives etc are added. However, due to the high acidity of sea buckthorn juice, it is inherently preservative. In addition, our product highlights the natural antioxidant properties and the high nutritional value of sea buckthorn, so we have not added substances to prevent the growth of harmful microorganisms during fermentation.

What was the final content of alcohol expressed in vol%?

Reply: Thank you for your thoughtful and careful comment. The alcohol content of the product at the end of fermentation was 0.45±0.05%, as we have added in subsection 2.3 of the MS. And the revision have been highlighted by using red colored text.

How did you monitored alcohol content to know when to stop fermentation? Explain

Reply: Thank you for your careful comments. We determined the time of cessation of fermentation based on the soluble solids content and the first method in GB 5009.225-2016 ″Determination of Ethanol Concentration in National Standard Liquor for Food Safety” to determine the alcohol content of the beverage to determine the time to stop fermentation.

How long fermentation did take? Explain.

Reply: Thank you for your careful comments. After a large number of experiments, we found that the alcoholic strength of the beverage could be reached after about 20 h of fermentation.

In the subsection 2.5. give detailed explanation of methods which were used.

Reply: Thank you for your thoughtful and constructive comment. We have made modifications in the text and the revision have been highlighted by using red colored text.

Content added to section 2.6: The method of Zhao et al. (2023) was referred to for ABTS radical scavenging activity (ABTS-SA) and DPPH radical scavenging activity (DPPH-SA) detection with slight modification. The ABTS stock solution was prepared by taking 0.20 mL of ABTS and adding 0.20 mL of potassium persulfate and reacting under dark conditions for 12 h. The solution was diluted with anhydrous ethanol (approx. 40-50 times) to A₇₃₄=0.70±0.02. Take 0.10 mL of sample solution and add 3.00 mL of ABTS reserve solution and shake well, let it stand at 35±2℃ for 10 min, and then measure the absorbance value (A_s) at 734 nm. Anhydrous ethanol was used to replace the ABTS reserve solution in the control group (A_d), and distilled water was used to replace the sample in the blank group (A₀). 20 μL of sample solution was added to 80 μL of deionized water and 100 μL of 0.20 mM DPPH solution, and placed at 37℃ for 45 min, and the absorbance value (A_s) was measured at 517 nm. Methanol was used as a control in-stead of DPPH solution (A_d), and distilled water was used as a blank instead of sample (A₀). Both ABTS+ and DPPH were calculated using the formula (1):

% clearance =(1-(A₀-(A_s-A_d)/A₀)) *100% (1)

% clearance =(1-(A_s-A_d)/A₀) *100% (2)

The above antioxidant tests were carried out using TAS-964 enzyme marker (Taian Technology Co., Ltd.) for absorbance value detection, and each sample was repeated three times.

In which units were expressed results in methods in subsection 2.5.

Reply: Thank you for your helpful and careful comments. The units of the DPPH-SA，ABTS-SA and OH-SA are % and the RP-CA of FSBJs is expressed in millimoles of ascorbic acid.