Investigation of wine fermentation of three-leaf cayratia (<i>Cayratia trifolia</i> L.) using <i>Saccharomyces cerevisiae</i> 2.1

Doan Thi Kieu Tien; Le Doan Quoc Binh; Huynh Thi Ngoc Mi; Nguyen Ngoc Thanh; Bui Hoang Dang Long; Ngo Thi Phuong Dung; Ha Thanh Toan; Huynh Xuan Phong

doi:10.12688/f1000research.129075.1

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Research Article

Investigation of wine fermentation of three-leaf cayratia (Cayratia trifolia L.) using Saccharomyces cerevisiae 2.1

[version 1; peer review: awaiting peer review]

Doan Thi Kieu Tien¹, Le Doan Quoc Binh², Huynh Thi Ngoc Mi¹, [...] Nguyen Ngoc Thanh², Bui Hoang Dang Long², Ngo Thi Phuong Dung², Ha Thanh Toan², Huynh Xuan Phong ²

Doan Thi Kieu Tien¹, Le Doan Quoc Binh², [...] Huynh Thi Ngoc Mi¹, Nguyen Ngoc Thanh², Bui Hoang Dang Long², Ngo Thi Phuong Dung², Ha Thanh Toan², Huynh Xuan Phong ²

PUBLISHED 21 Mar 2023

Author details Author details

¹ Faculty of Biological, Chemical and Food Technology, Can Tho University of Technology, Can Tho City, Vietnam
² Institute of Food and Biotechnology, Can Tho University, Can Tho City, Vietnam

Doan Thi Kieu Tien
Roles: Conceptualization, Formal Analysis

Le Doan Quoc Binh
Roles: Data Curation, Methodology

Huynh Thi Ngoc Mi
Roles: Methodology, Project Administration

Nguyen Ngoc Thanh
Roles: Resources, Validation

Bui Hoang Dang Long
Roles: Resources, Writing – Original Draft Preparation

Ngo Thi Phuong Dung
Roles: Conceptualization, Methodology

Ha Thanh Toan
Roles: Writing – Review & Editing

Huynh Xuan Phong
Roles: Investigation, Methodology, Supervision, Writing – Review & Editing

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Abstract

Background: Cayratia trifolia has been extensively studied for its bioactive components and medicinal properties. This study was carried out to evaluate the fermentation ability of Saccharomyces cerevisiae 2.1 yeast to determine suitable fermentation conditions. Methods: The initial sugar content for three-leaf cayratia fermentation and fermentation efficiency were calculated. The temperature for three-leaf cayratia fermentation, incubation time, and preliminary 1 liter of three-leaf cayratia wine fermentation were determined. All treatments showed that the total sugar content decreased after 9 days of fermentation compared to the initial level. Temperature during fermentation has a direct effect on yeast activity. Temperature increases the growth of yeast and speed of enzyme activity. The fermentation time changes depending on temperature, initial soluble solid contents, and yeast strains, leading to changes in the ethanol content after the end of fermentation.
Results: The results showed that the S. cerevisiae 2.1 strain was able to ferment at room temperature with an initial pH of 4.5, 24 °Brix, and a yeast density of 107 cells/mL. The appropriate fermentation conditions determined for S. cerevisiae 2.1 yeast were 24 °Brix and incubation at room temperature (28-33°C) with a fermentation time of 11 days.
Concusion: In 1 liter scale fermentation, three-leaf cayratia wine had 9.46% (v/v) and the fermentation efficiency was 90.34%. The wine produced had a unique color, flavor, and aroma that met the sensory evaluation of the Vietnamese standard TCVN-3217:79.

Keywords

Cayratia trifolia, ethanol fermentation, Saccharomyces cerevesiae, three-leaf cayratia wine

Corresponding author: Huynh Xuan Phong

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2023 Kieu Tien DT et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Kieu Tien DT, Quoc Binh LD, Ngoc Mi HT et al. Investigation of wine fermentation of three-leaf cayratia (Cayratia trifolia L.) using Saccharomyces cerevisiae 2.1 [version 1; peer review: awaiting peer review]. F1000Research 2023, 12:312 (https://doi.org/10.12688/f1000research.129075.1) First published: 21 Mar 2023, 12:312 (https://doi.org/10.12688/f1000research.129075.1) Latest published: 23 Sep 2025, 12:312 (https://doi.org/10.12688/f1000research.129075.5)

1. Introduction

Wine, an indispensable drink that contributes significantly to maintaining and supporting human health, is invested in various ingredients. In the past, France and Italy were the cradles of the wine industry. Countries like Australia and New Zealand have created wine brands with modern technology and high quality. In the Asian market, particularly in Vietnam, wine has been conquering the consumer thanks to its abundant production, competitive price, and novel taste. Wines are fermented alcoholic beverages made from various base ingredients, such as apple, banana, papaya, mango, apricot, pineapple, and jackfruit juice. These are classified as grape wine, fruit wine, berry wine, vegetable wine, plant wine, and raisin wine, as well as flavors from flowers and herbs. Typical wine, natural wines (9–14% alcohol), or dessert and appetizer wines (15–21% alcohol) contain ethyl alcohol, sugar, acids, higher alcohols, tannins, aldehydes, esters, amino acids, minerals, vitamins, anthocyanins, and so on (Amerine et al., 1980).

Three-leaf cayratia (Cayratia trifolia) has been extensively studied for its bioactive components and medicinal properties. In particular, thanks to the abundant source distributed throughout the Mekong Delta of Vietnam, its antioxidant and polyphenolic compound contents were not significantly changed after fermentation (Doan et al., 2018d). Additionally, its color and flavor are specific to three-leaf cayratia wine, particularly proven in our previous studies (Doan et al., 2018d, 2019b), and C. trifolia berries are becoming an expected source of winemaking materials from grapes in Vietnam. However, the isolated thermotolerant yeast from C. trifolia berries is mainly used to ferment three-leaf cayratia wine. At the same time, non-thermotolerant Saccharomyces cerevisiae is the primary source for winemaking at room temperature. Therefore, this study aimed to investigate and evaluate the fermentability of Saccharomyces cerevisiae to three-leaf cayratia juice isolated from rice wine starters. In addition, 1 liter of C. trifolia juice was fermented to complete the larger-scale fermentation process of three-leaf cayratia wine.

According to Ngo et al. (2005), 50 yeast strains have been isolated from wine yeast starters in the Mekong Delta, of which S. cerevisiae 2.1 has the highest fermentability. Since then, strain S. cerevisiae 2.1 has been selected for application in wine fermentation from three-leaf cayratia.

2. Methods

2.1 Materials

Wine fermentation of three-leaf Cayratia (Cayratia trifolia L.) using Saccharomyces cerevisiae 2.1 was conducted between 12/04/2022 and 25/10/2022. Three-leaf cayratia samples were collected from shiny ripe, dark black, and undamaged berries in the Mekong Delta, Vietnam. The fresh berries were brought to the Laboratory of the Food Biotechnology at the Biotechnology Research and Development Institute, Can Tho University. The selected un-crushed ripe samples were washed several times with tap water, rinsed with distilled water, and squeezed out of flesh into juice to conduct further experiments.

S. cerevisiae 2.1 strain was isolated from a rice wine starter and stored at the Laboratory of Food Biotechnology at the Biotechnology Research and Development Institute, Can Tho University (Ngo et al., 2005). A single colony of yeast was inoculated in Yeast Extract Peptone- Dextrose (YPD broth) (yeast extract 0.5%, peptone 0.5%, glucose 2.0%; sterilized at 121°C for 15 min) and shaken at 180 rpm at 30°C to 109 cells/mL of yeast inoculum level (using Haemocytometer – Neubauer).

2.2 Determination methods of three-leaf cayratia fermentation

2.2.1 Determination of initial sugar for three-leaf cayratia fermentation

Three-leaf cayratia juice was added to NaHSO₃ (140 mg/L) and left alone for two hours after being adjusted to 20, 24, and 28 °Brix (using sucrose provided by Bien Hoa Sugar Joint Stock Company) and pH 4.5 (Doan et al., 2018b, 2018d). 1 mL of S. cerevisiae was inoculated to 99 mL of three-leaf cayratia juice (107 cells/mL after inoculation) in a 250 mL Erlenmeyer flask. The flask was sealed with a water lock and incubated at room temperature (in triplicate). The changes in pH, °Brix, and ethanol contents were measured after 9 days of fermentation Nguyen (2007). The total sugar content of the fermentation process was analyzed according to Nielsen (2010), and fermentation efficiency was calculated.

Fermentation efficiency is calculated according to ethanol fermentation efficiency on the amount of sugar used, H(%)=(Et°*0.789*10)/(S*0.5111) where H: fermentation efficiency; Et°: alcohol content obtained (% v/v); 0.789: density of ethanol (0.789 g/cm³); 10: conversion factor; S: the actual amount of sugar used in glucose (following the phenol-sulfuric method); 0.5111: the grams of theoretical ethanol obtained from 1 gram of glucose.

2.2.2 Determination of temperature for three-leaf cayratia fermentation

Three-leaf cayratia juice was prepared with °Brix, selected from a previous experiment. After inoculation with 1 mL yeast culture, three-leaf cayratia juice was fermented at 25°C, room temperature (28–33°C), and 35°C for 9 days. The changes in pH, °Brix, and ethanol contents were determined as described above.

2.2.3 Determination of incubation time for three-leaf cayratia fermentation

Three-leaf cayratia juice was prepared with °Brix selected from the experiment as mentioned inSection 2.2.1, and incubated at the selected temperature from the experiment in Section 2.2.2, on days 7, 9, 11, and 13. The changes in pH, °Brix, and ethanol content were determined (see Section 2.2.1).

2.2.4 Preliminary 1 liter of the three-leaf cayratia wine fermentation

The initial sugar content, fermentation temperature, and fermentation time were determined based on the selective results of previous screening tests to ferment 1 liter of three-leaf cayratia juice (triplicate). Sensory evaluation of wine was based on the criteria of clarity, color, aroma, taste, and overall confidence according to the TCVN (Vietnamese Tiêu chuẩn Việt Nam) - Vietnam National Standard 3217:79, which is done through a sensory board consisting of 10 members at the Biotechnology Research and Development Institute Can Tho University. The changes in pH, °Brix, ethanol content, and total sugar content were determined (see Section 2.2.1).

2.2.5 Statistical analysis

The analyzed data were processed using Excel 2013 (RRID:SCR_016137) (Microsoft Inc., USA). The variance and the Lease Significant Difference (LSD) were analyzed using SPSS Version 27.0.1.0 (RRID:SCR_002865) and Statgraphics Centurion XV - https://www.statgraphics.com/ (Manugistics Inc., USA).

3. Results

3.1 The initial sugar of the three-leaf cayratia fermentation using S. cerevisiae 2.1

Three-leaf cayratia fermentation processes were conducted with an initial pH of 4.5 and an initial sugar content of 20, 24, and 28 °Brix, respectively. As to the results, all treatments show that the total sugar contents decreased after 9 days of fermentation compared to the initial level (Table 1). The ethanol content was the highest at 7.17% v/v in the 24 °Brix treatment, which is a significant difference compared to the 20 and 28 °Brix treatments. When the sugar concentration is too high, yeast cells shrink and die, lowering the alcoholic fermentation efficiency. In principle, the rate of fermentation and the maximum amount of ethanol decreased when the sugar concentration was increased. However, a particular type of yeast strain can be selected, conditioned to grow at higher sugar concentrations (>30% sugar), and adapted (Matei & Kosseva, 2017).

Table 1. The effects of °Brix on the fermentability of S. cerevisiae 2.1.

Initial °Brix	Initial sugar content (g/L)	Used sugar content (g/L)	Residual sugar content (g/L)	Fermentation efficiency (%)	Ethanol content (% v/v) at 20°C
20	142.23	116.67	25.56	82.47^b	6.23^b
24	187.98	121.63	66.35	91.00^a	7.17^a
28	229.47	115.60	113.87	75.13^c	5.64^b

The total sugar content of the samples was determined using the phenol-sulfuric acid method (Nielsen, 2010), measured at a wavelength of λ=490 nm. The linear regression equation was y=0.0095x+0.0049. The total sugar content after fermentation reflected the fermentation efficiency of Saccharomyces cerevisiae 2.1. The 24 °Brix treatment had the highest fermentation conversion efficiency of 91.00% produced by S. cerevisiae 2.1, 82.47% at 20 °Brix treatment, and 75.13% at 28°Brix treatment, which was significantly different from 5% (p<0.05). The fermentation conversion efficiency was proportional to the ethanol content (Table 2). The highest alcohol content (7.17 %) was produced in the 24 °Brix treatment. S. cerevisiae strains are tolerant to low pH, high sugar content, and high ethanol concentrations compared to other species, thus reducing the risk of bacterial contamination during industrial fermentation (Nevoigt, 2008).

Table 2. The effects of incubation time on fermentation.

Temperature (°C)	Initial °Brix	°Brix after fermentation	Ethanol content (% v/v) at 20°C
Room temp. (28–33°C)	24	12.17	6.94^a
25	24	14.17	6.16^b
35	24	16.83	4.28^c

3.2 Temperature for three-leaf cayratia fermentation

The ethanol content was significantly different depending on the incubation temperature (Doan et al., 2019a). Temperature during fermentation has a direct effect on yeast activity. In this study, the ethanol content was produced at 6.94% (v/v) at room temperature, higher than that of 25°C (6.16% v/v) and 35°C (4.28% v/v). The optimum temperature for the growth and multiplication of ordinary yeast is 30°C and is usually inhibited at temperatures higher than 32°C.

Temperature increases the growth of yeast and speed of enzyme activity. Because of the increased membrane fluidity, cell sensitivity to the toxic effects of alcohol increases with temperature. Thus, yeast viability may rapidly decline at temperatures above 20°C during wine fermentation. Normal cider production occurs at 20–25°C and lasts for 1–4 weeks (Waites et al., 2001).

3.3 Fermentation time for three-leaf cayratia wine

The fermentation time changes depending on temperature, initial soluble solid contents, and yeast strains, leading to changes in the ethanol content after the end of fermentation (Figure 1). The results showed the highest ethanol content at 11 days compared to 13, 9, and 7 days with 8.93%, 8.54%, 6.94%, and 6.23% v/v, respectively. There were no significant differences between 7 to 9 days and 11 to 13. However, these values were significantly different between days 7 and 11 (Table 3).

Figure 1. Three-leaf cayratia wine fermented by S. cerevisiae 2.1.

Table 3. The fermentation temperature results of S. cerevisiae 2.1.

Incubation time (day)	Initial °Brix	°Brix after fermentation	Ethanol content (% v/v) at 20°C
7	24	14.17	6.94^b
9	24	12.50	6.23^b
11	24	9.33	8.93^a
13	24	9.33	8.54^a

The end time of fermentation was inversely proportional to the degree of Brix after fermentation. The longer the time, the lower the °Brix, and the higher the ethanol concentration produced. Initially, when the ethanol concentration was quite low, the ethanol concentration began to increase, and the sugar content decreased.

3.4 Preliminary 1 liter of the three-leaf cayratia wine fermentation

Fermentability of fruit wine was tested with a volume of 1 liter, surveyed with a density of S. cerevisiae 2.1 at 107 cells/mL for 11 days at room temperature, and the juice was adjusted to pH 4.5 and 24 °Brix. From the above experiments, it can be seen that S. cerevisiae 2.1 produced an ethanol content of 9.49% (v/v), and the fermentation efficiency was quite high, reaching 90.34%.

After fermentation, the sensory properties were evaluated according to the criteria of TCVN 3217-79. Three-leaf wine was evaluated as good in clarity, color, aroma, and taste, reaching 4.4, 4.2, and 3.8 points, respectively. Sensory evaluation results show that the wine had no strange, cloudy smell, and the color and odor were very specific to the product. Compared to the Vietnamese standard TCVN-3217:79, the sensory quality assessment of left-sided wine received a good rating.

4. Discussion

In a previous study, we isolated 151 strains of yeast from 53 samples of C. trifolia in the Mekong Delta. Of these, 30 isolates were highly fermentative and produced ethanol concentrations between 6.0 and 9.9% (v/v) (Doan et al., 2018c). The fermentability of thermotolerant yeast isolates from three-leaf juice showed that the survey with 3.6 of the natural pH, 22 °Brix by adding sucrose, the initial yeast density of 106 cells/mL, and 7 days of incubation time at 37°C—Saccharomyces sp. CT3.2 has a fast filling time for the gas column in the Durham tube (after 12 h), and the ethanol content after fermentation was not high (5.4% v/v). In contrast, Saccharomyces sp. HG1.3 has a slower gas column filling time (after 18 h) but produces the highest ethanol content (9.9% v/v) (Doan et al., 2018b). Meanwhile, with initial fermentation conditions of pH 4.5, and 20 °Brix fermented at 35°C for 6 days, Saccharomyces sp. HG1.3 gave the ethanol content of post-fermentation three-leaf fruit wine 12.0% v/v (Doan et al., 2018d).

The fermentability of thermotolerant yeasts was investigated at pH 4.5, 22 °Brix, 107 cells/ml of the initial three-leaf juice, and at 37°C for 7 days. The ethanol contents of three-leaf wine produced by CM3.2, CM3.3, and BT1.2 were 8.95%, 7.01%, and 6.79% v/v, respectively (Torija et al., 2003; Doan et al., 2018a).

The fermentation of 1-liter results shows that the ethanol content met the Vietnam Standard 6-3:2010/BYT of the Ministry of Health for wine (Vietnam National Standard 3217:79, 1979), which is also similar to fermentation studies on watermelon wine (Nguyen, 2010; Ngo et al., 2011), pineapple wine (Nguyen et al., 2013), and sim wine (Nguyen, 2010). However, the concentration value is lower than that of fruit wine fermented by a tolerant yeast strain isolated from fruit but aged at 35°C; for example, the strain S. cerevisiae AG2.1 is 11.36% (v/v) (Doan et al., 2018c), and S. cerevisiae CM3.2 is 12.46% v/v (Doan et al., 2019b).

The fermentation of 1 L results shows that the ethanol content meets the Vietnam Standard 6-3:2010/BYT of the Ministry of Health for wine, which is also similar to fermentation studies on watermelon wine (Ngo et al., 2011), pineapple wine (Nguyen et al., 2013), and sim wine (Nguyen, 2010). However, the concentration value is lower than that of fruit wine fermented by a tolerant yeast strain isolated from fruit but aged at 35 °C; for example, the strain S. cerevisiae AG2.1 is 11.36% (v/v) (Doan et al., 2018c), S. cerevisiae CM3.2 is 12.46% v/v (Doan et al., 2019b).

5. Conclusion

This study aimed to test the fermentative capacity of the S. cerevisiae 2.1 strain isolated from a starter on three-leaf cayratia juice. However, microbiological and physicochemical criteria have not yet been implemented. The results showed that the S. cerevisiae 2.1 strain was able to ferment at room temperature with an initial pH of 4.5, 24 °Brix, and yeast density of 107 cells/mL. Preliminary 1 liter of fermentation for 11 days produced an ethanol content of 9.49% (v/v).

5.1 Ethical considerations

This study was approved by the research group at Can Tho University and Can Tho University of Technology. All authors mentioned in the manuscript have agreed to authorship, read and approved the manuscript, and provided consent for submission and subsequent publication of the manuscript. The authors declare no conflicts of interest.

Data availability statement

Underlying data

Figshare: Wine fermentation of three-leaf cayratia. figshare. Dataset, https://doi.org/10.6084/m9.figshare.22256188.v1 (Phong, 2023).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

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Doan TTK, Huynh MTN, Nguyen DD, et al.: Total polyphenol content and antioxidant capacity of Cayratia trifolia (L) Domin berries before and after fermentation using thermotolerant yeast Saccharomyces cerevisiae HG1.3, in Vietnamese. Vietnam Journal of Science and Technology. 2018d; 60(8): 44–60.
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Phong H: Investigaton of wine fermentation of three-leaf cayratia (Cayratia trifolia L.) using Saccharomyces cerevisiae 2.1. figshare. Dataset. 2023. Publisher Full Text
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Version 5

VERSION 5 PUBLISHED 21 Mar 2023

Author details Author details

¹ Faculty of Biological, Chemical and Food Technology, Can Tho University of Technology, Can Tho City, Vietnam
² Institute of Food and Biotechnology, Can Tho University, Can Tho City, Vietnam

Doan Thi Kieu Tien
Roles: Conceptualization, Formal Analysis

Le Doan Quoc Binh
Roles: Data Curation, Methodology

Huynh Thi Ngoc Mi
Roles: Methodology, Project Administration

Nguyen Ngoc Thanh
Roles: Resources, Validation

Bui Hoang Dang Long
Roles: Resources, Writing – Original Draft Preparation

Ngo Thi Phuong Dung
Roles: Conceptualization, Methodology

Ha Thanh Toan
Roles: Writing – Review & Editing

Huynh Xuan Phong
Roles: Investigation, Methodology, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

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Kieu Tien DT, Quoc Binh LD, Ngoc Mi HT et al. Investigation of wine fermentation of three-leaf cayratia (Cayratia trifolia L.) using Saccharomyces cerevisiae 2.1 [version 1; peer review: awaiting peer review]. F1000Research 2023, 12:312 (https://doi.org/10.12688/f1000research.129075.1)

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Javier Alonso-Del-Real, Instituto de Biomedicina de Valencia, Valencia, Spain; Instituto de Agroquimica y Tecnología de Alimentos, Paterna, Spain
Muazaz Azeez Hasan AL-Hadeethi, University of Baghdad, Baghdad, Iraq

Susan Abdul Raheem Hasan, University of Baghdad, Baghdad, Iraq
Shatha Abdullah Allaith, University of Kerbala Faculty of Agriculture (Ringgold ID: 125663), Kerbala, Iraq
Nguyen Khoi Hong Nguyen, Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam

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27 Sep 2025 | for Version 5

Susan Abdul Raheem Hasan, Biology, University of Baghdad, Baghdad, Iraq

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Microbiologist and expert in biological fermentation processes, with a particular focus on probiotics and plants.

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08 Sep 2025 | for Version 4

Nguyen Khoi Hong Nguyen, Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam

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Approved

I appreciate the efforts of the authors in the research process, however, if the research could add the following content, it would be more complete:
- The author's introduction also stated the purpose of the research, however, What are the benefits of Cayratia trifolia wine? Before and after fermentation, how have the bioactive components in Cayratia trifolia been published in previous studies? If the authors could add more to the overview, it would be better.
- In my personal opinion, the author's analysis and evaluation method needs to be presented in more detail.
- The explanation of the basis of the results is mainly based on the authors' previous research, if other studies are added, it would be more convincing.
- The conclusion has summarized the data achieved by the topic, if 1-2 sentences about the potential use or value that the research brings are added, it would be better.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

My area of expertise is food technology, particularly in processing sectors such as seafood, meat, fruits and vegetables, fermentation, and preservation technology. In my assessment, the content of this study is acceptable, as Cayratia trifolia represents a novel plant source. The research provides a dataset that could serve as a valuable reference foundation for future studies.

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23 Sep 2025

Huynh Xuan Phong, Can Tho University, Vietnam

On behalf of the authors, I would like to express our sincere gratitude to the Reviewer for the valuable comments.

1. The author's introduction also stated the purpose of the research, however, what are the benefits of Cayratia trifolia wine? Before and after fermentation, how have the bioactive components in Cayratia trifolia been published in previous studies? If the authors could add more to the overview, it would be better.
Thank you for your suggestions. The benefits from wine are demonstrated by many previous papers [Kumar et al., 2011; Rousdy et al., 2025] but we do not have the benefits from Cayratia trifolia wine directly. However, our previous study reported the bioactive compound from the Cayratia trifolia juice before and after fermentation [Doan et al., 2018d]. We add some more information in the Introduction and the source of these articles in the Reference.

2. In my personal opinion, the author's analysis and evaluation method needs to be presented in more detail.
Thank you for your comments. We added more details on the analysis and evaluation methods in a new section (Section 2.2.5) with references. We hope the information can provide a better understanding to the viewer and for the study.

3. The explanation of the basis of the results is mainly based on the authors' previous research, if other studies are added, it would be more convincing.
This is a great suggestion. We edit the discussion with added information on the results from previous research and other studies. We hope this can improve the quality of the discussion.

4. The conclusion has summarized the data achieved by the topic, if 1-2 sentences about the potential use or value that the research brings are added, it would be better.
Thanks for your comments. We edited the conclusion, which includes information on the value and contribution of applying S. cerevisiae 2.1 to the production of three-leaf caraytia wine.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

13 Views

02 Sep 2025 | for Version 4

Shatha Abdullah Allaith, University of Kerbala Faculty of Agriculture (Ringgold ID: 125663), Kerbala, Karbala Governorate, Iraq

13 Views Cite this report Responses(1)

Not Approved

1. The experimental design is not up to the required scientific standard, especially since it used a yeast isolate from a previous study but did not utilize it well to demonstrate the importance of the current study.
2. In paragraph 2.1, the researcher did not cite any references when using the YPD broth medium. Why? Why did he use a cell concentration of 10^9 at a temperature of 30°C without citing any references?
3. In paragraph 2.2.1, he relied solely on fermentation efficiency. It would have been preferable to study fermentation kinetics using a mathematical model, such as RSM or others, to study the economics of fermentation so that it could be applied commercially.
4. When measuring the optimum conditions for wine production, it would have been preferable to add it to the results of the previous research from which the yeast was isolated or to include it in a new study that reflects the scientific content of the topic and title of the research.
5. It would have been preferable for the statistical analysis to rely on the mean ± experimental error, as this provides a clearer explanation of the standard error of the experimental parameters.
6. Why did the researcher rely on Brix in the experiment without specifying the type of device used to measure which concentrations, and why? Especially since it is widely used in the fermentation industry, which includes sugar.
7. In Table 1, were the initial sugar content and residual sugar content estimated using the phenolsulfuric acid method? The method was not explained in the method.
8. In Table 2, how the ethanol content was calculated each time at 20°C is shown, while the optimal temperature used was room temperature. The ethanol content was not explained as to how it was calculated each time.
9. The discussion is very brief and unclear, and the research results were not compared well with other studies.
10. The conclusion did not mention the challenges and obstacles and finding appropriate solutions for them in the wine industry, despite the difficulties facing the fermentation process, nor did it mention how conditions are controlled at the laboratory level and transformed to a commercial scale.

I confirm that I have read this manuscript carefully, and based on my experience in the field of food technology and industrial fermentation, I decline to accept the research for the reasons stated above. I hope the researcher will reconsider the above points to improve the manuscript in the future and experimental error, as this provides a clearer explanation of the standard error of the experimental parameters.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

food technology and industrial fermentation

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Author Response

23 Sep 2025

Huynh Xuan Phong, Can Tho University, Vietnam

On behalf of the authors, I would like to express our sincere gratitude to the Reviewer for the valuable comments. The referee’s comments were very useful in improving the quality and readability of our manuscript. We have followed the comments and made revisions carefully, point by point.

1. The experimental design is not up to the required scientific standard, especially since it used a yeast isolate from a previous study but did not utilize it well to demonstrate the importance of the current study.
Thank you for your comments. We have carefully revised the experimental design and method description. We also provide a more comprehensive discussion with additional references to strengthen the argument. We hope the revision helps improve the quality of this study.

2. In paragraph 2.1, the researcher did not cite any references when using the YPD broth medium. Why? Why did he use a cell concentration of 10^9 at a temperature of 30°C without citing any references?
Thank you for your comments. We use the protocol applied in a previous study of our group (ref: 10.22144/ctujos.2024.372). We modified the text that supplies the reference to provide clear information on the protocol applied in this study.

3. In paragraph 2.2.1, he relied solely on fermentation efficiency. It would have been preferable to study fermentation kinetics using a mathematical model, such as RSM or others, to study the economics of fermentation so that it could be applied commercially.
In the scope of this study, we intended to conduct a trial fermentation on a laboratory scale. The focus of this study was to investigate the properties of fermentation products. The optimization by RSM should be conducted in further studies on a larger scale, which can be more suitable for scale-up practical fermentation in industries. However, we added a recommendation in the section for further studies to conduct RSM analysis for optimization of the conditions.

4. When measuring the optimum conditions for wine production, it would have been preferable to add it to the results of the previous research from which the yeast was isolated or to include it in a new study that reflects the scientific content of the topic and title of the research.
This is a valuable comment. We added the discussion on the results of the previous studies on S. cerevisiae 2.1 in comparison with this study in the discussion section. This can clearly help improve the scientific content of this research.

5. It would have been preferable for the statistical analysis to rely on the mean ± experimental error, as this provides a clearer explanation of the standard error of the experimental parameters.
We agree with your comment. we added the experimental error to all analyzed data in this study.

6. Why did the researcher rely on Brix in the experiment without specifying the type of device used to measure which concentrations, and why? Especially since it is widely used in the fermentation industry, which includes sugar.
We agree on the comments that the information on the device for measuring the Brix should be included in the paper. For that, we added information on the device and the method description in Section of this study 2.2.5. Besides, the Section “2.2.5 Analytical methods” was added to describe the methods of quantification of parameters employed in this study.

7. In Table 1, were the initial sugar content and residual sugar content estimated using the phenolsulfuric acid method? The method was not explained in the method.
This is a great recommendation. We added information on the phenol-sulfuric methods with a reference. Besides, we described the method in detail in section 2.2.5.

8. In Table 2, how the ethanol content was calculated each time at 20°C is shown, while the optimal temperature used was room temperature. The ethanol content was not explained as to how it was calculated each time.
The ethanol at 20°C was calculated by using the correction table. For that, measured ethanol contents at room temperature were converted to equivalent ethanol contents at 20°C. We added information on the ethanol exchanging method in Section 2.2.5 with a reference.

9. The discussion is very brief and unclear, and the research results were not compared well with other studies.
Thank you for your suggestion. We added new discussions in Section 4 to compare the obtained results with previous studies on the related topic. Besides, we add some recommendations for further studies to improve the practical application of the study. We hope that the discussion can help gain a better understanding and improve the quality of the paper on the results of this study.

10. The conclusion did not mention the challenges and obstacles and finding appropriate solutions for them in the wine industry, despite the difficulties facing the fermentation process, nor did it mention how conditions are controlled at the laboratory level and transformed to a commercial scale.
This comment is great. Since we did a trial fermentation and optimization of conditions at laboratory scale. We think that further studies can conduct further optimization using the RSM method at a larger scale, such as pilot and factory scale. That can help apply the results commercially. We added a recommendation in Section 3.4.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

16 Views

17 Mar 2025 | for Version 4

Javier Alonso-Del-Real, Instituto de Biomedicina de Valencia, Valencia, Spain; Instituto de Agroquimica y Tecnología de Alimentos, Paterna, Spain

16 Views Cite this report Responses(1)

Not Approved

Although authors address some of the concerns raised before, the paper still contain some important issues that must be reviewed.

Authors claim that moderate consumption of wine is healthy. As I pointed out in the previous round, recent evidence clearly suggest that no level of alcohol consumption is healthy. Please, correct appropriately.

Authors still hold that temperatures over 20ºC provoke defects in cell viability, which is in strong disagreement with evidence on S. cerevisiae temperature adaptation.

Also, authors should make the analysis of wine composition during the fermentation under the light of S. cerevisiae metabolic properties, exhaustively studied for a very long time.

Another thing that I find interesting, is the fact that fermentation is not conducted until dryness, and ethanol starts lowering down between days 11 and 13. Authors should discuss that.

Please, try to avoid trivial observations such as "The longer the time, the lower the °Brix, and the higher the ethanol concentration produced. Initially, when the ethanol concentration was quite low, the ethanol concentration began to increase, and the sugar content decreased."

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Laboratory scale wine fermentation by Saccharomyces cerevisiae

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Author Response

23 Sep 2025

Huynh Xuan Phong, Can Tho University, Vietnam

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

13 Views

05 Mar 2025 | for Version 4

Muazaz Azeez Hasan AL-Hadeethi, Botany, University of Baghdad, Baghdad, Baghdad, Iraq

Susan Abdul Raheem Hasan, Biology, University of Baghdad, Baghdad, Iraq

13 Views Cite this report Responses(0)

Approved

I have no further comments.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Microbiologist and expert in biological fermentation processes, with a particular focus on probiotics and plants.

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Reviewer Report

27 Views

22 Jun 2024 | for Version 3

Susan Abdul Raheem Hasan, Biology, University of Baghdad, Baghdad, Iraq

Muazaz Azeez Hasan AL-Hadeethi, Botany, University of Baghdad, Baghdad, Baghdad, Iraq

27 Views Cite this report Responses(1)

Approved With Reservations

The article (Investigation of wine fermentation of three-leaf cayratia (Cayratia trifolia L.) using Saccharomyces cerevisiae 2.1) investigates the optimal conditions for the fermentation process. It discusses each condition and whether it was appropriate to meet the best efficiency (the aim of the study). This purpose was cleared in the context in detail. The materials were mentioned with their sources clearly and appropriate to their need and use in methods. Some methods details were not mentioned, but the source was written so the reader or researcher could follow the references for some unclear steps in the processes of this work. All following parts of the article were transparent and agreed or disagreed with the indexed sources and references for results and conclusion. It also mentioned some further criteria, such as microbiological and physiochemical to be studied. Some issues need to be solved or cleared to improve the article:

Method in abstract missing details such as (volumes, weights, etc.) that were tested, which then were cleared in the main part of the manuscript.
It is crucial to emphasize the need for additional Statistical analysis, which will further strengthen the validity of the findings.
It is essential to discuss the effect of a lower amount of sugar, as this could potentially significantly impact the fermentation process.
Further information and details should be added to a conclusion as in the abstract.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

References

1. Doan T, Huynh M, Tran T, Nguyen T, et al.: The fermentation conditions of low alcoholic three-leaved (Cayratia trifolia (L.) Domin) cider using Saccharomyces cerevisiae HG1.3. Journal of Applied Biology & Biotechnology. 2024. Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Microbiologist and expert in biological fermentation processes, with a particular focus on probiotics and plants.

Respond to this report

Responses (1)

Author Response

27 Feb 2025

Huynh Xuan Phong, Can Tho University, Vietnam

1. Method in abstract missing details such as (volumes, weights, etc.) that were tested, which then were cleared in the main part of the manuscript.
This is a valuable comment. We clarified the information on the volumes of each fermentation trial (100 milliliters and 1 liter) in the abstract section.

2. It is crucial to emphasize the need for additional Statistical analysis, which will further strengthen the validity of the findings.
We agree with your comments. For that, we added Section 2.2.5 on Lines 87-91 to clarify the method of statistical analysis.

3. It is essential to discuss the effect of a lower amount of sugar, as this could potentially significantly impact the fermentation process.
A discussion on the effect of lower sugar amount on the fermentation was added on Lines 103-106. We also add a reference to clarify the explanation.

4. Further information and details should be added to a conclusion as in the abstract.
We revised the conclusion part of the abstract to include essential findings from our study. For that, we clarify that the result of this study provides information for further investigation conducted in the analysis of bioactive compounds produced by fermentation.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

27 Views

06 May 2024 | for Version 3

Javier Alonso-Del-Real, Instituto de Biomedicina de Valencia, Valencia, Spain; Instituto de Agroquimica y Tecnología de Alimentos, Paterna, Spain

27 Views Cite this report Responses(1)

Not Approved

The present paper aims to describe the suitability of a particular yeast strain isolated in a previous related work for the fermentation of Cayratia trifolia fruits. To that purpose authors conduct several fermentation experiments with different amount of initial sugars and temperature set up. Based on that, they determined the most suitable conditions in micro scale fermentation, and then scale up to pilot scale with a positive outcome.

Although the manuscript harbors potential interest from a biotechnological point of view, several issues need to be addressed.

First of all, some of the conclusions presented are not supported by the results or the experiments conducted. Also, The reader may have trouble finding the link between some of the
conclusions and the results supporting them. The authors may find these cases detailed below so that they can improve this aspect.

Moreover, authors should clearly explain the statistical treatment of the obtained data. In the paper, it is stated that three replicates have been used, but there is no indication about what are the values presented (presumably mean values). In addition, errors or standard deviations, as well as tests conducted to determine possible significant differences were not shown.

Also, the writing itself is an issue of concern, as it is sometimes difficult to follow. I would strongly recommend to consider a professional English editing service. Not only phrasing, but punctuation is confusing at some points. Also, the text is unnecessarily repetitive in some occasions, and ideas are not expressed in a cohesive and coherent way.

Some particular comments reflecting these general considerations are the following:

-In Abstract Methods section, the sentence "The temperature for three-leaf cayratia fermentation, incubation time, and preliminary 1 liter of three-leaf cayratia wine fermentation were determined" should be rephrased. Should "preliminary" be accompanying a noun?

-In general, Abstract Methods section reflects a content more suitable for a results section. Please, modify that section accordingly.

-The Conclusion Abstract section also has a content more suitable for a results section.

- The first sentence of the introduction about the impact of alcoholic beverages on health is not in agreement with WHO, based on several recent studies such as Anderson BO et al, 2023 [Ref 2]

-The word "fermentability" is used as a characteristic typical of yeast, but it would rather refer to a carbon source or a growth medium. Anyway, authors should provide an objective set of parameters defining quality of yeast in fermentation to justify the strain selection.

-Some background about in vitro fermentation of three-leaf cayratia, their properties and biotechnological potential should be furthered commented. Otherwise, the work would remain quite out of focus.

-In section 3.1, authors state that yeast die in the highest sugar concentration condition. However, results from a cell viability test should be provided to prove it. Anyway, another explanation would be a slower fermentation rate, probably because adjustment to higher sugar concentrations at the initial must normally requires a significant amount of time. In any case, it would be nice to have data of the dynamics of both the fermentation and the yeast growth.

-In section 3.2, authors claim that ordinary yeast optimal temperature is 30ºC, but they probably refer to S. cerevisiae. There is a wide range of optimal growth temperatures for other yeast. Also, they state that their growth is inhibited above 32ºC, but this is simply not correct [Refer Ref 1]. After that, authors affirm that temperatures above 20ºC could provoke yeast viability to lower down, which seems contradictory. All in all, discussion of the results are not appropriated in its current state.

-In section 3.3, the relationship between fermentation and temperature is introduced. However, the results presented do not correspond to anything about temperature. Also, the method to determine the end of the fermentation is not presented. It rather seems a serial sampling of the same fermentations at different time points.

-Figure 1 relevance is hard to interpret.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

References

1. Salvadó Z, Arroyo-López FN, Guillamón JM, Salazar G, et al.: Temperature adaptation markedly determines evolution within the genus Saccharomyces.Appl Environ Microbiol. 2011; 77 (7): 2292-302 PubMed Abstract | Publisher Full Text
2. Anderson BO, Berdzuli N, Ilbawi A, Kestel D, et al.: Health and cancer risks associated with low levels of alcohol consumption.Lancet Public Health. 2023; 8 (1): e6-e7 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Laboratory scale wine fermentation by Saccharomyces cerevisiae

Respond to this report

Responses (1)

Author Response

27 Feb 2025

Huynh Xuan Phong, Can Tho University, Vietnam

1. First of all, some of the conclusions presented are not supported by the results or the experiments conducted. Also, The reader may have trouble finding the link between some of the conclusions and the results supporting them. The authors may find these cases detailed below so that they can improve this aspect.
Thank you for your comments, we added discussion on Lines 103-106, 147-148
In this information, we have linked the data with corresponding discussions that improve the discussion quality.

2. Moreover, authors should clearly explain the statistical treatment of the obtained data. In the paper, it is stated that three replicates have been used, but there is no indication about what are the values presented (presumably mean values). In addition, errors or standard deviations, as well as tests conducted to determine possible significant differences were not shown.
This is a valuable comment, we clarified the statistical analysis methods on the information on Lines 87-91 of Section 2.2.5.

3. Also, the writing itself is an issue of concern, as it is sometimes difficult to follow. I would strongly recommend to consider a professional English editing service. Not only phrasing, but punctuation is confusing at some points. Also, the text is unnecessarily repetitive in some occasions, and ideas are not expressed in a cohesive and coherent way.
Thank you for your suggestion, we have read and made needed changes to improve the English writing of the whole manuscript.

4. In Abstract Methods section, the sentence "The temperature for three-leaf cayratia fermentation, incubation time, and preliminary 1 liter of three-leaf cayratia wine fermentation were determined" should be rephrased. Should "preliminary" be accompanying a noun?
We changed the word “preliminary 1-liter” into “trial fermentation in the 1-liter-scale of three-leaf cayratia wine” in the abstract section and the names of sections 2.2.4 (line 76) and 3.4 (Line 152).

5. In general, Abstract Methods section reflects a content more suitable for a results section. Please, modify that section accordingly.
Thank you for your suggestion, we have modified the methods of the abstract section and included only the needed methods for the study. The information related to results was relocated to the result part of the abstract.

6. The Conclusion Abstract section also has a content more suitable for a results section.
We relocated the information related to results from the conclusion to the results of the abstract section. The conclusion part is revised to include only the needed conclusion of the study. Besides, we have clarified that the results of this study can be utilized as a base for further studies conducted in exploring bioactive compounds generated from the fermentation.

7. The first sentence of the introduction about the impact of alcoholic beverages on health is not in agreement with WHO, based on several recent studies such as Anderson BO et al, 2023 [Ref 2]
We agree that wine has both benefits and harmfulness depending on the consumption amount of people. We have modified the introduction stating that moderate consumption of wine can benefit human health on Lines 2-3.

8. The word "fermentability" is used as a characteristic typical of yeast, but it would rather refer to a carbon source or a growth medium. Anyway, authors should provide an objective set of parameters defining quality of yeast in fermentation to justify the strain selection.
We changed the word “fermentability” into “fermentation ability” in the suitable sections of the whole study. The yeast S. cerevisiae 2.1 is demonstrated for the quality and fermentation ability in a previous study of Ngo et al. (2005). In this study, we applied that strain of yeast for the fermentation of three-leaf trifolia.

9. Some background about in vitro fermentation of three-leaf cayratia, their properties and biotechnological potential should be furthered commented. Otherwise, the work would remain quite out of focus.
Thank you for your comment. We added information on the introduction of vivo-fermentation of three-leaf trifolia wine on Lines 24-28 of Section 1 – Introduction.

10. In section 3.1, authors state that yeast die in the highest sugar concentration condition. However, results from a cell viability test should be provided to prove it. Anyway, another explanation would be a slower fermentation rate, probably because adjustment to higher sugar concentrations at the initial must normally requires a significant amount of time. In any case, it would be nice to have data of the dynamics of both the fermentation and the yeast growth.
We modified the text on Lines 99-100 which stated that “when the sugar concentration is too high, yeast fermentation is limited due to high osmotic pressure, lowering the alcoholic production”. Besides, we added one reference to clarify this point. In this test, we focus on ethanol production to assess the fermentation quality.

11. In section 3.2, authors claim that ordinary yeast optimal temperature is 30ºC, but they probably refer to S. cerevisiae. There is a wide range of optimal growth temperatures for other yeast. Also, they state that their growth is inhibited above 32ºC, but this is simply not correct [Refer Ref 1]. After that, authors affirm that temperatures above 20ºC could provoke yeast viability to lower down, which seems contradictory. All in all, discussion of the results are not appropriated in its current state.
We agree with your comments. For that, we modified the text on Lines 128-130 of Section 3.2 stating that the optimal temperature for the growth and fermentation of S. cerevisiae is 30-35°C. We also add one more reference to clarify our explanation.

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Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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[2] Doan TTK, Lu NH, Nguyen TN, et al.: Isolation and selection of thermotolerant yeasts for wine production from three-leaf cayratia (Cayratia trifolia L.), in Vietnamese. The Journal of Agriculture and Development. 2018a; 2: 55–64.

[3] Doan TTK, Vien YTH, Huynh PX, et al.: Selection of thermotolerant yeasts and application in wine production from three-leaf cayratia (Cayratia trifolia L.) in Hau Giang, in Vietnamese. Can Tho University Journal of Science. 2018b; 54(4B): 64–71.

[4] Doan TTK, Huynh THA, Nguyen TNM, et al.: Selection of thermotolerant yeasts for production of three-leaf cayratia (Cayratia trifolia L.) wine in Kien Giang, in Vietnamese. Science and Technology Journal of Agriculture and Rural Development. 2018c; 2: 54–62.

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Investigation of wine fermentation of three-leaf cayratia (Cayratia trifolia L.) using Saccharomyces cerevisiae 2.1

Abstract

Keywords

1. Introduction

2. Methods

2.1 Materials

2.2 Determination methods of three-leaf cayratia fermentation

3. Results

3.1 The initial sugar of the three-leaf cayratia fermentation using S. cerevisiae 2.1

Table 1. The effects of °Brix on the fermentability of S. cerevisiae 2.1.

Table 2. The effects of incubation time on fermentation.

3.2 Temperature for three-leaf cayratia fermentation

3.3 Fermentation time for three-leaf cayratia wine

Figure 1. Three-leaf cayratia wine fermented by S. cerevisiae 2.1.

Table 3. The fermentation temperature results of S. cerevisiae 2.1.

3.4 Preliminary 1 liter of the three-leaf cayratia wine fermentation

4. Discussion

5. Conclusion

5.1 Ethical considerations

Data availability statement

Underlying data

References

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