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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]
PUBLISHED 21 Mar 2023
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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

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 °BrixInitial sugar content (g/L)Used sugar content (g/L)Residual sugar content (g/L)Fermentation efficiency (%)Ethanol content (% v/v) at 20°C
20142.23116.6725.5682.47b6.23b
24187.98121.6366.3591.00a7.17a
28229.47115.60113.8775.13c5.64b

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 fermentationEthanol content (% v/v) at 20°C
Room temp. (28–33°C)2412.176.94a
252414.176.16b
352416.834.28c

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).

7ecbb5d8-bbde-4e40-a5e0-9d2c8bb19633_figure1.gif

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 fermentationEthanol content (% v/v) at 20°C
72414.176.94b
92412.506.23b
11249.338.93a
13249.338.54a

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.

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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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