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Agriculture and Food Sciences Research 
Vol. 12, No. 2, 116-122, 2025 

ISSN(E) 2411-6653/ ISSN(P) 2518-0193 
DOI: 10.20448/aesr.v12i2.7530 

© 2025 by the author; licensee Asian Online Journal Publishing Group 

 
 

 
 
 
Fermentation characteristics of yeasts isolated from apple and kiwi 

 
Hiroko Seki  

 

 
 
Department of Local Produce and Food Sciences, Faculty of Life and Environmental Sciences, University of 
Yamanashi, Takeda, Kofu, Yamanashi, Japan. 
Email: hiroko.sk@yamanashi.ac.jp  

 
Abstract 

This study investigated the fermentation capacity of two natural yeast strains, Metschnikowia pulcherrima and 
Candida apicola, isolated from apples and fermented kiwi syrup, respectively. Natural yeasts are increasingly 
valued for imparting complexity, unique aroma, and distinctive flavor to alcoholic beverages and baked goods. 
However, their fermentation performance is often weaker and less stable than that of commercial dry yeast. 
To evaluate optimal conditions, each strain was cultured on agar and inoculated into liquid media containing 
yeast extract, peptone, and glucose. Bread dough was then fermented with each strain under varying 
temperatures, and sugar types and concentrations. Fermentation efficiency was assessed by measuring dough 
height. Statistical analysis was performed using t-tests and one-way ANOVA on triplicate data following 
Fisher’s three principles. Results showed that M. pulcherrima performed best at 25 °C with 5.0% glucose, while 
C. apicola showed optimal fermentation at 35 °C with 5.0% sucrose. These findings indicate that tailoring 
fermentation conditions to specific natural yeast strains can significantly enhance the efficiency of alcohol and 
bread production, offering a promising alternative to commercial yeast in both artisanal and industrial 
applications. 

 
Keywords: Bread production, Candida apicola, Fermentation temperature, Fermentation, Metschnikowia pulcherrima, Sugar type. 

 
Citation | Seki , H. (2025). Fermentation characteristics of yeasts 
isolated from apple and kiwi. Agriculture and Food Sciences 
Research, 12(2), 116–122. 10.20448/aesr.v12i2.7530 
History:  
Received: 21 August 2025 
Revised: 16 September 2025 
Accepted: 26 September 2025 
Published: 10 October 2025 
Licensed: This work is licensed under a Creative Commons 

Attribution 4.0 License  
Publisher:  Asian Online Journal Publishing Group 

Funding: This study received no specific financial support.   
Institutional Review Board Statement: Not applicable. 
Transparency: The author confirms that the manuscript is an honest, accurate, 
and transparent account of the study; that no vital features of the study have 
been omitted; and that any discrepancies from the study as planned have been 
explained. This study followed all ethical practices during writing. 
Competing Interests: The author declares that there are no conflicts of 
interests regarding the publication of this paper. 

 
Contents 
1. Introduction .................................................................................................................................................................................... 117 
2. Materials and Methods ................................................................................................................................................................. 117 
3. Results .............................................................................................................................................................................................. 118 
4. Discussion ........................................................................................................................................................................................ 120 
5. Conclusion ....................................................................................................................................................................................... 121 
References ............................................................................................................................................................................................ 121 
 

 

 

 

 

 

 

 

 

 

mailto:hiroko.sk@yamanashi.ac.jp
https://creativecommons.org/licenses/by/4.0/
https://creativecommons.org/licenses/by/4.0/
https://www.doi.org/10.20448/aesr.v12i2.7530
https://orcid.org/0000-0003-2426-7554


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Contribution of this paper to the literature 
This study contributes to existing literature by investigating the fermentation characteristics of 
two natural yeasts. It provides new insights into the effects of temperature, sugar type, and sugar 
concentration on the fermentation capacity of these yeasts, thereby deepening our understanding 
of bread making and sake brewing. 

 
1. Introduction 

Alcohol production is primarily carried out through fermentation using Saccharomyces cerevisiae, although other 
natural yeasts are also commonly utilized. For instance, Torulaspora delbrueckii is used in beer production [1]. 
Additionally, Metschnikowia pulcherrima is applied in wine production [2], Schizosaccharomyces pombe in rum 
production, and Kluyveromyces marxianus in the fermentation of whey from cheese production to produce vodka and 
gin [3]. In recent years, bread production with natural yeast has garnered considerable interest [4]. Natural yeast 
is a type of fungus that thrives under diverse environmental conditions. Unlike bread made with commercial dry 
yeast, bread produced using natural yeast has a complex taste, as different yeasts impart unique aromas and flavors 
[5]. Currently, natural yeast bread made with strains isolated from dried grapes [6], sudachi petals, and fruits and 
flowers collected in Tokachi Region, Hokkaido, Japan, is commercially available [7]. Moreover, bread has been 
successfully produced using Lachancea fermentati, Lachancea kluyveri, and Torulaspora species isolated from rose 
cultivars grown in Fukuyama City, Hiroshima, Japan [8]. Saccharomyces rouxii, Saccharomyces bisporus, and 
Saccharomyces exigus have been isolated from grape juice, pineapple juice, and rice obtained from local markets in 
Savar, Bangladesh [9]. In bread production, yeast fermentation generates carbon dioxide, which causes wheat gluten 
to expand, and differences in yeast fermentation capacity directly affect bread quality. Commercial yeast has a strong 
fermentation capacity, resulting in a softer texture. In contrast, natural yeast is less stable, ferments slowly, and 
generally shows weaker activity [10]. Therefore, when producing bread with natural yeast, it is essential to adjust 
the sugar type and concentration as well as temperature to stabilize fermentation and improve efficiency. For 
example, bread can be successfully produced with Hanseniaspora meyeri when monosaccharides such as glucose and 
fructose are provided at fermentation temperatures of 25–28°C [11]. Kluyveromyces delphensis ferments dextrose and 
sucrose but not fructose or lactose, as demonstrated by sugar-specific fermentation tests [12]. Furthermore, S. 
cerevisiae has an optimum growth temperature of 25–30°C and a fermentation temperature of 45°C; it can utilize 
glucose, fructose, and sucrose but not in the presence of maltose and amylose [13]. Its ethanol production capacity 
is maximized at a sugar concentration of 125 g/L rather than 100 g/L. However, ethanol productivity decreases at 
concentrations above 150 g/L [14], suggesting that both sugar type and concentration substantially influence the 
fermentation efficiency of yeast. As different natural yeasts respond differently during fermentation, appropriate 
conditions should be selected for each yeast to ensure successful bread production. In this study, I focused on 
Metschnikowia pulcherrima [15], isolated from fermented apple syrup, and Candida apicola [16], isolated from 
fermented kiwi syrup. Metschnikowia pulcherrima has previously been isolated from the spontaneous fermentation of 
Tannat and Marselan grape musts in Concordia (Entre Rios, Argentina) [17], Cabernet Sauvignon wine in Ningxia, 
China [18], and Marastina wine [19]. Candida apicola has been isolated from naturally fermented Alorena green 
table olives [20], wine yeasts that ferment grapes with high sugar content [21], and cachaca (sugarcane wine) [22, 
23]. Here, the effects of fermentation temperature as well as the sugar type and concentration were assessed to 
determine the optimal fermentation conditions required by each species in bread production. The results of this study 
may have potential applications in the production of alcoholic beverages. 

 

2. Materials and Methods 
Metschnikowia pulcherrima (NRBC0863) was purchased from the NITE Biological Resource Center, and C. apicola 

was isolated from kiwi syrup (accession number LC878464, DNA Data Bank of Japan). To prepare each yeast 
solution, yeast monocultured on agar medium was inoculated into a liquid medium containing 0.25% yeast extract, 
0.50% peptone, and 0.10% glucose (for M. pulcherrima) or 5.0% glucose (for C. apicola). The inoculum was incubated 
at 30 °C for approximately 1 week. After incubation, the medium was removed using centrifugation (890 × g, 5 min, 
25 °C), and the yeast was weighed. Subsequently, the yeast was suspended in a 1% glucose solution to obtain a yeast 
solution with a final concentration of 0.78%–3.9%. 
 

2.1. Examination of the Optimal Fermentation Temperature for Each Yeast  
Yeast solution (3.0 g) and strong flour (3.0 g; Tomizawa Shouten Inc., Tokyo, Japan) were mixed in a test tube 

(1.5 cm φ × 18 cm) and placed in a hot water bath adjusted to 20–35 °C in a low-temperature cooker (Anova; Axia 

International Co., Ltd., Tokyo, Japan). The increase in dough height was measured every hour for 5 hours and 

calculated as the increase in dough height per 0.1 g of yeast. 
 

2.2. Investigation of the Optimal Sugar Type for Each Yeast  
Yeast solution (3.0 g), strong flour (3.0 g), and sugar (0.15 g; glucose, sucrose, starch, fructose, and maltose) were 

mixed in a test tube (1.5 cm φ × 18 cm) and placed in a hot water bath at 25°C (M. pulcherrima) and 35°C (C. apicola) 
for 12 hours, and the fermentation capacity was calculated as the increase in dough height per 0.1 g of yeast. 
 

2.3. Examination of the Effect of Sugar Concentration  
Yeast solution (3.0 g), strong flour (3.0 g), and sugar (0.03–0.15 g) were mixed in a test tube (1.5 φ × 18 cm) and 

placed in a hot water bath at 25°C (M. pulcherrima) and 35°C (C. apicola) for 12 hours. The fermentation capacity was 
calculated as the increase in dough height per 0.1 g of yeast. 
 

2.4. Statistical Analysis  
Data were obtained in triplicate using the same sample based on Fisher's three principles. The mean differences 

between groups were assessed using t-tests, and those among three groups were assessed using a one-way analysis 



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of variance. All statistical analyses were performed using Microsoft Excel (Microsoft Corporation, Redmond, WA). 
The significance level was set at p < 0.05. 

 
3. Results 
3.1. Examination of the Optimal Fermentation Temperature for Each Yeast  

Figure 1 shows the effect of different temperatures on M. pulcherrima fermentation. At 35°C, no change was 
observed in dough height after 1 hour; however, the dough height increased to 1.3 cm after 5 hours (p < 0.05). At 
30°C, the height increased to 1.6 cm after 5 hours (p < 0.05). At 25°C, no change was observed in dough height after 
2 hours; however, the dough height increased to 1.7 cm after 5 hours (p < 0.05). At 20°C, no change was observed in 
dough height after 1 hour, but the height slightly increased to 0.16 cm after 2 hours (p > 0.05). Thereafter, no changes 
were observed up to 5 hours. 
 

 
Figure 1. Effect of temperature on Metschnikowia pulcherrima fermentation. ● Indicates 35°C, ■ indicates 30°C, ◆ indicates 25°C, 

and ▲ indicates 20°C. 
Note: The fermentation capacity was calculated as the increase in dough height per 0.10 g of yeast per hour; the results are 

shown as an integrated value. Measurements were obtained in triplicate. The error bars indicate the standard deviation. 

 
Figure 2 shows the effect of temperature on C. apicola fermentation. At 35°C, no change was observed in dough 

height after 1 hour; however, the dough height increased to 2.0 cm after 5 hours (p < 0.05). At 30°C, the height 
increased to 1.9 cm after 5 hours (p < 0.05). At 25°C, no change was observed in dough height up to 4 hours, and the 
height slightly increased to 0.49 cm after 5 hours (p > 0.05). At 20°C, no change was observed up to 2 hours; 
nevertheless, the height slightly increased to 0.25 cm after 3 hours (p > 0.05) and remained unchanged for up to 5 
hours (p > 0.05). 
 

 
Figure 2. Effect of temperature on Candida apicola fermentation. ● Indicates 35 °C, ■ Indicates 30 

°C, ◆ Indicates 25 °C, and ▲ Indicates 20 °C. 
Note: The fermentation capacity was calculated as the increase in dough height per 0.10 g of yeast 

per hour; the results are shown as integrated value. Measurements were obtained in 
triplicate. The error bars indicate the standard deviation. 



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3.2. Investigation of the Optimal Sugar Type for Each Yeast  
Figure 3 shows the effects of various sugars on M. pulcherrima fermentation. The addition of glucose increased 

the dough height to 8.1 cm after 12 h (p < 0.05). The addition of sucrose did not alter the dough height until 3 h, but 
it increased to 0.17 cm after 4 h (p < 0.05). Thereafter, no change was observed in dough height until 6 h (p > 0.05), 
followed by an increase to 0.34 cm after 7 h (p < 0.05). No further change was observed until 12 h (p > 0.05). With 
starch addition, the height increased to 3.2 cm after 8 h (p < 0.05), then slightly increased to 3.3 cm after 12 h (p > 
0.05). With fructose addition, the height increased to 6.7 cm after 11 h (p < 0.05), but slightly decreased after 12 h (p 
> 0.05). With maltose addition, the height increased to 2.5 cm after 7 h (p < 0.05); however, no change was observed 
after 8 h (p > 0.05). A slight increase to 2.9 cm was observed at 9 h (p > 0.05). No further change was observed until 
12 h. 
 

 
Figure 3. Effect of various sugars on Metschnikowia pulcherrima fermentation. ● Indicates glucose, 

■ Indicates sucrose, ◆ Indicates starch, ▲ Indicates fructose, and 〇 Indicates maltose. 
Note: The fermentation capacity was calculated as the increase in dough height per 0.10 g of yeast per hour; 

the results are shown as integrated value. Measurements were obtained in triplicate. The error bars 
indicate the standard deviation. 

 
Figure 4 shows the effects of various sugars on C. apicola fermentation. The addition of glucose increased the 

dough height to 4.9 cm after 10 h (p < 0.05); however, no significant change was observed after 11 h (p > 0.05), 
followed by a slight increase to 5.2 cm after 12 h (p > 0.05). Sucrose increased the height slightly to 1.6 cm after 2 h 
(p > 0.05); the height remained unchanged until 3 h (p > 0.05), but increased to 7.7 cm after 12 h (p < 0.05). With 
starch addition, the height increased to 0.25 cm after 2 h (p < 0.05), remained unchanged until 6 h (p > 0.05), and 
then increased to 0.81 cm after 8 h (p < 0.05). Thereafter, it remained unchanged until 11 h (p > 0.05) and increased 
to 1.3 cm after 12 h (p < 0.05). With fructose addition, no change was observed until 4 h (p > 0.05), and the height 
increased to 1.7 cm after 9 h (p < 0.05). Thereafter, no change was observed until 11 h (p > 0.05). Subsequently, the 
height increased to 2.2 cm after 12 h (p < 0.05). With maltose addition, the height increased to 0.50 cm after 2 h (p < 
0.05), but did not change until 6 h (p > 0.05). Thereafter, it increased slightly to 1.2 cm after 9 h (p > 0.05), remained 
unchanged until 10 h (p > 0.05), and increased to 2.0 cm after 12 h (p < 0.05). 
 

 
Figure 4. Effect of various sugars on Candida apicola fermentation. ● Indicates glucose, ■ 

Indicates sucrose, ◆ Indicates starch, ▲ Indicates fructose, and 〇 Indicates maltose. 
Note: The fermentation capacity is calculated as the increase in dough height per 0.10 g of yeast per 

hour; the results are shown as integrated value. Measurements were obtained in triplicate. The 
error bars indicate the standard deviation. 

 



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3.3. Examination of the Effect of Sugar Concentration 
Figure 5 shows the effect of glucose concentration on M. pulcherrima fermentation. When 1.0% glucose was 

added, no change was observed in dough height after 1 hour, but the dough height increased to 6.9 cm after 11 hours 
(p < 0.05) and remained unchanged until 12 hours (p > 0.05). At a concentration of 2.0%, no change was observed in 
dough height after 1 hour; however, the height increased to 7.3 cm after 12 hours (p < 0.05). At a concentration of 
3.0%, no change was observed after 1 hour (p > 0.05), but after 12 hours, the height increased to 8.7 cm (p < 0.05). 
At a concentration of 4.0%, the height slightly increased to 0.12 cm after 3 hours (p > 0.05), followed by an increase 
to 8.7 cm after 12 hours (p < 0.05). Following the addition of 5.0% glucose, the height increased to 10 cm after 12 
hours (p < 0.05). 
 

 
Figure 5. Effect of glucose concentration on Metschnikowia pulcherrima fermentation. ● 

Indicates 1.0%, ■ Indicates 2.0%, ◆ Indicates 3.0%, ▲ Indicates 4.0%, and 〇 Indicates 5.0%. 
Note: The fermentation capacity was calculated as the increase in dough height per 0.10 g of yeast 

per hour; the results are shown as integrated value. Measurements were obtained in 
triplicate. The error bars indicate the standard deviation. 

 

Figure 6 shows the effect of sugar concentration on C. apicola fermentation. When sucrose was added at a 
concentration of 1.0%, no change in dough height was observed after 12 hours. Furthermore, at 2.0%, no change was 
observed in dough height up to 2 hours, but it increased slightly to 0.57 cm after 3 hours (p > 0.05) and remained 
unchanged after 12 hours (p < 0.05). At 3.0%, no change was observed after 1 hour, but the height increased slightly 
to 0.71 cm after 3 hours (p > 0.05) and remained unchanged until 12 hours (p > 0.05). Similarly, no change was 
observed in dough height at 4.0% until 1 hour; however, after an increase in dough height to 0.71 cm after 2 hours 
(p < 0.05), no further change was observed until 4 hours (p > 0.05). After 10 hours, the height increased to 6.0 cm (p 
< 0.05), and no change was observed after 12 hours (p > 0.05). At 5.0%, the height increased to 3.3 cm after 3 hours 
(p < 0.05), but no change was observed after 4 hours (p > 0.05). Thereafter, it increased to 11 cm after 12 hours (p < 
0.05). 

 

 
Figure 6. Effect of sucrose concentration on Candida apicola fermentation. ● Indicates 1.0%, ■ 

Indicates 2.0%, ◆ Indicates 3.0%, ▲ Indicates 4.0%, and 〇 Indicates 5.0%. 
Note: The fermentation capacity was calculated as the increase in dough height per 0.10 g of yeast per 

hour; the results are shown as integrated value. Measurements were obtained in triplicate. The error 
bars indicate the standard deviation. 

  

4. Discussion 
4.1. Examination of the Optimal Fermentation Temperature for Each Yeast 

In this study, the optimal temperature, sugar type, and concentration required for fermentation using M. 
pulcherrima and C. apicola as natural yeasts in bread production were investigated. After 5 hours of fermentation, M. 
pulcherrima and C. apicola showed high fermentation capacities at 25°C and 35°C, respectively; however, after 5 hours, 
they exhibited relatively high fermentation capacities at 30°C and 35°C, respectively. Metschnikowia pulcherrima 



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grows efficiently at temperatures in the range of 15–20°C [2]; whereas, C. apicola grows efficiently at 25–37 °C [12], 
indicating that it does not grow well in low-temperature environments. Metschnikowia pulcherrima has been isolated 
as a dominant fermentation species during the fermentation of Fiano di Avellino grapes at 9 °C in wine production 
[24], suggesting that it can survive at low temperatures and show high fermentation potential. In addition, C. apicola 
can survive at low temperatures while marginally maintaining its fermentation potential. Detailed reports on the 
optimal fermentation temperature of C. apicola are lacking. As a species similar to C. apicola, C. tropicalis has been 
reported to be heat-resistant and capable of producing ethanol from xylose [25]. Candida species may have a high 
fermentation capacity at relatively high temperatures, suggesting that it is difficult for C. apicola to ferment substrates 
at lower temperatures. 
 

4.2. Investigation of the Optimal Sugar Type for Each Yeast  
In this study, M. pulcherrima and C. apicola showed high fermentation capacities following the addition of glucose 

and sucrose, respectively. Glucose, a monosaccharide, is used by yeast in ethanol fermentation. In a previous study, 
31 types of S. cerevisiae isolates from palm wine were provided with glucose, galactose, fructose, sucrose, maltose, 
trehalose, and raffinose, either in combination or individually, to ferment a substrate, and glucose was used for 
fermentation in all tests [26]. Among the aforementioned sugars, glucose, galactose, and fructose are 
monosaccharides; sucrose, maltose, and trehalose are disaccharides; and raffinose is the only trisaccharide. As these 
disaccharides and the trisaccharide contain glucose, they can be fermented if the yeast produces enzymes that can 
break them down into glucose. Reportedly, the strength of sucrase activity varies among different species of S. 
cerevisiae [27]. Hence, the type of sugar available and the fermentation rate may differ among different yeast species. 
In this study, the fermentation capacity of M. pulcherrima for sucrose was weaker than that for maltose and starch 
(Figure 3). Metschnikowia pulcherrima uses glucose and not sucrose in the fermentation of soy whey and produces 
ethanol as a product [28-30], consistent with the findings of the present study, suggesting that M. pulcherrima does 
not produce sucrase or possesses weak sucrase activity. Alcohol fermentation has been reported to be slow with 
maltose [31], and as starch is maltose-bound, M. pulcherrima may have weak maltase and amylase activities. Candida 
apicola has weak maltase or amylase activity (Figure 4) and shows high fermentation efficiency for sucrose. In 
fermentation capacity tests by sugar type, C. apicola was reportedly able to ferment sucrose but not fructose [12]. 
Similar results were obtained in this study. Hence, C. apicola has high sucrase activity; however, it was also able to 
ferment the dough with fructose in this study. The glucose and fructose produced from sucrose through sucrase were 
used for fermentation. However, the slow onset of fermentation of glucose and fructose suggests that sucrose may 
promote fermentation by C. apicola. In addition, as C. apicola has been reported to tolerate high ethanol concentrations 
[21], it is possible that ethanol produced through fermentation promoted the fermentation of glucose and fructose. 
However, baking bread was the focus of this study, and therefore, the fermentation mechanism of these yeasts in 
baking is a subject for future research. 
 

4.3. Examination of the Effect of Sugar Concentration  
Metschnikowia pulcherrima and C. apicola showed the maximum fermentation efficiency at a sugar concentration 

of 5.0%. During fermentation, the fermentation capacity of C. apicola was affected by sugar concentration. In a 
previous study, fermentation by S. cerevisiae increased ethanol concentration with increasing glucose concentrations 
from 2.0% to 8.0% [32]. Furthermore, as Starmerella zemplinina (synonym Candida zemplinina) has been reported to 
grow faster when glucose is added at concentrations of 2.0% and 20% [21], fermentation efficiency may have 
increased with increasing glucose concentrations up to 20%. In contrast, the fermentation efficiency of Hanseniaspora 
guilliermondii reportedly decreases at sugar concentrations >300 g/L [14]. These results indicate that the effect of 
sugar concentration on fermentation varies depending on the yeast type. The yeasts investigated in this study showed 
the highest fermentation efficiency at a 5.0% sugar concentration, and it is highly probable that the efficiency can be 
increased by increasing the sugar concentration. However, as a considerable increase in sugar concentration 
significantly affects the flavor of bread, the effect of sugar concentration on flavor must be considered. 

This study has some limitations. As both wheat and water are used in bread production, the effects of wheat and 
the ions in water should be considered. However, these factors were not examined in this study; therefore, their 
influence on the fermentation capacity of M. pulcherrima and C. apicola should be investigated in future studies to 
clarify the conditions suitable for bread production. Moreover, the fermentation capacity of natural yeasts could 
change depending on the environment. The present study did not focus on this aspect, warranting future studies on 
the effects of environmental factors on the fermentation capacity of these yeasts. 
 

5. Conclusion 
      The effects of temperature, sugar type, and concentration on yeast fermentation capacity were investigated in 
this study. The optimal fermentation temperature was 25°C for M. pulcherrima and 35°C for C. apicola. The 
concentration and type of sugar that most enhanced the fermentation efficiency of each yeast were 5.0% glucose for 
M. pulcherrima and 5.0% sucrose for C. apicola. Overall, fermentation using M. pulcherrima and C. apicola under 
appropriate temperature and sugar concentration conditions could potentially enhance the efficiency of bread 
production. Given the limited research examining the use of these natural yeasts in food, further studies are required 
to ensure their safety, effectiveness, and hygienic handling. 
 

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https://doi.org/10.1094/CCHEM-03-17-0044-R
https://doi.org/10.47371/mycosci.2021.10.003
https://doi.org/10.3390/applmicrobiol2030040
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https://doi.org/10.1007/s10973-020-09629-4

