Isolation, identification, and brewing characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid

doi:10.21203/rs.3.rs-4842597/v1

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Isolation, identification, and brewing characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid

https://doi.org/10.21203/rs.3.rs-4842597/v1

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Pyracantha fortuneana (P. fortuneana) fruit, as a dual-purpose plant resource, is rich in nutrients; however, studies on the isolation and identification of yeast strains from P. fortuneana fruit and their brewing characteristics are scarce. To screen for high-quality yeast strains specifically for P. fortuneana wine from the fermentation liquid, this study employed traditional pure culture methods using WL nutrient agar for morphological identification of the isolated strains. Molecular biological identification of the yeasts was performed using molecular biology techniques. The brewing characteristics of the yeast strains were analyzed based on growth characteristics, sugar tolerance, alcohol tolerance, SO₂ tolerance, and acid tolerance, providing potential quality yeast resources for P. fortuneana wine production. The results indicated that a total of 12 yeast strains were isolated and purified. After identification, they were classified as Pichia kudriavzevii and Saccharomyces cerevisiae. Strain HJ-2 was able to grow normally under the conditions of 40°C, 15% alcohol by volume, 360 mg/L SO₂ mass concentration, pH 3.2, 400 g/L sucrose mass concentration, and 250 g/L glucose mass concentration. Strain HJ-6 exhibited normal growth under conditions of 40°C, 3% alcohol by volume, 360 mg/L SO₂ mass concentration, pH 3.2, 250 g/L sucrose mass concentration, and 300 g/L glucose mass concentration. Both strains HJ-2 and HJ-6 demonstrated strong ester production capability and low hydrogen sulfide production. Overall, strains HJ-2 and HJ-6 show potential for application in the industrial production of P. fortuneana wine.

Pyracantha fortuneana

Yeast

Isolation and Identification

Brewing characteristics

Pyracantha fortuneana (P. fortuneana), commonly known as firethorn, torch fruit, or rescue grain, is an evergreen wild shrub belonging to the genus Pyracantha Roem in the subfamily Maloideae of the Rosaceae family (Li et al. 2022). There are a total of 10 species of the Pyracantha genus worldwide, including Pyracantha fortuneana Li., Pyracantha atalantioides Stapf., Pyracantha crenulata Roem., Pyracantha angustifolia Schneid., Pyracantha densiflora Yü., Pyracantha inermis Vidal., Pyracantha koidzumii Rehd., Pyracantha crenulata var. kansuensis, Pyracantha coccinea, and Pyracantha coccinea var. lalandei, which are mainly distributed in East Asia and Southern Europe (Yao et al. 2020). As a dual-purpose wild fruit resource, P. fortuneana fruit is rich in nutritional components (Fig. 1). Studies have shown that P. fortuneana fruits contain amino acids, unsaturated fatty acids, and various mineral elements, which have effects such as delaying aging, maintaining blood pressure, and enhancing immunity. Regular consumption is beneficial to human health (Wang et al. 2020; Xu et al. 2013; Liu et al. 2023).

With the improvement in living standards, a healthy lifestyle has guided the transformation of alcoholic beverage production from grain-based spirits to fruit wines (Liu et al. 2022). Due to the inherent lack of volatile phenolic compounds in P. fortuneana fruit, the flavor of the resulting P. fortuneana fruit wine is often insufficient. Therefore, the parameter settings and control management during the fermentation process of P. fortuneana fruit wine present significant challenges (Mo et al., 2019). Currently, research on P. fortuneana is mostly focused on areas such as ecology (Wu et al. 2023), bonsai (Chen et al. 2021), fresh fruit consumption (Hong et al. 2019), chemical engineering (Yao et al. 2020), and medicinal uses (Suárez-Lepe et al. 2012), with limited studies on the isolation, identification, and brewing characteristics of its yeast strains.

Yeast plays a crucial role in the fermentation process of fruit wine, as its metabolic products influence the color, aroma, and taste of the wine, thereby determining the quality of the fruit wine (Hu et al. 2023). However, during the fruit wine brewing process, various factors such as pH, alcohol concentration, and temperature not only affect the growth and reproduction of yeast but may also cause fermentation to pause or terminate (Liu et al. 2020). Yeasts can be broadly categorized into two types: Saccharomyces cerevisiae and non-Saccharomyces. Non-Saccharomyces have poor alcohol metabolism capabilities but strong flavor compound production abilities, while wine yeasts, although limited in their capacity to produce various flavor compounds, exhibit strong fermentation performance and high alcohol production capabilities (Huang et al. 2022). In recent years, advancements in biotechnology have provided a solid foundation for the selection and breeding of superior yeast strains (Liao et al. 2020). The quality of yeast significantly impacts both the yield and quality of wine, as the characteristics of yeast directly affect the overall quality of fruit wine (Gong et al. 2023). Yeast selection is fundamental to the breeding of superior yeast strains; thus, isolating high-quality brewing yeast from P. fortuneana is a key step in producing high-quality, distinctive P. fortuneana fruit wine.

In summary, this study used naturally fermented P. fortuneana fruit liquid as the isolation source, employing enrichment culture, purification, molecular biological identification, and tolerance tests to select local specialized yeast strains suitable for brewing P. fortuneana fruit wine, with the aim of laying a foundation for the improvement of the overall quality of P. fortuneana fruit wine.

P. fortuneana fruit: Collected from Liaokuo Mountain, Qilin District, Qujing City, Yunnan Province, September 2023.

Commercial yeast (SY): Purchased from Angel Yeast Co., Ltd.; Fungicides, chloramphenicol, ampicillin, kanamycin, and agar powder (biochemical reagents): Beijing Solarbio Science & Technology Co., Ltd.; Glucose, sucrose, citric acid (food grade), sodium metabisulfite: ACS grade from Biological Engineering (Shanghai) Co., Ltd.

Malt extract solid medium, yeast extract peptone dextrose agar medium (YPD), YPD liquid medium, and WL nutrient agar: Qingdao Hi-tech Industrial Park Haibo Biotechnology Co., Ltd. All media were sterilized at 121°C under high pressure steam for 15 minutes before use.

BIGGY medium (bismuth sulfite glucose glycine yeast): Heated and boiled for no more than 1 minute, cooled to 45–50°C, poured into petri dishes without high-pressure sterilization.

Experimental methods

Preparation of P. fortuneana fruit fermentation liquid

Remove diseased and spoiled P. fortuneana fruits, crush them, and place them in sterile Erlenmeyer flasks. Allow natural fermentation at 28°C until a wine-like aroma is observed, stirring the mixture twice daily (morning and evening).

Isolation, purification, and morphological identification of yeast

Take 1 ml of the fermentation liquid and perform a dilution plate method. Plate the diluted solution onto malt extract solid medium and YPD solid medium containing antibiotics, and incubate at 28°C for 24 to 48 hours. Observe colony growth and select single colonies with typical yeast characteristics for streak purification on YPD plates. After activation for 24 hours in YPD liquid medium, inoculate the isolated yeast strains into WL agar medium and incubate at 28°C for 5 days. Observe and record the colony color and morphology, and prepare water mount slides for microscopic observation. Analyze and preliminarily classify the yeast based on asexual reproduction and cell shape.

Fermentation performance testing of yeast strains

Place inverted Durham tubes into YPD liquid medium, sterilize at 121°C under high pressure for 15 minutes, then inoculate with the preliminary screened yeast strains. Mix thoroughly to remove air bubbles, and incubate in a 28°C constant temperature incubator. Observe and record gas production and sediment formation in the Durham tubes over 24 to 72 hours, and measure the gas volume.

Sulfur hydrogen production characteristics of yeast strains

Under sterile conditions, inoculate activated yeast strains onto the surface of BIGGY medium. Seal and incubate upside down at 28°C for 3 days. Observe changes in colony color. The color progression from light to dark is: white, light brown, dark brown, and black, indicating H₂S production capability as none, low, moderate, and high, respectively (Zhou et al. 2020).

Determination of ester production ability of yeast strains

Under sterile conditions, inoculate the activated yeast strains onto the surface of YPD medium for ester production screening. Seal the plates and incubate upside down in a constant temperature incubator at 28°C for 3 days. Observe the size of the transparent zones around the colonies; a larger transparent zone indicates a stronger ability to produce esters, while a smaller transparent zone indicates a weaker ability.

Molecular biological identification of yeast strains

DNA extraction from strains: Inoculate the strain preserved in glycerol tubes into 5 mL of YPD liquid medium and culture on a shaking incubator at 180 r/min and 28°C for 14 hours, during which the strain is in the logarithmic growth phase. Collect the cells by centrifuging the culture in a 1.5 mL centrifuge tube (discard the supernatant). Use a fungal DNA extraction kit to extract the genomic DNA of the yeast as a template.

PCR amplification: Based on references (Wu et al. 2015; Monnin et al. 2014), perform PCR amplification of the target strain using internal transcribed spacer (ITS) primers ITSI (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTC - CGCTTATTGATATGC-3'). The PCR amplification system consists of 45 µL of 1× PCR MIX, 2 µL of forward primer ITS1, 2 µL of reverse primer ITS4, and 1 µL of DNA template. PCR reaction conditions are as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 1 minute, annealing at 50°C for 1 minute, and extension at 72°C for 2 minutes, followed by 30 cycles and a final extension at 72°C for 10 minutes, then cooling down to 4°C to stop the reaction. After the reaction, take 5 µL of the PCR product and perform electrophoresis on a 1% agarose gel at 110 V. Visualize and photograph under a 260 nm UV lamp, and then send the sample to Kunming Shuoqing Biotechnology Co., Ltd. for sequencing.

Phylogenetic tree construction: Compare the sequencing results with regional sequences in the GenBank database using BLAST, construct a phylogenetic tree, and analyze the taxonomic status of the strain.

Tolerance determination of yeast strains

Under sterile conditions, inoculate the activated strains at a 2% inoculum level into YPD liquid medium with varying pH values of 2.8, 3.2, 3.6, and 4.0, ethanol concentrations of 3%, 6%, 9%, 12%, and 15%, temperatures of 4°C, 15°C, 25°C, 30°C, and 40°C, and sucrose and glucose concentrations of 200, 250, 300, 350, and 400 g/L, as well as SO₂ concentrations of 60, 120, 180, 240, and 300 mg/L. Perform three parallel repetitions and incubate at 28°C on a shaking incubator at 180 rpm for 24 hours. After incubation, measure the optical density (OD) of the strains at a wavelength of 600 nm using a UV spectrophotometer.

Data processing and analysis

The results are expressed as "mean ± standard deviation."Physicochemical parameters were analyzed using EXCEL 2021 software, and the phylogenetic tree was constructed using MEGA X software.

Isolation and identification of yeast strains

Yeast strains were isolated and purified from the naturally fermented wild pyracantha fruit fermentation liquid. After culturing these strains on WL nutrient agar, they were initially classified into 12 groups based on their surface morphology and colony color, numbered HJ-1 to HJ-12. Figure 2 displays the colony characteristics and microscopic features of the yeast strains grown on WL nutrient agar. It is evident that there are differences among these 12 yeast strains in terms of colony morphology, cytological characteristics, and reproduction methods. In terms of colony morphology, they exhibit irregular round shapes with colors ranging from white to green. Specifically, strain HJ-1 has a wavy edge; strains HJ-3, HJ-9, and HJ-12 have petal-like shapes; strains HJ-2, HJ-4, HJ-5, and HJ-8 display concentric rings; while strains HJ-6, HJ-7, HJ-10, and HJ-11 have fringed edges. In terms of cytological characteristics, the cells are round or oval, primarily reproducing by budding.

Yeast fermentation performance

Yeast fermentation primarily involves the conversion of sugars into alcohol and carbon dioxide. The rate of CO₂ production indicates the fermentation speed of the yeast (Wang et al. 2023). Within 24 hours, yeast strains that fill the Durham tubes with gas exhibit strong fermentation capabilities. Yeast strains with slower fermentation rates do not meet the requirements for industrial production, and prolonged fermentation of fruit wine increases the risk of microbial contamination. As shown in Table 1, after 24 hours of fermentation, strains HJ-2 and HJ-3 produce gas and fill the Durham tubes, indicating the fastest gas production rate and the strongest fermentation ability. After 48 hours of fermentation, strains HJ-1, HJ-5, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 also fill the Durham tubes with gas, demonstrating relatively fast gas production and strong fermentation ability. After 72 hours of fermentation, strains HJ-4 and HJ-6 achieve gas volumes reaching 2/3 of the Durham tube, indicating slower gas production rates and weaker fermentation abilities.

Table 1

Gas production in the Durcham tubes.
Strain Number	24 h		48 h		72 h
Strain Number	Gas Production	Precipitation Status	Gas Production	Precipitation Status	Gas Production	Precipitation Status
HJ−1	+	White, Compact Precipitate	++	White, Compact Precipitate	+++	White, Compact Precipitate
HJ−2	+++	White, Compact Precipitate	+++	White, Compact Precipitate	+++	White, Compact Precipitate
HJ−3	+++	White, Compact Precipitate	+++	White, Compact Precipitate	+++	White, Compact Precipitate
HJ−4	++	White, Compact Precipitate	++	White, Compact Precipitate	++	White, Compact Precipitate
HJ−5	++	White, Compact Precipitate	+++	White, Compact Precipitate	+++	White, Compact Precipitate
HJ−6	++	White, Compact Precipitate	++	White, Compact Precipitate	++	White, Compact Precipitate
HJ-7	++	White, Loose Precipitate	+++	White, Loose Precipitate	+++	White, Loose Precipitate
HJ-8	+	White, Loose Precipitate	+++	White, Loose Precipitate	+++	White, Loose Precipitate
HJ-9	+	White, Loose Precipitate	+++	White, Loose Precipitate	+++	White, Loose Precipitate
HJ-10	+	White, Loose Precipitate	+++	White, Loose Precipitate	+++	White, Loose Precipitate
HJ-11	+	White, Loose Precipitate	+++	White, Loose Precipitate	+++	White, Loose Precipitate
HJ-12	++	White, Loose Precipitate	+++	White, Loose Precipitate	+++	White, Loose Precipitate
Note: "+, ++, +++" indicate that gas production reaches 1/3, 2/3, and all of the Duchenne tubule volume, respectively.

H₂S production capability

Wine yeast produces trace amounts of H₂S through sulfur metabolism, which has the odor of rotten eggs, garlic, or onions, negatively affecting the flavor profile of fruit wine. H₂S can combine with bismuth to form a black bismuth sulfide precipitate, and the H₂S production capability of the strains can be determined based on the color of the colonies on the selective medium BIGGY using bismuth as an indicator. Based on the levels of H₂S production, the strains can be categorized into six levels: white, light brown, brown, light brown, brown, and black (Ying et al. 2024). As shown in the results of Fig. 3, strains HJ-2, HJ-3, and HJ-5 display white and light brown colors, classifying them as low H₂S producers; strains HJ-6 and HJ-4 exhibit deep brown colors, categorizing them as mediumH₂S producers; while strains HJ-1, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 show dark brown colors, indicating them as high H₂S producers. Zheng et al. (2020) isolated four indigenous wine yeast strains from grape-growing regions to assess their H₂S production capabilities and found that strains WJ1, Q12, and S21 were medium H₂S producers, while strain S12 was a high H2S producer. Huang et al. (2024) isolated six non-wine yeast strains from naturally fermented goat milk fruit, where strains H1, H7, and H10 were white, strain H5 had light brown edges, and strains H9 and H12 were deep brown. This indicates that strains H1, H7, and H10 do not produce H₂S, strain H5 produces low levels of H₂S, and strains H9 and H12 produce high levels of H₂S. Compared to the above studies, this demonstrates certain differences in the H₂S production capabilities of yeast, further emphasizing the importance of yeast selection.

Ester production capability

Microbial secreted enzymes can catalyze the reaction between acids and alcohols to produce esters, which are further hydrolyzed into acids and alcohols, thereby enhancing the flavor of fruit wine (Yuan et al. 2023). By inoculating yeast strains into a medium containing butyric glycerol ester and utilizing the esterases produced by the yeast to hydrolyze butyric glycerol ester, a transparent zone is formed that can be used to evaluate the ester production capability of the yeast strains (Ying et al. 2024). As shown in Fig. 4, strains HJ-5, HJ-6, HJ-11, HJ-3, HJ-2, and HJ-8 exhibit significant transparent zones, indicating strong ester production capabilities; strains HJ-4, HJ-10, HJ-7, and HJ-12 have smaller transparent zones, indicating weaker ester production capabilities; while strains HJ-9 and HJ-1 show no obvious transparent zones, suggesting that these two yeast strains lack ester production capability. Long et al. (2024) assessed the ester production capabilities of five wine yeast strains from the fermentation liquor of Dian olive and found that the five strains exhibited varying ester production abilities, with strain LJM-10 demonstrating a strong ester production capacity. This indicates that there are certain differences in the ester production capabilities among different yeast strains.

Molecular biological identification

The 26S rDNA D1/D2 region within ribosomal DNA is commonly used for the molecular biological classification of yeast (Wang et al. 2024). PCR amplification was performed on the 12 selected yeast strains, and as shown in Fig. 5, the size of the 26S rDNA D1/D2 region in all 12 strains is approximately 600 bp, which matches the expected size. The sequencing results were compared with the NCBI database using BLAST (see Table 2), and a phylogenetic tree was constructed (see Fig. 6). Strains HJ-1, HJ-6, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 showed over 98% similarity to known strains of Pichia kudriavzevii AC1 (OP678979.1), Pichia kudriavzevii B-NC-13-OZ23 (KJ794697.1), Pichia kudriavzevii 11 (KR259307.1), Pichia kudriavzevii YZ4 (EU394711.1), Pichia kudriavzevii PEX-11 (MW990004.1), Pichia kudriavzevii 15 (KR259308.1), Pichia kudriavzevii L1 (EF126365.1), Pichia kudriavzevii feni48 (KM234455.1) and clustered with Pichia kudriavzevii CDA174Y2 (OQ568324.1), indicating that strains HJ-1, HJ-6, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 belong to Pichia kudriavzevii. Strains HJ-2, HJ-3, HJ-4, and HJ-5 showed over 99% similarity to known strains of Saccharomyces cerevisiae YC-D8 (OP644089.1), Saccharomyces cerevisiae QTX-D20 (OP644141.1), Saccharomyces cerevisiae QTX-D14 (OP644135.1), Saccharomyces cerevisiae YQY-F11 (OP644252.1)) and clustered with Saccharomyces cerevisiae UTAD97 (OQ305072.1), indicating that strains HJ-2, HJ-3, HJ-4, and HJ-5 belong to Saccharomyces cerevisiae. It is widely recognized that strains with the same genotype tend to exhibit similar brewing characteristics (Bi et al. 2023). Therefore, one strain from Pichia kudriavzevii and one from Saccharomyces cerevisiae with good fermentation performance, low H₂S production, and high ester production (HJ-2, HJ-6) were selected for tolerance testing.

Table 2

Sequencing results of 26S rDNA D1/D2 sequence of five strain of yeast.
Strain Code	Series Length	Homologous Strain	Homologous strain number	degree of similarity(%)	Genebanknumber
HJ−1	575 bp	Pichia kudriavzevii	AC1	99%	OP678979.1
HJ−2	585 bp	Saccharomyces cerevisiae	YC-D8	99%	OP644089.1
HJ−3	589 bp	Saccharomyces cerevisiae	QTX-D20	100%	OP644141.1
HJ−4	587 bp	Saccharomyces cerevisiae	QTX-D14	100%	OP644135.1
HJ−5	585 bp	Saccharomyces cerevisiae	YQY-F11	100%	OP644252.1
HJ−6	577 bp	Pichia kudriavzevii	B-NC−13-OZ23	98%	KJ794697.1
HJ−7	582 bp	Pichia kudriavzevii	11	100%	KR259307.1
HJ−8	581 bp	Pichia kudriavzevii	YZ4	99%	EU394711.1
HJ−9	565 bp	Pichia kudriavzevii	PEX−11	99%	MW990004.1
HJ−10	582 bp	Pichia kudriavzevii	15	99%	KR259308.1
HJ−11	570 bp	Pichia kudriavzevii	L1	99%	EF126365.1
HJ−12	580 bp	Pichia kudriavzevii	feni48	99%	KM234455.1

Tolerance of yeast strains

The pH of fruit wine typically ranges from 3.0 to 4.0, which necessitates the use of yeast with strong acid tolerance during the winemaking process; both excessively high and low pH levels can affect yeast activity (Ying et al. 2024). Therefore, in this study, commercial yeast SY was used as a control strain to assess the pH tolerance of the selected strains HJ-2 and HJ-6. The results are shown in Fig. 7 (A), where it can be observed that as the environmental acidity increases, the growth condition of the strains gradually weakens. At a pH of 2.8, commercial yeast SY was able to grow normally, while the OD₆₀₀ nm values of strains HJ-2 and HJ-6 were below 0.5, approaching zero, indicating that the increased acidity significantly inhibited their growth. Conversely, as the acidity level decreased from pH 3.2 to pH 4.0, the growth rates of commercial yeast SY and strains HJ-2 and HJ-6 showed an upward trend, indicating their effective adaptability to these conditions. This finding is consistent with the results of Long et al. (2024), which studied the pH tolerance of five yeast strains. Therefore, it can be concluded that strains HJ-2 and HJ-6 exhibit good tolerance to low pH environments.

Ethanol is the primary metabolic product of yeast, and the accumulation of excessive ethanol in fruit wine fermentation can exert inhibitory and toxic effects on the yeast involved in the fermentation process, potentially leading to yeast cell death. The ethanol tolerance of yeast is closely related to its fermentation performance (Gan et al., 2022). The ethanol tolerance of strains HJ-2, HJ-6, and commercial yeast SY was assessed, and the results are shown in Fig. 7 (B). Within the ethanol concentration range of 3–15%, the OD values of the yeast generally exhibited a downward trend as ethanol concentration increased. At an ethanol concentration of 3%, strains SY, HJ-2, and HJ-6 were able to grow normally. However, when the ethanol concentrations reached 6% and 9%, the growth of strain HJ-6 was inhibited, while strains SY and HJ-2 continued to grow normally. When the ethanol concentration increased to 12% and 15%, only strain HJ-2 grew normally, while the growth of strains SY and HJ-6 was inhibited. These results indicate that strain HJ-2 can tolerate ethanol concentrations of up to 15%, whereas the tolerance limits for strains SY and HJ-6 are 9% and 3%, respectively. Qin et al. (2022) found that the ethanol tolerance of the strain Hyphopichia burtonii Y9 was 9%, categorizing it as a strain with moderate ethanol tolerance. Additionally, Jiang et al. (2024) reported that five aromatic yeast strains selected from high-temperature Daqu exhibited ethanol tolerances of 7%. Comparing these findings, it can be concluded that the selected strain HJ-2 demonstrates high ethanol tolerance, making it a suitable strain for the production of hawthorn fruit wine.

Sugar is the primary substrate for ethanol production through fermentation and serves as the energy source for yeast. However, high sugar concentrations can inhibit yeast growth and reproduction, as high osmotic pressure may lead to water loss in yeast cells (Zhou et al. 2024). The glucose and sucrose tolerance of strains SY, HJ-2, and HJ-6 were tested, with results shown in Fig. 7 (C) and Fig. 7 (D). The commercial yeast SY exhibited minimal growth impact under various glucose and sucrose concentrations. Strain HJ-6 demonstrated tolerance when glucose concentration reached 300 g/L, while strain HJ-2 experienced varying degrees of inhibition as glucose concentration increased. When sucrose was used as the carbon source, strain HJ-2 maintained an OD₆₀₀ nm value greater than 1.0 at different sucrose concentrations, indicating strong tolerance to sucrose levels up to 400 g/L. These results highlight significant differences in sugar tolerance between strains HJ-2 and HJ-6 under different carbon sources. Zhao et al. (2023) isolated the strain F3 from naturally fermented mulberry juice, which exhibited the best tolerance, being able to withstand a maximum sugar concentration of 400 g/L. Mou et al. (2023) identified excellent strains B-6 and C-3 from Qingxiang Daqu that could tolerate glucose concentrations of 400 g/L. Compared to the aforementioned studies, the strains HJ-2 and HJ-6 selected in this research demonstrated good sugar tolerance.

In wine production, the addition of SO₂ in appropriate amounts can inhibit harmful microorganisms and provide antioxidant and color-preserving effects. However, higher concentrations of SO₂ can adversely affect yeast strain growth, thereby prolonging fermentation time, and high osmotic pressure can also impact the metabolic products of yeast during alcoholic fermentation (Hu et al. 2023). As shown in Fig. 7 (E), strains HJ-2, HJ-6, and SY exhibited normal growth under different SO₂ concentrations, with no significant changes, indicating that these strains can tolerate SO₂ concentrations up to 360 mg/L. Gong et al. (2023) identified four yeast strains, YC-E8, QTX-D17, QTX-D7, and YQY-E18, from 168 local brewing yeast strains in Ningxia, which could tolerate 250 mg/L SO₂. Zhang et al. (2022) isolated three excellent yeast strains, GL14, GP21, and GP24, from prickly pear, all of which could tolerate 300 mg/L SO₂. These findings indicate that strains HJ-2 and HJ-6 exhibit good SO₂ tolerance, meeting the standards for high-quality yeast strains.

Temperature has a significant impact on yeast reproduction, with the optimal growth temperature for yeast being between 25°C and 30°C. Temperatures exceeding 35°C can lead to cell death; however, certain yeast strains can survive at temperatures above 40°C (Lai et al. 2022). In this study, temperature tolerance tests were conducted on the selected strains, as shown in Fig. 7 (F), which displays the temperature tolerance of the three yeast strains. Under conditions of 4°C, 15°C, 25°C, 30°C, and 40°C, the OD values of the yeast strains initially increased and then decreased. At 4°C, the growth of commercial yeast SY, as well as strains HJ-2 and HJ-6, was inhibited. At 15°C and 25°C, strains SY and HJ-2 grew without inhibition, while strain HJ-6 was inhibited. At 30°C, all three yeast strains exhibited normal growth. At 40°C, strains SY and HJ-2 demonstrated better growth compared to strain HJ-6. These results indicate that the optimal temperature for strains SY, HJ-2, and HJ-6 is 30°C, with the ability to tolerate high temperatures of up to 40°C, which is consistent with the heat resistance findings for industrial yeast strains at 40°C.

In this study, 12 yeast strains were isolated from the fermentation liquid of wild thorny fruits. Based on 26S rDNA D1/D2 region gene sequence analysis, strains HJ-1, HJ-6, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 were identified as Pichia kudriavzevii, while strains HJ-2, HJ-3, HJ-4, and HJ-5 were identified as Saccharomyces cerevisiae. Two strains (HJ-2 and HJ-6) were selected for tolerance testing due to their good fermentation performance, high ester production, and low H₂S production. Strain HJ-2 was able to grow normally under the following conditions: 40°C, 15% (v/v) ethanol, 360 mg/L SO₂, pH 3.2, 400 g/L sucrose, and 250 g/L glucose. Strain HJ-6 grew normally under the conditions of 40°C, 3% (v/v) ethanol, 360 mg/L SO₂, pH 3.2, 250 g/L sucrose, and 300 g/L glucose. Both strains HJ-2 and HJ-6 exhibited strong ester production capabilities and low H₂S production. Gan et al. (2022) screened a strain of brewing yeast that could withstand pH 1.6, 18.2% ethanol concentration, 45°C temperature, and 250 mg/L SO₂. Tan et al. (2020) isolated two brewing yeast strains from ten yeast strains that could grow and reproduce under conditions of 16% ethanol, 500 g/L glucose, 250 mg/L SO₂, high temperature of 45°C, and low temperature of 5°C. Lei et al. (2024) isolated five non-brewing yeast strains with strong ester production capabilities from naturally fermented juice, among which strain Wa3 showed the best tolerance to high concentrations of alcohol, sugar, and SO₂. Comparing with the above studies, strains HJ-2 and HJ-6 demonstrate excellent characteristics for fruit wine brewing. In summary, strains HJ-2 and HJ-6 are expected to provide yeast resources for fruit wine production and possess significant application potential in the thorny fruit wine industry.

Author contributions

The experiments were designed and conducted by L. Z., Data analysis was performed by J. Y. Y., Q. F. X., X. B., X. G., L. F. Z., W. L. L., H. H. W., and L. Z. The project was conceived and supervised by L. Z. The paper was written by L. Z. and J. C. with contributions from all authors, in accordance with the approval of the manuscript.

Conflicts of interest

The authors declare that there are no conficts of interest.

Funding information

This work was supported by Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (Grant NO. 202301BA070001-081), Yunnan Fundamental Research Projects (Grant NO. 202301AU070012), Special Basic Cooperative Research Innovation Programs of Qujing Science and Technology Bureau & Qujing Normal University (Grant NO. KJLH2023ZD06, KJLH2022YB07), National College Students Innovation and Entrepreneurship Training Program, China (Grant NO. G202310684015, G202310684002).

Ethical approval

No experiments involving human participants or animals were conducted by any of the authors in this study.

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No competing interests reported.

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Isolation, identification, and brewing characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid

Status:

Version 1

Abstract

Figures

Introduction

Materials and reagents

Experimental methods

Isolation, purification, and morphological identification of yeast

Fermentation performance testing of yeast strains

Sulfur hydrogen production characteristics of yeast strains

Determination of ester production ability of yeast strains

Molecular biological identification of yeast strains

Tolerance determination of yeast strains

Data processing and analysis

Results and discussion

Isolation and identification of yeast strains

Yeast fermentation performance

H₂S production capability

Ester production capability

Molecular biological identification

Tolerance of yeast strains

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

Isolation, identification, and brewing characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid

Status:

Version 1

Abstract

Figures

Introduction

Materials and reagents

Experimental methods

Isolation, purification, and morphological identification of yeast

Fermentation performance testing of yeast strains

Sulfur hydrogen production characteristics of yeast strains

Determination of ester production ability of yeast strains

Molecular biological identification of yeast strains

Tolerance determination of yeast strains

Data processing and analysis

Results and discussion

Isolation and identification of yeast strains

Yeast fermentation performance

H2S production capability

Ester production capability

Molecular biological identification

Tolerance of yeast strains

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

H₂S production capability