Improving the Sensory Quality of Xinhui Citrus Wine by Mixed Yeasts Fermentation of Saccharomyces cerevisiae and Non-Saccharomyces

Abstract

Xinhui Chenpi, a China National Geographical Indication Product, faces unexploited pulp resources, resulting in resource waste and potential environmental pollution. This study employed Xinhui citrus pulp as a raw material to produce Xinhui citrus wine (XCW), focusing on enhancing the aroma of XCW by utilizing a multi-strains fermentation involving Saccharomyces cerevisiae and Lachancea thermotolerans. The effect of S. cerevisiae Red and/or L. thermotolerans L19 on the enological parameters, physicochemical characteristics and volatile compounds of XCWs were evaluated. The mixed strains fermentation demonstrated a significant increase in esters content, including 2,3-butanediol dinitrate, diethyl succinate, ethyl benzoate, ethyl lactate, and ethyl glycolate compared to those of mono fermentation (p < 0.05), thereby enhancing the fruity and floral aromas of the XCW. The key aroma compounds of relative odor activity value (rOAV) > 1, which are specific to mixed fermentation, were ethyl 2-methylbutyrate, ethyl benzoate, ethyl isovalerate, ethyl propionate, 1-penten-3-ol, and phenylethyl alcohol. These compounds could strengthen the fruity, flowery, and sweet scents of XCW. The findings align with sensory evaluations, suggesting that the mixed fermentation of S. cerevisiae Red and L. thermotolerans L19 to produce XCW might resolve the resource waste of Xinhui citrus pulp.

Keywords

citrus wine / enological parameters / lacancea thermotolerans / volatile aroma compounds / Xinhui Chenpi

References

[1] Adams, M. J., M. Buehner, and K. Chandrasekhar, et al. 1973. “Structure-Function Relationships in Lactate Dehydrogenase.” Proceedings of the National Academy of Sciences 70, no. 7: 1968–1972. https://doi.org/10.1073/pnas.70.7.1968.

[2] Antalick, G., K. Šuklje, J. W. Blackman, C. Meeks, A. Deloire, and L. M. Schmidtke. 2015. “Influence of Grape Composition on Red Wine Ester Profile: Comparison Between Cabernet Sauvignon and Shiraz Cultivars From Australian Warm Climate.” Journal of Agricultural and Food Chemistry 63, no. 18: 4664–4672. https://doi.org/10.1021/acs.jafc.5b00966.

[3] Balikci, E. K., H. Tanguler, N. P. Jolly, and H. Erten. 2016. “Influence of Lachancea thermotolerans on cv. Emir Wine Fermentation.” Yeast 33, no. 7: 313–321. https://doi.org/10.1002/yea.3166.

[4] Banilas, G., G. Sgouros, and A. Nisiotou. 2016. “Development of Microsatellite Markers for Lachancea thermotolerans Typing and Population Structure of Wine-Associated Isolates.” Microbiological Research 193: 1–10. https://doi.org/10.1016/j.micres.2016.08.010.

[5] Benito, Á., F. Calderón, and S. Benito. 2019. “The Influence of Non-Saccharomyces Species on Wine Fermentation Quality Parameters.” Fermentation 5, no. 3: 54. https://doi.org/10.3390/fermentation5030054.

[6] Benito, Á., F. Calderón, F. Palomero, and S. Benito. 2015. “Combine Use of Selected Schizosaccharomyces Pombe and Lachancea thermotolerans Yeast Strains as an Alternative to the Traditional Malolactic Fermentation in Red Wine Production.” Molecules 20, no. 6: 9510–9523. https://doi.org/10.3390/molecules20069510.

[7] Benito, S.2018. “The Impacts of Lachancea thermotolerans Yeast Strains on Winemaking.” Applied Microbiology and Biotechnology 102, no. 16: 6775–6790. https://doi.org/10.1007/s00253-018-9117-z.

[8] Benito, S., T. Hofmann, M. Laier, et al. 2015. “Effect on Quality and Composition of Riesling Wines Fermented by Sequential Inoculation With Non-Saccharomyces and Saccharomyces cerevisiae.” European Food Research and Technology 241, no. 5: 707–717. https://doi.org/10.1007/s00217-015-2497-8.

[9] Binati, R. L., G. Innocente, and V. Gatto, et al. 2019. “Exploring the Diversity of a Collection of Native Non-Saccharomyces Yeasts to Develop Co-Starter Cultures Forwinemaking.” Food Research International 122: 432–442. https://doi.org/10.1016/j.foodres.2019.04.043.

[10] Binati, R. L., W. J. F. Lemos Junior, G. Luzzini, D. Slaghenaufi, M. Ugliano, and S. Torriani. 2020. “Contribution of Non-Saccharomyces Yeasts to Wine Volatile and Sensory Diversity: A Study on Lachancea thermotolerans, Metschnikowia Spp. and Starmerella bacillaris Strains Isolated in Italy.” International Journal of Food Microbiology 318: 108470. https://doi.org/10.1016/j.ijfoodmicro.2019.108470.

[11] Chen, K., C. Escott, I. Loira, et al. 2018. “Use of Non-Saccharomyces Yeasts and Oenological Tannin in Red Winemaking: Influence on Colour, Aroma and Sensorial Properties of Young Wines.” Food Microbiology 69: 51–63. https://doi.org/10.1016/j.fm.2017.07.018.

[12] Comitini, F., M. Gobbi, P. Domizio, et al. 2011. “Selected Non-Saccharomyces Wine Yeasts in Controlled Multistarter Fermentations With Saccharomyces cerevisiae.” Food Microbiology 28, no. 5: 873–882. https://doi.org/10.1016/j.fm.2010.12.001.

[13] Costa, G. P., K. P. Nicolli, J. E. Welke, V. Manfroi, and C. A. Zini. 2018. “Volatile Profile of Sparkling Wines Produced With the Addition of Mannoproteins or Lees Before Second Fermentation Performed With Free and Immobilized Yeasts.” Journal of the Brazilian Chemical Society 29, no. 9: 1866–1875. https://doi.org/10.21577/0103-5053.20180062.

[14] Escribano, R., L. González-Arenzana, J. Portu, et al. 2018. “Wine Aromatic Compound Production and Fermentative Behaviour Within Different Non-Saccharomyces Species and Clones.” Journal of Applied Microbiology 124, no. 6: 1521–1531. https://doi.org/10.1111/jam.13735.

[15] He, W., S. Liu, P. Heponiemi, et al. 2021. “Effect of Saccharomyces cerevisiae and Schizosaccharomyces pombe Strains on Chemical Composition and Sensory Quality of Ciders Made From Finnish Apple Cultivars.” Food Chemistry 345: 128833. https://doi.org/10.1016/j.foodchem.2020.128833.

[16] Heitmann, M., E. Zannini, and E. Arendt. 2018. “Impact of Saccharomyces cerevisiae Metabolites Produced During Fermentation on Bread Quality Parameters: A Review.” Critical Reviews in Food Science and Nutrition 58, no. 7: 1152–1164. https://doi.org/10.1080/10408398.2016.1244153.

[17] Hong, K. Q., X. M. Fu, S. S. Dong, and D. G. Xiao, and J. Dong. 2019. “Modulating Acetate Ester and Higher Alcohol Production in Saccharomyces cerevisiae Through the Cofactor Engineering.” Journal of Industrial Microbiology & Biotechnology 46, no. 7: 1003–1011. https://doi.org/10.1007/s10295-019-02176-4.

[18] Hou, Y., R. Ao, H. Chen, et al. 2023. “Analysis of Change Rules of Trace Components in Banchengshaoguo Liquor With Different Sun Exposure Time Based on Sorbent Pen.” Liquor—Making Science & Technology 6: 17–27. 33. https://doi.org/10.13746/j.njkj.2022216.

[19] Hranilovic, A., W. Albertin, D. L. Capone, et al. 2021. “Impact of Lachancea thermotolerans on Chemical Composition and Sensory Profiles of Merlot Wines.” Food Chemistry 349: 129015. https://doi.org/10.1016/j.foodchem.2021.129015.

[20] Hranilovic, A., S. Li, P. K. Boss, et al. 2018. “Chemical and Sensory Profiling of Shiraz Wines Co-Fermented With Commercial Non-Saccharomyces Inocula.” Australian Journal of Grape and Wine Research 24, no. 2: 166–180. https://doi.org/10.1111/ajgw.12320.

[21] Hu, L., R. Liu, X. Wang, and X. Zhang. 2020. “The Sensory Quality Improvement of Citrus Wine Through Co-Fermentations With Selected Non-Saccharomyces Yeast Strains and Saccharomyces cerevisiae.” Microorganisms 8, no. 3: 323. https://doi.org/10.3390/microorganisms8030323.

[22] Juega, M., Y. P. Nunez, and A. V. Carrascosa, and A. J. Martinez-Rodriguez. 2012. “Influence of Yeast Mannoproteins in the Aroma Improvement of White Wines.” Journal of Food Science 77, no. 8: 499–504. https://doi.org/10.1111/j.1750-3841.2012.02815.x.

[23] Kapsopoulou, K., A. Mourtzini, M. Anthoulas, and E. Nerantzis. 2007. “Biological Acidification During Grape Must Fermentation Using Mixed Cultures of Kluyveromyces thermotolerans and Saccharomyces cerevisiae.” World Journal of Microbiology & Biotechnology 23, no. 5: 735–739. https://doi.org/10.1007/s11274-006-9283-5.

[24] Kelebek, H., S. Selli, A. Canbas, and T. Cabaroglu. 2009. “HPLC Determination of Organic Acids, Sugars, Phenolic Compositions and Antioxidant Capacity of Orange Juice and Orange Wine Made From a Turkish cv. Kozan.” Microchemical Journal 91, no. 2: 187–192. https://doi.org/10.1016/j.microc.2008.10.008.

[25] Li, H., L. Lin, Y. Feng, and M. Zhao. 2024. “Exploration of Optimal Preparation Strategy of Chenpi (Pericarps of Citrus reticulata Blanco) Flavouring Essence With Great Application Potential in Sugar and Salt-Reduced Foods.” Food Research International 175: 113669. https://doi.org/10.1016/j.foodres.2023.113669.

[26] Li, J., J. Zhang, Y. Yang, et al. 2021. “Comparative Characterization of Lipids and Volatile Compounds of Beijing Heiliu and Laiwu Chinese Black Pork as Markers.” Food Research International 146: 110433. https://doi.org/10.1016/j.foodres.2021.110433.

[27] Lima, N. M., G. F. Santos, T. de Jesus A. S. Andrade, et al. 2025. “Metabolic Signatures by LC-HRMS/MS of Jabuticaba (Plinia Cauliflora) Juice, Liqueur, and Wines Reveal the Wealthiest Sources of Bioactive Metabolites.” Talanta 287: 127602. https://doi.org/10.1016/j.talanta.2025.127602.

[28] Liu, C., M. Li, T. Ren, et al. 2022. “Effect of Saccharomyces cerevisiae and Non-Saccharomyces Strains on Alcoholic Fermentation Behavior and Aroma Profile of Yellow-Fleshed Peach Wine.” LWT 155: 112993. https://doi.org/10.1016/j.lwt.2021.112993.

[29] Liu, D., J. Chen, X. Li, et al. 2024. “Revealing the Microbial Contributions in Chili Paste Fermentation by Inoculating In Situ Microbiome.” LWT 191: 115632. https://doi.org/10.1016/j.lwt.2023.115632.

[30] Liu, S., Y. Lou, Y. Li, et al. 2023. “Comparison of Anthocyanin and Volatile Organic Compounds in Juices and Fruit Wines Made From Blood Oranges (Citrus sinensis L. Osbeck) at Different Maturity Stages.” Food Bioscience 56: 103194. https://doi.org/10.1016/j.fbio.2023.103194.

[31] Morata, A., C. Escott, M. Bañuelos, et al. 2019. “Contribution of Non-Saccharomyces Yeasts to Wine Freshness: A Review.” Biomolecules 10: 34. https://doi.org/10.3390/biom10010034.

[32] Napoli, C., M. L. Balestrieri, V. Sica, et al. 2008. “Beneficial Effects of Low Doses of Red Wine Consumption on Perturbed Shear Stress-Induced Atherogenesis.” Heart and Vessels 23, no. 2: 124–133. https://doi.org/10.1007/s00380-007-1015-8.

[33] Padilla, B., J. V. Gil, and P. Manzanares. 2016. “Past and Future of Non-Saccharomyces Yeasts: From Spoilage Microorganisms to Biotechnological Tools for Improving Wine Aroma Complexity.” Frontiers in Microbiology 7: 411. https://doi.org/10.3389/fmicb.2016.00411.

[34] Petitgonnet, C., G. L. Klein, C. Roullier-Gall, et al. 2019. “Influence of Cell-Cell Contact between L. thermotolerans and S. Cerevisiae on Yeast Interactions and the Exo-Metabolome.” Food Microbiology 83: 122–133. https://doi.org/10.1016/j.fm.2019.05.005.

[35] Petruzzi, L., V. Capozzi, C. Berbegal, et al. 2017. “Microbial Resources and Enological Significance: Opportunities and Benefits.” Frontiers in Microbiology 8: 995. https://doi.org/10.3389/fmicb.2017.00995.

[36] du Plessis, H. W., M. du Toit, J. W. Hoff, R. S. Hart, B. K. Ndimba, and N. P. Jolly. 2017. “Characterisation of Non-Saccharomyces Yeasts Using Different Methodologies and Evaluation of Their Compatibility With Malolactic Fermentation.” South African Journal of Enology & Viticulture 38, no. 2: 46–63. https://doi.org/10.21548/38-1-819.

[37] Rainieri, S., and I. S. Pretorius. 2000. “Selection and Improvement of Wine Yeasts.” Annals of Microbiology 50: 15–31. https://doi.org/10.1038/npre.2010.4381.1.

[38] Remize, F., J. L. Roustan, J. M. Sablayrolles, P. Barre, and S. Dequin. 1999. “Glycerol Overproduction by Engineered Saccharomyces cerevisiae, Wine Yeast Strains Leads to Substantial Changes in, By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase.” Applied and Environmental Microbiology 65, no. 1: 143–149. https://doi.org/10.1128/aem.65.1.143-149.1999.

[39] Renault, P., J. Coulon, G. de Revel, J. C. Barbe, and M. Bely. 2015. “Increase of Fruity Aroma During Mixed T. delbrueckii/S. cerevisiae Wine Fermentation Is Linked to Specific Esters Enhancement.” International Journal of Food Microbiology 207: 40–48. https://doi.org/10.1016/j.ijfoodmicro.2015.04.037.

[40] Sánchez-Palomo, E., J. A. Delgado, M. A. Ferrer, and M. A. G. Viñas. 2019. “The Aroma of La Mancha Chelva Wines: Chemical and Sensory Characterization.” Food Research International 119: 135–142. https://doi.org/10.1016/j.foodres.2019.01.049.

[41] San-Juan, F., V. Ferreira, J. Cacho, and A. Escudero. 2011. “Quality and Aromatic Sensory Descriptors (Mainly Fresh and Dry Fruit Character) of Spanish Red Wines Can Be Predicted From Their Aroma-Active Chemical Composition.” Journal of Agricultural and Food Chemistry 59, no. 14: 7916–7924. https://doi.org/10.1021/jf1048657.

[42] Selli, S.2007. “Volatile Constituents of Orange Wine Obtained From Moro Oranges (Citrus sinensis [L.] Osbeck).” Journal of Food Quality 30, no. 3: 330–341. https://doi.org/10.1111/j.1745-4557.2007.00124.x.

[43] Serio, F., G. Imbriani, M. Acito, et al. 2023. “Moderate Red Wine Intake and Cardiovascular Health Protection: A Literature Review.” Food & Function 14, no. 14: 6346–6362. https://doi.org/10.1039/d3fo01004j.

[44] Song, N.-E., M. K. Kim, K.-G. Lee, and H. W. Jang. 2021. “Analysis of Volatile Compounds in Rooibos Tea (Aspalathus linearis) Using Different Extraction Methods and Their Relationship With Human Sensory Perception.” Food Research International 141: 109942. https://doi.org/10.1016/j.foodres.2020.109942.

[45] Styger, G., B. Prior, and F. F. Bauer. 2011. “Wine Flavor and Aroma.” Journal of Industrial Microbiology & Biotechnology 38, no. 9: 1145–1159. https://doi.org/10.1007/s10295-011-1018-4.

[46] Sun, J., D. Zhao, F. Zhang, et al. 2017. “Joint Direct Injection and GC–MS Chemometric Approach for Chemical Profile and Sulfur Compounds of Sesame-Flavor Chinese Baijiu (Chinese liquor).” European Food Research and Technology 244, no. 1: 145–160. https://doi.org/10.1007/s00217-017-2938-7.

[47] Sun, N., Z. Gao, S. Li, X. Chen, and J. Guo. 2022. “Assessment of Chemical Constitution and Aroma Properties of Kiwi Wines Obtained From Pure and Mixed Fermentation With Wickerhamomyces anomalus and Saccharomyces cerevisiae.” Journal of the Science of Food and Agriculture 102, no. 1: 175–184. https://doi.org/10.1002/jsfa.11344.

[48] Valle, R. L. D., M. Carmen, R.-V. M. José, and S. F. María. 2022. “Utilization of Oenococcus oeni Strains to Ferment Grape Juice: Metabolic Activities and Beneficial Health Potential.” Food Microbiology 101: 103895. https://doi.org/10.1016/j.fm.2021.103895.

[49] Vukmanovic, S., J. Vitas, S. Kravig, et al. 2024. “Influence of Main Production Variables on Nutritional Characteristics of Winery Effluent Kombucha.” Chemical Industry & Chemical Engineering Quarterly 30, no. 4: 285–294. https://doi.org/10.2298/CICEQ.231002001V.

[50] Wang, C., A. Mas, and B. Esteve-Zarzoso. 2016. “The Interaction Between Saccharomyces cerevisiae and Non-Saccharomyces Yeast During Alcoholic Fermentation Is Species and Strain Specific.” Frontiers in Microbiology 7: 502. https://doi.org/10.3389/fmicb.2016.00502.

[51] Wang, F., Y. Gao, H. Wang, et al. 2021. “Analysis of Volatile Compounds and Flavor Fingerprint in Jingyuan Lamb of Different Ages Using Gas Chromatography-Ion Mobility Spectrometry (GC-IMS).” Meat Science 175: 108449. https://doi.org/10.1016/j.meatsci.2021.108449.

[52] Wang, J., Z. Wang, H. Gao, et al. 2024. “Metabolomics and Flavor Diversity in Cabernet Sauvignon Wines Fermented by Various Origins of Hanseniaspora uvarum in the Presence and Absence of Saccharomyces cerevisiae.” LWT 203: 116396. https://doi.org/10.1016/j.lwt.2024.116396.

[53] Wang, X., G. Fan, Y. Peng, et al. 2023. “Mechanisms and Effects of Non-Saccharomyces Yeast Fermentation on the Aromatic Profile of Wine.” Journal of Food Composition and Analysis 124: 105660. https://doi.org/10.1016/j.jfca.2023.105660.

[54] Webber, V., S. V. Dutra, F. R. Spinelli, Â. R. Marcon, and G. J. Carnieli, and R. Vanderlinde. 2014. “Effect of Glutathione Addition in Sparkling Wine.” Food Chemistry 159: 391–398. https://doi.org/10.1016/j.foodchem.2014.03.031.

[55] Wei, J., Y. Zhang, Y. Qiu, et al. 2020. “Chemical Composition, Sensorial Properties, and Aroma-Active Compounds of Ciders Fermented With Hanseniaspora osmophila and Torulaspora quercuum in Co- and Sequential Fermentations.” Food Chemistry 306: 125623. https://doi.org/10.1016/j.foodchem.2019.125623.

[56] Xu, A., Y. Xiao, Z. He, et al. 2022. “Use of Non-Saccharomyces Yeast Co-Fermentation With Saccharomyces cerevisiae to Improve the Polyphenol and Volatile Aroma Compound Contents in Nanfeng Tangerine Wines.” Journal of Fungi 8: 128. https://doi.org/10.3390/jof8020128.

[57] Yang, Y., H. Li, Y. Xia, et al. 2024. “Unraveling Physicochemical Characteristics, Microbial Community Structure and Functional Enzymes Governing Fatty Acid Ethyl Esters Formation During Chinese Rice Wine Brewing.” LWT 198: 116035. https://doi.org/10.1016/j.lwt.2024.116035.

[58] Yu, W., Y. Zhu, R. Zhu, et al. 2022. “Insight Into the Characteristics of Cider Fermented by Single and Co-Culture With Saccharomyces cerevisiae and Schizosaccharomyces pombe Based on Metabolomic and Transcriptomic Approaches.” LWT 163: 113538. https://doi.org/10.1016/j.lwt.2022.113538.

[59] Zhang, B., V. Ivanova-Petropulos, C. Duan, and G. Yan. 2021. “Distinctive Chemical and Aromatic Composition of Red Wines Produced by Saccharomyces cerevisiae Co-Fermentation With Indigenous and Commercial Non-Saccharomyces Strains.” Food Bioscience 41: 100925. https://doi.org/10.1016/j.fbio.2021.100925.

[60] Zhang, J. G., J. J. Wang, W. W. Zhang, et al. 2024. “Metabolomics and HS-SPME-GC–MS-Based Analysis of Quality Succession Patterns and Flavor Characteristics Changes During the Fermentation of Lycium barbarum and Polygonatum cyrtonema Compound Wine.” Food Research International 184: 114270. https://doi.org/10.1016/j.foodres.2024.114270.

[61] Zhao, Y., W. Zheng, H. Xie, Q. Luo, C. Zhao, and Z. Wang. 2024. “Effect of Different Yeasts on Quality and Volatile Components of Ribes odoratum Wendl. Fruit Wine.” Food and Fermentation Industries 50, no. 12: 142–150. https://doi.org/10.13995/j.cnki.11-1802/ts.036992.

[62] Zhu, X., L. E. Healy, O. Sevindik, et al. 2022. “Impacts of Novel Blanching Treatments Combined With Commercial Drying Methods on the Physicochemical Properties of Irish Brown Seaweed Alaria esculenta.” Food Chemistry 369: 130949. https://doi.org/10.1016/j.foodchem.2021.130949.

[63] Zou, J., J. Wang, W. Ye, et al. 2022. “Citri Reticulatae Pericarpium (Chenpi): A Multi-Efficacy Pericarp in Treating Cardiovascular Diiseases.” Biomedicine & Pharmacotherapy 154: 113626. https://doi.org/10.1016/j.biopha.2022.113626.

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2025 The Author(s). Food Bioengineering published by John Wiley & Sons Australia, Ltd. on behalf of State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology.