{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T06:48:43Z","timestamp":1785739723541,"version":"3.56.0"},"reference-count":36,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T00:00:00Z","timestamp":1785715200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Modern Agricultural Industry Technology System Construction Special Project","award":["CARS\u201418"],"award-info":[{"award-number":["CARS\u201418"]}]},{"name":"\u201cXingdian talent support plan\u201d of Yunnan Province","award":["XDYC-QNRC-2024-480"],"award-info":[{"award-number":["XDYC-QNRC-2024-480"]}]},{"name":"High\u2014Level Talent Introduction and Training Project of Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences","award":["GR2025001"],"award-info":[{"award-number":["GR2025001"]}]},{"award":["GR2025001"],"award-info":[{"award-number":["GR2025001"]}],"id":[{"id":"https:\/\/ror.org\/02z2d6373","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>To improve the feeding value of forage mulberry as a non-grain feed, solid-state mixed fermentation with Lactobacillus plantarum and Pichia membranifaciens (1:1) was optimized via single-factor tests and an L9 (34) orthogonal design. The optimal conditions (7 days, 28 \u00b0C, 30% inoculum, 1:1.2 solid-to-water ratio) reduced crude fiber (CF) content by 44.60%, and increased crude protein (CP), crude fat (EE), and total amino acids (TAA) by 35.77%, 68.85%, and 40.85%, respectively (p &lt; 0.01). Mechanistically, the organic acids produced by L. plantarum lowered the fermentation pH to 4.0\u20134.5. This pH change was likely associated with the observed 50.84% increase in yeast cellulase activity. Yeast metabolites appeared to promote the proliferation of lactic acid bacteria (LAB). This implies a potential synergistic interaction between the two strains under acidic conditions. The in vitro ruminal dry matter digestibility (DMD) and CP digestibility reached 72.30% and 78.60%, respectively. These values were significantly higher than those of the unfermented control (p &lt; 0.01). Therefore, this pH-enzyme synergistic fermentation strategy can effectively degrade anti-nutritional components. It also improves the feeding nutritional quality of mulberry forage for animal production.<\/jats:p>","DOI":"10.3390\/ani16152366","type":"journal-article","created":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T06:17:40Z","timestamp":1785737860000},"page":"2366","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimizing Solid-State Mixed-Strain Fermentation of Forage Mulberry for Improved Nutritional Quality via Anti-Nutritional Factor Degradation"],"prefix":"10.3390","volume":"16","author":[{"given":"Yanzhen","family":"Duan","sequence":"first","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Na","family":"Wen","sequence":"additional","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daodian","family":"Wang","sequence":"additional","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingying","family":"Yang","sequence":"additional","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sha","family":"Li","sequence":"additional","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yemei","family":"Yang","sequence":"additional","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Yang","sequence":"additional","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junrong","family":"Huang","sequence":"additional","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wen","family":"Yang","sequence":"additional","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9939-8412","authenticated-orcid":false,"given":"Pingping","family":"Li","sequence":"additional","affiliation":[{"name":"Sericultural and Apicultural Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,3]]},"reference":[{"key":"ref_1","first-page":"192","article-title":"Analysis of nutritional components of mulberry for feed and evaluation of metabolic energy and amino acid digestibility in laying hens","volume":"11","author":"Liu","year":"2026","journal-title":"China Feed"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1186\/s13068-023-02368-2","article-title":"Cellulase-lactic acid bacteria synergy action regulates silage fermentation of woody plant","volume":"16","author":"Du","year":"2023","journal-title":"Biotechnol. Biofuels Bioprod."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Su, Y., Yu, Q., Xi, Y., Rong, Y., Long, Y., Xie, Y., Sun, H., Hao, J., Yang, F., and Zheng, Y. (2025). Role of laccase and xylanase, with or without ferulic acid esterase-producing Lactiplantibacillus plantarum, on the aerobic stability, microbial composition and in vitro degradability of mulberry silage. BMC Microbiol., 25.","DOI":"10.1186\/s12866-025-04165-3"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"105988","DOI":"10.1016\/j.envexpbot.2024.105988","article-title":"Integrated transcriptomic and proteomic analyses reveal the impact of drought and heat stress combination on Morus alba","volume":"228","author":"Song","year":"2024","journal-title":"Environ. Exp. Bot."},{"key":"ref_5","first-page":"4303","article-title":"Effects of fermented feed mulberry powder on antioxi-dant capacity and intestinal function of Ningxiang pigs","volume":"31","author":"Ding","year":"2019","journal-title":"Chin. J. Anim. Nutr."},{"key":"ref_6","first-page":"146","article-title":"Research progress on fermented mulberry leaves and their application in animal production","volume":"47","author":"Duan","year":"2024","journal-title":"Feed Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"110398","DOI":"10.1016\/j.foodres.2021.110398","article-title":"The potential of fermentation on nutritional and technological improvement of cereal and legume flours: A review","volume":"145","author":"Heredia","year":"2021","journal-title":"Food Res. Int."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e00873","DOI":"10.1016\/j.btre.2024.e00873","article-title":"Valorization of agro-industrial waste through solid-state fermentation: Mini review","volume":"45","author":"Perwez","year":"2025","journal-title":"Biotechnol. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1111\/j.1439-0396.2012.01294.x","article-title":"In vitro fermentation characteristics and effective utilisable crude protein in leaves and green pods of Moringa stenopetala and Moringa oleifera cultivated at low and mid-altitudes","volume":"97","author":"Melesse","year":"2013","journal-title":"J. Anim. Physiol. Anim. Nutr."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Guo, Y., Peng, H., Wang, H., Li, S., Zhao, J., and Zhang, W. (2025). Cellulase over lactic acid bacteria in enhancing antioxidant capacity of mulberry silage via phenolic release. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1725406"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Cui, X., Yang, Y., Zhang, M., Jiao, F., Gan, T., Lin, Z., Huang, Y., Wang, H., Liu, S., and Bao, L. (2022). Optimized Ensiling Conditions and Microbial Community in Mulberry Leaves Silage With Inoculants. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.813363"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Meng, C., Xie, J., Chen, J., Xuan, J., Zeng, Z., Lai, M., Kang, X., Li, J., Liu, G., and Tu, J. (2025). Fermentation of Pediococcus pentosaceus JC30 Improves Phytochemical, Flavor Characteristics and Antioxidant Activity of Mulberry Leaves. Molecules, 30.","DOI":"10.3390\/molecules30081703"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Lelis, D.L., Morenz, M.J.F., Paciullo, D.S.C., Roseira, J.P.S., Gomide, C.A.M., Pereira, O.G., and Chizzotti, F.H.M. (2025). Effects of Lactic Acid Bacteria on Fermentation and Nutritional Value of BRS Capia\u00e7u Elephant Grass Silage at Two Regrowth Ages. Animals, 15.","DOI":"10.1038\/s41598-025-88367-9"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1111\/1751-7915.14184","article-title":"Current approaches on the roles of lactic acid bacteria in crop silage","volume":"16","author":"Guo","year":"2023","journal-title":"Microb. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Wu, B., Ren, T., Li, C., Wu, S., Cao, X., Mei, H., Wu, T., Yong, M., Wei, M., and Wang, C. (2024). Exploring the Fermentation Products, Microbiology Communities, and Metabolites of Big-Bale Alfalfa Silage Prepared with\/without Molasses and Lactobacillus rhamnosus. Agriculture, 14.","DOI":"10.3390\/agriculture14091560"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"556","DOI":"10.5713\/ab.21.0270","article-title":"Effect of lactic acid bacteria and yeast supplementation on anti-nutritional factors and chemical composition of fermented total mixed ration containing cottonseed meal or rapeseed meal","volume":"35","author":"Yusuf","year":"2022","journal-title":"Anim. Biosci."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Ma, L., Li, J., Zhao, W., Wang, J., Li, Y., Xiong, Y., He, Y., Chu, X., and Liu, Q. (2025). Key cellulase components synergizing with lactic acid bacteria to degrade alfalfa lignocellulose to improve lactic acid fermentation. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1566973"},{"key":"ref_18","unstructured":"Huo, Z.W. (2024). Effect of L. plantarum and Cellulase on the Silage Quality of Grape Branches and Mulberry Leaves. [Master\u2019s Thesis, Shihezi University]."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Liao, C., Tang, X., Li, M., Lu, G., Huang, X., Li, L., Zhang, M., Xie, Y., Chen, C., and Li, P. (2022). Effect of lactic acid bacteria, yeast, and their mixture on the chemical composition, fermentation quality, and bacterial community of cellulase-treated Pennisetum sinese silage. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.1047072"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Hao, L., Jiang, F., Wang, Y., Wang, H., Hu, H., You, W., Hu, X., Cheng, H., Wang, C., and Song, E. (2024). Formic acid enhances whole-plant mulberry silage fermentation by boosting lactic acid production and inhibiting harmful bacteria. Front. Microbiol., 15.","DOI":"10.3389\/fmicb.2024.1399907"},{"key":"ref_21","unstructured":"(2022). Determination of Crude Fiber Content in Feeds (Standard No. GB\/T 6434-2022)."},{"key":"ref_22","unstructured":"(2018). Determination of Crude Protein in Feeds\u2014Kjeldahl Method (Standard No. GB\/T 6432-2018)."},{"key":"ref_23","unstructured":"(2025). Determination of Crude Fat in Feed (Standard No. GB\/T 6433-2025)."},{"key":"ref_24","unstructured":"(2025). Determination of Crude Ash in Feed (Standard No. GB\/T 6438-2025)."},{"key":"ref_25","first-page":"1436","article-title":"Effects of Fermentation with Different Strains on the Nutritional Values of Calliandra calothyrsus Leaf Powder","volume":"47","author":"Cui","year":"2020","journal-title":"Chin. Anim. Husb. Vet. Med."},{"key":"ref_26","unstructured":"(2019). Determination of Amino Acids in Feeds (Standard No. GB\/T 18246-2019)."},{"key":"ref_27","first-page":"3148","article-title":"Optimization of solid state fer-mentation process for mulberry leaf powder and its degradation effect on anti nutritional factors","volume":"38","author":"Duan","year":"2026","journal-title":"Chin. J. Anim. Nutr."},{"key":"ref_28","first-page":"26","article-title":"Progress of Probiotic Fermented Feed in Livestock and Poultry Production","volume":"46","author":"Qiao","year":"2025","journal-title":"Feed Ind."},{"key":"ref_29","first-page":"2849","article-title":"Effects of Different Com-pound Additives on Quality and Microbial Diversity of Mixed Silage of Forage Mulberry and King Grass","volume":"51","author":"Chen","year":"2024","journal-title":"Chin. Anim. Husb. Vet. Med."},{"key":"ref_30","unstructured":"Liu, S.R. (2024). Effects of Combined Application of Different Bacterial and Enzyme Preparations on the Silage Quality of Protein Mulberry. [Master\u2019s Thesis, Tarim University]."},{"key":"ref_31","first-page":"83","article-title":"Effects of different silage additives on nutritional compo-nents, fermentation parameters and microorganisms of feed mulberry silage","volume":"45","author":"Hao","year":"2022","journal-title":"Feed Res."},{"key":"ref_32","first-page":"126-130","article-title":"Effects of different silage additives on nutritional components, fermentation indexes, and microbiota of mulberry forage silage","volume":"48","author":"Ding","year":"2025","journal-title":"Feed Res."},{"key":"ref_33","first-page":"8008","article-title":"Effects of Different Additives on Fermentation Quality, Aerobic Stability and Microbial Community of Forage Mulberry Silage","volume":"37","author":"Hu","year":"2025","journal-title":"Chin. J. Anim. Nutr."},{"key":"ref_34","unstructured":"Deng, Z.R. (2025). Effects of Additives on Silage Quality and Antibiotic Resistance Genes of Feed Mulberry. [Master\u2019s Thesis, Guizhou University]."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"109285","DOI":"10.1016\/j.bej.2024.109285","article-title":"Functional enzyme analysis and metabolic regulation mecha-nism of the combined microflora LXB in the degradation of lignocellulose","volume":"206","author":"Hao","year":"2024","journal-title":"Biochem. Eng. J."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Wang, Y., Gou, C., Chen, L., Liao, Y., Zhang, H., Luo, L., Ji, J., and Qi, Y. (2023). Solid-State Fermentation with White Rot Fungi (Pleurotus Species) Improves the Chemical Composition of Highland Barley Straw as a Ruminant Feed and Enhances In Vitro Rumen Digestibility. J. Fungi, 9.","DOI":"10.3390\/jof9121156"}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/15\/2366\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T06:42:45Z","timestamp":1785739365000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/15\/2366"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,3]]},"references-count":36,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["ani16152366"],"URL":"https:\/\/doi.org\/10.3390\/ani16152366","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,3]]}}}