Fermentation doi: 10.3390/fermentation12060282

Authors: Kuntao Xu Yuyang Sheng Yaoming Deng Hongtao Han Bin Zeng

Plant-derived bioactive compounds, such as polyphenols and saponins, possess significant pharmacological value. However, conventional extraction methods often suffer from low efficiency, poor bioavailability, and environmental burdens. Aspergillus-based biotransformation has emerged as a superior platform for overcoming these limitations due to their robust secretomes, versatile metabolic networks, and the GRAS (Generally Recognized as Safe) status of specific industrially relevant species (e.g., A. oryzae and A. niger). Existing literature frequently focuses on isolated compounds or general fungal processes. To fill this gap, this review systematically links specific Aspergillus enzymatic systems to an “enzymatic hydrolysis–transformation–synthesis” closed-loop framework, which is essential for industrial-scale valorization. In this review, we summarize recent advances in the biotransformation of phytochemicals by A. niger, A. oryzae, and A. nidulans. These fungi utilize specialized enzymes—including β-glucosidases, cellulases, and glycosidases—to enable precise hydrolysis, deglycosylation, and detoxification under mild conditions. We highlight representative transformations that demonstrate markedly enhanced bioactivity and solubility. Key examples include the conversion of polydatin to resveratrol (>90% yield) and ginsenoside Rb1 to ginsenoside compound K (94.4% conversion rate). Although industrial applications span the food, pharmaceutical, and cosmetic sectors, significant challenges persist in solid-state fermentation (SSF) scale-up, strain stability, target compound over-degradation, and downstream purification. Genetic engineering, process optimization and hybrid bioprocessing are highlighted as promising strategies to overcome these limitations and realize sustainable, high-value production of natural bioactive metabolites.

Fermentation doi: 10.3390/fermentation12060281

Authors: Junhao Yue Wanting Wu Fangfang Fan Weirui Zhao Sheng Hu Zhuhua Chan Lehe Mei Changjiang Lyu

γ-Aminobutyric acid (GABA), a vital bioactive component, is biosynthesized via the decarboxylation of L-glutamate (L-Glu) catalyzed by glutamate decarboxylase (GAD). However, the GADs from various sources commonly suffer from low thermal stability, which hampers their industrial applications. In this work, four ancestral sequences of GAD (Anc19, Anc20, Anc28, and Anc30) were designed via an ancestral sequence reconstruction (ASR) approach. Thereafter, the genes were synthesized and heterologously expressed in the probiotic Escherichia coli strain Nissle 1917 (EcN). Among all variants tested, Anc28 exhibited the highest catalytic performance. The Km and kcat values were determined to be 26.80 mM and 57.41 s−1, respectively, yielding a catalytic efficiency (kcat/Km) of 2.14 s−1mM−1, which was 2.71-fold higher than that of the wild-type enzyme. Meanwhile, compared with the wild-type GAD, Anc28 exhibited a 6.74 °C increase in T5015 and a 4.1-fold extension in t1/2 at 60 °C. Furthermore, the GABA synthesis system using dormant Escherichia coli Nissle (T7)/pET28a-gadBAnc28 cells as the biocatalyst and pure water as a sole medium was also constructed. Upon completion of the 4 h reaction, the GABA titer reached 307.53 g/L with a conversion ratio of 99.36%. The resulting engineered strains were successfully employed for the efficient biosynthesis of GABA.

Fermentation doi: 10.3390/fermentation12060280

Authors: Gilver Rosero-Chasoy Elda España-Gamboa Jesús Alejandro Vazquez-Barea José Martin Baas-López Tanit Toledano-Thompson Liliana Alzate-Gaviria Raúl Tapia-Tussell

Methane production from Brosimum alicastrum seed coat was evaluated using a logistic model through three alkaline concentrations (0.19 M, 0.26 M, and 0.28 M) and three enzymatic activity levels (3000 U mL−1, 5000 U mL−1, and 7000 U mL−1) as pretreatments. Laccase was produced through submerged fermentation using T. hirsuta Bm-2 fungi, while NaOH served as the alkaline agent. Enzymatic pretreatment resulted in the highest specific CH4 yield (427.43 ± 2.28 mL CH4/g VSadded), surpassing both alkaline pretreatment (235.61 ± 9.19 mL CH4/g VSadded) and the control (102.54 ± 5.55 mL CH4/g VSadded). Kinetic analysis of CH4 production indicated that cumulative CH4 production reached its stationary phase within 30 days of digestion. Moreover, enzymatic pretreatment exhibited the highest CH4 formation rate (0.15–0.16 h−1), except for the control, which had a slightly higher rate (0.22 h−1). The kinetic analysis revealed that the enzymatic pretreatment significantly improved the hydrolysis stage of Ramon’s seed coat, promoting higher cumulative CH4 production and leading to an increased specific CH4 yield.

Fermentation doi: 10.3390/fermentation12060279

Authors: Jiaying Xiong Meixia Chen Laiping Zhang Qi Zhou Zhenyu Huang Xiaobin Lin Xiaomin Fang Xiangdong Ye Weiping Zhu Wei Liu Aiqin Shi

In the original publication [...]

Fermentation doi: 10.3390/fermentation12060278

Authors: Hümeyra Çetin Babaoğlu Talha Demirci Nihat Akın Sultan Arslan Tontul

This study aimed to evaluate the technological, functional and nutritional effects of exopolysaccharide-producing lactic acid bacteria (LAB) strains, isolated from artisanal Tulum cheese as type II sourdough starters. The objective of this study was to improve bread quality, delay staling, and enhance bioactive properties, such as antioxidant capacity and estimated glycaemic index (eGI). Six LAB strains (Loigolactobacillus coryniformis, Lactiplantibacillus plantarum, Levilactobacillus brevis, Lacticaseibacillus paracasei, Lactobacillus helveticus, and Lacticaseibacillus rhamnosus) were individually used for sourdough fermentation. Bread samples were analyzed for pH, titratable acidity (TA), LAB counts, specific volume, colour, total phenolic content (TPC), antioxidant activity (DPPH and ABTS), starch digestibility, eGI, staling kinetics (Avrami model) and amylopectin retrogradation (DSC). Strain-dependent improvements in bread functionality were observed. L. brevis and L. coryniformis strains increased sourdough acidity to a greater extent, and resulting in lower pH values. Accordingly, bread produced with sourdough fermented by these strains exhibited higher specific volume than the control. Although higher ABTS radical scavenging activity and TPC were detected in sourdough bread compared to the control bread, no significant differences were observed among the breads in terms of total antioxidant activity measured by DPPH. L. rhamnosus significantly improved antioxidant activity and reduced the eGI. L. coryniformis, L. plantarum and L. brevis were the most effective at retarding staling, reducing the increase in hardness and limiting amylopectin retrogradation. This study is the first to demonstrate the functional potential of LAB strains from artisanal Tulum cheese as sourdough starters. These findings reveal the potential for developing clean-label bakery products with an extended shelf life and improved health-related functionality.

Fermentation doi: 10.3390/fermentation12060277

Authors: Yinglong Wang Shiguo Chen Keyu Lei Yunfeng Pu Yang Li Boqun Liu Xujie Hou

Musalais is a traditional fermented beverage of the Uyghur people in Xinjiang, China. Its production involves boiling grape juice at high temperatures to concentrate it and enhance its sugar content, followed by natural fermentation. However, this high-temperature concentration process leads to a significant loss of bioactive and flavor compounds, adversely affecting the quality of the final product. Adding composite ingredients may help mitigate this quality decline. This study compares Musalais new product with traditional Musalais. Phenolic analysis showed that total monomeric phenols were 182.36 mg·L−1 in the new product versus 14.76 mg·L−1 in traditional Musalais. Headspace solid-phase microextraction/gas chromatography–mass spectrometry (HS-SPME/GC-MS) identified 72 volatile compounds in the new product (total content of 569,848.88 μg·L−1) compared to 58 compounds (total content of 362,774.17 μg·L−1) in traditional Musalais. Compared to traditional Musalais, the new product exhibits a 24.14% increase in volatile compound variety and a 57.09% increase in total concentration, with more pronounced floral, fruity, and vinous aromas, as well as higher sensory scores. Non-targeted metabolomics suggests that the new product may have superior phenolic and volatile profiles.

Fermentation doi: 10.3390/fermentation12060276

Authors: Mengwei Li Qian Deng Lijuan Peng Fang Xie Qian Lin Huade Xie Chengjian Yang

Elephant grass–ramie mixed silage represents a promising strategy to valorize tropical forage resources for ruminant production, yet the dynamic changes in microbial community and fermentation quality during semi-commercial silo bag ensiling remain poorly understood. Elephant grass and ramie were co-ensiled at a 70:30 (w/w) ratio and stored at room temperature for 15, 30, and 45 days. Fermentation quality was evaluated by pH, lactic acid, acetic acid, and ammonia nitrogen, while bacterial and fungal communities were analyzed via 16S and ITS rRNA high-throughput sequencing in triplicate. The results revealed limited fermentation efficiency across all periods, characterized by relatively high pH (≥5.1), restricted lactic acid accumulation, and substantial butyric acid concentrations. Vertically, silage at day 30 exhibited a transient, relatively better acidification profile (lowest pH and highest lactic acid) compared to days 15 and 45, though still sub-optimal overall. Bacterial diversity increased significantly by day 45, concurrent with a marked reduction in the relative abundance of Firmicutes, whereas fungal diversity declined progressively throughout the ensiling period. Enterococcus was identified as the core functional bacterium closely correlated with key fermentation parameters, while most fungal taxa exerted negative effects on silage quality. These findings demonstrate that while a 30-day ensiling duration offers a relatively stable window under restricted acidification, the high fibrous nature and baseline composition of the mixed material present challenges for preservation. This study provides a transparent theoretical basis and underscores the critical need for technical interventions in semi-commercial elephant grass–ramie ensiling.

Fermentation doi: 10.3390/fermentation12060275

Authors: Cailian Wu Yajie Wang Minwei Zhang Nurgul Reheman Rui Zhang Xiaoying Zhu

This study investigated the effects of single-strain fermentation (using Lactobacillus paracasei XY1-4 and Saccharomyces cerevisiae XX1-2) and 1:1 mixed fermentation on the differential metabolites and in vitro cholesterol-lowering activity of Cyperus esculentus L. tuber milk. A non-targeted metabolomics approach based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied, and the dynamic changes in physicochemical properties and in vitro cholesterol-lowering rates during fermentation were determined to systematically compare the metabolite profiles and functional characteristics of tuber milk samples subjected to single-strain fermentation and mixed fermentation for 12 h and 24 h, respectively. The results showed that a total of 1085 differential metabolites were screened across all fermentation groups under the criteria of VIP ≥ 1, p < 0.05, and FC ≥ 2 or FC ≤ 0.5, which were primarily classified as lipids and lipid-like molecules as well as organic acids and their derivatives. Enrichment analysis of these differential metabolites identified ten metabolic pathways closely associated with fermentation progression and functional activity, including arginine biosynthesis, the tricarboxylic acid (TCA) cycle, and glutathione metabolism. Further correlation analysis demonstrated that key metabolites such as succinic acid and L-glutamic acid were significantly and positively correlated with the in vitro cholesterol-lowering rate. This study clarifies the effects of different fermentation treatments on the metabolome and functional properties of tuber milk, laying a solid theoretical foundation for the development and quality optimization of functional fermented Cyperus esculentus L. products.

Fermentation doi: 10.3390/fermentation12060274

Authors: Diana Karina Olvera-Rosales Jesús Guadalupe Pérez-Flores Luis Guillermo González-Olivares Juan Ramírez-Godínez Ilse Monroy-Rodríguez Raúl Eduardo López-Hernández Lizet Manzo-Martínez María Aline Manzo-Martínez Laura Berenice Olvera-Rosales Emmanuel Pérez-Escalante

The global market for fermented functional foods containing probiotics has expanded rapidly, driven by increasing consumer interest in health-oriented food products. However, despite their long history and recognized benefits, the international commercialization of these products is hindered by heterogeneous regulatory frameworks. This review examines the current global regulatory landscape governing probiotic fermented foods, with particular emphasis on key regions, including the United States, the European Union, Asia, Africa, Oceania, and Latin America. Differences in product classification, safety assessment, the permitted use of the term “probiotic”, and scientific substantiation requirements for health claims are discussed. Additionally, major regulatory barriers affecting international trade are analyzed, including non-tariff barriers, labeling restrictions, and variability in sanitary registration and approval processes. These challenges are further compounded by inconsistencies in evaluation methodologies and the lack of harmonized criteria for microbial characterization and functional validation. Together, these factors limit product standardization, increase development costs, and hinder innovation and market access, particularly for emerging probiotic strains. This review highlights the need for greater international harmonization and for integrating robust scientific evidence into regulatory frameworks to facilitate global trade while ensuring consumer safety and claimed product functionality.

Fermentation doi: 10.3390/fermentation12060273

Authors: Wangruixue Tang Zonghao Cheng Weide Li Pengsong Li Ming Chen Yujie Fu

Hydrogen molecules can serve as a promising clean energy supplier; conventional hydrogen production usually relies on fossil fuels and leads to intense greenhouse gas emissions. Significant emphasis has been placed on exploring sustainable and renewable hydrogen resources. Cyanobacteria can convert solar energy into hydrogen through oxygen-sensitive hydrogenases or nitrogenases. However, practical application remains severely constrained by oxygen-evolving photosynthesis, inefficient electron allocation, and the low metabolic priority of hydrogen production in cyanobacterial cells. In recent years, substantial progress has been achieved in understanding hydrogen metabolism and improving hydrogen production through physiological regulation, hydrogenase engineering, photosynthetic electron transport chain (PETC) reconstruction, metabolic engineering, and biohybrid systems. This review summarizes recent advances in cyanobacterial hydrogen production, with particular emphasis on hydrogen-producing pathways, key limiting factors, and current engineering strategies. Importantly, this review highlights that many currently reported strategies still provide only transient improvements because hydrogen production is constrained by system-level conflicts among photosynthesis, redox balance, carbon fixation, and cellular stability. In addition, emerging approaches including metagenomic resource mining, synthetic biology, AI-assisted engineering, biohybrid photoelectrochemical systems, and techno-economic optimization are discussed as potential directions for improving the efficiency, scalability, and practical feasibility of cyanobacterial hydrogen production technologies in the future.

Fermentation doi: 10.3390/fermentation12060272

Authors: Jing Shao Ying Yin Jianping Wen

Vancomycin is the drug of last resort against Staphylococcus aureus infections, yet its industrial production is constrained by the inherently low yields of wild-type strains. Through systematic optimization of culture medium and fermentation conditions, we increased vancomycin production by Amycolatopsis orientalis ATCC 19795 from 0.60 g/L to 2.28 g/L, a 3.8-fold improvement. Transcriptomic analysis revealed that this optimized process activated central carbon metabolism, precursor biosynthetic pathways, and the rpm ribosomal gene cluster, thereby enhancing both precursor availability and translational efficiency. Furthermore, the downregulation of genes involved in fatty acid and biotin synthesis redirected carbon flux toward central metabolism and the TCA cycle. This synergistic regulatory network drove a substantial increase in vancomycin production and provides a theoretical foundation for the rational metabolic engineering of industrial production strains.

Fermentation doi: 10.3390/fermentation12060271

Authors: Zhenwei Zhang Xiaoyu Liu Xiuwen Chen Hongzhen Zhu Qingyu Xu Lin Wei Jinjin Wei Mingxia Han Yifan Wang Muhammad Zahoor Khan Changfa Wang

Locally sourced roughages constitute the dietary foundation of donkey production in northern China, yet their fermentation behavior in the donkey hindgut remains poorly characterized. The present study employed in vitro batch cultures to compare the dry matter disappearance (IVDMD), gas production (GP) kinetics and short-chain volatile fatty acid (VFA) profiles of five locally available feedstuffs—peanut vine (PNV), soybean straw (SBS), wheat shell (WS), reed grass (RG) and bamboo leaf (BL)—when incubated separately with cecal or colonic microbial inocula obtained from Dezhou donkeys. After 40 h incubation, both IVDMD and total VFA concentrations ranked identically across the two hindgut segments: PNV > SBS > WS > RG > BL (p < 0.05), and all indices were consistently higher in the cecal than in the colonic fermentation system (p < 0.05). The asymptotic gas production (A) and the time required to reach half of A (T1/2) followed the same ranking as IVDMD (p < 0.01), indicating that feedstuffs with greater fermentable substrate availability sustained fermentation for longer periods. In contrast, the fractional gas production rate (c) and the average gas production rate (AGPR) in RG and BL exceeded those of PNV, SBS, and WS under cecal incubation (p < 0.05), reflecting rapid utilization of a small pool of readily fermentable components in these fibrous substrates. Regarding VFA stoichiometry, BL yielded the highest molar proportion of acetate and PNV the lowest in the colonic system (p < 0.05), whereas the propionate proportion followed the order PNV > SBS > WS > RG > BL (p < 0.01). Consequently, the acetate-to-propionate (A:P) ratio and the non-glucogenic-to-glucogenic (NGR) ratio were highest in BL (p < 0.05). The molar proportions of butyrate and branched-chain VFAs (BCVFAs) in WS, RG, and BL were greater than those in PNV (p < 0.05). Collectively, the five feedstuffs differed markedly in their fermentability, kinetic behavior, and VFA yield profiles, reflecting distinct energy-supply potentials for the donkey host. PNV and SBS exhibited superior overall in vitro fermentation performance and are therefore recommended as preferred roughage sources, whereas BL and RG may serve complementary roles by supporting hindgut epithelial health through elevated butyrate production. These findings provide a mechanistic basis for the rational selection and combination of locally sourced roughages to optimize feeding strategies and improve feed-use efficiency in donkey production.

Fermentation doi: 10.3390/fermentation12060270

Authors: Sema Nur Yildirim Hümeyra İspirli Enes Dertli

Limosilactobacillus reuteri is a lactic acid bacteria (LAB) recognized for its significant technological and functional properties. This species produces diverse metabolites, including reuterin, exopolysaccharides (EPSs), B-group vitamins, short-chain fatty acids (SCFAs), and bioactive peptides, which benefit food systems and host health. However, high strain-specificity and metabolite complexity present challenges in elucidating precise mechanisms of action of these metabolites for their functional and technological roles. This review provides a comprehensive perspective on the latest applications of Lmb. reuteri and its metabolites in food technology by focusing on its utilization in diverse matrices, including dairy, plant-based products, supplements, and edible packaging systems, where it serves to extend shelf life, enhance nutritional profiles, and improve sensory attributes. The strain-specific nature of Lmb. reuteri allows for tailored applications to meet technological requirements. As consumer demand for clean-label and health-promoting foods increases, Lmb. reuteri stands out as a critical LAB species, for the development of next-generation functional foods and preservation strategies. Understanding the potential roles of this species in human and animal health and food safety will help us to build a roadmap for future research and industrial implementation.

Fermentation doi: 10.3390/fermentation12060269

Authors: Alonso R. Poma Ticona Heber E. Ramirez-Arua Roberto Castellanos Jéssica P. Silva Artur Carvalho Stranz Amparo Iris Zavaleta Igor Polikarpov Eliane F. Noronha Pedro R. Vieira Hamann

Starch-degrading enzymes are key biocatalysts in industrial applications, particularly when derived from thermophilic microorganisms with potential to operate under elevated temperatures. In this study, three recombinant starch-degrading enzymes were heterologously produced, purified, and biochemically characterized: an α-amylase from Geobacillus kaustophilus, and an α-glucosidase and a type I pullulanase from Geobacillus sp. G4, a thermophilic strain isolated from a geothermal field in southern Peru. The three enzymes were successfully expressed in soluble form in Escherichia coli and purified by one-step affinity chromatography. Biochemical characterization showed that α-glucosidase and α-amylase displayed optimum activity at pH 6–7, whereas pullulanase exhibited a broader pH profile, retaining high activity up to pH 9. All three enzymes reached maximum activity at 60 °C, although their thermal stability profiles differed markedly, with pullulanase showing the highest thermostability. Metal ion assays revealed enzyme-dependent effects, with pullulanase being stimulated by Ca2+ and Mg2+, while α-amylase and α-glucosidase showed limited responses to divalent ions. Kinetic analysis using soluble potato starch indicated that α-amylase had the most favorable catalytic profile, with the lowest Km and the highest catalytic efficiency among the three enzymes. Functional hydrolysis assays demonstrated that all enzymes were active on soluble starch and pretreated potato peel, while the enzymatic mixture consistently released the highest concentration of reducing sugars. These results expand the biochemical knowledge of thermophilic amylolytic enzymes from Geobacillus and support their potential use in future enzymatic systems for the conversion of starch-rich residues.

Fermentation doi: 10.3390/fermentation12060268

Authors: José Rubén Morones-Ramírez

This focused review examines fermentation and fermentation-integrated microbial platforms that convert two regionally relevant substrate classes, Latin American agro-industrial residues and concentrated CO2 streams, into high-value bioproducts. The review is not intended as a complete survey of all biomass valorization routes in Latin America. Instead, it evaluates platform–feedstock–product combinations with clear translational relevance for regional biorefineries, with emphasis on literature from 2020–2025 and on earlier benchmark studies only when they define current technical performance limits. Latin America and the Caribbean combine high-volume sugarcane, agave, coffee, citrus, banana, cacao, and tuber-processing residues with biogenic CO2 from ethanol fermentation and industrial point sources from cement, lime, and oil-and-gas operations. The technical opportunity is therefore not residue abundance alone, but the rational coupling of residue chemistry, CO2-source quality, locally isolated microbial strains, and process architectures that can be scaled under regional constraints. We compare phototrophic CO2-fixing modules based on cyanobacteria and microalgae, chemoautotrophic gas fermentation using Cupriavidus necator and related systems, heterotrophic yeast platforms including Rhodotorula spp. and Yarrowia lipolytica, and bacterial platforms for PHAs, bacterial cellulose, and organic acids. The core technical analysis focuses on substrate conditioning, hydrolysate inhibition, oxygen- and gas-transfer constraints, light delivery, C/N control, mixed-sugar utilization, metabolic engineering, reactor configuration, downstream processing, and quantitative reporting metrics. One fermentation-integrated laboratory case study—the Synechocystis sp. PCC 6803–Rhodotorula mucilaginosa UANL-001L CO2-to-carotenoid relay—and one explicitly defined non-fermentative boundary case on peel-extract-derived coating films are used to illustrate two different aspects of regional biorefinery design: dual-feedstock microbial conversion and low-CapEx product-fit decisions for agro-industrial residues. We conclude that Latin America’s strongest near-term position is in technically disciplined, product-specific biorefineries that integrate local feedstock chemistry with engineered or locally adapted chassis, rather than in generic biomass-to-product claims.

Fermentation doi: 10.3390/fermentation12060267

Authors: Alejandra Velasco-Pérez José Vian Hector Puebla Mariana Rodríguez-Jara Adán Cabal-Prieto Rocío Solar-González Javier Emanuel Bulbarela-Marini Tania García-Herrera Jesús Atenodoro-Alonso Emmanuel de Jesús Ramírez-Rivera

Anaerobic digestion (AD) of food waste is strongly influenced by substrate characteristics and operating conditions, particularly substrate composition, particle size, and substrate-to-inoculum (S/I) ratio. In this study, a modified ADM1-R3 model, incorporating a surface-based disintegration kinetics approach, was developed to evaluate the simultaneous influence of these factors on methane production. Sensitivity analysis identified the disintegration and hydrolysis constants as the most influential parameters, while principal component analysis supported a sequential calibration strategy. The model was calibrated using literature data and verified against independent datasets, achieving R2 values of 0.9967 (calibration) and 0.9745–0.9880 (verification). Simulation results showed that optimal performance was observed at low S/I ratios (0.5–1) and intermediate particle sizes (1.4–4 mm), with maximum yields of 419, 744, and 581 mL·g−1 VS for carbohydrate-rich, protein- and lipid-rich, and mixed substrates, respectively. Overall, the model provides a consistent framework for analyzing AD behavior and identifying favorable operational conditions, although further validation under unstable operating conditions is required to fully assess its predictive capability.

Fermentation doi: 10.3390/fermentation12060266

Authors: Jose Bueno-Mancebo Adriana Artola Raquel Barrena Antoni Sánchez Teresa Gea

Although solid-state fermentation (SSF) has long been used in food production in various traditional contexts, it is now emerging as a particularly promising strategy for the development of functional food ingredients from plant materials and agro-industrial side streams. This review examines recent advances in the application of SSF to enhance the nutritional, functional, sensory, and technological properties of food matrices. Current evidence indicates that SSF can increase the bioactive potential of plant-based substrates by promoting the release and biotransformation of phenolic compounds, while also improving antioxidant capacity, protein digestibility, and techno-functional performance. In addition, the process may support the formation of food-relevant metabolites, including vitamins, peptides, organic acids, and other secondary compounds, while reducing selected antinutritional, allergenic, and undesirable constituents. These compositional changes are often accompanied by modifications in aroma, volatile profiles, visual attributes, and, more recently, gut microbiota-related effects. Attention is given to the use of fungal-based processes for the valorization of cereals, legumes, fruit by-products, and other underutilized substrates. The review also addresses the growing industrial interest in SSF, especially in relation to mycelium-based foods, alternative proteins, functional ingredients, and feed applications. Despite its clear potential, the broader implementation of SSF will require further research and development to support its effective translation into food applications.

Fermentation doi: 10.3390/fermentation12060265

Authors: Jenny Ji-Chan Hung Teresa Pei-Ju Tsai Ethan I. Lan

Escherichia coli Nissle 1917 (EcN) has transformed from a traditional probiotic into a versatile microbial cell factory through innovations in genomic tools and metabolic engineering. This review summarizes recent progress in utilizing EcN for biochemical synthesis. First, the development of genetic editing tools is systematically discussed, highlighting how these methods serve as the foundation for metabolic rewiring. Second, we examine EcN bioproduction capabilities, including its application as in situ Live Biotherapeutic Products (LBPs) for targeted disease interventions and its use in the ex vivo biosynthesis of pharmaceuticals and nutraceuticals. Third, optimization strategies for fermentation processes, focusing on diverse carbon source assimilation and industrial scale-up parameters, demonstrate the potential of this strain for commercial production. Through these advancements, EcN emerges as a practical platform for next-generation biomanufacturing and precision medicine.

Fermentation doi: 10.3390/fermentation12060264

Authors: Baoyu Peng Bifeng Chen Zhaozhao Dai Jinwen Chen Lang Hu Lelei Wen Changchun Li

Rice wine fermentation involves complex biochemical dynamics that challenge traditional empirical control, highlighting the need for precise analytical characterization. This narrative review synthesizes the technological evolution of metabolomics from a descriptive tool to a driver of intelligent biomanufacturing. The progression from first-generation compositional profiling to third-generation strategies integrating high-resolution mass spectrometry, real-time sensing, multi-omics approaches, and artificial intelligence is delineated. This evolution has shifted research focus from static component cataloging to dynamic pathway elucidation, enabling deeper interpretation of flavor biosynthesis, functional metabolite formation, and accumulation of safety-related metabolites. Furthermore, this review critically analyzes how multi-omics integration reveals microbiome-metabolite interactions and provides mechanistic targets for quality regulation. Despite these advances, a gap remains between laboratory-scale analytical capabilities and industrial implementation. Key translational bottlenecks are identified, and a future roadmap toward AI-driven digital twin systems and real-time adaptive control is proposed. This framework positions metabolomics not merely as an analytical technique, but as a key foundation of next-generation smart fermentation strategies.

Fermentation doi: 10.3390/fermentation12060263

Authors: Bruna Lise Tusset Iago Mocelin Lorenza Corti Villa Alice Elvira Teixeira dos Santos Rafael de Matos Lívia Kmetzsch Fernanda Cortez Lopes

Microbial pigments are secondary metabolites that represent promising alternatives to synthetic colorants, offering advantages even over other natural sources. These pigments can be produced independently of seasonality and at low cost, especially when using agro-industrial residues as substrates, and their production can be optimized. Bioprospecting of microorganisms in unexplored environments offers valuable opportunities to discover safer and more efficient pigment producers. Brazil harbors vast biodiversity across multiple biomes, providing a rich reservoir for such discoveries. Biomes such as the Atlantic Forest, Pampa, Pantanal and Coastal Marine are still poorly explored with respect to the bioprospecting of pigment-producing microorganisms, representing a valuable opportunity for the discovery of novel pigments. However, several bottlenecks still hinder the regulatory approval of microbial pigments, particularly those produced by filamentous fungi, due to the frequent co-production of mycotoxins. To overcome these challenges, genetic engineering tools are crucial for eliminating mycotoxin co-production. CRISPR-Cas9, CRISPRi and CRISPR-Cpf1 have become the most widely used techniques for this purpose. Another key application of CRISPR is the enhancement of pigment yields, which can accelerate the industrial adoption of microbial pigments. Together, these two strategies, bioprospecting new environments and genetic engineering, can significantly speed up the transition from synthetic pigments to safer and more eco-friendly microbial alternatives.

Fermentation doi: 10.3390/fermentation12060262

Authors: Fotini Drosou Panagiotis Tataridis Vassilis G. Dourtoglou Vassiliki Oreopoulou

Yeasts are among the intrinsic factors affecting the quality of beer due to their impact on flavor. Apart from a long and rich brewing tradition of natural fermentations, involving different yeast and bacterial species in regions such as Belgium, non-Saccharomyces yeasts have not been the preferred industrial inoculation strains in both winemaking and brewing sectors. This is mainly due to their slower fermentation rates, lower alcohol tolerance, and limited ability to complete fermentation. This review explores the brewing characteristics of Torulaspora delbrueckii strains, focusing on the aromatic profile of the produced beers, in comparison with the well-known Saccharomyces cerevisiae brewing strains. The effect of fermentation parameters, as well as the use of mono- and mixed cultures with S. cerevisiae, is discussed. Specific T. delbrueckii strains have demonstrated the ability to ferment wort sugars and produce alcohol levels of up to 4–6% (v/v). The main volatile compounds produced include higher alcohols, such as isoamyl and phenyl ethyl alcohol, as well as esters and their acid precursors. Among these, ethyl hexanoate and ethyl octanoate are particularly important due to their contribution to fruity aromas. Mixed fermentations involving T. delbrueckii and S. cerevisiae have been shown to enhance ester production, resulting in improved aromatic complexity in the final product.

Fermentation doi: 10.3390/fermentation12060261

Authors: Manuel Malfeito-Ferreira

The winemaking sector is currently experiencing a period of deep transformation, driven by the convergence of traditional oenological knowledge with advances in biotechnology, microbial ecology, analytical chemistry, and sustainable production systems [...]

Fermentation doi: 10.3390/fermentation12060260

Authors: Xinyue Wang Yuhang Zheng Xinyu Wang Boran Zhang Wanli Sha Wenlong Dong Baishuang Yin

Research has demonstrated that the presence of synthetic preservatives tends to disrupt the balance of the gut microbiota, thereby posing a significant threat to food safety and human health. The present study investigates the antibacterial activity of a novel bacteriocin-like produced by Latilactobacillus sakei (L. sakei) against Escherichia coli (E. coli), as well as its mechanism of action. This study aims to validate the significant antibacterial effect of bacteriocin-like YY-1 produced by L. sakei W-1 through dialysis combined with Tricine-SDS-PAGE analysis, and to determine its molecular weight. The results of the study indicate that the molecular weight of the bacteriocin-like is less than 2.7 kDa. Moreover, this bacteriocin-like YY-1 exhibits broad-spectrum antimicrobial properties, demonstrating antibacterial activity against E. coli, S. Typhimurium, A. baumannii, P. mirabilis, S. aureus, L. monocytogenes. Furthermore, bacteriocin-like YY-1 exhibits optimal antibacterial activity at a pH of 4.0, with its activity gradually diminishing as pH increases. It is completely inactivated by trypsin treatment, while papain and proteinase K treatments significantly reduce its antibacterial activity. Additionally, this bacteriocin-like YY-1 has been demonstrated to inhibit the growth of E. coli and disrupt its normal development. The Viable/Necrotic Cell Stain and SEM observations confirmed that the bacteriocin-like compound induces cell death by forming distinct pores through the disruption of the cell membrane’s structural integrity. In summary, YY-1—a bacteriocin-like substance characterised by its low molecular weight (<2.7 kDa), broad-spectrum activity, and pore-forming mechanism—is a highly promising natural alternative to synthetic preservatives, capable of mitigating the damage caused by chemical additives to the gut microbiota.

Fermentation doi: 10.3390/fermentation12060259

Authors: Faqinwei Li Zheng Wang Chaoyang Wei Qiuhui Li Naoto Shimizu Yongheng Yuan

The overuse of mineral fertilizers has brought about numerous matters such as deteriorating soil health, crop safety concerns, and environmental pollution. The global requirements for effective waste handling and sustainable agricultural production have been growing continuously. Therefore, integrated nutrient management method might be a key way to achieve circular agriculture, such as replacing chemical fertilizers with organic fertilizers. In modern agriculture, digestate that is a byproduct of anaerobic digestion as a fertilizer is becoming increasingly favored as a viable method for improving crop yield and quality. However, the application of digestate in agriculture have not yet been fully explored. This review addresses a knowledge gap by synthesizing current research on digestate as a fertilizer. Firstly, the physical–chemical and biological properties of digestate are discussed. Following that, this review focuses on its specific impact on crop growth and quality. Lastly, it outlines the challenges faced in the application of digestate and looks ahead to future trends. With appropriate policy support and technological innovation, digestate holds promise for advancing environmental sustainability. This review aims to provide direction and reference for future research on the application of digestate.

Fermentation doi: 10.3390/fermentation12060258

Authors: Jingxin Jiang Mingyao Wang Huibo Luo Dan Huang

Issatchenkia orientalis Y1 is an important functional microorganism during the fermentation of strong-flavor Baijiu and may influence microbial community structure, community assembly mechanisms, and microbial functional potential predicted from sequencing data. In this study, a mixed-culture fermentation system was established using fermented grains and Huangshui from strong-flavor Baijiu production. I. orientalis Y1, isolated from fermented grains, was inoculated into this system as a perturbation treatment to investigate its effects on microbial diversity, community composition, and functional profiles, as well as its influence on microbial community assembly pathways. The results showed that inoculation with I. orientalis Y1 significantly altered microbial diversity and increased community complexity. The relative abundances of Paenibacillus, Aspergillus, and Acetobacter decreased, with the peak abundance of Paenibacillus declining from 62.8% to 40.2% and that of Acetobacter decreasing from 27.8% to 19.1%, while Aspergillus remained consistently less abundant than in the control group throughout fermentation. In contrast, the relative abundances of Issatchenkia and Lactobacillus increased, with their peak abundances rising from 15.0% to 23.9% and from 7.6% to 27.0%, respectively. In addition, it increased the contribution of deterministic processes in community assembly, with the deterministic proportion rising from 31.1% to 35.5% in bacterial communities and from 28.4% to 48.8% in fungal communities, while the contribution of stochastic processes decreased. These changes suggest that the microbial community became more controllable and functionally more stable after inoculation. Meanwhile, the overall predicted metabolic activity of the microbial community declined. In conclusion, the addition of I. orientalis Y1 reshaped microbial community structure, influenced microbial community assembly processes, and altered the correlations between dominant microorganisms and metabolic pathways.

Fermentation doi: 10.3390/fermentation12060257

Authors: Sebastian Andrade Claudia García Samanta Iturralde Jorge Delgado-Noboa Verónica Pinos-Vélez Mónica Abril-González Angelica Vele-Salto

The urgent need to reduce greenhouse gas emissions has driven the search for sustainable alternatives to fossil fuels. In this context, cocoa residues emerge as a promising feedstock for bioethanol production. This study evaluated the influence of a catalytic biorefinery treatment on the bioethanol production potential from cocoa pod husks. Both raw and catalytically treated biomass were characterized using SEM, pore size distribution analysis, and TGA. Subsequently, enzymatic hydrolysis was performed using various cellulase and hemicellulase loadings, followed by anaerobic fermentation with Saccharomyces cerevisiae. Bioethanol production was modeled using the modified Gompertz equation. The results evidenced changes in the structure and composition of the lignocellulosic matrix following catalytic treatment, increasing surface area and reducing hemicellulose content. Although total sugar release during hydrolysis was comparable between the two samples, the biomass processed via the catalytic biorefinery promoted higher sugar consumption and bioethanol concentration, reaching 3.36 g/L with a yield of 112 g kg−1 of dry biomass. The kinetic model showed a strong fit (R2 between 0.94 and 0.97). These findings demonstrate that the integration of catalytic biorefinery, enzymatic hydrolysis, and fermentation constitutes a viable alternative for the valorization of cocoa residues.

Fermentation doi: 10.3390/fermentation12060256

Authors: Weiqiang Song He Dong Xingzhao Zhou Sen Zong Xinyu Liu Zhiyong Tian Jiakun Du Yulin Li Xun Chen Wenge Li Meiling Hou Yanzi Xiao

This study aimed to screen high-yield and high-quality sorghum–sudangrass hybrid varieties suitable for popularization and cultivation in the Hulunbuir region through a comprehensive evaluation of silage fermentation quality and bacterial community. Comparative analyses were conducted on six sorghum–sudangrass varieties, namely, sorghum–sudangrass hybrid 3 (SS1), sorghum–sudangrass hybrid 4 (SS2), sorghum–sudangrass hybrid 5 (SS3), sorghum–sudangrass hybrid 7 (SS4), sorghum–sudangrass hybrid 8 (SS5), and Super Sugar–Dwarf Sorghum (SS6). The harvested sorghum–sudangrass at the late milk stage was chopped, vacuum-sealed in polyethylene bags (30 cm × 40 cm), and subjected to 60 days of ensiling fermentation. The results revealed significant differences among different varieties (p < 0.05). SS2 achieved the highest fresh herbage yield of 78.26 t/hm2, while the fresh yield of SS4 was 57.39 t/hm2, approximately 26.7% lower than that of SS2. However, SS4 exhibited superior quality, with the thickest stem diameter of 20.26 mm (p < 0.05). It exhibited the highest crude protein content, reaching 5.72% DM; its silage pH was relatively low, at only 3.71 (p < 0.05); its fiber content was significantly reduced (acid detergent fiber, ADF = 40.03% DM, p < 0.05); and it was rich in Lactococcus and Lactobacillus (p < 0.05). The ensiling process of SS4 highly significantly decreased bacterial diversity (p < 0.01) and shifted the dominant bacterial phylum from Proteobacteria to Firmicutes. Functional prediction indicated that the bacterial community of SS2 possessed the highest predicted abundance of functional genes related to cellulase and beta-glucosidase (p < 0.05). Our findings suggest that SS2 should be prioritized for production systems aiming for maximum biomass accumulation, whereas SS4 is recommended as a superior variety for high-quality animal nutrition systems where fermentation stability and nutrient preservation are paramount.

Fermentation doi: 10.3390/fermentation12060255

Authors: Maria Isabella Lima Garção Joachim Müller Andreas Lemmer

Two-stage anaerobic digestion systems are extensively researched for enhancing process stability and phase separation when processing complex organic materials. Scaling from laboratory setups to pilot plants necessitates engineering modifications to ensure operational feasibility. In this study, a laboratory-scale system comprising a 100 L horizontal CSTR and a packed-bed reactor was scaled up 100-fold. The design separates solid and liquid retention times, with fibers retained in the first stage while liquids and volatile fatty acids flow into the second. Fiber retention in the lab was achieved using a 100 µm sieve dividing the CSTR into two chambers, allowing prolonged lignocellulosic degradation. During scale-up, a filtration and recirculation system was introduced, able to return the fibers to the first reactor through a 1000 µm edge-gap filter, which separates liquids for the second reactor and recycles undegraded fibers. An economic analysis indicated a scale-up exponent of 0.396, indicating that unit costs decrease with plant size and demonstrating economies of scale. Laboratory-based mass balance estimates biogas production at approximately 16.3 m3 daily at the pilot scale, equivalent to 90 kWh. The modular system aims to be transferred to small farms, promoting cost-effective biogas from manure and local residues to support decentralized renewable energy in agriculture.

Fermentation doi: 10.3390/fermentation12060254

Authors: Ramón J. Ceballos-Zúñiga Miguel Ladero

Waste from the fruit juice industry presents high sugar and phenolic contents, high humidity and biological activities and cumbersome disposal or low-added valorization. Orange-peel waste (OPW) represents 35–55% w/w of processed fruit, with oranges being the main citric crop. OPW saccharification leads to sugar-rich hydrolysates that can be further processed via fermentative and catalytic routes. In this work, OPW enzymatic hydrolysis was studied via batch and fed-batch processing using either a 50 mM citrate buffer or a 9 g/L NaCl solution with pH control by adding CaCO3 to ensure high enzyme activity across the enzymatic process. Preliminary runs showed that particle size of 3.4 mm diameter and a 300 r.p.m. stirring speed, a six-blade Rushton turbine and wall baffles were adequate to reach high sugar yields in batch. Further scale-up in batch at medium solid loading (12.5% w/w) and fed-batch operation at high-solid loading (20% w/w) led to high yields and glucose and fermentable sugars (up to 74 and 136 g/L, respectively, when using the saline solution and CaCO3 as pH-controlling agent, in only 50 h; notably shorter and higher than when using the citrate buffer). Fractal kinetic models have been shown to accurately represent the compositional change across all batch and fed-batch conditions, highlighting NaCl reaction medium and alkali-driven pH control as the most appropriate approach to achieve high yields at low process times, a promising result for further developments at demonstration and industrial scales using automatic pH control.

Fermentation doi: 10.3390/fermentation12060253

Authors: Anderson Luiz de Lucca Bento Raizza Fátima A. Tulux Rocha Marcelo Vedovatto Jocely Gomes de Souza Fábio José Carvalho Faria Luís Carlos Vinhas Ítavo Anuzhia Paiva Moreira Andréa Roberto D. Lopes Souza Gumercindo Loriano Franco

The use of copaiba oil (COP) in ruminant nutrition is relatively recent, and the results reported in the literature remain controversial. This study evaluated the effects of increasing dietary concentrations of COP on ruminal fermentation in steers. Five rumen-cannulated steers were assigned to a 5 × 5 Latin square design and received the following treatments: Control (0 g kg−1 of COP), 1.25 g kg−1 COP, 2.50 g kg−1 COP, and 3.75 g kg−1 COP dry matter (DM), and monensin (positive control; 40 mg kg−1 DM in the concentrate). Animals were fed a 50:50 forage:concentrate diet. Copaiba oil supplementation (1.25 to 3.75 g kg−1 DM) did not affect ruminal pH, the concentrations of NH3-N, or propionate (mmol L−1; p > 0.05). Similarly, COP had no effect on intake or nutrient digestibility (p > 0.05). In contrast, monensin increased (p ≤ 0.05) the concentrations of NH3-N and propionate (mmol L−1). Overall, COP supplementation did not modify ruminal fermentation under the conditions of this study. However, further studies are needed to assess its effects in diets with higher forage proportions, which better represent grazing systems.

Fermentation doi: 10.3390/fermentation12060252

Authors: Ruicheng Mao Quanmin Sun Zexin Xie Yifa Wang Fang Luo Xiangmeng Ma Zhanbo Hu

Insufficient oxidative capacity can limit humification during municipal sludge composting. This study comparatively evaluated two Fenton-like amendment systems, a homogeneous copper-based treatment (CH) and a heterogeneous nano-iron-based treatment (NFH), for their effects on composting performance, humification-related indices, spectroscopic characteristics, and bacterial community succession. Both amended treatments improved composting performance relative to the control, reaching higher peak temperatures (68.5 °C for CH and 70.3 °C for NFH) and prolonging the thermophilic phase. NFH also showed stronger moisture removal, with the final moisture content decreasing to 58.1%, compared with 65.1% in CH and 64.1% in the control. CH showed the highest apparent humic acid accumulation (1173 mg kg−1), whereas NFH exhibited spectroscopic features commonly associated with lower E4/E6 ratios and more pronounced humic-like fluorescence characteristics. Ultraviolet–visible spectroscopy (UV–Vis), Fourier transform infrared spectroscopy (FTIR), and excitation–emission matrix fluorescence spectroscopy (EEM) analyses collectively indicated progressive transformation toward more aromatic and humified organic matter in the amended treatments. Bacterial community succession also differed across treatments, and several enriched taxa, including Rhodanobacter and Thermobifida, showed positive associations with reactive oxygen species (ROS)-related variables and humification indices. These results describe treatment-linked dynamics in humification and suggest corresponding changes in microbial succession during sludge composting, with potential implications for process outcomes.

Fermentation doi: 10.3390/fermentation12060251

Authors: Roberta Comunian Luigi Chessa

The growing demand to harmonize food safety with preservation of autochthonous sensory phenotypes has catalyzed a paradigm shift in starter culture microbiology [...]

Fermentation doi: 10.3390/fermentation12050250

Authors: Pamela J. Welz Amrita Ranjan Thandekile Mthethwa Marilize le Roes-Hill

Microbial polyhydroxyalkanoates (PHAs) are biodegradable biopolymers that are gaining traction as replacements for conventional petroleum-based plastics. In this study, sugar utilization, growth and polyhydroxybutyrate (PHB) and polyhdroxyvalerate (PHV) production in synthetic and real hydrolysates of Canola fines (SHCF, RHCF) by Gordonia lacunae BS2T were evaluated: (i) in SHCF under different C:N ratios and O2 availability, and (ii) in SHCF and RHCF (50% and 100%) under shaking v/s static conditions with limited or non-limited O2. The bacterium was able to utilize glucose, cellobiose, arabinose, and xylose. Athough O2 limitation reduced growth, higher measured concentrations of 3-hydroxyvalerate (3HV) were achieved under O2 limitation, translating into slightly higher 3-hydroxybutyrate (3HB)+3HV yields (15.4 ± 2.36%wt.wt.) than under non-O2 limited conditions (12.4 ± 2.26%wt.wt.). Notably, 50% RHCF was the most suitable medium for growth and PHB+PHV production, while 100% RHCF was the least suitable. The 3HV+3PV concentration (0.35 g/L), 3HV fraction (24%), and yield (15.4%wt.wt.) in 50% RHCF were highest under static, O2-limited conditions, corresponding with negligible sugar utilization (1.6 mg/day.100 mL−1 glucose) and suggesting alternative metabolic pathways using other substrates in the RHCF for growth. Nuclear magnetic resonance results indicated that Gordonia lacunae BS2T produces a desirable co-polymer (PHBV), paving the way for ongoing research using this bacterium.

Fermentation doi: 10.3390/fermentation12050249

Authors: Büşra Çınar Deniz Koçan Fatma Şahmurat

Probiotic yeasts are increasingly proposed as adjuncts in fermented dairy products, but their behavior in kefir is still poorly described. This proof-of-concept study examined the effect of Saccharomyces boulardii CNCM I-745 supplementation on kefir produced with two traditional grain cultures and two industrial direct-vat-inoculation cultures during 21 days of cold storage at 4 °C. Microbiological, physicochemical, and sensory parameters were monitored on days 1, 7, 14, and 21. The starter culture type was the main source of variation, with traditional grain kefirs showing higher microbial counts and better sensory scores than industrial cultures. S. boulardii did not change pH, titratable acidity, Lactococcus spp., or total mesophilic aerobic bacteria, indicating that it can be added without disturbing the established kefir microbiota or its acidification pattern. The probiotic yeast increased the total yeast count, slightly modulated Lactobacillus spp., and gave a small improvement in taste–aroma scores. In the yeast-free industrial culture, S. boulardii maintained viable counts above 6 log CFU/g throughout storage, showing that it can act as the sole yeast source in kefir matrices that lack indigenous yeast. Traditional grain kefirs kept a more stable overall sensory quality across 21 days than industrial cultures. The multivariate analysis confirmed two largely independent quality dimensions, one related to lactic acid bacteria and acidity and another to sensory perception. The study supports the use of S. boulardii as a probiotic adjunct in kefir and provides preliminary effect-size information for future, adequately replicated trials.

Fermentation doi: 10.3390/fermentation12050248

Authors: Federico Podversich Jorge Bonilla Urbina Callie Coble Zachary K. F. Smith Warren C. Rusche Rebecca O’Sullivan Mark J. Leggett Sophie L. Parker-Norman Ana Clara B. Menezes

Feed additives based on essential oils (EOs) have emerged as a potential alternative to ionophores for diets with elevated grain inclusion. Also, on some occasions, EOs have been used in combination with monensin, with variable results. A metabolism trial was conducted using a 2 × 2 factorial arrangement of treatments, evaluating supplementation with (A) monensin sodium (0 mg/steer daily vs. 400 mg. steer daily) and (B) a blend of EOs (eugenol, linalool, anethole, and cinnamaldehyde, 0 g/d vs. 14 g/d). Four Red Angus steers (BW = 435 ± 9.0 kg) with ruminal and duodenal cannulas were used, and the study was conducted as a Latin square with four periods of 28 days each. Ruminal fermentation and nutrient digestibility at different levels (ruminal, intestinal, and total tract) were determined. The EOs increased total tract starch digestibility (p = 0.05) by 4.5% and propionate concentration (p = 0.03) by 30.9%. Furthermore, EOs decreased acetate (p = 0.04) by 7.4% as well as the acetate to propionate ratio (p = 0.03). In conclusion, our results suggest that it is safe to combine this EO blend with monensin for feedlot diets. The EO blend improved starch digestibility and increased efficiency of the ruminal fermentation end-products, which suggests it could be beneficial in diets based upon grain.

Fermentation doi: 10.3390/fermentation12050247

Authors: Annabella Juhász-Erdélyi Márta Huszár Attila Farkas Gergely Maróti Roland Wirth Márk Szuhaj Zoltán Bagi Kornél L. Kovács Etelka Kovács

Lignocellulose is degraded in the rumen by diverse microorganisms. This study aimed to select the top ruminal microbes associated with an anaerobic fungus (AF) capable of forming consortia that facilitate biogas production from wheat straw. The workflow included the following steps: (1) batch reactors, divided into three compartments with porous membrane bags containing wheat straw, were assembled. The outermost compartment was inoculated with freshly collected rumen content. The first microbes colonizing the wheat straw in the innermost compartment within 72 h were identified. (2) Synthetic consortia were assembled comprising the following identified microbes: an anaerobic fungus (AF) (Neocallimastix lanati); methanogenic archaea (M) (Methanobrevibacter ruminantium or Methanobrevibacter gottschalkii); bacteria (B) (Butyrivibrio hungatei or Succinoclasticum ruminis). (3) Wheat straw was subjected to 7-day pretreatments with these synthetic consortia. (4) The pretreated straw served as substrate in biochemical methane potential (BMP) tests that used a biogas reactor digestate as the inoculum. The pretreated straw produced elevated biomethane yields; nonetheless, this process needs further optimization. The cross-kingdom AF + M + B consortia increased methane production by 35–70%, and superior volatile fatty acid production was confirmed via HPLC. The results suggest novel strategies for advanced practical biogas/biomethane technologies.

Fermentation doi: 10.3390/fermentation12050246

Authors: Yuan Shi Jianhua Cheng Litao Hu Jialiang Lin Yan Wang Hao Huang Zihao Yu Chunlu He Wenjie Xu Wuxia Chen Yichen Fan Weikang Cui Yuan Ban Shaonian Chang Haiyang Ye Haifeng Huang

Grape seeds are a major by-product of grape processing and a rich source of polyphenolic compounds, yet their value remains underutilized. In this study, 12 lactic acid bacteria (LAB) strains were evaluated in a grape seed-based fermentation system to compare their tolerance, metabolic performance, and ability to promote polyphenol release. Among them, Lactiplantibacillus plantarum FBL002 showed the best overall performance. The strain maintained strong viability and metabolic activity at 5% grape seed concentration and released polyphenols more effectively than the other tested strains. The resulting fermentation broth also showed pronounced intracellular antioxidant activity. To clarify the basis of this phenotype, we further combined metabolomic, genomic, and transcriptomic analyses. Fermentation caused substantial shifts in phenolic metabolites, characterized by a decrease in glycosylated forms and an increase in more bioactive aglycones. Genome annotation revealed an enrichment of β-glucosidase-related genes in FBL002, and transcriptomic analysis showed that these genes were markedly upregulated during fermentation. This pattern was closely associated with the enhanced release of polyphenols. Together, these findings identify β-glucosidase as a key driver of grape seed polyphenol biotransformation by FBL002 and support the sustainable, high-value use of grape seeds in functional foods and cosmetic applications.

Fermentation doi: 10.3390/fermentation12050245

Authors: Yang Qiu Tongyi Wang Qiao Yang Xiaoxue Liu Chen Song Renpeng Du

Exopolysaccharides (EPSs) produced by lactic acid bacteria (LAB) are natural high-molecular-weight polymers secreted extracellularly during growth. They possess unique rheological properties and emulsifying stability and may exhibit prebiotic-related functionalities. In food systems, EPSs exhibit multiple functional values. In recent years, driven by the global “Clean Label” movement and increasing consumer demand for natural and healthy foods, EPSs, as safe and traceable natural food-grade prebiotics, have attracted extensive attention in the dairy industry. This review summarizes EPSs’ structure, properties, and mechanisms in dairy systems. It focuses on their functional effects and mechanisms in typical dairy products such as yogurt, cheese, and ice cream, and analyzes the technical bottlenecks limiting large-scale production, including low yield, high cost, and challenges in separation and purification. This review further outlines several promising research directions for EPS research. These include strain modification via synthetic biology strategies, fermentation optimization using high-throughput screening technologies, and targeted application based on structure–function relationships. It aims to provide systematic theoretical references and practical guidance for the efficient development and innovative application of EPSs in the food industry.

Fermentation doi: 10.3390/fermentation12050244

Authors: Johannes Delgado-Ospina Carlos David Grande-Tovar Maria del Pilar Garcia-Mendoza Luis Gabriel Poveda-Perdomo Fabián Felipe Fernández-Daza Clemencia Chaves-López

Colombian palm wine is a traditional fermented beverage produced from the sap of Attalea butyracea, whose microbiota and biochemical features remain poorly characterized. A comprehensive analytical framework was applied to palm wine samples from three Andean producers. This included the determination of proximate composition, total phenolic content, and antioxidant activity, alongside a dual microbiological approach: traditional plate counting and high-throughput 16S rRNA/ITS metabarcoding. PICRUSt2 was employed to predict KEGG-based metabolic pathways to elucidate the microbial functional potential. The wines exhibited a low pH (3.35–3.65), a variable ethanol content (1.62–8.40 g/L), high residual sugars, moderate microbial loads, and limited antioxidant activity (as measured using the ABTS and DPPH assays). Analysis using high-throughput sequencing revealed high bacterial community diversity, dominated by Liquorilactobacillus nagelii, Limosilactobacillus fermentum, Limosilactobacillus panis, Lacticaseibacillus casei, and Zymomonas mobilis alongside the yeast Saccharomyces cerevisiae. Functional profiling revealed a significant enrichment in metabolic pathways related to carbohydrates, amino acids, and cofactors/vitamins, as well as xenobiotic biodegradation and metabolism. These findings provide the first integrated microbiological and physicochemical characterization of Colombian palm wine and highlight its biotechnological potential.

Fermentation doi: 10.3390/fermentation12050243

Authors: Qian Li Jianjian Niu Shanquan Wang Xiao Wang

Poly(β-L-malic acid) (PMLA) has attracted considerable industrial attention due to its promising applications in biomedicine, bioplastics, and environmental fields. However, its biosynthesis is highly dependent on elevated dissolved oxygen (DO) levels, while the simultaneous production of pullulan represents a major obstacle. This study introduces a novel strategy to enhance PMLA production in Aureobasidium melanogenum by selectively inhibiting pullulan biosynthesis. We demonstrate that excessive pullulan accumulation severely impairs fermentation performance by significantly reducing oxygen transfer efficiency—an uncharacterized bottleneck in PMLA production. To address this, an ARTP-induced mutant, designated No. H13, was generated, exhibiting an 82.1% reduction in pullulan synthesis. This metabolic shift led to an 86.93% increase in the oxygen mass transfer coefficient (KLa), ultimately enhancing PMLA yield by 72.1% to 45.0 g/L with a specific production of 1.09 g/g. Transcriptomic analysis suggested a potential redirection of carbon flux toward PMLA biosynthesis through coordinated up-regulation of glycolysis and TCA cycle genes, alongside down-regulation of gluconeogenesis and pullulan-exporting ABC transporters. This work presents an alternative to enzymatic approaches by employing a consolidated mutagenesis strategy to reconfigure metabolic networks, offering a strategy for PMLA overproduction.

Fermentation doi: 10.3390/fermentation12050242

Authors: Huanjun Yuan Fen Yin Bingxin Liu Jingjing Yang Jieru Nan

This study investigated the production of poly(3-hydroxybutyrate) (PHB) using mixed microbial cultures (MMCs) with lignocellulosic hydrolysates as a carbon source. Single-factor experiments were conducted to examine the effects of substrate concentration, C/N ratio, and pH on PHB synthesis. The highest PHB yield (612.35 mg/L) was achieved at a substrate concentration of 1700 mg/L (R1700), with an effective C/N ratio of approximately 31 and pH 7.0. Nitrogen limitation and neutral pH were favorable for PHB production. Microbial community analysis via 16S rDNA sequencing revealed Chryseobacterium as the dominant genus in all reactors. These findings provide insights into the efficient conversion of waste lignocellulose into biodegradable PHB using MMCs.

Fermentation doi: 10.3390/fermentation12050241

Authors: Mingyue Di Mengjian Liu Wenshuai Zeng Mei Xu Zhanlin Ma Dong Xu Yong Chen

This study aimed to evaluate the effects of dietary supplementation with a defined blend of phytochemicals (DBP) composed of carvacrol, thymol, and cinnamaldehyde on the growth performance, slaughter performance, rumen fermentation, microbial diversity, and blood physiological and biochemical parameters of sheep. Twenty-four healthy male Altay lambs, aged six months, were randomly assigned to three groups: (1) fed a basal diet (CON), (2) basal diet with 400 mg/kg DM of DBP (DBP1), and (3) basal diet with 800 mg/kg DM of DBP (DBP2). Results show that DBP supplementation had no significant effect on growth or slaughter performance (p > 0.05). The molar proportion of acetate and the acetate-to-propionate ratio increased linearly, and the molar proportions of propionate and valerate decreased linearly (p < 0.05). DBP supplementation had no significant effect on rumen bacterial α-diversity; however, in the DBP1 group, the relative abundances of Succinivibrionaceae UCG-002, Prevotellaceae UCG-004, Sphaerochaeta, Monoglobus, and Moryella were significantly increased, whereas in the DBP2 group, the relative abundances of Coprococcus and U29-B03 were significantly increased (p < 0.05). DBP exhibited a significant quadratic effect on interleukin-2 and superoxide dismutase activity (p < 0.05). In conclusion, although the DBP altered the rumen microbial community structure and rumen fermentation pattern in sheep to some extent, it showed minimal efficacy in improving growth performance, slaughter performance, immune function, and antioxidant status. Further large-scale studies are warranted to determine the optimal inclusion level and timing of this phytochemical blend in sheep diets.

Fermentation doi: 10.3390/fermentation12050240

Authors: Qi Wei Pengcheng Chen Dan Wu Pu Zheng

L-carnitine is a crucial quaternary ammonium compound widely used in the pharmaceutical, food, and feed industries. Microbial biosynthesis of L-carnitine, compared with chemical synthesis, offers milder conditions, higher stereoselectivity, and a lower environmental impact. However, highly efficient strains and mechanistic insights into the bioconversion of γ-butyrobetaine (γBB) to L-carnitine remain limited. This study focuses on strain WQ-1, a newly screened strain capable of converting γBB to L-carnitine. Based on morphological, physiological, and phylogenetic analyses of 16S rRNA and housekeeping genes, the strain was identified as Ensifer sp. WQ-1. Under the condition of 30 °C, initial pH 8.5, 10% inoculum, 6 g/L initial γBB, shake-flask fermentation reached molar conversion rate of 88%. In a 5 L bioreactor fed-batch fermentation, the L-carnitine titer achieved 13.98 g/L with a 78.7% molar conversion rate. Genomic analysis revealed a 6.97 Mb genome harboring 6568 protein-coding genes, including candidates for quaternary ammonium transport, CoA-dependent transformation, and transcriptional regulation. Comparative transcriptomics identified 58 differentially expressed genes, highlighting the significant upregulation of genes related to acyl-CoA activation, dehydrogenation, carnitine metabolism, and thioester hydrolysis in the presence of γBB. Multi-omics analyses support a putative CoA-dependent metabolic pathway for conversion of γBB to L-carnitine in Ensifer sp. WQ-1.

Fermentation doi: 10.3390/fermentation12050239

Authors: Alan Portal D’Almeida Aida Aguilera Infante-Neta Maria Rosiene Antunes Arcanjo Tiago Lima de Albuquerque

Probiotics and prebiotics in dairy products have gained increasing attention due to their potential health benefits and functional properties. Probiotics are beneficial microorganisms that help maintain intestinal microbiota balance, while prebiotics are non-digestible compounds that stimulate the growth of beneficial gut bacteria. Their incorporation into dairy foods has been associated with improved digestive health, nutrient absorption, and product functionality. However, challenges related to microbial survival during processing and storage, interactions with the dairy matrix, and strain-specific limitations remain significant. This review presents a bibliometric analysis of recent scientific advances involving probiotics and prebiotics in dairy products. The bibliometric analysis revealed a marked increase in publications over the last decade, with research concentrated on gut microbiota modulation, functional dairy foods, fermentation technologies, and health-promoting effects. The results also indicate the relevance of bacterial groups such as lactic acid bacilli and Bifidobacterium, as well as the growing interest in synbiotics and bioactive compounds. Additionally, emerging technologies, including microencapsulation, ohmic heating, and ultrasound, are discussed as promising strategies to improve probiotic stability, functionality, and industrial application in dairy systems. Overall, the findings highlight that the successful development of probiotic and prebiotic dairy products depends on the integration of strain selection, matrix compatibility, and emerging technologies to ensure stability, functionality, and industrial applicability.

Fermentation doi: 10.3390/fermentation12050238

Authors: Franco Van de Velde Micaela Albarracín Raúl E. Cian Silvina R. Drago

This study evaluated the impact of lactic acid fermentation on microbiological and nutritional quality, bioactive compound profile, and bioactive properties of mashed navy beans (MNB). Lactic Acid Bacteria (LAB) viability and microbiological quality of fermented mashed navy beans (FMNBs) were maintained for up to 28 days at 4 °C. Fermentation improved protein quality while reducing trypsin inhibitor activity. Additionally, fermentation enhanced the extractability of phenolic compounds, especially of bound forms. Proteolytic activity during fermentation generated low-molecular-weight peptides enriched in hydrophobic residues. Although antioxidant capacity remained comparable between samples, fermented samples exhibited higher angiotensin-converting enzyme inhibitory (ACE-I) activity (IC50 ACE-I = 0.635 ± 0.043 and 0.413 ± 0.002 mg solids mL−1 for MNBs and FMNBs, respectively). Simulated gastrointestinal digestion enhanced both antioxidant (ABTS•+) and antihypertensive potential. ECA-I inhibition was higher in the fermented sample dialysates (D), with IC50 values of 0.160 ± 0.005 and 0.117 ± 0.003 mg solids mL−1 for MNB-D and FMNB-D, respectively, due to the increased dialyzability of phenolic compounds and the presence of hydrophobic low-molecular-weight peptides in FMNB-D. Furthermore, FMNB-D exhibited competitive ACE-I inhibition. These findings demonstrate that lactic fermentation is an effective strategy to enhance the nutritional and health-promoting properties of legume-based foods.

Fermentation doi: 10.3390/fermentation12050237

Authors: David Willian Bertan Eliana Setsuko Kamimura Célia Quintas

Table olives, particularly traditionally fermented cracked-style green olives, rely on natural microbial activity without chemical debittering, with fungi playing key roles; in contrast, arbutus berry fermentation remains less characterized in terms of microbial functionality. This study investigated the enzymatic and antibacterial potential of fungal isolates from both systems. A total of 84 isolates belonging to Aureobasidium, Candida, Cryptococcus, Saccharomyces, Pichia, Issatchenkia, Torulaspora, and Sporobolomyces were screened for hydrolytic enzymes (pectinases, amylases, cellulases, xylanases, lipases, proteases, tannases, and β-glucosidases) using selective media, and for antibacterial activity against major foodborne pathogens. Isolates from arbutus fermentation showed no relevant enzymatic or antibacterial ability. In contrast, several isolates from olive fermentation exhibited significant functional traits. Aureobasidium pullulans demonstrated broad enzymatic capacity, producing amylases, esterases, and tannases, along with lipid hydrolysis, but also expressed cellulase, pectinase, and protease abilities. Cryptococcus spp. displayed interesting profiles, with low cellulolytic and pectinolytic capacity and higher phenolase, esterase, and lipase capacities. Antibacterial activity was observed exclusively against Gram-positive bacteria, particularly Staphylococcus aureus and Listeria monocytogenes, mainly among Candida membranifaciens, Cryptococcus spp., and A. pullulans. Overall, table olive fermentation isolates showed promising biotechnological potential for food preservation and quality enhancement, whereas arbutus isolates appeared to have limited functional relevance.

Fermentation doi: 10.3390/fermentation12050236

Authors: Jonas P. Souza Miquéias G. dos Santos Henrique M. Fogarin Sâmilla G. C. Almeida Gisele C. A. Santos Débora D. V. Silva Érica R. Filletti Kelly J. Dussán

Xylitol is a five-carbon sugar alcohol of industrial interest due to its applications as a food sweetener and sugar substitute. In this study, artificial neural networks combined with a genetic algorithm were evaluated as a data-driven approach for modeling and exploring xylitol production by Spathaspora boniae and Spathaspora brasiliensis during fermentation of sugarcane bagasse hemicellulosic hydrolysate. The dataset comprised 20 experimental points obtained from a face-centered central composite design, using urea, yeast extract, peptone, and ammonium sulfate as input variables. The neural network models showed high goodness-of-fit, with R2 values of 0.9952 for S. boniae and 0.9930 for S. brasiliensis. Experimental validation of the optimized conditions resulted in xylitol production of 11.54 ± 0.52 g L−1 for S. boniae and 9.29 ± 0.24 g L−1 for S. brasiliensis. Comparison with response surface methodology showed that both approaches provided strong predictive performance, although the statistical model predicted the optimum conditions more accurately. For S. boniae, however, the ANN-GA approach identified an alternative condition associated with lower nitrogen supplementation and higher experimental xylitol production. Given the limited dataset, this study should be regarded as a proof-of-concept for the application of data-driven optimization tools to xylitol fermentation. The results indicate that ANN-GA can complement classical statistical methods by helping to identify alternative operating conditions in bioprocess optimization.

Fermentation doi: 10.3390/fermentation12050235

Authors: Guanhua Jiao Haoyu Tian Junqing Wang Nan Li Kaiquan Liu Piwu Li Fengyong Lu Qi Wang Ruiming Wang Peng Du

Daqu is the core saccharifying and fermenting agent in Baijiu production and a pivotal factor in flavor formation. Challenges that often hinder traditional strong-flavor Daqu brewing include low enzymatic activity and insufficient aroma. Therefore, we have developed a novel Daqu brewing system. Furthermore, we investigated the differences in flavor profiles between traditional and novel Daqu by performing headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). We comparatively analyzed the microbial communities, metabolic functions, and flavor compositions in the two Daqu types via absolute quantitative metagenomics. Functional microorganisms were significantly enriched in the novel Daqu, which exhibited enhanced carbohydrate metabolism and a highly robust acidic environment owing to the fostering of core functional genera such as Aspergillus, Saccharomyces, and Pediococcus. This significantly increased the aldehyde and organic acid levels, which resulted in pronounced aldehydic and acidic sensory characteristics. Carbohydrate-Active EnZyme (CAZy) profiling confirmed the significantly elevated abundance of glycoside hydrolases (GHs) and glycosyltransferases (GTs) in novel Daqu, which improved starch bioconversion and synthesis of flavor precursors. Thus, this study shows that novel Daqu promotes ethanol accumulation and the synthesis of flavor compounds like acetals by strengthening the core microbiota and metabolic networks. These findings provide a theoretical foundation for enriching the aromatic complexity of Baijiu.

Fermentation doi: 10.3390/fermentation12050234

Authors: Gyoo Taik Kwon So Mi Kim Jae In Jung Cho Yeon Park Hyeji Hwang Il-Jun Kang

Chronic inflammation contributes to various metabolic and immune disorders. Plant-derived phytochemicals and fermented foods have attracted attention as dietary modulators of inflammation. This study evaluated the anti-inflammatory potential of sword bean (Canavalia gladiata) extract (CG) and its Lacticaseibacillus paracasei SKH 003-fermented derivative (CGF) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Cells were treated with CG or CGF (0–400 µg/mL) with or without LPS (0.1 µg/mL). Both CG and CGF significantly attenuated LPS-induced inflammatory responses while maintaining high cell viability. The extracts reduced nitric oxide (NO) and prostaglandin E2 (PGE2) production, suppressed mRNA expression of iNOS, COX-2, TNF-α, IL-6, IL-1β, MCP-1, and CXCL10, and upregulated IL-1Ra. Notably, CGF showed broader and stronger suppressive effects on most pro-inflammatory mediators, cytokines, and chemokines than unfermented CG, whereas IL-1Ra induction was comparable between the two extracts. Western blot analysis revealed that CGF inhibited the phosphorylation of NF-κB p65 and all three major MAPKs (p38, JNK, ERK), whereas CG showed limited effects on MAPK activation. These findings demonstrate that fermentation with the specific strain L. paracasei SKH 003 enhances the anti-inflammatory activity of sword bean extract by simultaneously targeting NF-κB and MAPK signaling pathways. Consequently, CGF holds significant potential as a functional food ingredient for managing macrophage-mediated inflammatory responses.

Fermentation doi: 10.3390/fermentation12050233

Authors: Hai Huang Jinsong Zhao Yue Deng Jingcheng Liu Liping Xu Hui Lv

Baijiu, a traditional Chinese distilled spirit with profound cultural and economic significance, faces long-standing challenges in standardization, quality consistency, and skill inheritance due to its empirical production model. The rapid advancement of artificial intelligence (AI) and multi-omics technologies is driving a paradigm shift in Baijiu research from experience-driven to data-driven approaches. This review systematically summarizes the current state of AI applications across the entire Baijiu industry chain. Common AI methods including traditional machine learning, deep learning, multimodal data fusion, and emerging paradigms such as explainable AI (XAI), genome-scale metabolic models (GEMs), and few-shot learning are critically examined. Key bottlenecks—data silos, small sample sizes, model interpretability, and the tension between technology and tradition—are discussed in depth. Future directions are proposed, including multimodal fusion, digital twins, hybrid mechanistic–data modeling, closed-loop control, human–machine collaboration, standardization, and ethical governance. This review provides a comprehensive framework for integrating AI into Baijiu research and offers references for intelligent transformation in other fermented food systems.

Fermentation doi: 10.3390/fermentation12050232

Authors: Jiaying Xiong Meixia Chen Laiping Zhang Qi Zhou Zhenyu Huang Xiaobin Lin Xiaomin Fang Xiangdong Ye Weiping Zhu Wei Liu Aiqin Shi

Lignocellulose is the most abundant renewable biomass on Earth, and its efficient bioconversion is critical for achieving carbon neutrality, substituting fossil resources, and advancing sustainable biomanufacturing. However, furfural, a dominant inhibitor generated during lignocellulosic pretreatment, severely compromises microbial metabolism and fermentation performance. To date, no systematic review has comprehensively integrated the mechanisms of furfural-induced microbial toxicity with corresponding stress tolerance strategies. This review elaborates on three core themes: the multi-pathway toxic effects of furfural, intrinsic microbial tolerance mechanisms, and advanced strategies for constructing a high-tolerance microbial chassis. Despite considerable progress, several research gaps persist, including poorly understood synergistic or antagonistic interactions between furfural and other hydrolysate inhibitors, insufficient integration of adaptive laboratory evolution, rational design, and random mutagenesis in anti-inhibitor research, and limited understanding of trade-offs between furfural tolerance and industrial fermentation robustness. Future efforts should address these gaps through combinatorial stress simulation, multi-omics profiling, and the “evolve–elucidate–engineer” paradigm, thereby enabling the scalable and stable application of lignocellulosic biomanufacturing.

Fermentation doi: 10.3390/fermentation12050231

Authors: Luana Milena Pinheiro Rodrigues Alberto Jefferson da Silva Macêdo Edson Mauro Santos Daniele de Jesus Ferreira Juliana Silva de Oliveira Paulo da Cunha Tôrres Mateus Lacerda Pereira Lemos Guilherme Medeiros Leite Chrislanne Barreira de Macêdo Carvalho Arthur Herculano Araújo Geovergue Rodrigues de Medeiros João Paulo de Farias Ramos Anderson de Moura Zanine

Microbial inoculants are widely used to improve the fermentation and aerobic stability of silages, particularly in sorghum, which is susceptible to deterioration; therefore, this study evaluated the effects of Lentilactobacillus buchneri (Lb), alone or combined with Lentilactobacillus hilgardii (Lb + Lh), on the fermentation profile, microbial stability, chemical composition, and aerobic stability of whole-plant sorghum silage. A completely randomized design was adopted in a 3 × 3 factorial scheme, with three fermentation periods (20, 60 and 100 days) and three microbial inoculants (control, Lb and Lb + Lh), with five replicates per factorial treatment; the fermentation parameters, chemical composition, microbial populations, and aerobic stability were evaluated. A interaction (p < 0.05) between inoculants and fermentation periods was observed for pH, organic acids, microbial counts, and aerobic stability; inoculated silages showed increased lactic acid bacteria, higher acetic and propionic acid production, and inhibition of yeasts and molds, especially at 100 days, resulting in improved aerobic stability at 60 and 100 days. The microbial diversity was lower in inoculated factorial treatments, with predominance of Lentilactobacillus, while the control showed a higher abundance of undesirable microorganisms; Kazachstania was the predominant fungal genus. In conclusion, inoculation improves the fermentation quality, microbial stability, and aerobic stability of sorghum silage, reducing losses.

Fermentation doi: 10.3390/fermentation12050230

Authors: Yuzhang Wu Jingjing Zhao Shiqiao Zhang Xiaoli Fu Changfeng Gong Yingdong Pan Lele Li Muxi Xia Manjin Wang Xiangyong Wang Zhiyu Zhu

To elucidate the regulatory mechanism of straw domestication on Jiang-flavor Daqu quality, this study systematically tracked variations in physicochemical properties and flavor compounds between Daqu fermented with aged versus fresh straw. Results showed that moisture content in aged-straw Daqu remained above 18.2% during the early fermentation stage (days 0–9), significantly higher than the fresh-straw group. This moisture retention was accompanied by thermodynamic differentiation: aged-straw Daqu exhibited a “delayed peak with gradual decline” pattern (peak temperature 46.8 °C on day 6, maintaining a high-temperature plateau from days 3 to 9), whereas fresh-straw Daqu followed an “early peak with rapid decline” trajectory (peaking at 50.7 °C on day 3 before deteriorating quickly). Total acidity was significantly elevated in the aged-straw group (1.9 vs. 1.0 mmol/10 g, p < 0.05). However, this acidic environment was associated with lower activities of starch-hydrolyzing enzymes, resulting in comparatively lower diastatic and liquefying powers. Flavor profiling identified 1539 volatile compounds. Redundancy analysis revealed moisture, temperature, and liquefying power as key driving factors, explaining 44.24% of variance (p = 0.002). Although the overall flavor architecture remained similar between groups, characteristic compounds differed markedly. Aged-straw Daqu was enriched with derivatives from Maillard reactions and lipid oxidation, contributing to a more substantial flavor foundation. In contrast, fresh-straw Daqu tended to accumulate primary alcohols and exogenous residues. Collectively, aged straw was associated with greater flavor complexity and typicality of Jiang-flavor Daqu, likely through optimization of microenvironmental homeostasis, without altering the fundamental flavor framework.

Fermentation doi: 10.3390/fermentation12050229

Authors: Fabian Herrmann Anna Stock Anne Oppelt Emelie Petzel Dirk Weuster-Botz

The efficient production of microbial oils from agricultural residues and acetic acid has recently been shown with Cutaneotrichosporon oleaginosus. However, around 50% of the carbon is released as CO2 during aerobic yeast oil production. Anaerobic fermentation of CO2 and H2 with A. woodii enables the carbon-efficient production of acetate. The semi-continuous autotrophic production of acetate with A. woodii was studied in a stirred-tank bioreactor with continuous gassing, where the time of the repeated batch processes was adjusted to the batch process time for microbial oil production (6–7 days). Eight repeated batch processes with 80% medium exchange were performed with A. woodii within 48 days. After adaptation of the A. woodii cells, 48.29 ± 0.35 g L−1 acetate was achieved in the last four repeated batch processes with 70% H2 and 30% CO2 gassing. Acetic acid was extracted from the clarified and acidified fermentation broth with ethyl acetate, yielding 94.3% (w/w). Based on our process performance data with A. woodii and previously published data with C. oleaginosus, it was shown that, for providing enough acetic acid for microbial oil production, a 3.08 times higher bioreactor capacity is needed for the gas fermentation compared to the aerobic yeast fermentation. The lack of CO2 produced by C. oleaginosus may be compensated for by increasing the sugar supply (hydrolysate) during yeast oil production, or by the additional use of other biogenic CO2 sources. Thus, CO2-neutral production of microbial oils from sugars or hydrolysates of agricultural residues is possible by reusing the CO2 produced in the aerobic yeast oil production for the autotrophic production of acetic acid, which is fully recycled as an additional carbon source for yeast oil production.

Fermentation doi: 10.3390/fermentation12050228

Authors: Diana B. Muñiz-Márquez Christian I. Cano-Gómez Fabiola Veana José Manuel Sánchez-González María Luisa Carrillo-Inungaray Cristóbal N. Aguilar Jorge E. Wong-Paz

This study evaluates fungal-assisted extraction by solid-state fermentation (FAE-SSF) as a green alternative for recovering phenolic compounds from Moringa oleifera leaves and compares it with conventional maceration, focusing on their effects on antimicrobial and antioxidant properties. FAE-SSF was carried out using Aspergillus niger, and phenolic compounds were quantified as total polyphenols (hydrolysable and condensed tannins), followed by purification and characterization by HPLC-ESI-MS. Biological activities were assessed through antibacterial, antifungal, and DPPH assays. FAE-SSF increased total phenolic content to 20.3 ± 1.7 mg TP/g dry basis at 96 h, representing a 1.53-fold increase compared to maceration (13.3 ± 0.3 mg TP/g db at 24 h). However, maceration showed higher productivity due to shorter extraction time. FAE-SSF extracts exhibited improved antibacterial activity against Staphylococcus aureus, while no activity was observed against Shigella sp., and antifungal activity was lower compared to maceration. Antioxidant activity was also reduced in FAE-SSF extracts (39 ± 7%) compared to maceration (71 ± 4%). HPLC-ESI-MS analysis revealed that maceration preserved a greater diversity of phenolic compounds, whereas FAE-SSF induced biotransformation and reduction of key flavonoids. These results indicate that FAE-SSF enhances phenolic recovery but alters chemical composition and bioactivity, highlighting the importance of process optimization depending on the desired functional properties.

Fermentation doi: 10.3390/fermentation12050227

Authors: Manuel Schnitter Clara Vitaggio Matteo Pollon Valentina Caraci Filippo Amato Riccardo Savastano Laura Girolli Onofrio Corona

Yeast selection plays a strategic role in winemaking, influencing not only the quality and style of the final product but also the expression of the cultivar. This study evaluated the impact of selected Saccharomyces cerevisiae strains on the fermentation of three white grape cultivars grown in Western Sicily: Grillo, Catarratto, and Moscato Giallo (Vitis vinifera L.). A standardized vinification protocol was applied to assess the fermentative performance and effects on the chemical composition, aromatic profile, and sensory profile. Alcoholic fermentation kinetics, major analytical parameters, free and glycosylated volatile compounds, and sensory attributes were monitored. Significant differences were observed among the yeast strains in their fermentation dynamics and production of secondary metabolites. Notably, certain strains enhanced the aromatic expressions of the cultivars, particularly in Moscato Giallo, modulating the free and glycosylated terpene profiles. This approach to fermentation highlights the potential to optimize wine quality through yeast selection, aligning the strain performance with the specific needs of each cultivar. Furthermore, the use of efficient yeast strains may reduce reliance on additives, contributing to more sustainable and economically viable winemaking.

Fermentation doi: 10.3390/fermentation12050226

Authors: Federica Meanti Chiara Rossetti Chiara Mussio Annalisa Rebecchi Dordoni Roberta Luigi Lucini Lorenzo Morelli

The growing over-75 population has increased the demand for functional foods tailored to the nutritional needs of the elderly. Within the AURA project, an innovative oat okara sourdough was developed to produce bread with enhanced nutritional and functional properties. Breads were produced using oat okara sourdough, oat sourdough, and wheat sourdough for comparison. All samples were subjected to microbiological, physical-chemical, technological, and metabolomic analysis. In addition, bread digestibility was evaluated. The results showed that oat okara flour is an excellent fermentable substrate, yielding sourdoughs with high counts of lactic acid bacteria and yeasts. The breads made with oat okara and oats were softer and brownish due to the oat presence and higher relative yeast. Moreover, oat okara bread exhibited a lower proportion of rapidly digestible starch (RDS) and a higher proportion of slowly digestible starch (SDS), suggesting potential benefits for post-prandial glycaemic control. Metabolomic profiling highlighted lipids, particularly steroidal glycosides (saponins) and fatty acyls, as discriminant metabolites in fermented samples, suggesting enhancement of bioactive compounds through sourdough fermentation. Overall, the use of oat okara in sourdough represents a sustainable approach to upcycle agro-industrial by-products while producing nutritionally valuable bakery products aligned with circular economy principles.

Fermentation doi: 10.3390/fermentation12050225

Authors: Muhammad Salman Farid Muhammad Imran Hussain Saba Akhtar Aniqa Abbas Mahwish Tanveer Sania Khalid Izabela Dmytrów Łukasz Łopusiewicz

The rising global demand for functional, “clean-label” fermented foods has driven intense interest in versatile microbial starter cultures. Levilactobacillus brevis is an obligately heterofermentative lactic acid bacterium that is highly valued for its robust environmental adaptability and exceptional capacity to synthesize bioactive metabolites, notably γ-aminobutyric acid (GABA) and exopolysaccharides (EPS). This review comprehensively evaluates the recent progress in L. brevis applications across major food fermentations. In dairy systems, L. brevis is most effective in co-cultures, where partner starters compensate for limited proteolysis and acidification, enabling improved texture, aroma profiles, and GABA enrichment. In fermented meats, selected strains contribute to nitrite reduction, flavor formation, and bioprotection, supporting nitrite-reduced strategies while maintaining sensory quality. In fish and seafood fermentations, L. brevis shows promise for controlling spoilage indicators and biogenic amines (notably histamine) in high-salt environments, although strain compatibility in mixed cultures is product-dependent. In plant-based matrices, outcomes are strongly constrained by acidity and nitrogen limitation; however, optimized fermentation can enhance phenolic bioaccessibility, generate high GABA levels, and enable emerging precision-biofortification approaches. Despite these functional advantages, its industrial application is frequently constrained by strain-specific technological limitations, and its use often necessitates synergistic co-culture systems, particularly in challenging matrices. Ultimately, this review highlights current research gaps and proposes future directions, including multi-omics integration and targeted strain evolution, to overcome sensory trade-offs and fully harness the biotechnological potential of L. brevis in next-generation functional foods and agricultural byproduct valorization.

Fermentation doi: 10.3390/fermentation12050224

Authors: Mark Korang Yeboah Ahmad Addo Nana Yaw Asiedu

Consolidated bioprocessing (CBP) has been widely studied as an integrated route for converting biomass into biofuels and bioproducts, yet most quantitative modeling work has focused on ethanol as a single response. Because CBP systems can generate multiple products and co-products, this study develops a literature-derived benchmark for multi-product CBP modeling using a standardized dataset assembled from published endpoint experiments. Product prediction is formulated as both an observed-only product-wise problem and a joint multi-output problem, allowing direct comparison under study-aware grouped validation. The modeling space integrates biomass composition, pretreatment descriptors, microbial and consortium characteristics, reactor information, operating conditions, and engineered categorical descriptors of feedstock, pretreatment family, and process configuration. Predictive performance was strongly product-dependent and was shaped by target support and missing-label structure. The observed-only product-wise formulation consistently outperformed the joint missing-as-zero multi-output strategy, indicating that naive zero-filling of unreported products is not well suited to sparse literature-derived CBP data. Among the evaluated products, butanol showed the clearest predictive signal, ethanol was only moderately learnable, and the sparsest co-products remained too weakly supported for strong quantitative inference. Overall, this study provides a benchmark for multi-product CBP modeling and clarifies both the potential and the current limitations of literature-derived data for broader data-driven biorefinery analysis.

Fermentation doi: 10.3390/fermentation12050223

Authors: Rangling Li Yankun Gao Weiming Shao Peng Liu Haihong Zhang Chi Zhang Hui Sun

Anaerobic digestion is crucial for safe treatment and energy recovery from municipal sludge. However, seasonal variations in sludge physicochemical properties challenge the continuous, stable operation of anaerobic digestion systems. To investigate the seasonal variations in characteristics of municipal sludge and their impact, this study collected sludge samples from a Beijing plant over a year, analyzed their properties and microbial communities, and evaluated their biogas potential through four-week batch anaerobic digestion tests. The results demonstrated that spring sludge exhibited the highest organic matter (68.7% of total solids, TS), including soluble proteins, sugars, and lipids, while the lignocellulose content peaked in autumn (17% TS). These fluctuations were primarily driven by variations in rainfall, temperature, and human activities. The microbial community shifted significantly: Proteiniclasticum and other hydrolytic bacteria were dominant in spring, whereas Candidatus_Microthrix was notably enriched in winter. Consequently, the biochemical methane potential (BMP) was highest in spring (342.5 mL/g volatile solids) and lowest in autumn (255.8 mL/g volatile solids). Spearman’s correlation analysis indicated a significant positive correlation between BMP and soluble protein content, and a weak negative correlation with cellulose content. These findings provide essential data support for seasonal regulation of sludge anaerobic digestion systems, facilitating strategies to achieve stable biogas production.

Fermentation doi: 10.3390/fermentation12050222

Authors: Natasha Petrenko Huey Yi Loh Julia Baroni Alves Tyler Thomas Arturo Rodezno Gomez Julia T. da Silva Wendela Wapenaar Kirsty Bardoul Genevieve D’Souza Terry E. Engle

This study aimed to evaluate the effect of bromoform (CHBr3) dose on in vitro rumen fermentation and on CHBr3 and dibromomethane (CH2Br2) concentrations in solution and the gas cap. In vitro treatments consisted of CHBr3 (DOSE: 0, 1, 10, 100, 1000, 10,000 µg of CHBr3), with five replicates per DOSE at each time-point (TIME: 0, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 12, 24, 48, and 72 h). The 10,000 µg CHBr3 DOSE inhibited fermentation completely and was removed from the dataset. The acetate:propionate ratio, nitrogen, and methane (CH4) produced per gram of DMD decreased as DOSE increased (p = 0.01). As the DOSE increased, CH4 decreased, and H2 increased in a dose-dependent manner (p < 0.01). The CHBr3 concentration dropped below the detection limit within 3 h of incubation. Dibromomethane concentrations for DOSE 1000 and 100 µg of CHBr3 increased in solution and gas cap beginning at 0.25 h and 1 h post incubation and plateaued by hour 3 and 5, respectively (p < 0.01). The addition of CHBr3 alters the molar proportion of volatile fatty acids, decreases CH4, and increases H2 production, and it is dehalogenated to CH2Br2 within 3 h of incubation in an in vitro system.

Fermentation doi: 10.3390/fermentation12050221

Authors: Chutikarn Somsin Nattanon Chinchusak Aran Incharoensakdi Saranya Phunpruch

Hydrogen (H2) is a promising clean energy carrier with the potential to partially replace fossil fuels. Biological H2 production using microorganisms offers an environmentally friendly alternative. The halotolerant cyanobacterium Aphanothece halophytica can produce H2 under nitrogen-deprived and dark anaerobic conditions. In this study, a co-culture strategy was investigated to enhance H2 production. Five bacterial strains were screened for their ability to improve H2 production when co-cultivated with A. halophytica. Among them, Staphylococcus aureus significantly enhanced H2 production, achieving a maximum rate of 11.11 ± 0.18 µmol H2 g−1 dry weight h−1. Optimization of the bacterial partner revealed that S. aureus cells harvested at 12 h in the mid-logarithmic phase with an OD600 of 4.0 were the most effective. An inoculum ratio of A. halophytica to S. aureus of 4:1 further enhanced H2 production, increased bidirectional hydrogenase activity, and reduced O2 accumulation. Under optimal conditions (0.945 mmol C-atom L−1 glucose, 0.25 M NaCl, pH 7.4, and 35 °C), the maximum H2 production rate reached 132.49 ± 4.45 µmol H2 g−1 dry weight h−1, approximately 5.5-fold higher than that under normal conditions. The co-culture achieved a cumulative H2 yield of 3248.51 ± 88.11 µmol H2 g−1 dry weight after 48 h.

Fermentation doi: 10.3390/fermentation12050220

Authors: Carlos A. Guerra André F. Guerra Lucas M. Costa

This study aimed to develop a biotechnological process for producing a lactic-fermented tomato ingredient (Solanum lycopersicum) capable of acting as a natural reddish colorant and enhancing microbiological stability in fresh pork sausage, reducing dependence on cochineal carmine, whose market price has fluctuated substantially. The bioprocess was conducted at industrial scale using a 10% tomato flour solution subjected to enzymatic hydrolysis with pectinases to release lycopene, followed by co-culture fermentation with Lacticaseibacillus paracasei ATCC 25302 and Pediococcus acidilactici ATCC 8042 to convert sugars into lactic acid. The antimicrobial potential of the ingredient was assessed through minimum inhibitory concentration assays using the Computational Microbial Density Scanning method against microbiota isolated from fresh pork sausage. A dose-dependent inhibitory effect was observed, with significant growth reduction from 2%. The fermented ingredient was then applied at 2% (w/w) in fresh pork sausage, partially or fully replacing cochineal carmine. Instrumental color analysis showed that 2% enabled a 50% reduction in cochineal carmine without compromising color. Microbiological stability evaluated using the MicroLab_ShelfLife method revealed a substantial reduction in microbial growth rates in treated groups. Overall, lactic-fermented tomato can partially replace cochineal carmine while preserving sensory color and providing an antimicrobial function, thereby enhancing product stability and shelf-life.

Fermentation doi: 10.3390/fermentation12050219

Authors: Tomoki Kono Yi-Chung Lai Bang-Yuan Chen Meng-I Kuo

This study investigated the fermentation kinetics, microbial community succession, and potential functional metabolite formation in Symbiotic culture of bacteria and yeast (SCOBY)-mediated fermentation using pomelo peel substrates. Pomelo peel substrates were prepared using 1% and 6% (w/w) SCOBY combined with 10 g and 25 g pomelo peel and fermented at 30 °C for 25 days. The results showed that higher SCOBY inoculum significantly accelerated acid production, resulting in a rapid decrease in pH and an increase in titratable acidity. Total soluble solids continuously decreased due to microbial utilization of sugars. The highest lactic acid bacteria count (6.04 log CFU/mL) and total viable count (7.23 log CFU/mL) were observed in S6-P25 at day 25. Bioactive compound analysis revealed that total flavonoid content reached its maximum in S6-P25 at day 20 (15.34 ± 0.70 mg RE/g dry weight, DW), while the highest total phenolic content was found in S1-P25 (151.5 ± 1.29 mg GAE/g DW), suggesting that a lower SCOBY level may favor polyphenol production. Antioxidant activity (DPPH and TEAC) increased with fermentation time and was highest in S6-P25. Microbiome analysis demonstrated that Firmicutes was the dominant phylum, with Apilactobacillus ozensis accounting for 99% of the relative abundance, indicating strong microbial selection and its potential role in acid production and fermentation ability. This microbial structure was consistent with the improved fermentation performance and enhanced bioactive properties observed in the pomelo peel substrates. These findings highlight SCOBY fermentation as a promising biotechnological strategy for converting citrus processing by-products into fermented ingredients for food applications.

Fermentation doi: 10.3390/fermentation12050217

Authors: Sergio Hernández-Suárez Jennifer López-Sánchez Julio César García-Martínez Paulina Gutiérrez-Macías Odín Rodríguez-Nava

Garden pruning waste from Cynodon sp. is a lignocellulosic resource with high lignin content, which limits anaerobic digestion efficiency. White-rot fungi degrade biomass through solid-state fermentation (SSF). The efficacy of these organisms, however, depends on the balanced removal of lignin and the subsequent preservation of fermentable carbohydrates. The present study evaluated the effect of SSF durations (8, 21, and 36 days) with Trametes hirsuta on enzymatic activity and subsequent biogas production. Laccase activity increased progressively, reaching 983.84 U/L at 36 days, while manganese and versatile peroxidases peaked at 21 days. Fungal-pretreated samples exhibited reduced methane yields, with a maximum of 225.32 NmL/gVS at 8 days, compared with untreated biomass (381.66 NmL/gVS). The total lignin content increased across treatments, suggesting the formation of pseudo-lignin during autoclave sterilization, while glucose and xylose decreased. These results underscore the complexity of optimizing fungal pretreatment and highlight the need to balance fermentation time to preserve carbohydrates while modifying lignin structure.

Fermentation doi: 10.3390/fermentation12050218

Authors: Chenchen Fang Jiaqing Wang Changwei An Wenzhong Huang Xingjiang Liu Mengcan He Fengchen He Shuang Ma

Abundant non-medicinal byproducts of Polygonatum sibiricum and Gentiana scabra are severely underutilized, resulting in resource waste and environmental burden. A previous study confirmed that triple-microbial co-fermentation enhances their antibacterial activity, yet the temporal metabolic mechanism and optimal process parameters remain unclear due to endpoint-only metabolomics limitations. This study aimed to optimize the staged solid-state fermentation (SSF) system for maximum antibacterial activity, verify the triple-microbial consortium’s synergistic enhancement effect, and elucidate the dynamic metabolic mechanism via time-series metabolomics. A staged SSF strategy was established: Aspergillus niger monoculture (0–48 h) followed by Bacillus subtilis and Saccharomyces cerevisiae co-culture (48–72 h). Key parameters were optimized via single-factor experiments and a Box–Behnken design. Under optimal conditions, inhibition zones against Staphylococcus aureus and Escherichia coli reached 20.8 ± 0.3 mm and 17.6 ± 0.2 mm, respectively, with a 17.5% increase in S. aureus inhibition and markedly improved E. coli inter-batch consistency. Time-series untargeted LC-MS/MS metabolomics (2681 identified metabolites) revealed a three-stage metabolic relay model driving antibacterial enhancement: 0–48 h shikimate pathway activation for phenolic precursor accumulation; 48–60 h dipeptide conversion and ABC transporter enrichment initiating antibacterial synthesis; 60–72 h metabolic flux redirected to indole alkaloid biosynthesis for complex antibacterial compound accumulation. This work provides a mechanistic paradigm for the high-value valorization of herbal byproducts, with applications in natural antibacterial agents and functional feed additives.

Fermentation doi: 10.3390/fermentation12050216

Authors: Viktoria Aleksandrovna Semenova Svetlana Anatolyevna Kishilova Viktoria Aleksandrovna Leonova Vera Anatolyevna Mitrova Irina Vladimirovna Rozhkova Anastasia Valeryevna Kosareva Vladislav Konstantinovich Semipyatnyi Natalya Sergeevna Pryanichnikova Aram Genrikhovich Galstyan

Fermented dairy products with probiotic and functional properties are a promising matrix for modulation of the human microbiome. The functionality of such products will depend not only on the technological properties of the lactic acid bacteria included in the starter culture but also on the combined effects of metabolites, enzymatic activity, stress tolerance, and strain-specific adaptation mechanisms. The aim of this work was to conduct a comprehensive analysis of Lactobacillus strains to facilitate the design of microbial consortia for the development of fermented products with diverse functional properties. Twenty Lactobacillus strains from different species were investigated using microbiological, physicochemical, and biochemical methods to evaluate antagonistic activity against opportunistic microorganisms and to assess changes in amino acid and organic acid profiles, vitamin content, fatty acid composition, and enzymatic activity. Additionally, proteomic analysis was performed to create a matrix of functional complementarity of the studied strains, representing proteins associated with antimicrobial activity, bacteriocin transport, resistance to oxidative stress, surface structure formation, and adhesion. It was shown that the studied strains exhibit pronounced functional heterogeneity, demonstrating the feasibility of scientifically based selection of strains to create next-generation fermented dairy products with predictable properties.

Fermentation doi: 10.3390/fermentation12050215

Authors: Warllisson Yarli Santos Paulino João Victor Oliveira Nascimento da Silva Carlos Eduardo de Farias Silva Larissa Rodrigues Macário Francine Pimentel de Andrade Albanise Enide da Silva Renata Maria Rosas Garcia Almeida Brígida Maria Villar da Gama

Cheese whey, a byproduct of the dairy industry, has a high organic load and nutrient availability, associated with parameters such as chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP), representing an environmental problem when improperly disposed, and even considering the traditional biological wastewater treatment (secondary treatment), a polishing step (tertiary treatment) could be required in order to meet legislation parameters of discharge in water bodies. This study evaluated the efficiency of co-cultivation between the microalga Tetradesmus obliquus and the yeast Saccharomyces cerevisiae during the tertiary (advanced) treatment of dairy effluent. The process was operated in batch mode to optimize the COD:N ratio and, subsequently, in semicontinuous mode applying the volumetric replacement rates (VRRs) of 40% and 60%. In the batch stage, the COD:N ratio of 20 stood out as the most balanced in terms of nutritional requirement, achieving removal rates of 85.49% for COD, 96.23% for total Kjeldahl nitrogen (TKN), and 100% for TP. In the semicontinuous system, a VRR of 40% optimized nitrogen (91.67%) and phosphorus (95.93%) recovery while COD was also removed (71.68%). The pH remained stable within the range of 7.0 to 7.5 at the end of the process, indicating self-buffering of the consortium. Biomass production reached 915 mg·L−1 (dry cell weight) in batch operation mode and 720 mg·L−1 in semicontinuous mode (VRR of 40%). The results confirmed that the T. obliquus and S. cerevisiae co-cultivation constitutes a stable and sustainable strategy for nutrient recovery during dairy wastewater treatment, aligning with the principles of circular bioeconomy.

Fermentation doi: 10.3390/fermentation12050214

Authors: Chunyan Cheng Tingting Wei Shimin Lin Yuxin Qin Hongrong Lu Lu Wei Lijuan Du Qinju Sun Lingling Liao Jianzong Meng

(1) Background: Flavored craft beer is favored for its diverse and distinctive aroma compounds; however, traditional fermentation processes are often plagued by poor yeast flocculation, which leads to substantial beer losses and compromised production efficiency. Yeast immobilization technology has emerged as a promising strategy to improve fermentation performance, shorten the primary fermentation period, and mitigate beer loss. (2) Methods: In this study, a natural material–based carrier was developed for the immobilization of yeast, and its application in mango craft beer fermentation was systematically investigated. The optimal fermentation conditions were screened, and the physicochemical properties, nutritional composition, and volatile flavor profiles of the resulting mango craft beer were comprehensively evaluated. (3) Results: The results showed that the maximum mass gain of yeast after immobilization on the natural carrier reached 13.3%. Compared with free yeast, the immobilized yeast exhibited a 1.58-fold higher average extract consumption rate and a 1.39-fold higher alcohol production rate based on the overall fermentation system, while the primary fermentation period was shortened by approximately 33%. Under the optimized fermentation conditions, the mango craft beer achieved a sensory score of 81 points, with a β-carotene retention rate of 91.25%. Furthermore, the mango craft beer exhibited a more diverse profile of volatile flavor compounds and enhanced nutritional composition compared with the control. (4) Conclusions: Overall, fermentation conditions were optimized using Response Surface Methodology (RSM) based on Box–Behnken Design (BBD). Natural immobilization carrier developed in this study effectively enhanced yeast fermentation efficiency and shortened the primary fermentation cycle, and these findings demonstrate its significant potential for cost reduction and efficiency enhancement in the production of flavored craft beer, providing a practical technical support for the industrial application of natural carrier-based yeast immobilization technology.

Fermentation doi: 10.3390/fermentation12050213

Authors: Tanapon Mattayaruk Yotsapon Yangngam Seangla Cheas Chanon Suntara Metha Wanapat Chanadol Supapong Areerat Lunpha Ruangyote Pilajun Payungsuk Intawicha Anusorn Cherdthong

Corn dust is an abundant agro-industrial by-product with potential as an alternative energy source. Its use in animal feeding, however, is restricted by high fiber content and low digestibility. This study evaluated the effects of non-starch polysaccharide (NSP) enzymes and yeast (Candida tropicalis KKU20) on the chemical composition, fermentation characteristics, and microbial populations of fermented corn dust. The experiment was conducted using a completely randomized design with a 3 × 2 factorial arrangement plus an additional control treatment. Factor A consisted of three levels of enzyme supplementation (0.02%, 0.04%, and 0.06% of dry matter), and Factor B consisted of yeast supplementation (without yeast or with C. tropicalis KKU20, approximately 1 × 1013 cells/g of inoculum). The control treatment consisted of fermented corn dust without enzyme or yeast supplementation. Samples were fermented for 15 days prior to analysis. Yeast inoculation increased crude protein and non-fiber carbohydrate contents while reducing neutral detergent fiber, acid detergent fiber, and acid detergent lignin (p < 0.05). Significant enzyme × yeast interactions were observed for several components, particularly fiber fractions (p < 0.05). The reduction in fiber was more pronounced when enzymes were combined with yeast. Predicted energy values, including metabolizable and digestible energy, were increased following yeast supplementation (p < 0.05). Fermentation characteristics were mainly affected by yeast. Yeast-treated samples exhibited higher pH and ammonia–nitrogen concentrations, indicating increased nitrogen turnover during fermentation. In contrast, lactic and propionic acid concentrations were higher in treatments without yeast, while yeast inoculation was associated with lower acetic acid and slightly higher butyric acid levels. Microbial analysis indicated interactions between treatments for lactic acid bacteria populations, reflecting competition for available substrates. No coliform bacteria were detected, indicating acceptable hygienic quality. Overall, yeast inoculation modified the chemical composition of corn dust, particularly by increasing crude protein and reducing fiber fractions, while NSP enzymes contributed to fiber degradation, especially when combined with yeast. However, these changes reflect compositional modification rather than confirmed feeding value, and further evaluation under rumen or in vivo conditions is required.

Fermentation doi: 10.3390/fermentation12050212

Authors: Kessara Seneesrisakul Oijai Khongsumran Krittiya Pornmai Ee Ling Yong Malinee Leethochawalit Sumaeth Chavadej

Microaeration has been applied to enhance anaerobic digestion (AD), although the underlying mechanisms remain unclear. This work proposes that improving methanogenic activity can be achieved by alleviating micronutrient deficiencies and enhancing digestibility. The microaeration technique was employed to enhance the methanogenic activity of cassava wastewater (CW) both with and without added cassava residue (CR) and to improve CR digestibility in a continuous stirred tank reactor (CSTR) at 37 °C. The sole CW had the optimal COD loading rate of 1.71 kg/m3d. The addition of CR at 1000 mg/L to the CW resulted in the greatest methanogenic improvement of 88% compared with the sole CW and provided the greatest digestibility of CR. Under the optimal specific O2 dosage rate (3 mL/LRd), the improvements in CH4 yields were 251% and 140% in comparison to those of the sole CW and the CW with added CR, respectively. Additionally, it achieved substantial improvements in digestibility for the cellulose (59%), hemicellulose (61%), and remaining starch (67%) fractions of added CR. However, lignin degradation remained unaffected, a potential area for future optimization. This work opens new avenues for enhancing biogas production from wastewater by adding agricultural residue in conjunction with microaeration.

Fermentation doi: 10.3390/fermentation12050211

Authors: Khalida Shahni Banaraj Haobam Oinam Ibochouba Singh Keisham Shanta Devi Soibam Thoithoisana Devi Nanaocha Sharma Kshetrimayum Birla Singh

Fermented foods hold a significant position in global culinary traditions, particularly within ethnic and traditional diets. They are widely consumed for their distinctive flavors, textures, and health-promoting attributes. Although extensive research exists on fermentation processes, comprehensive insights into the nutraceutical potential and mechanistic health benefits of these foods remain limited. This review highlights key fermented products traditionally consumed in the north-eastern region of India including Hawaijar, Soibum, Ngari, alongside global counterparts such as Natto, Chongkukjang, Miso, Kefir, Tempeh, Kimchi, Kombucha, and Sauerkraut. These foods are rich in bioactive compounds (phenolics, peptides, organic acids, and exopolysaccharides), probiotic microorganisms, and essential nutrients that collectively contribute to their antioxidant, anti-inflammatory, antidiabetic, and cardioprotective effects. Recent in vitro and in vivo studies demonstrate that regular consumption of such foods may support the prevention and management of chronic conditions, including diabetes, cardiovascular diseases, obesity, gastrointestinal disorders, and neurodegenerative diseases. However, mechanistic studies remain insufficient to fully elucidate the synergistic interactions between microbial metabolites, host metabolism, and gut microbiota modulation. The review therefore emphasizes the biochemical and therapeutic mechanisms underlying ethnic fermented foods, advocating for advanced metabolomic and molecular approaches to validate their health-promoting efficacy. This review provides a timely and integrative perspective by critically evaluating preclinical and clinical evidence, highlighting mechanistic insights, translational gaps, and future research priorities. These insights will support the development of functional food formulations and reinforce the integration of traditional fermented foods into modern dietary strategies for disease prevention and overall well-being.

Fermentation doi: 10.3390/fermentation12050210

Authors: Chatchol Kongsinkaew Chutipol Tangsattayatithan Supenya Chittapun Parivat Phiphatbunyabhorn Tunyaboon Laemthong Mariena Ketudat-Cairns Soisuda Pornpukdeewattana Awanwee Petchkongkaew Theppanya Charoenrat

Seaweed bioactive extraction generates de-extracted residual solids that remain carbohydrate-rich but are often underutilized. This study developed an integrated valorization route for Gracilaria fisheri spent biomass to produce fermentable sugars for β-carotene production by Rhodotorula paludigena CM33. Reducing sugar production was optimized using response surface methodology (Box–Behnken design) by varying reaction time, sulfuric acid concentration, and biomass loading at 90 °C. The predicted optimum (47.39 min, 2.50% (w/v) H2SO4, and 7.13% (w/v) biomass) yielded 22.41 g/L reducing sugars and was validated experimentally at 22.22 ± 0.19 g/L, indicating that the model reliably predicted reducing sugar production. The optimized condition was scaled up in a 22 L bioreactor with sequential acid hydrolysis followed by enzyme-assisted hydrolysis, increasing reducing sugars from ~30 to ~40 g/L. FTIR and SEM analyses indicated progressive modification of the carbohydrate matrix across processing stages. Batch cultivation of R. paludigena on the hydrolysate showed that ammonium sulfate supplementation significantly increased biomass, whereas β-carotene titers were not significantly different. Repeated-batch operation on non-supplemented hydrolysate sustained production over four cycles with β-carotene titers of 13.75–17.27 mg/L, demonstrating the operational feasibility of the hydrolysate-based system. Overall, this work demonstrates a practical seaweed biorefinery approach to upgrade G. fisheri spent biomass into sugars and carotenoid-rich yeast biomass.

Fermentation doi: 10.3390/fermentation12050209

Authors: Nancy Nayeli Domínguez-Alfaro Mónica Cristina Rodríguez-Palacio Diana Guerra-Ramírez Patricia Castilla-Hernández

Microalgae and cyanobacteria are photosynthetic microorganisms capable of removing nutrients from eutrophic waters and producing biomass. Therefore, the aim of this study was to evaluate the bioremediation performance of three microalgae and one cyanobacterium native to Lake Xochimilco and to assess their potential for biofuel production (biodiesel and biogas) from biomass generated. In photobioreactors, ammonium (96.61–97.06%), nitrate (82.4–100%), and phosphate (83.95–89.71%) were effectively removed from the lake water. The specific growth rates ranged from 0.041 to 0.144 d−1 and biomass productivities from 0.016 to 0.049 g L−1 d−1, with high biomass yield on the substrate. The estimated CO2 fixation rates ranged from 0.024 to 0.092 g L−1 d−1. Chlorella sp. achieved the highest yield of fatty acid methyl esters (FAMEs) with 91.24% of the extracted lipids. Overall, saturated FAMEs were predominant in the biodiesel; however, the presence of monounsaturated FAMEs such as methyl palmitoleate and methyl oleate enhances their fluidity and oxidative stability. Synechocystis sp. and Chlorella sp. produced the most biogas using biomass after lipid extraction, at 429.5 L kg−1 VS and 404.9 L kg−1 VS, respectively, with over 60% biomethane. These strains represent a sustainable and promising possibility for water bioremediation and generating biofuels.

Fermentation doi: 10.3390/fermentation12040208

Authors: Thomas Bintsis Sofia Lalou Stylianos Exarhopoulos Ioanna Voulgaridi Fani Th Mantzouridou

The effect of cold smoking on the physicochemical, microbiological, and aromatic properties of Formaella-type cheese has not been previously investigated. In this study, experimental Formaella-type hard cheeses (≤38% moisture) were produced using a multistep high-temperature cooking process and subjected to weak (20 min) and intense (60 min) cold smoking, alongside an unsmoked control. Cheeses were analyzed before and after smoking and during refrigerated storage (up to 90 days). Smoking significantly influenced pH, water activity, and colour parameters, with intensively smoked cheeses exhibiting lower pH, reduced lightness (L*), and increased redness (a*) and yellowness (b*). Microbiological analyses revealed low viable counts across all samples, attributed to severe cooking steps and vacuum storage. Smoking, particularly at high intensity, significantly reduced total mesophilic counts and enterococci, while Enterobacteriaceae, staphylococci, yeasts, and moulds were not detected after manufacture. The dominant microbiota consisted mainly of lactic acid bacteria, identified by MALDI-TOF MS, including Enterococcus durans, Ent. faecium, Leuconostoc lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Lacticaseibacillus rhamnosus, and Lactobacillus curvatus. Headspace-SPME-GC-MS analysis identified 75 volatile compounds, with free fatty acids, ketones, aldehydes, and lactones as the predominant groups. Smoking introduced characteristic phenolic and furan derivatives associated with smoky aroma. Overall, smoking intensity modulated microbial dynamics and aroma development without compromising microbiological quality.

Fermentation doi: 10.3390/fermentation12040207

Authors: Rujirek Nopgason Tanapawarin Rampai Thanaporn Dechpreechakul Kobkul Laoteng Siwaporn Wannawilai

Thraustochytrids are promising alternatives for the production of docosahexaenoic acid (DHA; C22:6 n-3), a long-chain polyunsaturated fatty acid with health benefits. For practical application of this oleaginous microorganism, an efficient cultivation method to enhance DHA production is required, which relies on several factors that support cell growth, lipid accumulation, and lipid turnover. In this study, the robust submerged fermentation of an acid- and high-temperature-tolerant strain of Aurantiochytrium limacinum was investigated. Under controlled temperature and acidic conditions (pH 4.5 and 30 °C), glucose and peptone were the best carbon and nitrogen sources for enhancing biomass and DHA production, respectively, with a glucose concentration of 60 g/L and a C/N ratio of 24 being optimal for DHA production. Applying an aeration rate of 2 vvm and an agitation speed of 300 rpm using a combination of a ring sparger and pitch-blade impeller in a stirred-tank bioreactor improved DHA production using intermittent fed-batch fermentation. The highest DHA titer was obtained at 3.01 g/L, and the DHA content in biomass was 10.69% (w/w) after intermittent feeding of cassava starch hydrolysate as the substrate.

Fermentation doi: 10.3390/fermentation12040206

Authors: Marlene Höller Enes Demiray Katrin Krause Erika Kothe

The genus Streptomyces is a major driver of the soil microbial community. These filamentous, exospore-producing bacteria are copious producers of bioactive compounds that are not only used as antibiotics but also affect the soil microbial community in composition and activity. With an average of about 30 different bioactive compounds produced per species, the bacteria use complex regulatory mechanisms that respond to environmental as well as community signals. Understanding these mechanisms will be useful in harnessing the full potential of Streptomyces in biotechnology, e.g., to tackle the antibiotic resistance crisis. This includes the discovery of new antibiotics that are not produced under standard laboratory conditions, as well as being able to modulate the signaling cascades to produce other biotechnology products. As an example, the genus Streptomyces, as one of the few bacterial and archaeal taxa, produces cobalamin de novo through both the oxic and anoxic biosynthesis pathways. This feature adds to the importance of this genus for the soil microbial communities, as well as for applications in fermentation.

Fermentation doi: 10.3390/fermentation12040205

Authors: Guangyu Yan Ying Li Meng Liu Zhaomin Sun Feifei Gong Lei Yu

Metallothionein (MT) is a multifunctional metal-binding protein with broad applications in medicine, healthcare, and food industries, but its large-scale use is limited by inefficient industrial synthesis. To address this and obtain optimal fermentation parameters for large-scale MT production, this study used the recombinant marine-derived MT-producing Pichia pastoris strain SMD1168-MT. We first optimized the strain’s growth and induced fermentation conditions, then constructed a Back Propagation (BP) neural network model for in-depth parameter optimization and accurate MT expression prediction. Results showed the optimal growth conditions for SMD1168-MT were: 30 °C, initial pH 8.0, shaking speed 220 r/min, and 4% inoculum size. The BP model exhibited high accuracy (training set: R2 = 0.8430, MAE = 0.0129, RMSE = 0.0175; validation set: R2 = 0.8337, MAE = 0.0144, RMSE = 0.0174). Combined with Particle Swarm Optimization (PSO), the optimal fermentation conditions were: 7.7% methanol, initial OD600 8.2, 240 r/min, 50 h induction, and 125 μmol/L Zn2+. Validation confirmed MT expression reached 0.2141 mg/mL (2.93-fold). This study demonstrates that the BP neural network effectively optimizes recombinant P. pastoris-based marine-derived MT fermentation, improving yield and providing a basis for industrial scale-up.

Fermentation doi: 10.3390/fermentation12040204

Authors: Sofia Mendo Beatrice Zignego Francesca Bonazza Fabio Masotti Sara Casati Sofia Vanerio Roberto Foschino Alessio Battistini Ivano De Noni

Growing environmental and food security concerns have increased interest in circular strategies to valorize agri-food by-products. Sunflower-seed press cake (SSPC), a protein-rich residue from oil extraction, is largely underutilized despite its high nutritional and functional value. This study aimed to develop a fermented plant-based food prototype (PBFP) from SSPC using Lactococcus lactis B12 and Penicillium camemberti, evaluating microbiological safety, chemical characteristics, and sensory acceptability. A blend containing 40% SSPC and 60% water was autoclaved, inoculated, and ripened for 4 weeks under controlled temperatures. Microbial counts, pH evolution, free amino acids, biogenic amines, volatile organic compounds (VOCs), cyclopiazonic acid (CPA) content, and sensory attributes were evaluated using cultural techniques, HPLC, HS-SPME/GC-MS, LC–ESI–MS/MS (QTRAP 4000), and sensory evaluation. L. lactis efficiently acidified the matrix (pH ≈ 4.5–4.9), ensuring microbial food safety, with high LAB counts (~109 CFU/g) and absence of pathogens (Listeria monocytogenes and Salmonella spp.) and hygienic markers < 2 log CFU/g (B. cereus, E. coli, and Enterobacteriaceae). Free amino acids decreased during fermentation, and no histamine or tyramine was detected. VOC analysis revealed diacetyl, acetoin, 2,3-butanediol, and 1-octen-3-ol, contributing to mild dairy-like notes. CPA was detected at 0.48 ng/g, well below levels reported in cheeses. Sensory evaluation showed no significant differences in overall intensity between inoculated and control blends, although qualitative descriptors indicated subtle changes in aroma and texture. These results demonstrate the feasibility of safely producing a fermented plant-based prototype from SSPC. Future studies should explore longer ripening times, additional microbial consortia, and strategies to enhance texture and aroma complexity.

Fermentation doi: 10.3390/fermentation12040203

Authors: Huiying Zhang Kai Lou Gulinizier Nueraihemaiti Yuanyuan Chen Yan Gao Jun Zeng Qing Lin Xiangdong Huo

To explore the appropriate supplementation rate of yeast culture (YC) in Kazakh sheep during fattening, the effects of different YC supplementation rates on rumen fermentation parameters and microbial community were studied through in vitro rumen fluid fermentation experiments. A 0.40 g high-concentrate diet was used as the fermentation substrate, and five groups were added with YC at 0% (CK), 1.25% (YC1), 2.5% (YC2), 3.75% (YC3) and 5% (YC4) of dietary dry matter, respectively. Anaerobic fermentation was carried out for 48 h in 60 mL fermentation broth. The results showed that the 48 h GP and microbial crude protein (MCP) concentration in all YC supplementation groups were significantly higher than those in the CK group (p < 0.05). The concentrations of total volatile fatty acids (TVFA) and propionate in the YC1 and YC2 groups were significantly increased and the A/P ratio in the two groups was significantly decreased (p < 0.05). The Multi-factor Comprehensive Evaluation Index (MFAEI) calculation indicated that 1.25% was appropriate. The YC1 and YC2 groups significantly increased the richness and diversity of rumen bacterial communities (Chao1 and Shannon indices, p < 0.05), and significantly increased the relative abundance of Bacteroidota and NK4A214_group (p < 0.05), while significantly decreasing the relative abundance of the potential pathogenic bacterium Campylobacter (p < 0.05). Ustilago abundance was significantly suppressed in all the YC-supplemented groups (p < 0.05). The most effective YC supplementation rate among the tested doses was 1.25% according to the MFAEI and key microbial indicators. The results suggest that dietary supplementation of 1.25% YC (dry matter basis) may beneficially modulate rumen fermentation parameters under in vitro conditions, providing a reference for further in vivo studies on its application in fattening Kazakh sheep.

Fermentation doi: 10.3390/fermentation12040202

Authors: Chih-Feng Wang Chih-Chung Wu Yi-Jou Chung Cui-Rou Huang Ying-Chen Lu

Obesity is a major global health concern, and functional fermented foods have attracted increasing attention for their potential metabolic benefits. Grain–oolong tea fermented beverage (GOFB), produced through a two-step spontaneous fermentation process, is rich in fermentation-derived bioactive compounds; however, its effects on adipogenesis remain unclear. In this study, we investigated the effects of GOFB on adipogenesis-related phenotypes in 3T3-L1 adipocytes. The results showed that GOFB exhibited antioxidant activity in vitro and significantly reduced intracellular reactive oxygen species and lipid peroxidation in MDI-induced adipocytes. GOFB treatment was associated with reduced cell proliferation, lipid accumulation, and triacylglycerol content in 3T3-L1 adipocytes. In addition, GOFB was associated with attenuated adipogenic responses, accompanied by reduced expression of genes related to RAS, ERK, c-Myc, cyclin D1, SREBP-1c, PPAR-γ, C/EBP-α, NCoR1, and FAS. Collectively, these findings suggest that GOFB is associated with attenuated adipogenic responses in 3T3-L1 adipocytes and support its potential application as a functional fermented beverage.

Fermentation doi: 10.3390/fermentation12040201

Authors: María de la Luz Herrera-Estrada José Sandoval-Cortés Carlos N. Cano-González Teresinha Gonçalves da Silva José L. Martínez-Hernández Miguel A. Aguilar-González Juan A. Ascacio-Valdés Mónica L. Chávez-González Cristóbal N. Aguilar

Carotenoids are natural pigments of high industrial value, with recognized antioxidant properties, and are widely used in the food, cosmetic, and pharmaceutical industries. Oleaginous yeasts, such as Rhodotorula glutinis, represent a promising alternative for the sustainable production of these compounds through submerged fermentation, compared to their extraction from plant sources or chemical synthesis. This study aimed to optimize culture conditions to maximize biomass and carotenoid production in R. glutinis P4M422. To this end, the effects of various culture factors, including light, carbon-to-nitrogen (C/N) ratio, temperature, pH, and glycerol addition, on cell growth and pigment biosynthesis were evaluated. The results showed that agitation speed and C/N ratio are key variables in system performance, significantly influencing both growth and carotenoid accumulation. Under the established optimal conditions (210 rpm, C/N ratio of 50, red light, and 30 °C), a maximum volumetric yield of 343.1 mg/L and a productivity of 4.8 mg/L/h were achieved, representing a substantial improvement in process efficiency. These values position the R. glutinis P4M422 strain as a competitive alternative for the biotechnological production of carotenoids. Taken together, these findings confirm the efficiency of submerged culture as a platform for obtaining high-value-added biopigments and reinforce the potential of microbial fermentation systems as a sustainable, scalable, and controllable strategy for their production.

Fermentation doi: 10.3390/fermentation12040200

Authors: Fatmanur Mavi Ayça Uras Fatma Ersöz Burcu Emine Tefon-Öztürk Mehmet İnan Cüneyt Dinçer Demet Yıldız-Turgut Orçun Çınar Muharrem Gölükcü Ayhan Topuz Aysun Türkanoğlu-Özçelik

Amylase enzyme catalyzes the breakdown of starch by acting on the α-1,4 glycosidic bond. The use of amylases is common in areas such as baking and fruit juice production in the food industry, as well as in the detergent, textile, and paper industries. Due to their broad industrial applicability, the recombinant production of amylases has received increasing attention in recent years. In this study, the production of Aspergillus niger α-amylase enzyme was investigated for the first time in Pichia pastoris under the control of the ethanol-inducible synthetic ADH2 (SNT5) promoter. A codon-optimized A. niger α-amylase gene was expressed extracellularly in the P. pastoris MK115-PDI strain. Optimal production conditions were 24 °C and pH 6.0. In a 5 L bioreactor, total secreted protein reached 2.2 g/L and enzyme activity reached 44,062 U/mL. The recombinant enzyme was characterized and showed optimal activity at 60 °C and pH 7. In apple juice assays, the enzyme hydrolyzed starch and demonstrated suitability for juice clarification, although performance depended on enzyme concentration. Overall, these results indicate that the SNT5 synthetic promoter enables efficient recombinant α-amylase production in P. pastoris and represents a promising alternative to conventional promoter systems for industrial enzyme manufacturing.

Fermentation doi: 10.3390/fermentation12040199

Authors: Denis Atroshenko Alexandra Roslova Anastasia Yakobson Diana Markova Diana Golovina Vladimir Tishkov

Thermotolerant methylotrophic yeast Ogataea parapolymorpha is a promising host for high-temperature bioprocesses, yet the effects of carbon source and exogenous carbohydrates on their heat response remain poorly understood. We investigated how growth on glucose, glycerol, or methanol, short-term thermoadaptation (45 °C, 2 h), and supplementation with trehalose, sucrose, maltose, or xylose affect thermotolerance (55 °C, 30 min) and intracellular trehalose content. Thermoadaptation increased survival on all carbon sources and was accompanied by substantial trehalose accumulation in glucose- and glycerol-grown cells, but only minor trehalose accumulation in methanol-grown cells. Carbohydrate supplementation improved survival only in methanol-grown cultures. Under these conditions, trehalose, sucrose, and maltose increased intracellular trehalose levels, whereas xylose enhanced survival without a comparable increase in trehalose. These results show that the heat-stress response of O. parapolymorpha is strongly carbon source-dependent and that the protective effects of carbohydrate supplementation in methanol-grown cells cannot be explained by trehalose accumulation alone.

Fermentation doi: 10.3390/fermentation12040198

Authors: Marina Savić Milana Vujičić Lato Pezo Đorđe Vojnović Anita Milić Aleksandra Tepić Horecki Zdravko Šumić

Cabbage has been cultivated in Serbia for centuries, especially in the Vojvodina Province. Fermentation enhances its functional properties, as fermented cabbage contains live lactic acid bacteria with proven health benefits. Besides improving functional properties, fermentation modifies the sensory characteristics and chemical composition of cabbage while extending its shelf life. This study aimed to investigate the use of antioxidants—ascorbic and citric acid—in various concentrations during fermentation and their effect on the nutritional and sensory properties of the final product. The experiment was carried out under industrial conditions over 45 days. The addition of these acids influenced both the chemical composition and sensory acceptance of the fermented cabbage. Among the tested samples, LK15 (fermented with 0.025% citric acid) showed the best results in terms of sensory quality and nutritional value. It had the highest total phenolic content (419.05 ± 16.01 mg GAE/100 g dry matter) and high antioxidant activity as determined by the ABTS method (0.1224 mg/g). The results highlight that the use of citric and ascorbic acid in cabbage fermentation can effectively enhance product quality, suggesting potential for further research and application in industrial fermentation to improve both nutritional and sensory attributes.

Fermentation doi: 10.3390/fermentation12040197

Authors: Anahid Esparza-Vasquez Sara Saldarriaga-Hernandez Rosa Leonor González-Díaz Tomás García-Cayuela Danay Carrillo-Nieves

Brewer’s spent grain (BSG) is an abundant lignocellulosic by-product whose valorization can support circular bioeconomy strategies. This study evaluated BSG bioconversion by Aspergillus oryzae ATCC 10124 under solid-state fermentation (SSF) to produce lignocellulolytic enzymes and release second-generation (2G) sugars relevant to biorefinery applications. SSF was monitored over 0–10 days, and FPase, endo-cellulase, β-glucosidase, xylanase, mannanase, amylase, and ligninolytic enzyme activities were quantified. Enzymatic crude extracts were further assessed in SDS-PAGE analysis. Glucose, cellobiose, xylose and arabinose release and consumption were tracked throughout fermentation, and substrate transformation was supported by FTIR. The secretome exhibited a predominantly hydrolytic profile, with maximal hemicellulolytic and cellulolytic activity around days 2–4, as well as sustained amylase activity. Ligninolytic activity was not detected. Sugar profiles indicated rapid early hydrolysis of glucose, followed by progressive pentose release. The stabilization and decline were consistent with fungal uptake. Changes in the carbohydrate fingerprint and SDS–PAGE banding supported structural polysaccharide remodeling and hydrolytic protein secretion. Thus, this SSF platform confirmed certain potential for low-cost cellulolytic and hemicellulolytic enzyme generation. However, because sugar accumulation was temporary and followed by consumption, this system is best interpreted as a biological pretreatment and enzyme-generation step that supports subsequent downstream valorization.

Fermentation doi: 10.3390/fermentation12040196

Authors: Yuki Iwasaki Yuto Mine Zen-ichiro Kimura

Robust genetic tools are a prerequisite for causal, perturbation-based tests of redox physiology in acetogens. Here we establish practical genetic entry points for Sporomusa sphaeroides DSM 2875 under strictly anaerobic handling. We first attempted genome editing via double-crossover allelic exchange targeting pyrF using a non-replicative pUC19-based knockout construct and 5-fluoroorotic acid counterselection. Diagnostic PCR identified ΔpyrF candidates with the expected size shifts, demonstrating that homologous recombination is technically feasible in DSM 2875; however, the ΔpyrF genotype exhibited severe growth defects and could not be stably maintained over repeated passages, indicating a key limitation of a pyrF-based workflow under our current conditions. We then evaluated multiple E. coli–anaerobe shuttle plasmids for introduction and maintenance. Among the tested vectors, pJIR751 reproducibly yielded erythromycin-resistant transformants after prolonged incubation and supported serial passaging on selective media. Plasmid retention was confirmed by diagnostic PCR from liquid cultures in all tested isolates. Importantly, this maintainable plasmid platform enables genetically grounded perturbation-and-rescue experiments under electrode- or Fe0-assisted conditions, allowing mechanistic hypotheses in bioelectrochemical acetogenesis to be tested causally rather than inferred from phenotypes alone. Together, these results define current practical boundaries for S. sphaeroides genetics and establish pJIR751 as a practical foundation for downstream genetic manipulation in bioelectrochemical studies.

Fermentation doi: 10.3390/fermentation12040195

Authors: Martin Gierus

Microbial fermentation is a fundamental biological process that shapes the transformation of nutrients, energy flow, and ecosystem functioning across a wide range of biological systems [...]

Fermentation doi: 10.3390/fermentation12040194

Authors: José Juan Buenrostro-Figueroa Sergio Huerta-Ochoa Cristóbal Noé Aguilar María Isabel Reyes-Arreozola Francisco José Fernández Lilia Arely Prado-Barragán

The increasing demand for environmentally sustainable and efficient laundry detergents has prompted the exploration of innovative biotechnological solutions. This study aims to integrate solid fermentation and by-product valorization for high-quality proteases suitable for laundry detergents. Of 486 strains isolated from fruit by-products, 9 were selected for their proteolytic activity, but only 3 showed proteolytic activity in the presence of detergent components. Strain M17, identified as Yarrowia lipolytica (Yl), proved to be the most effective in producing proteolytic extracts with activity similar to that found in commercial detergents. The produced proteases were incorporated into laundry detergent formulations, and their enzyme activity was compared with that of commercial laundry detergents. The results showed that the proteolytic extracts have enzyme activity similar to that of commercial laundry detergents. Culture media were developed to enhance protease production using fruit by-products. The highest activity (43.71 U (g dm)−1) was achieved at C/N = 20.04, while the best productivity (1.37 U (g dm·h)−1) at pH 7.0 and 30 °C was observed. The results demonstrate that culture media based on fruits and vegetable by-products enhance protease yield and activity. This approach not only reduces waste but also adds value to natural resources through an environmentally friendly process. This study underscores the potential of combining solid-state fermentation with by-products. Using Yl in combination with fruit and vegetable by-products is a practical, eco-friendly method for producing high-quality proteases for laundry detergents. This green enzyme innovation offers significant promise for advancing the detergent proteolytic enzymes and promoting sustainable practices in by-product management.

Fermentation doi: 10.3390/fermentation12040193

Authors: Dongmei Wang Fei Wang Ping Tang Lei Wang Yusheng Xie Maosen Xiong Qian Luo Yanping Luo Dan Huang Lei Yang

Microbial communities are the fundamental determinants of Nongxiang Daqu quality. In this study, we systematically investigated the assembly and succession mechanisms of microbial communities during Nongxiang Daqu fermentation. Our findings reveal that this ecological succession is primarily driven by deterministic processes, encompassing dynamic environmental variables and interspecific microbial interactions. Significant stage-specific temporal variations in the community structure were observed, and biomarkers identified via a random forest model further corroborated these dynamic successional patterns. Both the neutral community model and Modified Stochasticity Ratio (MST) tests demonstrated that community assembly is dominated by deterministic processes, the influence of which intensifies as fermentation progresses. Notably, the fungal community exhibited a more pronounced response to these deterministic environmental selections than the bacterial community. Furthermore, co-occurrence network analysis, Mantel tests, and redundancy analysis (RDA) collectively illustrated that microbial interactions and environmental factors—specifically temperature, humidity, oxygen, carbon dioxide, and acidity—synergistically regulate this succession. Crucially, the rates of change in these environmental parameters directly dictated the pace of microbial turnover. Among these, oxygen and acidity had the greatest influence: oxygen accounted for 17.32% and 29.05% of the effects on fungi and bacteria, respectively, while acidity accounted for 16.77% and 25.23%, respectively. Time-series forecasting indicated that community structural assembly and stabilization predominantly conclude within the initial 30 days of fermentation. Ultimately, this study uncovers the ecological driving forces shaping the Nongxiang Daqu microbiome, providing a vital theoretical foundation for the targeted regulation of Daqu microecology and the enhancement of product quality.

Fermentation doi: 10.3390/fermentation12040192

Authors: Xiaosi Lin Liping Xiao Jingru Xiao Congjie Dai

Cycloheptylprodigiosin is a promising anticancer candidate that induces cancer cell death accompanied by severe Golgi stress. Although the soybean oil-based optimized MB2216 medium produced a total prodiginine titer approximately three times that of the basal MB2216 medium, the overall production level remained limited. In addition, a substantial fraction of the pigments partitioned into floating oil droplets, hindering efficient recovery by simple centrifugation. In this study, a novel medium was rationally formulated based on genomic insights derived from homology analysis of conserved biosynthetic genes involved in cycloheptylprodigiosin production in Spartinivicinus ruber MCCC 1K03745T. Sequential optimization through single-factor experiments, full factorial designs, steepest ascent experiments and response surface methodology identified an optimal medium consisting of peptone (5 g/L), yeast extract (1 g/L), peanut meal (7.611 g/L), and L-Proline (0.695 g/L) prepared in seawater at pH 7.6. Under the optimized conditions, the total prodiginine titer reached 53.33 mg/L, which was 11.37 times that of the basal MB2216 medium and 3.29 times that of the soybean oil-based MB2216 medium. Moreover, the pigment-associated biomass could be efficiently recovered by centrifugation. This study provides a genomics-informed strategy for improving prodiginine production in S. ruber and facilitates downstream pigment recovery.

Fermentation doi: 10.3390/fermentation12040191

Authors: Yukun Zhang Manabu Ishikawa Shunsuke Koshio Saichiro Yokoyama Na Jiang Jiayi Chen Yiwen Tong Xiaoxiao Zhang

To address the challenge of depleting traditional feed resources, this study aimed to biovalorize sweet potato waste (SPW), a major byproduct of the Japanese shochu industry, into a high-value functional animal feed. An innovative two-stage solid-state fermentation (SSF) was employed, featuring an initial aerobic stage with Aspergillus oryzae for substrate degradation, followed by an anaerobic stage with Lactobacillus plantarum for nutritional enhancement. To optimize this complex, multi-variable process, the predictive performance of Artificial Neural Network (ANN) and Random Forest (RF) machine learning models was compared based on an augmented experimental dataset (N = 80). To ensure statistical robustness and prevent data leakage, a repeated k-fold cross-validation strategy was implemented. The RF model demonstrated significantly superior accuracy and reliability than the ANN model, particularly in predicting the primary metric, crude protein (R2 = 0.61 ± 0.04 vs. R2 = 0.12 ± 0.15). Subsequently, the validated RF model was integrated with a Constrained Differential Evolution (CDE) algorithm for global parameter optimization. The optimized process was predicted to yield a final product with a crude protein content of 25.0%, alongside significant increases of 114.1% in total amino acids and 123.9% in essential amino acids. These projections were experimentally validated in vitro, confirming the model’s accuracy with a relative error of less than 5%. Furthermore, comprehensive biochemical assays demonstrated a massive degradation of anti-nutritional factors and significant enhancements in total phenolic content and antioxidant activity. This study provides a scientifically validated, data-driven framework for the valorization of SPW. It confirms the superior efficacy of ensemble learning methods for optimizing complex bioprocesses with limited data, offering a contribution to the development of a circular bioeconomy and sustainable feed resources.

Fermentation doi: 10.3390/fermentation12040190

Authors: Yaiza Rodríguez Juan Manuel Del Fresno Carmen González Antonio Morata

Climate change presents a challenge for global viticulture due to rising temperatures and water stress, which accelerate grape ripening, increase sugar levels, and reduce acidity. This compromises wine quality and microbial stability, resulting in higher reliance on sulfur dioxide (SO2). However, SO2 can inhibit desirable fermentations, including those carried out by non-Saccharomyces yeasts, which are key biotechnological tools for climate adaptation due to their ability to modulate acidity, aroma, and ethanol. Therefore, alternative disinfection methods are needed to control wild microbiota without hindering inoculated yeasts. This review critically analyzes ozone (O3) as a non-thermal disinfection technology for winemaking. It examines the antimicrobial mechanism of ozone, its efficacy against wine-related microorganisms, its impact on the physicochemical and aromatic parameters of grapes, and its practical viability. Ozone effectively reduces spoilage-causing microbiota, achieving inactivation of approximately 3–4 log CFU/mL for yeasts, while preserving crucial grape compounds and providing a favorable environment for novel fermentation biotechnologies. Compared to other emerging technologies and SO2, ozone offers a balanced profile: effective disinfection, minimal residues, cost-effectiveness, and compatibility with sustainable winemaking. Ozone is emerging as a promising alternative to facilitate controlled fermentations and improve wine quality among the current climatic and oenological challenges.

Fermentation doi: 10.3390/fermentation12040189

Authors: Nisa Nur Hacıbayramoğlu Haktan Aktaş

As probiotic microorganisms must remain viable at a certain level throughout the shelf life of fermented foods, various plant-based prebiotics are added to fermented dairy products. Pea (Pisum sativum L.) is a remarkable food source due to its prebiotic properties, high phenolic content and antioxidant capacity. In this study, fermented milks containing different proportions of pea fiber powder (0%, 0.5%, 1%, 1.5% and 2%) were produced using Limosilactobacillus reuteri ACC27, which has probiotic potential, and Streptococcus thermophilus 212S. The addition of pea fiber powder promoted the growth of Limosilactobacillus reuteri ACC27, increasing viable cell counts by approximately 1 log CFU/g compared to the control during storage. In addition, the fermentation time was shortened by approximately 30 min in samples containing pea fiber. Malic (84.07–175.58 mg/kg), lactic (11,670.45–13,791.66 mg/kg), acetic (145.12–240.53 mg/kg) and benzoic acids (17.07–20.34 mg/kg) were detected in all samples. Furthermore, pea fiber supplementation improved physicochemical properties by reducing syneresis and modifying water release behavior, while also increasing viscosity. The addition of pea fiber also enhanced total phenolic content and antioxidant capacity of the samples. The results of the principal component analysis revealed that the addition of pea fiber powder was associated with potentially improved functional attributes and enhanced probiotic viability under the studied conditions.

Fermentation doi: 10.3390/fermentation12040188

Authors: Gabriella Siesto Rocchina Pietrafesa Antonio Caporusso Giorgia La Rocca Grazia Alberico Vito Valerio Angela Capece

Olive mill wastewater (OMWW) is a highly polluting agro-industrial effluent characterized by elevated organic load, low pH, and high concentrations of phenolic compounds responsible for its phytotoxicity and dark coloration. In this study, 41 non-conventional yeast strains belonging to the University of Basilicata Yeast Collection (UBYC), were tested for both the oleaginous potential traits and OMWW detoxification capacity in comparison to two commercial oleaginous controls, Yarrowia lipolytica ATCC 46483 and Lipomyces tetrasporus Li-0407. Primary screening in synthetic medium under nitrogen-limited conditions revealed widespread intracellular lipid accumulation. Quantitative analysis showed lipid contents above 20% (w/w) in some strains, with Candida tropicalis AII122 (33.3%) and Pichia manshurica ML-3 (29.4%) exhibiting the highest values in synthetic medium. The cultivation of eight selected strains in synthetic medium supplemented with 15% (v/v) of OMWW reduced intracellular lipid accumulation, with the highest value of 6.48% for the 2R1 strain. Levels of phenol reduction and color removal were highly different among all the analyzed strains, and C. tropicalis AII122 achieved the highest phenolic reduction and decolorization ability. These findings demonstrate that indigenous non-conventional yeasts represent a source of natural biodiversity, supporting sustainable waste valorization strategies based on the use of selected microorganisms within a circular bioeconomy framework.

Fermentation doi: 10.3390/fermentation12040187

Authors: Jesús Delgado-Luque Álvaro García-Jiménez Juan Carbonero-Pacheco Juan C. Mauricio

Alcoholic fermentation in winemaking is a complex bioprocess governed by physicochemical parameters such as temperature, density, pH, CO2 and redox potential, which critically affect yeast metabolism and wine quality. This review provides an integrated analysis of fermentative dynamics and emerging sensorization technologies, highlighting how their combined implementation enables real-time monitoring and advanced control in precision enology. Advances in conventional physicochemical sensors, spectroscopic techniques (NIR/MIR/UV-Vis) and non-conventional devices (e-noses, electronic tongues) integrated into IoT platforms enable continuous data acquisition, overcoming traditional manual sampling limitations. Predictive modeling, including kinetic models, machine learning approaches (e.g., Random Forest, XGBoost) and model predictive control (MPC/NMPC), supports anomaly detection, optimization of enological interventions and energy-efficient thermal management, while virtual sensors based on Kalman filters improve the estimation of non-measurable states (e.g., biomass, ethanol kinetics). Despite current challenges in calibration and interoperability, these innovations foster sustainable and reproducible winemaking under climate variability and pave the way for digital twins and semi-autonomous fermentation systems.

Fermentation doi: 10.3390/fermentation12040186

Authors: Caroline E. Hartner Mark A. Eiteman

Mevalonate is a biochemical precursor to a wide range of isoprenoids. Because the mevalonate pathway uses three moles of acetyl–CoA, native pathways which metabolize acetyl–CoA, including citrate synthase, strongly compete with mevalonate synthesis. Our hypothesis is that modifications in citrate synthase, with the aim of reducing this enzyme’s activity, can result in increased mevalonate. Previous research has demonstrated that citrate synthase variants can increase generation of acetyl–CoA-derived products from glucose, but research has not evaluated citrate synthase variants with other common carbon sources like xylose and glycerol. Using five variant strains with chromosomal modifications of citrate synthase, we first compared the growth of these variants with wild-type Escherichia coli on glucose, xylose, or glycerol. In general, any particular modification in citrate synthase (GltA) led to the greatest effect on growth rate in glucose-grown cells. Because the GltA[Y87N D101D* P208L] and GltA[A267T] variants showed the greatest effect on growth using glycerol, we selected these two variants to study the formation of mevalonate from glycerol by E. coli with an introduced mevalonate pathway. Controlled batch processes at the 1.3 L scale demonstrated significantly increased mevalonate production in variants compared to the wild-type background, with the GltA[A267T] attaining 7.3 g/L mevalonate in 16.5 h from 30 g/L glycerol. Nitrogen-limited or phosphorus-limited fed-batch processes using the GltA[A267T] variant performed similarly, and generated over 12 g/L mevalonate in 24–32 h at a yield of 0.24 g/g. This study demonstrates that GltA variants offer a means to generate acetyl–CoA-derived products from glycerol.

Fermentation doi: 10.3390/fermentation12040185

Authors: Fanny Claire Capri Enrico Tornatore Andrea Firrincieli Gemma Fernánez-García Rosa Alduina Angel Manteca Alessandro Presentato

Actinomycetes are among the richest sources of bioactive secondary metabolites in biotechnology, owing to their remarkable metabolic diversity. Although the genus Streptomyces has been extensively explored and has yielded many clinically important antibiotics, rare actinomycetes remain comparatively underinvestigated. In this study, Kitasatospora sp. SeTe27, isolated from uncontaminated soil in Sicily (Italy), was investigated for its antibacterial activity and fermentation-driven enhancement of secondary metabolite production. The strain inhibited Staphylococcus aureus ATCC 25923, prompting physiological and genomic analyses. Spore conditioning was evaluated in four media (R5A, GYM, TSB, and YEME) to enhance antibiotic production. Conditioned cultures exhibited markedly increased antibacterial activity in TSB and YEME, moderate production in R5A, and no detectable activity in GYM. Whole-genome sequencing revealed an 8.5 Mb genome (73.5% GC) containing 48 biosynthetic gene clusters (BGCs), including NRPS, PKS, terpene, and hybrid pathways. Several clusters showed high similarity to known antibiotic-associated BGCs, such as clifednamide- and phenazine-related pathways, while numerous orphan clusters indicated significant unexplored biosynthetic potential. These findings identify Kitasatospora sp. SeTe27 as a promising antimicrobial producer and demonstrate that spore conditioning in complex media is an effective strategy to enhance antibiotic production in rare actinomycetes.

Fermentation doi: 10.3390/fermentation12040184

Authors: Emma Mani-López Beatriz Mejía-Garibay Ricardo H. Hernández-Figueroa Aurelio López-Malo

Phenyllactic acid (PLA), a natural antimicrobial metabolite produced by lactic acid bacteria (LAB), has emerged as a key compound for biopreservation in food systems. The aims of this review are to summarize the main findings on LAB-producing strains, the effects of primary PLA precursors, the impacts of culture conditions on PLA production, antimicrobial activity, mechanisms of action, quantification and analysis methods, food applications, regulatory status, and the challenges in PLA production and applications. In this review, the quorum sensing role in PLA production and multi-omics strain improvement was revised. Applications in dairy, bakery, fruits, vegetables, meat, and fish products as well as active packaging are analyzed, demonstrating their effectiveness in controlling microbial spoilage and pathogens while preserving sensory quality. Its broad-spectrum antifungal and antibacterial activities make it particularly attractive as a clean-label alternative to synthetic preservatives, contributing to both food safety and extended shelf life. Finally, current limitations and future research needs are outlined, particularly in optimizing PLA production and establishing its role as a sustainable and effective tool for food safety management.

Fermentation doi: 10.3390/fermentation12040183

Authors: Zorica Lelova Temelkova Helena Baša Česnik Andreja Vanzo Klemen Lisjak

This study aimed to determine the effects of diammonium phosphate (DAP) and yeast autolysates (organic nutrients) added during alcoholic fermentation on the content and profile of aroma compounds in Sauvignon Blanc wines. Sequential additions of either DAP or organic nutrients were applied mainly during the first half of fermentation, increasing yeast assimilable nitrogen (YAN) from an initial 124 mg N/L to final concentrations of 208 and 209 mg N/L for DAP and yeast autolysates, respectively. Control musts were fermented without nutrient supplementation. All treatments were fermented using commercial yeast strain. Varietal thiols, ethyl and acetate esters, higher alcohols, glutathione (GSH), and YAN were monitored at early, mid, and late stages of fermentation, as well as in the final wines. Varietal thiols were formed at early stages of fermentation in all treatments; however, concentrations of both 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) were higher in wines supplemented with organic nutrients comparing to DAP and control. Compared to the control, DAP and organic nutrient supplementation increased ethyl ester concentrations in wine by 40.2% and 26.9%, respectively. Both nutrient treatments also resulted in higher acetate ester concentrations, while total higher alcohols were reduced by 19.1% with DAP and 12.1% with organic nutrients. No significant differences in GSH concentrations were observed among treatments. Sensory analysis revealed that wines supplemented with DAP achieved the highest scores for tropical aroma, varietal aroma, and overall quality. Overall, sequential supplementation with either inorganic or organic nitrogen positively influenced fermentation kinetics and aroma compound composition, resulting in improved varietal expression of Sauvignon Blanc wines. However, in low-YAN musts, DAP had a greater impact than organic nitrogen sources and should therefore be considered a key strategy for ensuring an adequate yeast nitrogen status.