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.

Fermentation doi: 10.3390/fermentation12040182

Authors: Zhaorui Gu Xiaojing Li Freddie Moore Anil Kumar Jamithireddy Steven Bates Nicholas J. Harmer

Microorganisms are increasingly being used for the industrial production of raw materials for food, chemical products and pharmaceuticals. The unconventional yeast Yarrowia lipolytica has a rising profile as a platform for industrial biotechnology. It has attractive physiological and metabolic properties, including high terpene and lipid production, high tolerance to complex environments, and amenability to genetic modification. Y. lipolytica naturally produces sufficient levels of cytosolic acetyl-CoA and malonyl-CoA to achieve lipid accumulation. Engineering biology methods allow transformation of these native metabolites into synthetic precursors for high-value compounds such as terpenes and flavonoids. Gene-editing, expression, and regulation tools have been developed for Y. lipolytica, facilitating improvement in bio-manufacturing yields for this chassis. This review summarizes natural product yields in Y. lipolytica and strategies for improving productivity. We highlight morphological engineering, metabolic engineering, and adaptive laboratory evolution as key strategies that can be used to improve the future yield, productivity and controllability of target molecules for Y. lipolytica engineering.

Fermentation doi: 10.3390/fermentation12040180

Authors: Luiza N. C. Silva Isabela F. Carrari Ícaro R. R. Castro Giulia B. C. Leite Amanda M. Cezar Eduardo M. Paula Marcos I. Marcondes

The rumen simulation technique (RUSITEC®) is a known model for research in rumen microbiology and fermentation. However, our research group observed inconsistencies in gas production across trials. This study investigated the effects of different gas outlet locations on digestibility, ruminal fermentation, gas production, and microbial protein synthesis. Fifteen fermenters tested three different gas outlet locations within the RUSITEC® equipment: (1) gas outlet directly on the effluent vessel for output liquid (EV); (2) gas outlet directly on fermenter cap (F); and (3) gas outlet on both effluent vessel and fermenter cap (EVF). Data were analyzed using a completely randomized design in SAS (v. 9.4) with the MIXED procedure, and significance was set at p < 0.10. Results showed that altering the gas outlet location did not affect nutrient digestibility (p > 0.10), microbial protein synthesis (p > 0.10), and volatile fatty acid (VFA) production when expressed on a molar basis (p > 0.10). However, total gas production (p = 0.108) was higher in the EVF group and ammonia nitrogen produced in the fermenter was higher in group F (p = 0.081). Furthermore, methane (CH4) production was underestimated when the gas outlet location was in just one of the locations when compared to the EVF group (p = 0.006). VFA proportion was also affected, with lower acetate (p = 0.005) and higher butyrate (p = 0.014) for group EV. These results indicate that the location of the gas outlet is an important methodological factor affecting fermentation measurements in the RUSITEC system, with outlets positioned in both the effluent and fermenter vessels enhancing gas recovery.

Fermentation doi: 10.3390/fermentation12040181

Authors: Azize Atik İlker Atik Gökhan Akarca

In this study, kefir production was investigated using both commercial kefir cultures and kefir grains, with milk treated at different UV-C doses and flow rates. The flow rate was set to 25 or 50 mL/min, and doses of 43.2 and 21.6 J/mL were applied at each flow rate, respectively. In all samples subjected to UV-C treatment, pH values decreased during storage, while % titratable acidity values increased. The kefir samples produced with UV-C-irradiated milk showed increased hardness and consistency, while cohesion and the index of viscosity decreased. The highest effect was observed in samples produced with kefir grain and at a flow rate of 50 mL/min. Lactic acid bacteria, Streptococcus/Lactococcus, and yeast counts in kefir samples produced from UV-C-treated milk increased. Flow rate affected the increase in microorganism counts. The physicochemical, textural, and microbiological changes during storage were more pronounced in kefir samples produced with kefir grains than with powdered cultures. The organic acid levels of kefir samples produced from milk treated with UV-C decreased compared to those of control samples. Furthermore, organic acid values increased during storage in all samples. As the flow rate increased, the amount of organic acids formed decreased (except for malic and formic acid levels).

Fermentation doi: 10.3390/fermentation12040179

Authors: Chatchai Kaewpila Pongsatorn Gunun Nikom Srikacha Chanon Suntara Waroon Khota

In ruminant nutrition, the lignocellulosic complex is a primary constraint limiting the utilization of dietary fiber. The objective of this study was to evaluate the effects of inoculating lignin-degrading bacteria (LDB) isolated from the ruminant rectum on in vitro rumen fermentation characteristics. Rectal fecal samples were collected from healthy beef cattle, dairy cattle, buffaloes, and goats (n = 4 per species) using the grab sampling technique. Twenty-eight bacterial colonies were isolated through enrichment and screening on media containing sodium lignosulfonate. Lignin degradation efficiency was assessed spectrophotometrically, while laccase activity was determined using a 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation assay. Seven isolates exhibiting ligninolytic activity (1.4–5.6% degradation efficiency) were selected to evaluate their effects on in vitro rumen fermentation using a completely randomized design with four replicates. LDB treatments were standardized to a concentration of 2.4 × 105 colony-forming units/mL of rumen fluid medium, while the control received an equal volume of a 0.85% sterile NaCl solution. A rice straw-based total mixed ration served as the substrate, with rumen fluid collected from beef cattle. All treatments were incubated for 48 h. Notably, isolate BC3 consistently enhanced in vitro dry matter digestibility (23.1%), total gas production (18.6%), and total volatile fatty acid concentrations (13.2%) relative to the control and other LDB isolates (p < 0.01). All seven LDB isolates were identified as Gram-negative, rod-shaped, facultative anaerobic bacteria that exhibit catalase activity and tolerate moderately acidic conditions. Phylogenetic tree analysis based on 16S rRNA gene sequencing identified isolate BC3 as being closely related to Escherichia coli strains. These findings demonstrate that the ruminant hindgut is a promising source of LDB with the functional potential to enhance feed digestibility and fermentation end-products in the rumen. Future research should prioritize in vivo trials to evaluate the safety and efficacy of LDB as a direct-fed microbial, specifically focusing on its impact on animal performance and health.

Fermentation doi: 10.3390/fermentation12040178

Authors: Tanitpan Pongjongmit Thitima Norrapoke

The effects of substituting pelleted diets manufactured from cassava, chaya, and mulberry leaves for concentrate on growth performance, feed intake, rumen fermentation, and microbial protein synthesis in beef cattle were evaluated. Four beef cattle (initial BW: 250 ± 50 kg) were assigned to four treatments: a control diet (T1) and diets in which 50% of the concentrate was replaced with cassava leaf pellets (T2), chaya leaf pellets (T3), or mulberry leaf pellets (T4). The data were analyzed using a 4 × 4 Latin square with animal as a period effect as appropriate. Rumen volatile fatty acids were determined by means of HPLC, and microbial protein synthesis was assessed using urinary purine derivatives. Cattle fed cassava leaf pellets (T2) showed the greatest average daily gain (0.79 kg/d) compared with the control (0.50 kg/d; p < 0.05). Compared with T1, T4 exhibited a higher ruminal propionate proportion and total VFA concentration, which was associated with a lower acetate-to-propionate pattern, suggesting reduced methanogenic potential. No adverse health effects were observed, as indicated by hematocrit and blood urea nitrogen values within normal ranges. Microbial protein production increased in the leaf-pellet treatments, with T4 showing the highest efficiency. Overall, cassava, chaya, and mulberry leaf pellets can partially replace concentrate while maintaining growth performance and improving rumen fermentation efficiency in beef cattle.

Fermentation doi: 10.3390/fermentation12040177

Authors: Ying Xu Tingting Xu Tao Jiang Xiaoyi Wang Peipei Zhao Kuidong Xu Xuekui Xia Lixin Zhang

Plasmid copy number (PCN) is a key factor limiting the expression level of heterologous proteins in yeast. Static strategies for enhancing PCN, such as reducing the transcriptional intensity of selection markers or increasing selection pressure, only maintain PCN at a single fixed level and struggle to achieve dynamic, precise, and reversible copy number regulation. This study established a dynamic plasmid copy number regulation strategy based on CRISPR interference (CRISPRi). Flexible control of PCN was achieved by designing specific guide RNAs (gRNAs) and integrating them into the inducible CRISPRi system. Optimization of the gRNA target site, inducer concentration, and induction timing resulted in a >2-fold increase in the fluorescence intensity of yeast-enhanced green fluorescent protein (yeGFP) compared with the group without induction. Using naringenin synthesis as proof-of-concept, this regulatory tool was applied to modulate the expression of chalcone synthase (CHS), the rate-limiting enzyme in naringenin biosynthesis. Finally, the yield of naringenin increased by 35.62% under the optimal induction conditions.

Fermentation doi: 10.3390/fermentation12040176

Authors: Liwen Lu Lin Su Qingjing Huang Xiao Zou Bangmeng Zhou Jun Kang Yang Li Jiamin Zhang Jie Cheng

Currently, the production methods for L-threonine (L-Thr) mainly include chemical synthesis, protein hydrolysis, and microbial fermentation. Among these, microbial fermentation has become an important method for the industrial production of L-Thr, owing to its advantages of abundant raw material sources, environmental friendliness, and high product purity. In recent years, gene editing, synthetic biology, and artificial intelligence have been integrated to significantly improve the synthesis efficiency and production stability of L-Thr and its derivatives through the rational design of metabolic networks, dynamic regulation of fermentation processes, and intelligent optimization of strain performance. This review systematically summarizes the progress of research on the biosynthesis of L-Thr and its derivatives, with emphasis on elucidating synthetic pathway regulation methods based on genetic engineering and metabolic engineering strategies, and summarizes the latest research developments in the synthesis of its derivatives, aiming to provide systematic references for efficient biomanufacturing in this field.

Fermentation doi: 10.3390/fermentation12040175

Authors: Seong-Shin Lee Seong-Uk Jo Hyun Sang Kim Ma-Ro Lee Su-Hyun An Hwan-Ku Kang

The present study was conducted to demonstrate the effects of Codium fragile extract on methane production using in vitro and in vivo experiments. An in vitro batch experiment was conducted to evaluate different inclusion levels of Codium fragile extract (0, 0.25, and 0.5% of substrate dry matter). Methane production significantly decreased in the 0.5% treatment (p < 0.05), whereas dry matter digestibility and total volatile fatty acid concentration were not significantly affected (p > 0.05). Based on the in vitro results, an in vivo feeding experiment was conducted using a 0.5% inclusion level of Codium fragile extract on Hanwoo steers. Methane emissions were significantly decreased by approximately 10% in steers fed Codium fragile extract (p < 0.05). In contrast, rumen fermentation characteristics, feed intake, average daily gain, and blood parameters were not significantly different between the treatments (p > 0.05). These results demonstrate that a dietary additive with 0.5% Codium fragile extract effectively reduced methane emissions without negatively affecting rumen fermentation and growth performance in Hanwoo steers.

Fermentation doi: 10.3390/fermentation12040174

Authors: Siwaporn Wannawilai Jutamas Anantayanon Thanaporn Dechpreechakul Kobkul Laoteng Sukanya Jeennor

Given the therapeutic potential of bioactive cordycepin in medical and healthcare products, precision fermentation using an engineered strain of Aspergillus oryzae was performed to enhance cordycepin production. To understand and predict the dynamics of cell growth and cordycepin production in this fungal strain, mathematical modeling of submerged fermentation was applied. The effects of different nitrogen sources (yeast extract, peptone, (NH4)2SO4, NH4Cl, NaNO3, and KNO3) and carbon sources (glucose and cassava starch hydrolysate, CSH) on cell growth and cordycepin production were evaluated under submerged fermentation conditions. The results showed that organic nitrogen sources significantly enhanced biomass formation and cordycepin production compared with inorganic nitrogen sources. Among them, yeast extract provided the best performance, yielding the highest biomass (13.63–15.99 g/L) and cordycepin titer (1.24–1.72 g/L). In contrast, nitrate-based nitrogen sources supported cell growth but resulted in negligible cordycepin production. Under optimized conditions in a bioreactor, both glucose and CSH supported fungal growth, although CSH promoted higher biomass formation while glucose favored cordycepin biosynthesis. The kinetic model demonstrated that the growth of engineered A. oryzae was well described by the logistic growth model (R2 > 0.88). The cordycepin production profiles were well fitted by the Luedeking–Piret model (R2 > 0.99), indicating a mixed growth-associated product with kinetic constants α and β representing growth-associated and non-growth-associated production, respectively. Overall, the developed kinetic model provides a quantitative framework for describing cell growth, substrate utilization, and cordycepin formation, offering guidance for process optimization and scale-up of cordycepin production in engineered fungal systems.

Fermentation doi: 10.3390/fermentation12040173

Authors: Lorena Vieira Bentolila de Aguiar Larissa Batista do Nascimento Soares Giovanna Lima-Silva Daiane Barão Pereira Vítor Alves Pessoa Aldenora dos Santos Vasconcelos Roberta Pozzan Josilene Lima Serra Ceci Sales-Campos Larissa Ramos Chevreuil Walter José Martínez-Burgos

The global functional food market continues to expand, and edible mushrooms are emerging as high-value ingredients due to their rich nutritional profile, particularly their high protein content, balanced amino acid composition, and dietary fiber. This growing industrial interest is reflected in the registration of more than 322 patents in the past five years according to the Derwent Innovation patent database. Recent advances include the integration of precision mycology (PM) and omics-based approaches, such as CRISPR-Cas9, into solid-state fermentation and submerged fermentation, enabling improvements in natural umami flavor and bioactive composition. Innovative products, including meat analogues with fibrous textures, functional beverages such as kombucha and juices, and fermented dairy products such as yogurts and cheeses, have been formulated to deliver prebiotic, antioxidant, and immunomodulatory properties. Future trends indicate a shift towards the production of high-value nutraceutical peptides and biomass, together with the adoption of artificial intelligence (AI) and the Internet of Things (IoT) to enhance bioreactor automation and scalability. Nevertheless, significant challenges remain, including regulatory constraints, the scarcity of clinical validation in humans, and the need for strict control over the bioaccumulation of heavy metals in mushroom-derived raw materials. Addressing these gaps will be critical for advancing regulatory frameworks, improving industrial standardization, and supporting the translational development of mushroom-based functional foods.

Fermentation doi: 10.3390/fermentation12040172

Authors: Kevin Edward Schulz Paula Hegmann Bastian Dreher Lena Regenauer Carlota Delso Muniesa Wolfgang Frey Katrin Ochsenreither Anke Neumann

Industrial production of single-cell oils (SCOs) by oleaginous yeasts relies predominantly on nitrogen limitation, which constrains process flexibility when nitrogen-rich substrates are used. Although phosphate limitation has been reported as an alternative lipid induction strategy, its process-level performance relative to nitrogen limitation remains insufficiently resolved under controlled reactor-scale conditions. In this study, phosphate-limited, nitrogen-limited and nutrient-replete cultivations of Cutaneotrichosporon oleaginosum ATCC 20509, Saitozyma podzolica DSM 27192, Scheffersomyces segobiensis DSM 27193 and Apiotrichum porosum DSM 27194 were benchmarked in 2.5 L stirred-tank reactors operated under identical media compositions and process parameters. Biomass formation, lipid titres, specific lipid production rates, biomass composition and fatty acid profiles were systematically compared. Nitrogen limitation resulted in the highest lipid titres, reaching up to 9.2 g L−1 (A. porosum), while maximum lipid titres under phosphate-limited conditions reached 5.0 g L−1 (C. oleaginosum) and nutrient-replete conditions 3.9 g L−1 (A. porosum), respectively. The highest specific lipid production rate under nitrogen limitation was 0.0028 g gCDW−1 h−1 (S. podzolica), while phosphate limitation yielded a maximum of 0.0037 g gCDW−1 h−1 (S. podzolica). These results demonstrate that phosphate limitation can decouple cellular lipid productivity from biomass formation and represents a process-relevant alternative for SCO production from nitrogen-rich feedstocks.

Fermentation doi: 10.3390/fermentation12040171

Authors: Louis Levinger Monisha Sherpa Julia Gelman Mariapia Dibonaventura Rabindra Mandal

Fermentation is a type of biological process conducted domestically or commercially to preserve foods and beverages, produce alcohol, add nutritional value and improve aroma and flavor. The natural fermentation of flour in water to obtain a leaven for baking, lately scrutinized in the laboratory with the application of metagenomic methods, has been ubiquitous since the dawn of civilization. Commercially, single culture or defined mixtures of microorganisms are used for their predictability, but regularly fed two-domain microorganism cultures are favored in less industrialized and domestic operations. Fungi principally produce the carbon dioxide responsible for leavening. The bacteria produce acid in the bread commonly known as sourdough for its aroma and flavor. A leaven made by fermentation using flour and water can be stored while it is dormant. We studied a mature culture that is fed twenty-fold with water and flour by incubating it for 24 h, sampling it regularly for pH measurements, and plating it. The colonies were suspended for micrography and DNA extraction for PCR and Sanger sequencing. The metagenomic DNAs were analyzed for bacterial and fungal composition. The proportions of the plant and microbial DNA endogenous to the flour decline rapidly, and the predominant bacteria and fungi in mature leaven propagate, without overlap between the respective microbiomes.

Fermentation doi: 10.3390/fermentation12040170

Authors: Yufeng Chen Hongbo Zhang Haolong Wu Xueqin He

This study investigated the impacts of diverse aeration processes (continuous aeration vs. intermittent aeration) and aeration rates on the aerobic composting process. The key properties examined include temperature, oxygen dynamics, lignocellulose degradation, humification, and the functional potential of carbohydrate-active enzymes (CAZymes) based on metagenomic analysis. Among all the treatments, continuous aeration at a low rate (CA_1.5) attained the highest level of lignocellulose degradation by balancing the thermophilic duration and oxygen supply. Conversely, intermittent aeration (IA_3) led to superior humus stabilization, with the ratio of humic acid to fulvic acid (H/F) increasing by 118.45% in comparison to the initial level. Low total ventilation in CA_1.5 and IA_3 facilitated an increase in the abundance of glycosyl transferases (GTs) genes. Notably, intermittent aeration (IA_3) synergistically augmented the activities of glycoside hydrolases (GHs) and GTs, propelling the efficient conversion of lignocellulose into stable humic substances. In conclusion, the aeration process influenced the functional potential of microbial CAZymes, thus exerting an influence on both the composting efficiency and the quality of the final product.

Fermentation doi: 10.3390/fermentation12030169

Authors: Zerrin Polat Bilge Sayın Mükerrem Kaya Güzin Kaban

The microbial production of value-added lipids by oleaginous yeasts has attracted considerable interest as a sustainable alternative to conventional lipid sources. In this study, the effects of selected fermentation parameters on biomass production, lipid production, and fatty acid composition of Yarrowia lipolytica YB-423 were investigated using flaxseed and chia seed oils as carbon sources. A Taguchi method was employed to evaluate and optimize the influence of temperature, fermentation time, nitrogen concentration, and oil supplementation. The results showed that nitrogen availability was the dominant factor governing biomass formation. The highest lipid production was achieved at 21 °C after 6 days of fermentation in the absence of an added nitrogen source supplemented with 10 mL/L oil, resulting in lipid contents of 62.53% and 64.61% for flaxseed and chia seed oils, respectively. Lipid content was primarily influenced by nitrogen concentration and oil supplementation, while temperature and fermentation time showed secondary but significant effects. Beyond total lipid production, fatty acid profiling demonstrated that both oil sources supported PUFA-rich lipid production; however, chia seed oil resulted in a broader variation in α-linolenic acid (ALA) content across fermentation conditions. The highest ALA content reached 67.40% at 14 °C after 4 days of fermentation under 30 mL/L chia seed oil supplementation. Additionally, ALA levels reached approximately 62% at 7 °C under higher chia seed oil concentrations (20–30 mL/L). In contrast, flax seed oil yielded relatively stable ALA levels, generally ranging between 45% and 56%, depending on fermentation parameters.

Fermentation doi: 10.3390/fermentation12030168

Authors: Yuke He Suttavadee Junyakul Nachon Raethong Massalin Nakphaichit Solange I. Mussatto Wanwipa Vongsangnak

Limosilactobacillus fermentum KUB-D18 is a probiotic strain with significant potential in food fermentation and health promotion, yet the systems-level mechanisms underlying its physiological robustness remain elusive. To elucidate the metabolic remodeling strategies operating across growth phases, we developed an integrated framework combining genome-scale metabolic modeling (GSMM) with transcriptomics. A high-quality metabolic model for L. fermentum KUB-D18, designated iYH640 and comprising 640 genes, 1530 metabolites, and 1922 reactions, was constructed and validated against experimental growth data. Specifically, in vitro assays measuring biomass and glucose concentrations showed a maximum specific growth rate of 0.2696 h−1 and a glucose uptake rate of 11.75 mmol gDCW−1 h−1, providing physiological constraints for the model. Using transcriptome-regulated flux balance analysis (TR-FBA), gene expression profiles from the logarithmic phase (L-phase) and stationary phase (S-phase) were integrated to quantify growth phase-specific metabolic flux distributions. These simulations revealed a distinct transcription-driven metabolic shift, in which the organism moves from a proliferation-oriented metabolic state with active central carbon metabolism and macromolecule synthesis to a maintenance-oriented state. This S-phase is characterized by reduced flux through anabolic pathways together with the selective preservation of redox balance and nucleotide homeostasis. Collectively, these results provide a quantitative explanation of how L. fermentum KUB-D18 balances growth and maintenance, offering a mechanistic basis for improving its stability and functional performance in industrial probiotic applications.

Fermentation doi: 10.3390/fermentation12030167

Authors: Ronnie Coêlho de Andrade Marco Antonio Previdelli Orrico Junior Giuliano Reis Pereira Muglia Isabele Paola de Oliveira Amaral Ana Carolina Amorim Orrico Mábio Silvan José Da Silva

This study evaluated the effects of essential oils, such as limonene (LIM) and a cinnamaldehyde–carvacrol blend (CCB), on the fermentation, chemical composition, in vitro digestibility, and aerobic stability of corn silage stored for 150 and 200 days. Treatments included a control, CCB (100 and 200 mg/kg DM), and LIM (100 and 200 mg/kg DM). Essential oils improved fermentation by increasing lactic acid (up to 7.46% of DM) and reducing proteolysis (NH3-N: 0.46–0.59% of total N). Limonene, particularly at 100 mg/kg DM, enhanced the lactic:acetic acid ratio (up to 3.07), better preserved non-fiber carbohydrates (≈32.7%), and increased in vitro DM digestibility (up to 81.5%) compared to the control (≈76.0%). The CCB treatment raised acetic acid concentrations (up to 3.04% of DM). Extending storage to 200 days reduced DM recovery (≈84.0%) versus 150 days (92.5%). Treated silages showed greater aerobic stability, with a lower pH increase after 72 h of air exposure, most notably in the LIM 200 treatment (pH 4.10) compared to the control (pH 5.40). Essential oils, particularly limonene, effectively improve the fermentative quality, nutritional value, and aerobic stability of corn silage under prolonged storage.

Fermentation doi: 10.3390/fermentation12030166

Authors: Sheng Gao Yinuo Li Quan Cui Chuanzhuang Guo Jianbin Wang Junlin Li Ting Wang Piwu Li Jing Su Ruiming Wang Nan Li Junqing Wang Han Fan

D-allulose is a rare low-calorie sugar with considerable health benefits and industrial potential. Compared with chemical synthesis and free enzyme catalysis, microbial production using engineered cells offers a low-cost and highly stable solution. Therefore, we investigated the reaction pathway underlying the synthesis of D-allulose from D-glucose. Specifically, the enhancement of glucose isomerase-catalyzed reactions and their role in D-allulose synthesis were evaluated. First, a mutant strain with significantly increased glucose isomerase from Anoxybacillus kamchatkensis G10 (AGGI) expression was obtained through ultraviolet mutagenesis combined with high-throughput flow cytometry. A 4.55-fold increase in AGGI activity and a D-fructose conversion yield of 51.2% were obtained. A dual-enzyme pathway was subsequently constructed by co-expressing AGGI and D-allulose 3-epimerase (DAEase) in the optimized host. After balancing the catalytic requirements of both enzymes through optimization of reaction conditions, CRISPR-associated transposase was employed to efficiently integrate the glucose transporter gene galP into the genome, further enhancing substrate supply. The final engineered Escherichia coli strain achieved a D-allulose conversion rate of 15% from 20 g/L D-glucose. This demonstrates the crucial role of glucose isomerase in microbial D-allulose production and advances the optimization and development of D-allulose synthesis strategies using D-glucose as a substrate.

Fermentation doi: 10.3390/fermentation12030165

Authors: Kun-Qiang Hong Xiao-Meng Fu Xi-Yu Shu Zi-Zhen Liu Yi-Jia Wang Zhi-Jian Tan

Cofactors are small molecules or ions that participate in enzymatic reactions as essential carriers of electrons, atoms, or functional groups, thereby governing cellular redox balance and energy metabolism. In the yeast Saccharomyces cerevisiae, the availability of cofactors such as NAD(H), NADP(H), CoA, and acetyl-CoA directly influences the flux through biosynthetic pathways leading to aroma-active compounds. Esters and higher alcohols are the two most important families of volatile flavor compounds in fermented alcoholic beverages. Their synthesis is intimately linked to the intracellular levels and ratios of these cofactors. This review summarizes recent progress in cofactor engineering strategies aimed at modulating the production of esters, higher alcohols, and 2,3-butanediol in S. cerevisiae. We discuss the underlying metabolic pathways, highlight key studies that manipulate cofactor pools to redirect carbon flux, and examine emerging tools (e.g., riboswitches, fine-tuned promoter systems) that enable precise cofactor balancing. Finally, we outline future challenges and opportunities for applying cofactor engineering to design yeast cell factories with tailored flavor profiles.

Fermentation doi: 10.3390/fermentation12030164

Authors: Chenxi Xia Xueting Zhang Li Lu Ning Xie Chaoyang Lu Wenzhe Li Quanguo Zhang

The high-efficient utilization technology of organic waste can alleviate the dual pressures of energy and the environment. The study investigated the effects of substrate ratio, substrate concentration, and temperature on biohydrogen yield, and further optimized the process conditions for co-digestion of food waste and fecal sludge as substrates for biological hydrogen production. The results of batch mode experiments show that when the ratio of food waste to fecal sludge is 5:1, substrate concentration is 60 g/L, and fermentation temperature is 40 °C, the system achieves maximum cumulative hydrogen production of 183 mL (equivalent to 32 mL/g VS). The response surface methodology (RSM) indicates that substrate ratio, substrate concentration, and temperature all exert remarkably significant effects on hydrogen yield (p < 0.01). In addition, the synergistic interaction between substrate ratio and temperature significantly influences hydrogen production performance (p < 0.05). This study elucidates the synergistic mechanism of key process factors in the co-digestion of food waste and fecal sludge for biohydrogen production. The findings provide a theoretical basis for the engineering application of organic waste to hydrogen technologies.

Fermentation doi: 10.3390/fermentation12030163

Authors: Xiang Zhang Qiulin Wei Yanbo Tang Fuqiang Yu Zhenqiang Wu Xiaofei Tian

Hypocrellin A (HA) represents a pharmaceutically important perylenequinone photosensitizer produced by Shiraia bambusicola. However, submerged fermentation remains constrained by filamentous morphological characteristics and inherent mass transfer limitations. Although microparticle-enhanced cultivation (MPEC) has demonstrated efficacy in filamentous fungal systems, the molecular mechanisms by which physical cues, such as microparticle-induced shear stress, reprogram fungal metabolism remain largely unexplored. This work systematically optimizes SiO2-based MPEC parameters for S. bambusicola GDMCC 60438, including particle dimensions, temporal addition protocols, and solid loading. Mechanistic investigations integrated pellet morphology analysis, membrane lipid composition, intracellular redox status, energy/precursor markers, and RNA-seq transcriptomic profiling with qRT-PCR validation. Under optimized conditions (10% w/v SiO2, 30 mesh, added at 6 h), HA yield reached 41.76 ± 5.02 mg/L, representing a 3.65-fold increase over controls. MPEC shifted morphology toward smaller, more porous pellets with denser internal structure, accompanied by increased membrane fluidity (unsaturated/saturated fatty acid ratio from 1.54 to 2.63), elevated ROS levels with antioxidant enzyme activation, and enhanced acetyl-CoA and ATP accumulation. Transcriptomic analysis identified 206 differentially expressed genes enriched in oxidative phosphorylation, carbon metabolism, and stress responses, with upregulation of PKS-related biosynthetic genes and major facilitator superfamily transporters. This work establishes an integrated mechanistic framework linking particle-induced morphological changes to metabolic reprogramming through oxidative stress and subsequent transcriptional activation of the HA biosynthetic pathway, providing rational design principles for MPEC strategies in filamentous fungi.

Fermentation doi: 10.3390/fermentation12030162

Authors: Mingzhu Li Hongchen Fan Mingshou Lü

Sodium salt is critical for determining the quality of fermented vegetables, with particular influence on their physicochemical properties, textural characteristics, and food safety. However, the relatively high salt levels in traditional fermented products do not align with the growing consumer preference for low-salt, health-oriented foods. Simply reducing salt levels compromises the storage stability, edibility, and processability of fermented vegetables. Therefore, lowering salt content while maintaining product quality poses a significant challenge for the fermented vegetable industry. Effective quality control in low-salt fermented vegetable production requires a comprehensive understanding of the role salt plays in product quality. In this review, we outline the functions of salt in fermented vegetables and detail the current applications and technological developments in salt-reduction strategies; we focus on sodium substitutes, biological salt-reduction approaches, and emerging non-thermal technologies. The current challenges in low-salt fermentation processes are also discussed, providing valuable theoretical and practical guidance for the high-quality processing of low-salt fermented vegetable products.

Fermentation doi: 10.3390/fermentation12030161

Authors: Yu Su Haojin Peng Leiyu Feng Yinguang Chen

The bioconversion of propionate, a well-known intermediate of anaerobic digestion (AD), to methane is energetically unfavorable under standard conditions, which typically occurs in the syntrophy of bacteria and methanogens via methylmalonyl-CoA (MMC) and the dismutation pathway. Since the latter, which is reported only in Smithella, possessed a thermodynamic advantage over the former, enriching Smithella and promoting the syntrophic interaction and metabolism of the microbiota are important for improving AD efficiency. In this study, lysine-modified Fe3O4 (Lys@Fe3O4) significantly enhanced the bioconversion of propionate to methane. The methane yield and the maximum methane production rate (Rmax) in a Lys@Fe3O4 reactor were 278.7% and 271.7% of Blank, and the corresponding values were 201.9% and 201.6% of bare Fe3O4, respectively. The metaproteomic results indicated that Lys@Fe3O4 increased not only the abundance of Smithella but also the expression of cell surface and adhesion proteins, thereby promoting syntrophic interaction between Smithella and methanogens and facilitating electron and acetate transfer from Smithella to methanogens. Moreover, the expression of quorum-sensing proteins was enhanced, benefiting the cooperation of Smithella and its associated bacterium (Syntrophomonas). Furthermore, the expressions of key enzymes related to metabolism and electron transfer in propionate oxidation, butyrate oxidation, CO2-reductive methanogenesis and acetoclastic methanogenesis were all significantly upregulated. The results are of great significance for maintaining low propionate concentration and stability of AD.

Fermentation doi: 10.3390/fermentation12030160

Authors: Xiaoying Zhou Mona Correa Dino Athanasiadis Veronica Benites Bryce Doherty Lucy Edy Christy Piamonte Gener Eliares Marvin Cornejo Ting Gong Leon Parker Manuel Oliveira Walter Rakitsky James Casey Lippmeier Jessica M. Walter Frédéric Destaillats

Palmitoleic acid (POA; 16:1 n-7 or cis-9 16:1) is a bioactive monounsaturated fatty acid (FA) with emerging metabolic and skin-health relevance, yet conventional botanical and animal sources provide limited and variable levels. Here we report on the development of a high-yield POA product platform in the heterotrophic microalga Prototheca moriformis through targeted genetic engineering. A Δ9-fatty acid desaturase from Macadamia integrifolia (MiSAD1618) was integrated using a phosphite-based selection system. Primary screening identified stable transformants producing up to 54% POA of total fatty acids, compared to 0.8% in the parental strain. In 1 L shake-flask cultivation, POA reached up to 58.2% of total fatty acids. In a 1 L fed-batch fermentation, the engineered strain accumulated 47.8 g/L of lipids with 43.5% POA after 96 h of fermentation, corresponding to 20.8 g/L of POA. GC–MS analysis of 4,4-dimethyloxazoline (DMOX) derivatives confirmed that the major 16:1 isomer was 16:1 n-7 (Δ9). Together, these results establish P. moriformis as a scalable fermentation platform for producing POA-rich oil and highlight its potential as an efficient alternative source of POA, providing a foundation for further strain and process optimization toward commercial production.

Fermentation doi: 10.3390/fermentation12030159

Authors: José Eduardo Malfeito-Ferreira Manuel Malfeito-Ferreira

The effect of alcohol on health is a controversial topic when it comes to the moderate or conscious consumption of fermented beverages. The recent claim by the World Health Organisation (WHO) and the European Heart Network (EHN) that the safe level of alcohol consumption is zero has compromised the efforts of the fermentation scientific community in developing healthier and more sustainable beverages. Therefore, the objective of this review was to assess the scientific background for such a claim that appears to be the result of recent scientific evidence. Using the meta-analytic data supporting WHO and EHN guidelines, it was possible to demonstrate that fermented beverages (e.g., wine and beer) have lower effects compared to spirits, that some population ethnicities have higher sensitivity to alcohol, and that drinking patterns influence the outcomes. Moreover, higher relative risks associated with younger individuals are mostly related to injuries (e.g., car accidents, self-inflicted injuries) and not with diseases. Sequential WHO studies produced significantly higher limits and emphasized that preventive policies should be tailored to populations at higher risk. In conclusion, the statement that “all alcohol is hazardous” has no scientific background and should be understood under the perspective that “one drink is too many and one thousand is never enough” used in alcoholism prevention. Fermentation researchers should continue their efforts on the promotion of healthier lifestyles, sustainable development and on the preservation of cultural heritage under the responsible drinking perspective.

Fermentation doi: 10.3390/fermentation12030158

Authors: Yiwei Jin Feng Chen Jiang Cao

Industrial spore production of the probiotic Heyndrickxia coagulans is hindered by its generally low and highly variable sporulation efficiency across strains. To address this, we selected the representative model strain ATCC 7050 and applied an integrated strategy combining statistical medium optimization with genomic analysis. Key factors (glucose, yeast extract, CaCl2) were screened and optimized using Plackett–Burman and Box–Behnken designs, yielding an optimal formulation that achieved 1.84 × 108 spores/mL in a bioreactor, consistent with the model prediction. Further genomic analysis revealed 112 sporulation-associated genes and identified key homologous genes related to spore resistance and germination. Among them, the successful identification of spoVA, which is implicated in calcium-dipicolinate transport in bacilli, allowed us to hypothesize why calcium ions play a critical role. This work not only enhances the spore yield of a model strain but also provides a framework to tackle the widespread sporulation variability in H. coagulans for industrial applications.

Fermentation doi: 10.3390/fermentation12030157

Authors: Iva Šikuten Ivana Kosi Ivana Tomaz Ana Jeromel Darko Preiner

The wine aromatic profile is influenced by complex interactions between grapevine genotype and enological practices. Thus, the aim of this study was to investigate the combined effects of grapevine clones and yeast strains on the volatile composition and sensory properties of wines produced from the Croatian indigenous variety Moslavac. Wines from five registered Moslavac clones (PUS-017, PUS-026, PUS-030A, PUS-087, and PUS-111) were produced using two commercially available yeast strains (Lalvin QA23 and Zymaflore Xarom). Significant effects of both clone and yeast strain were observed, particularly for yeast-derived compounds, such as isoamyl alcohol, phenylethyl alcohol, and medium-chain fatty acids. Ester production was generally enhanced by the Xarom yeast strain, although clone differences were also observed. Grape-derived volatile compounds differed significantly among clones, with wines from clones PUS-030A and PUS-087 having higher concentrations of norisoprenoids and terpenes, while PUS-017 wines consistently displayed lower concentrations of volatile compounds. Furthermore, PCA and MLF analyses revealed a clear differentiation between clones, with the yeast strain having a secondary modulatory effect. The sensory results were consistent with chemical data, demonstrating that clonal selection plays a key role in defining aromatic expression and typicity of Moslavac wines.

Fermentation doi: 10.3390/fermentation12030156

Authors: Mengyao Li Zequn Zhang Jingyu Chen Hui Sun Fuping Lu Yihao Liu Yihan Liu

Phosphatidylserine (PS), a valuable phospholipid, is widely used in food, pharmaceutical and cosmetic industries. Its enzymatic synthesis, catalyzed by phospholipase D (PLD) via transphosphatidylation under mild conditions, has drawn considerable attention. However, the industrial use of free PLD is limited by poor stability, difficult recovery, and high cost. To address these challenges, a ternary composite carrier—integrating the flexibility of chitosan, the stability of cellulose, and the macroporosity of agarose—was constructed for immobilizing the PLD from Streptomyces antibioticus (saPLD). The resulting saPLD@chitosan–cellulose–agarose biocatalyst demonstrated enhanced immobilization efficiency, catalytic performance, and stability across varying pH and temperatures. After eight consecutive batches of usage, the PS yield of saPLD@chitosan–cellulose–agarose reached over 60% of that from the first batch. Thus, this study established a new method for preparing immobilized saPLD, and developed a robust and promising platform for the efficient and sustainable production of PS.

Fermentation doi: 10.3390/fermentation12030155

Authors: Michael Vuma Moses M. Ratsaka Julius T. Tjelele Thomas Langa Bhutikini D. Nkosi Ingrid M. M. Malebana

The study evaluated the effects of replacing soybean meal (SBM) with marula oilcake (MOC) at equal inclusion (10% fresh weight) levels in whole-crop maize silage treated with or without lactic acid bacteria inoculants on fermentation characteristics, nutritive value, aerobic stability, and in vitro nutrient degradability. Maize was ensiled with SBM or MOC in a non-iso-nitrogenous 2 × 3 factorial design and either inoculated or uninoculated with Lalsil Fresh or Sil-All 4×4 for 90 days. Protein sources differed significantly (p < 0.05). The MOC showed high DM, EE, GE, and ADL, whereas SBM had high CP, ash, and IVOMD. Fibre fractions (aNDF and ADF) were similar (p > 0.05). The SBM control showed significantly high (p < 0.05) LA, NH3-N, CP, IVOMD, propionic acid, and early gas production, indicating efficient fermentation. The SBM + Lalsil maintained low pH, and early OM, CP, and GE degradability. The SBM + Sil-All achieved the highest (p < 0.05) OM, NDF, and ADF degradability and acetic acid production than other treatments. The MOC control showed low (p < 0.05) pH, high fibre and GE, reduced butyric acid, and low 48 h gas production, indicating slower fermentation but improved stability. The MOC + Lalsil had high (p < 0.05) DM, low CO2 and yeasts and moulds, and the highest (p < 0.05) CP degradability, propionic acid, and peak gas production at 12 h. The MOC + Sil-All showed high (p < 0.05) GE and WSC with peak GE degradation at 12 h, but low NDF degradability and reduced gas production. Overall, SBM improved degradability and fermentation efficiency, particularly with Sil-All, whereas MOC enhanced energy density and aerobic stability, with Lalsil optimising protein utilisation. Matching inoculant type to protein source is essential to optimise silage quality and rumen fermentation. Further research should assess different inoculant inclusion rates and include a maize-only control, and evaluate protein source inclusion under iso-nitrogenous conditions to allow more accurate comparisons.

Fermentation doi: 10.3390/fermentation12030154

Authors: Kevin Edward Schulz Paula Hegmann Bastian Dreher Marina Schreidl Katrin Ochsenreither Anke Neumann

Yeasts such as Cutaneotrichosporon oleaginosum can convert low-value side streams into single-cell oils with fatty acid profiles comparable to vegetable oils. Crude glycerol (CG), a byproduct of biodiesel production, offers a cost-effective substrate, but its variable impurity load often causes strong growth inhibition. In this study, two untreated industrial CG batches were characterized and evaluated in 2.5 L and 19 L stirred-tank fermentations. Direct batch cultivation on CG resulted in no measurable growth, whereas an adapted stepwise feeding strategy effectively mitigated early inhibition and restored biomass formation, metabolic activity, and lipid accumulation. In 2.5 L cultivations, apparent growth rates up to 0.51 h−1 and volumetric productivities up to 0.22 g L−1 h−1 were achieved, with lipid contents of ~30% and oleate-dominated fatty acid profiles. Fatty acid profiles remained oleate-dominated (~53–55% C18:1). Transition-scale (19 L) repeated-batch fermentations confirmed process robustness across > 640 h of operation, during which lipid content (~30–36%) and fatty acid composition (oleate ~51–53%) remained stable despite pronounced substrate-batch variability and increasing nitrogen limitation. These results demonstrate that untreated CG can be reliably valorized for lipid production using scalable feeding strategies without prior detoxification. This closes a gap between laboratory-scale feasibility studies and process-oriented, multi-cycle operation on industrial-grade feedstocks, confirming that feeding-driven inhibition control can ensure robust performance without substrate purification.

Fermentation doi: 10.3390/fermentation12030153

Authors: Qing Wu Heyu Zhang Shihua Xin Jianhong Guo Xiaoping Yang Qi Wang Haitian Fang

The current understanding of microbiota–flavor correlations in Chinese sourdough steamed bread is predominantly derived from the central provinces, with comparatively limited investigation in northeastern and northwestern regions. This study bridges this gap by analyzing traditional starters from Heilongjiang (HLJ) and Ningxia (TX) versus an industrial starter (JM) through integrated metagenomics and untargeted metabolomics. HLJ was dominated by Limosilactobacillus fermentum (14.75%), while TX featured a synergistic Lactiplantibacillus plantarum–Fructilactobacillus sanfranciscensis consortium. Metabolic pathway analysis revealed enhanced glycolysis, amino acid metabolism, and glycerophospholipid transformation driving flavor biosynthesis and dough rheology improvement, supported by nitrogen-metabolizing Bradyrhizobium spp. (6.00–6.61%). Core pathway enrichment established molecular foundations for region-specific flavors: HLJ generated sulfury/pungent notes via the enzymatic conversion of pentyl glucosinolate to isothiocyanates, whereas TX developed caramel–roasted aromas through stachyose/xylose-derived Maillard reactions forming 2-(methylthiomethyl)furan. Both consortia exhibited higher bitterness and lower umami than JM, with HLJ showing marginally higher umami and lower bitterness than TX. These findings elucidate the microbial mechanisms underlying regional flavor differentiation.

Fermentation doi: 10.3390/fermentation12030152

Authors: Rudolphus Antonius Timmers Enrique Pérez Zapatero Fernán Berride García Miriam Barrazón Peña Miguel Ángel Sánchez-Gatón Dolores Hidalgo

To address the limitations of conventional anaerobic digestion (AD), this study explored the integration of microbial electrolysis cells (MECs) with AD to improve biogas production and process stability. While AD is a proven technology for renewable energy recovery from waste, it can suffer from volatile fatty acid accumulation and reduced efficiency. The hybrid MEC–AD system leverages electro-methanogenesis to enhance methane yields and overall system performance. This research evaluated the effects of different electrode materials (graphite plate vs. graphite felt) and applied voltages (0.5 V and 0.7 V) on biogas output, methane content, and operational stability. Results showed that MEC–AD systems significantly outperformed conventional AD, with the highest biogas production reaching 239 ± 3 mL/gVS·d—an increase of up to 162% using graphite felt electrodes at 0.5 V. Internal resistance was also markedly lower with graphite felt (19 Ω/m2) compared to graphite plates (1120 Ω/m2). Furthermore, the pH of the MEC–AD system with graphite felt electrodes was maintained within the optimal range (6.8–7.0), avoiding the acidification seen in control systems. These findings underscore the promise of MEC–AD systems for advancing circular bio-economy initiatives and carbon neutrality. Further work is needed to refine electrode materials and reactor design for improved scalability and efficiency.

Fermentation doi: 10.3390/fermentation12030151

Authors: Laura-Dorina Dinu Rely-Andreea Tudor Teodora-Otilia Alexiu Emanuel Vamanu

Kombucha, a traditionally fermented tea, has gained increasing scientific and commercial interest due to its sensory quality and bioactive metabolites profile associated with different health-related activities. Recent research highlights the value of enriching traditional and honey kombucha with plant-based biomolecules to create new functional beverages with enhanced functional and nutraceutical properties, improved flavor, and chemical stability. Therefore, this study aimed to review and update the research on the enrichment of kombucha with these natural biomolecules that have been shown to expand the spectrum of health-promoting activities (e.g., antioxidant, antimicrobial, anticancer, and anti-aging), while also enhancing the physicochemical stability of raw kombucha. Yet this innovation must be navigated with a thoughtful understanding of safety, biochemical stability, and sensory evaluation. Thus, this review strongly advocates that the integrative enrichment approach presents a promising strategy for developing next-generation functional beverages with synergistic nutritional and therapeutic benefits. Further controlled studies are needed to elucidate the mechanistic interactions between the kombucha’s microbiome and these added bioactive substrates, as well as to optimize formulations for targeted health applications.

Fermentation doi: 10.3390/fermentation12030150

Authors: Ji Yin Pingping Liu Shiwei Wang Changtao Wang Dongdong Wang Jiachan Zhang Dan Zhao Meng Li

Heyndrickxia coagulans is widely used for industrial L-lactic acid production, but carbon catabolite repression (CCR) and its link to fermentative metabolism remain poorly understood. A ccpA deletion mutant (ΔccpA) and a complementation strain (C-ccpA) were constructed to investigated the physiological, enzymatic, and transcriptomic consequences of CcpA loss. Deletion of ccpA completely abolished glucose-mediated CCR, enabling simultaneous glucose–xylose co-utilization, and triggered a marked shift from L-lactic to mixed-acid fermentation, with an 82.5% reduction in lactate titer accompanied by 24.1-fold and 51.6-fold increases in acetate and formate, respectively. Enzyme activity assays showed that L-lactate dehydrogenase activity was reduced by half, whereas acetate kinase activity increased nearly six-fold. Transcriptomic analysis revealed downregulation of ldhL and upregulation of pflB and ackA. Scale-up fermentation in a 5 L bioreactor confirmed that the wild type directed 90.2% of carbon flux to lactate (yield, 0.95 g/g glucose), compared with only 24.5% in the mutant. All phenotypes were fully restored upon complementation. These results demonstrate that CcpA is as an indispensable dual regulator of both CCR and L-lactic fermentation, providing a foundation for rational metabolic engineering of H. coagulans.

Fermentation doi: 10.3390/fermentation12030149

Authors: Balaji Prasath Barathan Yuping Su Ying Wang

We evaluated the algicidal properties of Bacillus Ba3 fermentation broth combined with clay for harmful algae bloom (HAB) control through in situ enclosure experiments in Suao Bay, China. It was indicated by the results that the combination significantly reduced HAB abundance, turbidity and phosphorous in water without affecting zooplankton and small fish. The treatment achieved 99.8% (Phase 1) and 100% (Phase 2, with sediment) removal rates for harmful dinoflagellates, primarily Prorocentrum donghaiense and Karenia mikimotoi, while demonstrating high taxonomic selectivity, allowing beneficial diatom populations such as Chaetoceros spp. to remain resilient. This synergy is attributed to clay acting as a physical carrier that brings adsorbed algicidal metabolites into direct, prolonged contact with algal membranes. This method shows promise for prolonged dinoflagellate control and may offer an economical and environmentally sound approach to HABs. More research is needed to establish its action on a wider scale in marine environments.

Fermentation doi: 10.3390/fermentation12030148

Authors: Mihye Park Sun Mee Lee

The roots of Panax ginseng are well known for their bioactive properties, while its berries have recently attracted attention for their pharmacological potential. This study investigated whether fermentation with Lactiplantibacillus plantarum enhances the antioxidant properties of ginseng roots and berries and their protective effects against oxidative stress in vitro. Fermentation significantly increased total polyphenol, flavonoid, and saponin contents and promoted the conversion of major ginsenosides (ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Rb2), which are relatively less bioavailable, into minor ginsenosides (ginsenoside Rh1, ginsenoside Rg2, and ginsenoside Rg3) with enhanced biological activity and bioavailability. Fermented extracts exhibited higher radical-scavenging activities in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays than non-fermented extracts. In tert-butyl hydroperoxide (t-BHP)-stimulated Chang liver cells, fermented extracts reduced intracellular reactive oxygen species (ROS) generation, inhibited lipid peroxidation, restored the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, and enhanced antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT). These results demonstrate that L. plantarum-mediated fermentation effectively enhances the antioxidant and cytoprotective potential of ginseng roots and berries, supporting their application as functional food ingredients.

Fermentation doi: 10.3390/fermentation12030147

Authors: Viyils Sangregorio-Soto Edgar Yesid Mayorga Lancheros Renata De La Hoz

Scaling up microalgae cultivation is key to commercial viability. Over the past two decades, the market value of microalgae has expanded exponentially, driven by their applications in the pharmaceutical, nutraceutical, cosmetic, and animal feed industries. High-value compounds such as omega-3 fatty acids, proteins, and pigments are in strong demand. However, supply remains constrained by suboptimal cultivation practices and high harvesting costs. Despite decades of progress in process modeling, control, and optimization, industrial adoption is still limited by dynamic cultivation conditions influenced by weather variability, biological adaptation, and integration challenges. Technical barriers, including limited data accuracy, modest control performance, and the fragility of low-cost devices, further restrict optimization efforts. In response, we examined recent advances in control, optimization, and automated machine learning applied to microalgae cultivation. We propose an automated architecture built on a closed-loop supervisory layer that embeds machine learning within the control loop, enabling real-time monitoring, prediction, and adaptive actuation. This approach aligns with real-time optimization and distributed control system practices, integrating system identification, controller optimization, fault diagnosis and tolerance, and perception to achieve autonomous, uncertainty-aware operation.

Fermentation doi: 10.3390/fermentation12030146

Authors: Mariana Muñoz-Santacruz Silvia Luna-Suárez Nelly Ramírez-Corona Aurelio López-Malo Jocksan I. Morales-Camacho

Brazzein is a sweet-tasting protein with high stability across a wide range of pH and temperature conditions. This study aimed to develop a simplified peptone-based medium (PSM) for the recombinant expression of brazzein in Pichia pastoris X-33 and to evaluate the effect of two inoculum concentrations (5%, 10%, and 15%) on cell growth and protein production in flask fermentations. Subsequently, fermentation was scaled up to a 2 L bioreactor using PSM and a 10% inoculum, achieving a yield of 0.196 g·L−1 after 216 h of induction. These results demonstrate that the PSM medium promotes robust biomass growth and efficient brazzein expression, representing a cost-effective alternative to conventional complex media. Additionally, the effect of pH (5.0, 5.5, and 6.0) and temperature (20, 25, and 28 °C) on brazzein production was evaluated, revealing that fermentation at pH 5.0 and 28 °C resulted in the highest protein concentration (0.422 g·L−1, unpurified). Finally, kinetic models based on the Monod and Luedeking–Piret equations were developed to describe the relationship between biomass formation, substrate consumption, and recombinant protein production.

Fermentation doi: 10.3390/fermentation12030145

Authors: Eda Kılıç Kanak Suzan Öztürk Yılmaz

This study investigated the development of a sugar-free prebiotic and probiotic dark chocolate formulated with 39.76% cocoa mass, 41.856% cocoa butter, 14.8% inulin, and 1.6% stevioside. To enhance the stability of the probiotic yeast Saccharomyces boulardii, a microencapsulation technique using a cocoa powder, Na-alginate, and fructooligosaccharides (10:1:1 ratio) matrix was employed. The physicochemical properties and probiotic viability were monitored over a 120-day storage period at 25 °C. A significant increase in pH values was observed during storage (p < 0.05), while water activity (aw) values significantly decreased (p < 0.05). Encapsulation provided superior protection, with encapsulated samples retaining 2.51 log CFU/g more probiotics than unencapsulated samples after 120 days. Furthermore, in vitro gastrointestinal digestion assays conducted after one day of storage demonstrated the protective efficacy of the matrix; while unencapsulated probiotics suffered a drastic reduction of 6.42 log CFU/g under gastric conditions (pH 3, 2 h), the encapsulated probiotics showed a significantly lower reduction of only 3.99 log CFU/g. These results confirm that the Na-alginate/FOS-based encapsulation significantly improves the resilience of S. boulardii against both storage conditions and gastrointestinal stress, making this sugar-free prebiotic chocolate an effective delivery vehicle for probiotic yeasts.

Fermentation doi: 10.3390/fermentation12030144

Authors: Ignacio Guarda Catalina Ardiles Sebastián Dehnhardt-Amengual Isidora Achiardi-Letelier Wladimir Mardones

Sexual reproduction in yeasts is a fundamental biological process that promotes genetic recombination and phenotypic diversification, enabling adaptation to fluctuating and stressful environments. Sporulation and subsequent mating generate novel allele combinations that enhance evolutionary potential; however, many domesticated industrial strains exhibit reduced sporulation capacity, limiting their use in breeding programs and constraining the generation of new diversity. This represents one of the major bottlenecks for improving yeast performance in industrial fermentations, particularly under the harsh conditions characteristic of bioethanol production. In this study, we exploited meiotic recombination and mass-mating strategies to expand genetic and phenotypic diversity in S. cerevisiae. By mass-mating haploid spores derived from genetically distinct parental strains, we generated highly heterogeneous hybrid populations in a single step, overcoming the limitations imposed by conventional breeding approaches, such as micromanipulation. These populations were subsequently screened to identify strains with enhanced fermentative performance and increased tolerance to industrial stressor media associated with bioethanol production. Our results demonstrate that sexual reproduction combined with mass-mating represents an efficient strategy to unlock hidden genetic potential and generate superior industrial yeast phenotypes. This work highlights the value of utilizing the natural reproductive biology of S. cerevisiae to accelerate strain improvement and develop robust yeasts adapted to challenging fermentation environments.

Fermentation doi: 10.3390/fermentation12030143

Authors: Xiya Cao Linan Duan Yurou Ren Hao Liang Kexin Li Xinyao Guo Jiali Wang Junmei Ma Junnan Xu

Enhancing the acid tolerance of Lactiplantibacillus plantarum is essential for improving its fermentation performance and metabolic activity under acidic conditions, thereby strengthening its probiotic functionality. In this study, the glutamate decarboxylase gene (gadA) and the arginine decarboxylase gene (speA) from Alicyclobacillus acidoterrestris DSM 3922T were heterologously expressed in L. plantarum WCFS1 to enhance its acid resistance. Recombinant expression vectors pMG36e-gadA and pMG36e-speA were constructed and introduced into L. plantarum WCFS1 via electroporation. The acid tolerance, cell membrane integrity, intracellular pH, ATP content, gene expression profiles, and enzyme activities of the recombinant L. plantarum WCFS1-gadA and WCFS1-speA were systematically evaluated. The results demonstrate that both recombinant strains exhibited significantly higher acid tolerance than the control strains. Under acid stress, the expression of gadA and speA was up-regulated, accompanied by enhanced activities of glutamate and arginine decarboxylases. In addition, the recombinant strains maintained higher intracellular pH and ATP levels compared with the control strain. Furthermore, the fermentative activity results support their potential applicability in fruit juice fermentation. Collectively, the heterologous expression of gadA and speA effectively improved the acid tolerance of L. plantarum, providing both mechanistic insights into acid stress adaptation and a theoretical basis for developing industrially robust, acid-resistant probiotic strains.

Fermentation doi: 10.3390/fermentation12030142

Authors: Sergi Maicas Ismail Moukadiri

Moroccan lben is a traditional spontaneously fermented milk widely consumed across the Maghreb. In this review, we synthesize data on spontaneously fermented milks from Morocco and the wider Maghreb–Middle Eastern region to infer the likely microbiota of Moroccan lben, with particular emphasis on dominant lactic acid bacteria such as Lactococcus lactis, Streptococcus thermophilus, Leuconostoc mesenteroides and lactobacilli sensu lato, alongside yeasts including Kluyveromyces marxianus and Saccharomyces cerevisiae. These communities drive a staged fermentation in which early mesophilic lactic acid bacteria (LAB) rapidly acidify the milk and initiate coagulation, intermediate heterofermentative LAB and yeasts generate key aroma compounds and mild effervescence, and late acid-tolerant lactobacilli contribute to flavor refinement and microbiological stability. We summarize how these bacteria and fungi collectively shape physicochemical, sensory and safety attributes through pH reduction, organic acid and bacteriocin production, proteolysis, and volatile formation, and discuss potential nutritional and health-related effects associated with bioactive peptides and putative probiotic strains. Finally, we identify major research gaps, including the need for high-resolution, culture-dependent and culture-independent studies, systematic safety assessments, and rational design of starter and adjunct cultures that reproduce traditional sensory profiles while improving process control.

Fermentation doi: 10.3390/fermentation12030141

Authors: Mmaphuti Abashone Ratau Oluwaseun Peter Bamidele Victoria Adaora Jideani Victor Ntuli Shonisani Eugenia Ramashia

The rising demand for plant-based, lactose-free functional beverages amid gut health concerns positions finger millet (FM, Eleusine coracana) as a promising substrate. This study assessed sprouting and fermentation inoculum effect: dairy starters (Streptococcus thermophilus and Lactobacillus bulgaricus) or backslopping with commercial Mageu on microbial growth, fermentation dynamics, nutrition, antioxidants, color, and texture of FM beverages. Microbial growth increased modestly over 48 h OD600 = 0.169–0.201, peaking in non-sprouted FM with dairy starters (ND) at OD600 = 0.201). ND showed the fastest pH decline (ΔpH = 2.19), while sprouted FM with dairy starters (SD) or backslopping (SB) had controlled acidification. Total titratable acidity increased from 0.14 to 0.66%, with the highest total soluble solids in sprouted substrates (SD = 11.26 °Brix; SB = 10.97 °Brix). Proximate analysis revealed SB had high crude fiber (2.86%) and SD highest protein (4.02%). Sprouted beverages excelled in minerals (SB Ca = 27.00 mg/100 g; SD Ca = 25.75 mg/100 g), while ND or non-sprouted FM fermented spontaneously (NS) had high Fe (4.31%, 2.65%) and K (48.08%, 38.32%). ND showed peak antioxidants: phenolics 10.54 µg/mL, DPPH 87.80%, FRAP 21.24 µM Fe2+/g, ABTS 79.09%. Sprouted beverages displayed distinct color (L* = 37.67–39.65, C* = 25.94–27.03) versus commercial Mageu (L* = 57.89, C* = 14.50) and favorable texture (firmness 12.78–13.40 g, secondary peak force ~−7.2 g). Controlled fermentation of sprouted FM yields nutrient-dense, antioxidant-rich, vegetarian beverages with superior attributes, affirming its functional potential.

Fermentation doi: 10.3390/fermentation12030140

Authors: Jürgen J. Heinisch Hans-Peter Schmitz

Hanseniaspora uvarum, formerly known as Kloeckera apiculata, is the predominant yeast species in grape musts for most wine fermentations worldwide. Despite its important impact on wine quality, its genetics has only been studied in some detail within the past decade, and methods for targeted manipulations first emerged in 2021. Since then, they have been improved and extended not only with respect to the wide applications of H. uvarum in beverage industries and as an environmental control agent, but also as tools in basic genetic research. In this review, the latest developments and future perspectives are summarized.

Fermentation doi: 10.3390/fermentation12030139

Authors: Emma Pelizza Giulia Bertazzoli Eleonora Troiano Renato Leal Binati Veronica Gatto Agata Czyżowska Sandra Torriani Giovanna E. Felis

Metschnikowia pulcherrima is increasingly valued in sustainable vitiviniculture for its dual role as a biocontrol agent and as a contributor to wine quality. However, a coordinated dual-purpose selection strategy has not yet been systematically implemented for this species. This study aimed to identify native strains with combined wine-related traits and biocontrol potential by screening a collection of 179 isolates for key phenotypic traits—β-glucosidase and β-lyase activities, hydrogen sulfide (H2S) production, and pulcherrimin biosynthesis—and assessing their genotypic diversity. Dereplication yielded 106 unique strains, from which five with the most favorable wine-related traits and distinct genotypic profiles were selected for subsequent evaluation of antagonistic potential. Safety-related traits, including growth at 37 °C, invasive growth, pseudohyphal formation, and proteolytic activity, were assessed to exclude virulence-associated behaviors. Antagonistic activity against Botrytis cinerea was evaluated through in vitro dual-culture assays and in vivo grape-berry inoculations, revealing strain- and pathogen-dependent inhibition, with volatile-mediated effects generally exceeding direct-contact interactions. Among the tested strains, NLSFS4 showed strong and consistent biocontrol potential. Microvinification trials further confirmed its oenological relevance, demonstrating the ability to modulate wine aroma composition while preserving fermentation performance. Overall, this study highlights the substantial functional diversity within M. pulcherrima and identifies a promising native strain for integrated use in wine fermentation and biological control in sustainable production systems.

Fermentation doi: 10.3390/fermentation12030137

Authors: Bin Zhang Xichun Wang Zhuzhong Yin Sheng Li Song Xie Xing Peng Huaqun Yin Delong Meng

Biological fermentation drying is an eco-friendly method for food waste treatment. It reduces waste mass and volume effectively. Microbial interactions drive drying efficiency. Yet these interactions remain unclear. Here we show that the inoculation of thermophilic strain and enrichment improved drying efficiency by 24.58% to 30.09%. The temperature comprehensive index and crude fat degradation rate in food waste were increased. The total nitrogen content was reduced by inoculation of thermophiles. The bacterial community was primarily composed of Proteobacteria, Bacteroidetes, and Firmicutes, with increased abundances of Proteobacteria, Actinobacteria, and Cyanobacteria. The fungal community included Ascomycota, Basidiomycota, Glomeromycota, and Chytridiomycota. Thermophilic Bacillus inoculation enhanced bacterial diversity, stabilized the fungal network, and influenced the dominant species in the bacterial-fungal cross-domain network at different stages of bio-drying. Environmental factors such as moisture content and conductivity significantly affected the size and complexity of the network. The study highlights the potential benefits of microbial inoculation and underscores the importance of understanding microbial dynamics and environmental factors in this process.

Fermentation doi: 10.3390/fermentation12030138

Authors: Cecilia Naveira-Pazos María C. Veiga Christian Kennes

The ability of Rhodotorula toruloides DSM 4444 to metabolize low-cost carbon sources such as fatty acids was comprehensively studied. This organism is shown, for the first time, to simultaneously accumulate microbial oils (biofuel precursors) and carotenoids from acetic acid obtained from CO2 fermentation. This fatty acid is typically the single end product of acetogenic bioconversion of one-carbon gas pollutants (e.g., CO2 and CO). In the first set of experiments, different aerobic fermentations were carried out in automated bioreactors, with acetic acid in one case and with glucose, a more conventional carbon source, as a control, in another bioreactor. R. toruloides consumed around 80 g/L substrate under both conditions. Maximum lipid content (27.2% g/g dry weight) was reached from 38 g/L glucose, while carotenoid content was higher with acetic acid (1.4 mg/g cell after 54.1 g/L acetic acid consumed), representing a 40% increase compared to glucose (1.0 mg/g cell after 64.2 g/L glucose consumed). Additionally, in the second set of assays, a fermented broth produced by Acetobacterium woodii from CO2 fermentation, containing residual nutrients and metabolites, was tested. Despite its complex composition, R. toruloides grew and produced carotenoids (up to 0.141 mg/g), showing potential adaptability. To the best of our knowledge, this is the first report on a greenhouse gas-based biotechnological process as a promising sustainable alternative for the valorization of pollutants, e.g., gas emissions, their bioconversion to VFAs, such as acetic acid, and subsequent fermentation of the carboxylic acid into microbial oils, as a source of renewable energy, as well as carotenoids as a high-value nutraceutical product.

Fermentation doi: 10.3390/fermentation12030136

Authors: Liang Zhao Jialin Liu Liang Zhang Zhenbiao Luo Qulai Tang Jingjing Zhao Qing Ji Xinye Wang

Incorporating plant-based additives was a promising approach for modulating the microbial ecosystems of fermentation starters. This study investigated how adding Kuding tea (20% wt/wt) influenced the assembly and succession of fungal communities during Jiang-flavored Daqu production, compared to traditional wheat-based Daqu. Using amplicon sequencing of the ITS1 region and integrated measurements of endogenous factors, we analyzed community dynamics across a 40-day fermentation period. Results showed that tea addition significantly increased fungal diversity and altered succession trajectories. Community assembly shifted from stochastic towards deterministic processes, with homogeneous selection increasing from 0.47 in wheat-based Daqu to 0.62 in tea-added Daqu. Temporal species accumulation was stronger (STR exponent z: 0.565 vs. 0.436), while compositional turnover slowed (TDR slope w: −0.539 vs. −0.626). Random forest models revealed tea-specific fungal drivers and stronger correlations with endogenous factors (e.g., reducing sugar and moisture). We concluded that Kuding tea appears to function predominantly as an environmental filter that enhanced deterministic selection, stabilized community succession, and restructured the key microbial–physicochemical relationships, providing a potential strategy for steering Daqu fermentation.