Fermentation

21 pages, 1740 KB

Open AccessArticle

Isolation, Characterisation and Vitamin B12 Production Optimization of P. freudenreichii from Turkish Traditional Kars Gravyer

by Akif Emre Kavak, Zerya Beyza Alimoğlu, Akın Özdemir and Enes Dertli

Fermentation 2025, 11(12), 704; https://doi.org/10.3390/fermentation11120704 - 18 Dec 2025

Abstract

In this study, nine different Propionibacterium freudenreichii strains were isolated from Kars Gravyer produced by traditional methods in Turkey and identified by sequencing the 16S–23S intergenic region using species-specific primers. The isolated strains were examined in vitro for the presence of the β-galactosidase [...] Read more.

In this study, nine different Propionibacterium freudenreichii strains were isolated from Kars Gravyer produced by traditional methods in Turkey and identified by sequencing the 16S–23S intergenic region using species-specific primers. The isolated strains were examined in vitro for the presence of the β-galactosidase enzyme, autoaggregation ability, sensitivity against eight selected antibiotics and survivability under harsh conditions in order to determine their potential probiotic properties. After probiotic potentials were evaluated, an experimental design was made to optimize the production of vitamin B12 in a 3 L glass bioreactor P. freudenreichii NUV774. While all strains showed similar resistance (92–98%) to gastric juice (0.3% pepsin, pH 3.0), they showed resistance to intestinal fluid (0.1% pancreatin, 0.3% bile salt, pH 8.0) between 60% and 92%. It was determined that the viability after 3 and 6 h of incubation in 0.5% and 1% bile salt differed between strains. All isolates exhibited resistance to ciprofloxacin, ampicillin, and trimethoprim–sulphamethoxazole; however, most were sensitive to ofloxacin. Overall, P. freudenreichii strains showed resistance to the gastrointestinal tract, tolerance to pH 3.0, and high tolerance to bile salts. As a result of optimization, maximum vitamin B12 production was found to be 156.8 mg/L. The optimum operating conditions were calculated as temperature = 36.9 °C, aeration = 2.430 vvm, and agitation = 159.120 rpm. Hence, P. freudenreichii, as future probiotic strain candidates, will offer an alternative source to Lactobacillus, Bifidobacterium and some Bacillus spp. In addition, this study denoted that the alteration of the production of active vitamin B12 by P. freudenreichii occurs in a strain-dependent manner. Full article

(This article belongs to the Special Issue Microbial Metabolism Focusing on Bioactive Molecules)

18 pages, 2462 KB

Open AccessArticle

Fe/Mn-Modified Biochar Facilitates Functional Microbial Enrichment for Efficient Glucose–Xylose Co-Fermentation and Biohydrogen Production

by Jianing Fan, Jiwen Wu, Ji Zhao, Hongsheng Hao, Yange Yu, Guangli Cao and Nanqi Ren

Fermentation 2025, 11(12), 703; https://doi.org/10.3390/fermentation11120703 - 18 Dec 2025

Abstract

Biohydrogen production can be derived from low-value lignocellulosic biomass; however, in many biohydrogen producing systems, xylose is utilized less efficiently than glucose, which limits overall substrate conversion. To address this issue, Fe/Mn-modified biochar was employed to enhance dark fermentation of glucose–xylose mixed sugars, [...] Read more.

Biohydrogen production can be derived from low-value lignocellulosic biomass; however, in many biohydrogen producing systems, xylose is utilized less efficiently than glucose, which limits overall substrate conversion. To address this issue, Fe/Mn-modified biochar was employed to enhance dark fermentation of glucose–xylose mixed sugars, and its performance was compared with other inoculum treatments. The biochar addition achieved a hydrogen yield of 2.57 ± 0.10 mol-H₂/mol-sugar, representing 14.6% enhancement over untreated controls, while enabling complete substrate utilization across varying xylose proportions. Biochar supplementation also reduced the lag phase by 24.4% and increased hydrogen productivity by 47.3% in mixed-sugar cultivation. Integrated analyses of the experimental data revealed the dual role of Fe/Mn-modified biochar in constructing conductive extracellular polymeric substance networks and directing metabolic flux toward high-yield butyrate pathways. This work establishes Fe/Mn-biochar as a multifunctional microbial engineering tool that alleviates carbon catabolite repression and promotes the enrichment of hydrogen-producing bacteria (HPB), thereby providing a practical and effective strategy for enhanced biohydrogen production from lignocellulosic biomass. Full article

(This article belongs to the Section Industrial Fermentation)

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20 pages, 5398 KB

Open AccessArticle

Bioaugmentation Versus pH Adjustment in High-Load Food Waste Anaerobic Digestion: Divergent Microbial Responses and Methanogenesis Regulation

by Chenyu Piao, Zhe Wang, Keqian Zhao, Mengfei Du and Ke Wang

Fermentation 2025, 11(12), 702; https://doi.org/10.3390/fermentation11120702 - 18 Dec 2025

Abstract

High organic loading is known to destabilize anaerobic digestion (AD). This study compared bioaugmentation and pH adjustment under increasing organic loading rate (OLR: 2.0, 4.0 and 6.0 gVS L⁻¹ d⁻¹), focusing on the responses of microbial structure, metabolic pathways, and [...] Read more.

High organic loading is known to destabilize anaerobic digestion (AD). This study compared bioaugmentation and pH adjustment under increasing organic loading rate (OLR: 2.0, 4.0 and 6.0 gVS L⁻¹ d⁻¹), focusing on the responses of microbial structure, metabolic pathways, and energy metabolism. Results demonstrated that bioaugmentation maintained stable methane production of 400.54 ± 10.08 and 374.15 ± 24.32 mL·g-VS⁻¹ at 4.0 and 6.0 gVS L⁻¹ d⁻¹, respectively, whereas control and pH-adjusted reactors failed at 4.0 gVS L⁻¹ d⁻¹. The acidified system restored methane yield from 86.30 to 382.13 mL·g-VS⁻¹ after bioaugmentation, whereas pH adjustment and feeding cessation were ineffective, failing to produce methane within 25 days. Microbial analysis showed bioaugmentation enriched Methanosarcina, enhanced hydrogenotrophic/methylotrophic methanogenesis, and strengthened syntrophy with syntrophic propionate-oxidizing bacteria (SPOB), reducing volatile fatty acid accumulation via reinforced syntrophic propionate/butyrate oxidation. Upregulation of osmoregulatory (nha, kdp, proP) and energy metabolism genes (eha, mvh, hdr) maintained osmotic balance and energy supply under high load. In contrast, pH adjustment downregulated SPOB and propionate oxidation genes, causing persistent acid inhibition. This study elucidated the distinct regulatory effects of bioaugmentation and pH adjustment on high-load AD systems, providing actionable strategies for both maintaining operational stability in high-load reactors and recovering methanogenesis in acid-inhibited systems. Full article

(This article belongs to the Special Issue 10th Anniversary of Fermentation: Feature Papers in Section "Industrial Fermentation")

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15 pages, 915 KB

Open AccessArticle

Silymarin in the Diet of Dairy Cows and Its Impacts on Liver Health, Ruminal Fermentation, Productive Performance, and Milk Quality

by Pablo Vinicius Novakoski, Luisa Nora, Guilherme Luiz Deolindo, Gilnei Bruno da Silva, Daiane Manica, Margarete Dulce Bagatini and Aleksandro Schafer da Silva

Fermentation 2025, 11(12), 701; https://doi.org/10.3390/fermentation11120701 - 18 Dec 2025

Abstract

The search for natural alternatives that improve the productive efficiency and metabolic state of dairy cows has driven the use of phytogenic compounds such as silymarin, a flavonolignan extracted from Silybum marianum L. Gaertn with recognized antioxidant, anti-inflammatory, and hepatoprotective properties. This study [...] Read more.

The search for natural alternatives that improve the productive efficiency and metabolic state of dairy cows has driven the use of phytogenic compounds such as silymarin, a flavonolignan extracted from Silybum marianum L. Gaertn with recognized antioxidant, anti-inflammatory, and hepatoprotective properties. This study evaluated the effects of silymarin supplementation on the productive performance; milk composition; and ruminal, hematological, biochemical, and oxidative parameters of lactating Jersey cows kept in a confined system with robotic milking. Twelve cows (230 ± 30 days in lactation; 22 ± 3.5 kg/day of milk) were distributed in a crossover design, receiving a control diet (GCON) or a diet supplemented with 5 g/cow/day of silymarin (GSIL) for 28 days in each stage. Silymarin intake did not alter dry matter intake, feed efficiency, or average milk production (p > 0.05), but it increased milk fat content (4.27 × 4.02%; p = 0.05) and, consequently, milk production corrected for 4% fat (24.4 × 23.2 kg/day; p = 0.05). In the rumen environment, cows in the GSIL group showed higher concentrations of acetic acid (57.4 × 48.4 nmol/L), and total short-chain fatty acids (100.2 × 89.4 nmol/L; p = 0.01). Regarding the biochemical profile, silymarin reduced gamma-glutamyltransferase and aspartate aminotransferase activities, as well as haptoglobin levels, indicating a hepatoprotective effect, combined with a lower inflammatory response in the liver. Oxidative status was improved by decreased levels of TBARS (lipid peroxidation) and reactive oxygen species, as well as myeloperoxidase activity in the serum of cows fed silymarin (p ≤ 0.05), but there was no difference between groups for superoxide dismutase activity and glutathione levels. The inclusion of silymarin in the diet of lactating Jersey cows improved the antioxidant and hepatic profile, increased milk fat content, and favored ruminal fermentation, suggesting metabolic and productive benefits in confined systems with high physiological demands. Full article

(This article belongs to the Special Issue Research Progress of Rumen Fermentation)

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20 pages, 2429 KB

Open AccessReview

Epipolythiodioxopiperazines: From Chemical Architectures to Biological Activities and Ecological Significance—A Comprehensive Review

by Qingqing Zhang, Mingyang Jia, Hongyi Li, Tingting Shi, Ying Xu, Taili Zhao, Lixin Zhang, Peipei Zhao and Xuekui Xia

Fermentation 2025, 11(12), 700; https://doi.org/10.3390/fermentation11120700 - 17 Dec 2025

Abstract

Epipolythiodioxopiperazines (ETPs), characterized by a diketopiperazine (DKP) core bridged by disulfide or polysulfide bonds, exhibit exceptional structural diversity and functional adaptability. This review comprehensively explores their multifaceted properties, covering chemical structural characteristics, therapeutic application potential, and ecological functional value. Structural diversity arises from [...] Read more.

Epipolythiodioxopiperazines (ETPs), characterized by a diketopiperazine (DKP) core bridged by disulfide or polysulfide bonds, exhibit exceptional structural diversity and functional adaptability. This review comprehensively explores their multifaceted properties, covering chemical structural characteristics, therapeutic application potential, and ecological functional value. Structural diversity arises from variations in the core DKP scaffold, sulfur bridge connectivity patterns, and additional modifications. Biosynthesis involves initial DKP assembly, enzyme-catalyzed sulfur incorporation and oxidation to form the signature sulfur bridge of ETPs, diversification by tailoring enzymes, and distinct regulatory mechanisms. ETPs possess diverse biological activities, including cytotoxicity, antitumor activity, antimicrobial properties, and immunomodulatory functions. From an ecological standpoint, ETPs mediate fungal–host interactions and influence competition and symbiosis within fungal communities. Furthermore, this review also addresses the current challenges and outlines future research directions. In summary, as a class of significant compounds spanning the fields of chemistry, biology, medicine, and ecology, ETPs deserve focused attention for their research value and application prospects. Full article

(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)

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16 pages, 3996 KB

Open AccessArticle

FTIR Spectroscopy, a New Approach to Evaluating Caseinolytic Activity of Probiotic Lactic Acid Bacteria During Goat Milk Fermentation and Storage

by Juan José Carol Paz, Ana Yanina Bustos and Ana Estela Ledesma

Fermentation 2025, 11(12), 699; https://doi.org/10.3390/fermentation11120699 - 17 Dec 2025

Abstract

Goat milk can be a vehicle for beneficial microorganisms, such as probiotic lactic acid bacteria (LAB). During lactic fermentation, the hydrolysis of milk proteins can improve their nutritional properties and sensory attributes and even have beneficial health effects. The objective of this study [...] Read more.

Goat milk can be a vehicle for beneficial microorganisms, such as probiotic lactic acid bacteria (LAB). During lactic fermentation, the hydrolysis of milk proteins can improve their nutritional properties and sensory attributes and even have beneficial health effects. The objective of this study was to evaluate the caseinolytic activity of LAB strains with probiotic potential and to monitor the changes induced by fermentation and during storage in milk components using Fourier transform infrared (FTIR) spectroscopy. First, the proteolytic activity of 36 LAB strains isolated from dairy products was qualitatively assessed. Then, 17 strains with probiotic potential and moderate to high proteolytic activity were selected for further analysis. Casein proteolysis was found to be strain-dependent, with a decrease in total protein concentration ranging from 28% to 87% and an increase in amino acids ranging from 29% to 88%. Furthermore, a notable difference was observed in the amide bands in the FTIR spectra between the beginning and end of incubation, showing a decrease in the intensities of the bands attributed to proteins. In fermented goat milk, LAB growth resulted in a final count between 0.62 and 2.6 log CFU/mL, a 0.29 to 2.0 drop in pH, and lactic acid production between 0.20 and 1 g/L. FTIR spectra revealed time-dependent modifications in amide I and II bands accompanied by a marked reduction in carbohydrate content and an increase in lactic acid signal. After 21 days of storage, the viability of the strains, pH, and lactic acid in the fermented milks were not substantially modified. These results highlight the potential of lactic fermentation with strains selected for their probiotic potential as an approach to producing value-added goat milk products, as well as the usefulness of FTIR spectroscopy for characterizing complex systems such as goat milk. Full article

(This article belongs to the Special Issue Advances in Functional Fermented Foods)

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17 pages, 676 KB

Open AccessArticle

Bioyogurt Enriched with Provitamin A Carotenoids and Fiber: Bioactive Properties and Stability

by Camila Bernal-Castro, Ángel David Camargo-Herrera, Carolina Gutiérrez-Cortés and Consuelo Díaz-Moreno

Fermentation 2025, 11(12), 698; https://doi.org/10.3390/fermentation11120698 - 16 Dec 2025

Abstract

Recent research has focused on yogurts supplemented with plant-derived and apiculture ingredients to enhance functional properties. This study evaluates the symbiotic potential of provitamin A carotenoids, dietary fiber, and oligosaccharides from carrots, mangoes, and honeydew honey in probiotic-enriched bioyogurt. Formulations were assessed during [...] Read more.

Recent research has focused on yogurts supplemented with plant-derived and apiculture ingredients to enhance functional properties. This study evaluates the symbiotic potential of provitamin A carotenoids, dietary fiber, and oligosaccharides from carrots, mangoes, and honeydew honey in probiotic-enriched bioyogurt. Formulations were assessed during fermentation (45 °C ± 1 °C for 5 h) and refrigerated storage (4 °C ± 1 °C for 21 days). Probiotic and starter culture viability was determined using pour-plate counts on MRS agar. Physicochemical parameters including pH, titratable acidity, total soluble solids, water-holding capacity, and antioxidant metrics (total phenolics and carotenoids) were analyzed. After 21 days of storage, the probiotic culture (VEGE 092) reached 10.26 log CFU/mL and the starter culture (YOFLEX) achieved 8.66 log CFU/mL, maintaining therapeutic thresholds. Total carotenoid content increased significantly (p < 0.05) from 2.15 to 3.96 µg β-carotene/g, indicating synergistic interactions between lactic acid bacteria and plant-derived bioactive compounds. These findings demonstrate that combining plant-derived carotenoids, prebiotic fibers, and honeydew oligosaccharides effectively maintains probiotic viability and enhances antioxidant stability throughout fermentation and refrigerated storage, supporting the development of functional dairy products with improved nutritional profiles. Full article

(This article belongs to the Special Issue Lactic Acid Bacteria in Functional Foods: From Microbiology to Health Benefits)

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18 pages, 7100 KB

Open AccessArticle

Intraspecific Diversity of Saccharomyces cerevisiae Associated with Traditional Fermented Beverages in Chiapas, Mexico

by Maritza Tawas-Penagos, Ruth Percino-Daniel, José Alberto Narváez-Zapata, René Quezada-Romero, Anne Christine Gschaedler-Mathis and Alma Gabriela Verdugo-Valdez

Fermentation 2025, 11(12), 697; https://doi.org/10.3390/fermentation11120697 - 16 Dec 2025

Abstract

Traditional fermented beverages from Chiapas, Mexico, represent an important source of microbial diversity, particularly of Saccharomyces cerevisiae. In native strains isolated from traditional fermented beverages, Saccharomyces cerevisiae has been observed to display distinct morphological and physiological traits; therefore, the aim of this [...] Read more.

Traditional fermented beverages from Chiapas, Mexico, represent an important source of microbial diversity, particularly of Saccharomyces cerevisiae. In native strains isolated from traditional fermented beverages, Saccharomyces cerevisiae has been observed to display distinct morphological and physiological traits; therefore, the aim of this study was to evaluate the population growth and the tolerance of twenty isolates to different stress factors such as temperature, osmotic pressure, and high ethanol concentrations, as well as the genetic variability through interdelta analysis, and to determine whether these physiological and molecular characteristics are associated with the type of beverage and the locality of origin. Differences were observed in tolerance to various factors, including high ethanol concentrations and elevated temperatures, as well as in the production of volatile compounds, with Taberna and Mezcal isolates showing notable performance. These isolates were able to withstand temperatures ranging from 43 to 45 °C and ethanol concentrations of up to 17% in Mezcal and Pox isolates, and 15% in Taberna isolates. High concentrations of isoamyl acetate and higher alcohols such as isoamyl alcohol were detected. In addition, the genetic variability of the isolates was evaluated, and its relationship with the type of beverage and the geographical origin of production was explored, including isolates obtained from Taberna, Mezcal, Pox, and Chicha de Chilacayote. Intraspecific variability was assessed through a retrotransposon-based analysis of the interdelta region using different primer combinations (δ1-δ2, δ12-δ21, and δ12-δ2). The generated banding patterns were analyzed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), which enabled the identification of molecular variability patterns among the isolates. Furthermore, a UPGMA analysis was performed using physiological and compound production data, revealing a relationship between these characteristics and the geographical origin of the isolates. The results revealed a high degree of intraspecific variability, which was associated with both the type of beverage and the locality of origin of the isolates. Full article

(This article belongs to the Special Issue Yeasts as Microbial Cell Factories: Diversity, Biotechnology Potential and Applications)

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14 pages, 895 KB

Open AccessArticle

Adding Digestive Enzymes to Anaerobic Co-Digestion of Cattle Manure and Industrial Corn Grain Waste

by Laís Medeiros Cintra, Roberta Passini, Luana Alves Akamine, Kedinna Dias de Sousa, Frank Freire Capuchinho, Sérgio Botelho de Oliveira and Silvia Robles Reis Duarte

Fermentation 2025, 11(12), 696; https://doi.org/10.3390/fermentation11120696 - 16 Dec 2025

Abstract

Brazil is one of the world’s largest producers of grains and cattle, activities that generate a large amount of organic waste, which has high potential for biogas and methane production. Cattle manure (CM) and industrial waste from corn processing are substrates with significant [...] Read more.

Brazil is one of the world’s largest producers of grains and cattle, activities that generate a large amount of organic waste, which has high potential for biogas and methane production. Cattle manure (CM) and industrial waste from corn processing are substrates with significant potential for biogas and methane generation, particularly through the process of anaerobic co-digestion (AcoD). This study aimed to assess the biogas and methane yield, as well as the stability of the AcoD process involving CM and corn grain residues (CG) derived from a grain processing agroindustry, in conjunction with the application of an enzyme complex. The experiment was conducted in plug-flow biodigesters, with a total volume of 28 L, under a semi-continuous feeding regime (OLR = 0.84 g vs. L d⁻¹) at ambient temperature. The findings indicated increases in daily biogas and methane production for AcoD, without the addition of enzymes, of 52.1% and 44.4%, respectively, in comparison to CM mono-digestion. The incorporation of the enzyme complex did not yield beneficial effects, irrespective of the substrate composition. The utilization of enzymes in semi-continuous biodigesters to enhance methane yields necessitates further investigation to achieve favorable outcomes and validate its efficiency. Full article

(This article belongs to the Special Issue The Future of Fermentation Technology in the Biorefining Process: 3rd Edition)

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27 pages, 778 KB

Open AccessReview

Yeast-Derived Biomolecules as Green Nanoplatforms for Sustainable Lignocellulosic Biorefineries

by Fabio P. Sanchez Vera, Naiara J. Clerici, Gabriela A. Lourenço, Sara B. Santa Rita, Kiara A. Garcia Bustos, Eduardo Florez Martinez, Guilherme O. Silva, Paulo R. Franco Marcelino, Julio César dos Santos and Silvio S. da Silva

Fermentation 2025, 11(12), 695; https://doi.org/10.3390/fermentation11120695 - 16 Dec 2025

Abstract

Yeast-derived biomolecules are redefining the boundaries of green nanotechnology. Biosurfactants, exopolysaccharides, enzymes, pigments, proteins, and organic acids—when sourced from carbohydrate-rich lignocellulosic hydrolysates—offer a molecular toolbox capable of directing, stabilizing, and functionalizing nanoparticles (NPs) with unprecedented precision. Beyond their structural diversity and intrinsic biocompatibility, [...] Read more.

Yeast-derived biomolecules are redefining the boundaries of green nanotechnology. Biosurfactants, exopolysaccharides, enzymes, pigments, proteins, and organic acids—when sourced from carbohydrate-rich lignocellulosic hydrolysates—offer a molecular toolbox capable of directing, stabilizing, and functionalizing nanoparticles (NPs) with unprecedented precision. Beyond their structural diversity and intrinsic biocompatibility, these biomolecules anchor a paradigm shift: the convergence of biorefineries with nanotechnology to deliver multifunctional materials for the circular bioeconomy. This review explores: (i) the expanding portfolio of metallic and metal oxide NPs synthesized through yeast biomolecules; (ii) molecular-level mechanisms of reduction, capping, and surface tailoring that dictate NP morphology, stability, and reactivity; (iii) synergistic roles in intensifying lignocellulosic processes—from enhanced hydrolysis to catalytic upgrading; and (iv) frontier applications spanning antimicrobial coatings, regenerative packaging, precision agriculture, and environmental remediation. We highlight structure–function relationships, where amphiphilicity, charge distribution, and redox activity govern resilience under saline, acidic, and thermally harsh industrial matrices. Yet, critical bottlenecks remain: inconsistent yields, limited comparative studies, downstream recovery hurdles, and the absence of comprehensive life-cycle and toxicological evaluations. To bridge this gap, we propose a translational roadmap coupling standardized characterization with real hydrolysate testing, molecular libraries linking biomolecule chemistry to NP performance, and integrated techno-economic and environmental assessments. By aligning yeast biotechnology with nanoscience, we argue that yeast-biomolecule-driven nanoplatforms are not merely sustainable alternatives but transformative solutions for next-generation lignocellulosic biorefineries. Full article

(This article belongs to the Special Issue Yeast Biotechnology in Valorization of Waste and By-Products)

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14 pages, 1643 KB

Open AccessArticle

Use of Pichia manshurica as a Starter Culture for Spontaneous Cocoa Fermentation in Southern Bahia, Brazil

by Adriana Barros de Cerqueira e Silva, Eric de Lima Silva Marques, Rachel Passos Rezende, Cristiano Santana, Angelina Moreira Freitas, Maria Clara Bessa Souza, Carine Martins dos Santos, Adriana Cristina Reis Ferreira, Marianna Ramos Soares, Alberto Montejo Díaz, Ádanny Maia da Cruz Santos, Luan Melo Andrade, Louise Pereira Ramos, Carla Cristina Romano, João Carlos Teixeira Dias and Sérgio Eduardo Soares

Fermentation 2025, 11(12), 694; https://doi.org/10.3390/fermentation11120694 - 16 Dec 2025

Abstract

To improve cocoa fermentation and the quality of its final products, microbial cultures with potential as starters were investigated. Yeasts were considered a promising option due to their adaptability to biotechnological processes and ease of laboratory manipulation. From 185 strains previously isolated from [...] Read more.

To improve cocoa fermentation and the quality of its final products, microbial cultures with potential as starters were investigated. Yeasts were considered a promising option due to their adaptability to biotechnological processes and ease of laboratory manipulation. From 185 strains previously isolated from spontaneous cocoa fermentation, those producing protease, amylase, and cellulase were identified. Strain CII87b (Pichia manshurica) exhibited the most favorable results and was evaluated for cytotoxicity using the MTT assay, showing no adverse effects. This culture was subsequently inoculated into freshly harvested cocoa almonds during the secondary (winter) harvest. The inoculum accelerated and increased the average fermentation temperature from 25 to 50 °C, reduced internal mold incidence, decreased defect rates, increased total fermentation, and resulted in a more desirable pH compared to the control. These findings demonstrate that the use of P. manshurica CII87b as a starter culture in winter harvests can improve fermentation efficiency and product quality, offering a biotechnological tool with potential benefits for cocoa producers and the chocolate industry. Full article

(This article belongs to the Special Issue 10th Anniversary of Fermentation: Feature Papers in the "Fermentation for Food and Beverages" Section)

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12 pages, 307 KB

Open AccessArticle

Evaluation of Plant Essential Oils as Natural Alternatives to Monensin in In Vitro Ruminal Fermentation

by Amelia Barbosa Lima, Kemmily Lima de Almeida, Bruna Eduarda Teixeira de Lima, Khalid Haddi, Ludmila Couto Gomes Passetti, Gustavo Leão Rosado and Cláudia Braga Pereira Bento

Fermentation 2025, 11(12), 693; https://doi.org/10.3390/fermentation11120693 - 14 Dec 2025

Abstract

Extensive growth promoter use in livestock production has raised concerns about their role in selective pressure on resistant microorganisms, driving interest in natural alternatives such as essential oils (EOs). This study aimed to evaluate the effects of tea tree, holy wood, and citronella [...] Read more.

Extensive growth promoter use in livestock production has raised concerns about their role in selective pressure on resistant microorganisms, driving interest in natural alternatives such as essential oils (EOs). This study aimed to evaluate the effects of tea tree, holy wood, and citronella EOs on in vitro ruminal fermentation. The study follows a completely randomized design with the following five treatments: control, monensin (5 μM), tea tree EO (50 mg/L), holy wood EO (50 mg/L), and citronella EO (50 mg/L), each conducted in triplicate. Incubations were performed at 39 °C for 48 h in the rumen fluid collected from fistulated cattle fed a 20:80 forage-to-concentrate diet. Notably, EOs exhibited no significant effects on pH, microbial protein production, total volatile fatty acids, or in vitro dry matter digestibility (p > 0.05). Tea tree and holy wood EOs enhanced deamination activity, and all treatments increased ammonia concentration compared with that in the control. Monensin treatment increased acetate concentration and reduced in vitro neutral detergent fiber digestibility; holy wood EO exhibited a similar trend. Altogether, the findings of this study suggest that EOs can selectively modulate the ruminal microbiota, influencing nitrogen metabolism and fermentation patterns without impairing rumen stability. Full article

(This article belongs to the Special Issue Ruminal Fermentation: 2nd Edition)

19 pages, 306 KB

Open AccessArticle

In Vitro and In Situ Evaluation of White Mulberry (Morus alba) Pomace and Leaf: Fermentation Kinetics, Digestibility, and Potential as Alternative Ruminant Feed Sources

by Zekeriya Safa İnanç and Huzur Derya Arik

Fermentation 2025, 11(12), 692; https://doi.org/10.3390/fermentation11120692 - 12 Dec 2025

Abstract

Mulberry (Morus alba) by-products represent underutilized feed resources with potential for ruminant nutrition. This study evaluated the rumen fermentation kinetics and rumen digestibility of dried mulberry pomace (MP) and leaf (ML) to determine optimal inclusion strategies in dairy cattle diets. Mulberry [...] Read more.

Mulberry (Morus alba) by-products represent underutilized feed resources with potential for ruminant nutrition. This study evaluated the rumen fermentation kinetics and rumen digestibility of dried mulberry pomace (MP) and leaf (ML) to determine optimal inclusion strategies in dairy cattle diets. Mulberry pomace (MP) and mulberry leaf (ML) were sun-dried and incorporated at 50% substitution levels into total mixed rations (TMR) with varying concentrations (30%, 35%, 40%, 45%, and 50%) of neutral detergent fiber (NDF). This created ten treatment groups: 30NP through 50NP (pomace-supplemented, where the number represents basal TMR NDF%) and 30NL through 50NL (leaf-supplemented), plus control groups containing only MP or ML and five basal TMR controls (30N through 50N). Rumen fluid was collected from two non-lactating Holstein cows fitted with ruminal cannulas. Chemical analysis revealed that ML contained 19% crude protein and 27.4% NDF, while MP contained 14.9% crude protein and 35.8% NDF. The highest gas production was observed in the 45NP (43.20 mL) and 50NL (43.50 mL) groups. Results demonstrated that MP achieved optimal fermentation when combined with 40–45% NDF TMR (maximum total volatile fatty acid (VFA): 88.86 mmol/L in 40NP at 48 h), whereas ML performed best with 45% NDF TMR (45NL: 88.03 mmol/L total VFA), indicating these as the most promising treatment combinations for ruminant feeding systems pending in vivo validation. Acetate proportions were higher in ML groups (84–96%), while propionate ratios were elevated in MP groups. Both materials maintained optimal ruminal pH (6.2–6.8). In vitro NDF digestibility was significantly higher for ML, with differences increasing from 2.97% at 2 h to 16.44% at 240 h. In situ degradation of MP was nearly complete at 48 h, while ML reached maximum degradation at 24 h. These findings indicate the potential of MP and ML as valuable alternative feed sources for ruminants, particularly in TMRs containing 40–45% NDF. Full article

(This article belongs to the Special Issue Ruminal Fermentation: 2nd Edition)

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12 pages, 2908 KB

Open AccessArticle

An Intelligent Strategy for Colony De-Replication Using Raman Spectroscopy and Hybrid Clustering

by Xinli Li, Mingyang Liu, Jiaqi Sun and Su Wang

Fermentation 2025, 11(12), 691; https://doi.org/10.3390/fermentation11120691 - 12 Dec 2025

Abstract

Efficient de-redundant colony picking is essential to accelerating strain screening in fermentation microbiology. Conventional random picking is inefficient, exhibits high redundancy, and often misses low-abundance but valuable strains. To address this, we present a high-efficiency de-redundant selection strategy based on colony Raman spectroscopy [...] Read more.

Efficient de-redundant colony picking is essential to accelerating strain screening in fermentation microbiology. Conventional random picking is inefficient, exhibits high redundancy, and often misses low-abundance but valuable strains. To address this, we present a high-efficiency de-redundant selection strategy based on colony Raman spectroscopy and a hybrid clustering algorithm. We directly acquire colony Raman spectra and combine the complementary strengths of k-means and hierarchical clustering (HCA) to achieve both balanced global partitioning and sensitivity to low-abundance taxa. Systematic application on pure colonies and complex plate settings shows that, by picking only 12–26% of colonies, the method attains 80–100% species coverage. Relative to manual random picking and image-based feature selection, picking efficiency increased by 116.8% and 44.5%, respectively, substantially shortening the screening cycle and reducing workload. Overall, Raman-guided hybrid clustering substantially reduces redundant picking and improves detection of low-abundance strains. It provides practical support for efficient strain discovery, library construction, and process optimization. Full article

(This article belongs to the Section Fermentation Process Design)

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19 pages, 3721 KB

Open AccessArticle

Effect of Bulking Agent Particle Size on Garden Waste–Dairy Manure Composting: Relationship Between Microbial Community Dynamics and Physicochemical Factors

by Qian Liu, Zhike Liu, Shaohan Ma, Le Li, Qing Hao, Shiyu Liu, Mingyi Lu and Yanhua Li

Fermentation 2025, 11(12), 690; https://doi.org/10.3390/fermentation11120690 - 12 Dec 2025

Abstract

Garden waste is a solid waste produced by plant littering or pruning. Improper disposal can easily pollute the environment. The addition of bulking agents (BAs) can improve the efficiency of organic waste composting. In this study, garden waste and dairy manure were used [...] Read more.

Garden waste is a solid waste produced by plant littering or pruning. Improper disposal can easily pollute the environment. The addition of bulking agents (BAs) can improve the efficiency of organic waste composting. In this study, garden waste and dairy manure were used as raw materials, and easily available and recyclable branches were used as bulking agents to realize the synergistic resource utilization of the two. Three treatments were set up in the experiment, and 10% crushed branches, 1 cm branches, and 3 cm branches were added to the raw materials, respectively. The results showed that compared with the control group (adding crushed branches), the addition of 1 cm branches and 3 cm branches increased the cellulose degradation rate by 13.16–13.33% and the hemicellulose degradation rate by 18.24–23.86%. The monitoring results of CO₂ release showed that the cumulative CO₂ release of the treatment groups with 1 cm and 3 cm branches was 78.56 L and 102.17 L, respectively, which was significantly higher than that of the crushed branches (67.24 L), indicating that the addition of 1 cm and 3 cm branches increased microbial activity and degradation efficiency. Microbial diversity analysis further showed that in the treatment group with 1 cm branches, the number of nodes in the co-occurrence network increased by 24.11% and 2.84%, respectively, compared with the crushed branches and 3 cm branches, and the number of edges increased by 44.25% and 19.72%, forming the most abundant and complex microbial community, which verified its promotion effect on the composting process from the microbial level. In summary, this study recommends the use of branches with a particle size of 1 cm as BAs for garden waste composting. Full article

(This article belongs to the Special Issue Resource Recovery and Microbial Transformation of Organic Solid Waste)

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20 pages, 2939 KB

Open AccessArticle

Caproate Production from Yellow Water Fermentation: The Decisive Roles of Electron Donors

by Kai Shen, Xing Chen, Jiasheng Shi, Xuedong Zhang, Yaya Sun, He Liu, Salma Tabassum and Hongbo Liu

Fermentation 2025, 11(12), 689; https://doi.org/10.3390/fermentation11120689 - 12 Dec 2025

Abstract

Caproate is a valuable medium-chain fatty acid (MCFA) that is found to be extensively used in biofuel production, food preservation, and the pharmaceutical industries. Short-chain fatty acids (SCFAs) from waste streams can be upgraded sustainably through their biological synthesis via anaerobic chain elongation. [...] Read more.

Caproate is a valuable medium-chain fatty acid (MCFA) that is found to be extensively used in biofuel production, food preservation, and the pharmaceutical industries. Short-chain fatty acids (SCFAs) from waste streams can be upgraded sustainably through their biological synthesis via anaerobic chain elongation. However, caproate production is frequently limited in real-world systems due to low carbon conversion efficiency and a lack of electron donors. In this study, we developed a two-stage fermentation strategy employing yellow water—a high-strength organic wastewater from liquor manufacturing—as a novel substrate. During primary fermentation, Lactobacillus provided endogenous electron donors by converting the residual carbohydrates in the yellow water into lactic acid. Nano zero-valent iron (NZVI) was introduced to the secondary fermentation to enhance power reduction and electron flow, further promoting caproate biosynthesis. The caproate production increased significantly due to the synergistic action of lactic acid and NZVI, reaching a maximum concentration of 20.41 g·L⁻¹ and a conversion efficiency of 69.50%. This strategy enhances carbon recovery and electron transport kinetics while lowering dependency on expensive external donors like hydrogen or ethanol. Microbial community analysis using 16S rRNA sequencing revealed enrichment of chain-elongating bacteria such as Clostridium kluyveri. These findings demonstrate the feasibility of employing an integrated fermentation–electron management technique to valorize industrial yellow water into compounds with added value. This study offers a scalable and environmentally sound pathway for MCFA production from waste-derived resources. Full article

(This article belongs to the Special Issue Fermentation of Organic Waste for High-Value-Added Product Production—2nd Edition)

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16 pages, 2154 KB

Open AccessArticle

Low-Cost Production Process of Saccharomyces cerevisiae Yeast for Craft Beer Fermentation

by Jessica Rodríguez, Domenica Villavicencio, Alys Raza, Fernanda Hernández-Alomía, Carlos Bastidas-Caldes and María Alejandra Cruz

Fermentation 2025, 11(12), 688; https://doi.org/10.3390/fermentation11120688 - 12 Dec 2025

Abstract

The production of craft beer depends on the quality and availability of yeast. However, many small breweries in developing countries face high costs due to their reliance on imported yeast strains. Developing efficient and low-cost propagation methods is therefore essential for sustainable production. [...] Read more.

The production of craft beer depends on the quality and availability of yeast. However, many small breweries in developing countries face high costs due to their reliance on imported yeast strains. Developing efficient and low-cost propagation methods is therefore essential for sustainable production. A lager-type Saccharomyces cerevisiae strain (SC-Lager2) was propagated using both synthetic and low-cost alternative media. The latter was formulated with malt extract as a carbon source and yeast extract obtained from brewery by-products as a nitrogen source. A Plackett–Burman design identified significant factors influencing growth (p < 0.05), and a full factorial design (2⁴) optimized conditions. Growth kinetics and biomass yield were validated at laboratory (2 L) and pilot (83 L) scales. Maltose, yeast extract, zinc sulfate, and agitation significantly affected cell density and viability (p < 0.05). Under optimized conditions, 100% viability, a maximum cell density of 1.4 × 10¹⁰ cells/mL, and a biomass yield of 10 g/L were achieved values that were statistically higher (p < 0.05) than those obtained with the synthetic medium. The maximum specific growth rate (μ_max) increased by 52%, while doubling time decreased by 39%. Overall, the use of agro-industrial by-products reduced medium costs by approximately 65% compared to conventional synthetic formulations. The proposed low-cost medium provides a scalable, economical, and sustainable solution for yeast propagation, reducing production costs while maintaining high cell viability and performance. This approach supports the autonomy and competitiveness of the craft beer sector in developing regions. Full article

(This article belongs to the Special Issue Development and Application of Starter Cultures, 2nd Edition)

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19 pages, 877 KB

Open AccessArticle

The Influence of Winemaking Processes on the Formation of Biogenic Amines in Wine

by Karolina Kostelnikova, Mojmir Baron, Michal Kumsta and Jiri Sochor

Fermentation 2025, 11(12), 687; https://doi.org/10.3390/fermentation11120687 - 11 Dec 2025

Abstract

This study investigated the influence of different winemaking processes, particularly fermentation type and must clarification, on the formation of biogenic amines (BA) in Sauvignon wine. The experiment investigated seven methods of vinification combining spontaneous and controlled alcoholic and malolactic fermentation. The concentrations of [...] Read more.

This study investigated the influence of different winemaking processes, particularly fermentation type and must clarification, on the formation of biogenic amines (BA) in Sauvignon wine. The experiment investigated seven methods of vinification combining spontaneous and controlled alcoholic and malolactic fermentation. The concentrations of six biogenic amines (histamine, tyramine, tryptamine, phenylethylamine, putrescine, and cadaverine) were determined using a HILIC-LC-MS/MS. Statistical evaluation confirmed the significant effect of alcoholic and malolactic fermentation, maturation stage, and must processing on the overall amine profile of the wine (p < 0.001). The total BA content in all the variants was low and well below values considered to pose a health risk. Histamine and tryptamine were only detected in trace amounts (<0.1 mg/L), whereas putrescine and tyramine exhibited the greatest variability. Higher concentrations were recorded in variants that underwent malolactic fermentation, particularly in combination with clarified must. In contrast, whole-mash fermentation produced the lowest BA concentrations, possibly due to factors associated with extended skin and seed contact. These findings indicate that the choice of fermentation strategy significantly affects the formation of biogenic amines in wine. Full article

(This article belongs to the Special Issue Applications of Microbial Biodiversity in Wine Fermentation)

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21 pages, 2080 KB

Open AccessArticle

Fermentation of Wheat Bread with Lactiplantibacillus plantarum: Study of Changes in Acrylamide and Microbiological Spoilage During Packaging at Different Temperatures

by Almas Zhanbolat, Ulbala Tungyshbayeva, Zhanat Iskakova, Maryna Mardar, Raushangul Uazhanova, Maxat Iztileuov, Sholpan Amanova, Bakhytkul Assenova, Raushan Izteliyeva, Sagynysh Aman and Farida Amutova

Fermentation 2025, 11(12), 686; https://doi.org/10.3390/fermentation11120686 - 10 Dec 2025

Abstract

The safety and shelf life of wheat bread depend not only on recipe formulation and fermentation but also on post-baking handling, particularly the packaging stage. This study focused on evaluating the effect of the temperature of the bread crumb at the moment of [...] Read more.

The safety and shelf life of wheat bread depend not only on recipe formulation and fermentation but also on post-baking handling, particularly the packaging stage. This study focused on evaluating the effect of the temperature of the bread crumb at the moment of packaging (30, 40, and 45 °C) on acrylamide content and microbiological spoilage during storage. Wheat bread samples prepared with 5, 10, and 15% Lactiplantibacillus plantarum sourdough were compared to control bread without sourdough. The results revealed that packaging at elevated temperatures (40–45 °C) led to higher residual acrylamide levels and accelerated mold growth due to condensation and increased humidity inside polyethylene bags. In contrast, packaging at 30 °C significantly reduced acrylamide formation, limited microbial proliferation, and extended the shelf life of bread up to 7 days while maintaining acceptable sensory qualities. The combined effect of sourdough concentration and packaging temperature demonstrated that the optimal conditions for ensuring safety and extending shelf life are the use of 5–10% sourdough and packaging at 30 °C. These findings underline the critical role of sourdough content and packaging temperature in controlling chemical contaminants and microbiological spoilage in bread production. Full article

(This article belongs to the Section Fermentation for Food and Beverages)

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