Editor’s Choice Articles

In this study, we cloned and characterized three bacterial laccases from strains of the species Pediococcus parvulus, Lacticaseibacillus paracasei, and Lactococcus lactis isolated from wine and cheese and evaluated their biogenic amine degradation abilities in the presence/absence of artificial 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) or natural (epicatechin) mediator compounds. Although some recombinant bacterial laccases have been characterized and found to be biological tools for degrading biogenic amines with or without the use of mediators, no prior research has investigated the role of natural mediators, like phenolic substrates found in wine and certain vegetable foods, in the degradation of biogenic amines. The three recombinant bacterial laccases exhibited sigmoidal kinetics and had similar molecular mass but varied in k_0.5, k_cat, and specific activity toward ABTS. They are acidophilic and have an optimal temperature of 28 °C. However, they exhibit low thermal stability at temperatures higher than 37 °C. The three laccases were capable of degrading dopamine without the use of mediators, while the other amines were not degraded. The presence of ABTS enhanced the degradation of dopamine and tyramine, but the addition of epicatechin did not improve their degradation. This study presents a comparison of the laccases’ biogenic amine-degrading efficiency using different mediators. This is the first time such a comparison has been made. Full article

This paper evaluates the effects of an alternative hopping technique, called dip hopping, on beer. This technique involves infusing hops in hot water (or in a portion of wort) and subsequently combining the infusion with the wort (after wort cooling) directly in the fermenter when the yeast is added for fermentation. The reference beers were produced employing the “traditional” late hopping technique, and the experimental beers were produced using the dip hopping technique. A variety of hops with a significant concentration of essential oil and a strain of yeast with high β-glucosidic activity capable of releasing aromatic molecules from precursors supplied by hops were used. The samples were analysed in terms of alcohol content, degree of attenuation, colour, and bitterness. Sensory analysis and gas chromatography analysis were also performed. The data showed statistically significant differences between the reference beers and the experimental beers, with the latter featuring greater hints of citrus, fruity, floral, and spicy aromas. As an overall effect, there was an increase in the olfactory and gustatory pleasantness of the beers produced with the dip hopping technique. Full article

Natural starter cultures, characterised by undefined microbiota, can contribute to the technological process, giving peculiar characteristics to artisanal fermented foods. Several species have a long history of safe use and have obtained Qualified Presumption of Safety (QPS) status from the European Food Safety Authority (EFSA), whereas others (non-QPS) could represent a potential risk for consumers’ health and must undergo a safety assessment. In this work, the biodiversity, at species and strain level, by pulsed-field gel electrophoresis (PFGE) and (GTG)₅ rep-PCR, of an undefined natural starter culture, in frozen and lyophilized form, obtained from ewe’s raw milk avoiding thermal treatment or microbial selection, was investigated. The culture was constituted by different biotypes of Enterococcus durans, Enterococcus faecium, Enterococcus faecalis, and Lacticaseibacillus paracasei. Streptococcus oralis and Streptococcus salivarius were also found, over species belonging to the Streptococcus bovis–Streptococcus equinus complex (SBSEC), like Streptococcus gallolyticus subsp. macedonicus, Streptococcus lutetiensis, and Streptococcus equinus. Molecular investigation on virulence and antibiotic resistance genes, as well as minimum inhibitory concentration (MIC) determination, revealed that all the non-QPS strains can be considered safe in the perspective of using this culture for cheesemaking. The obtainment of a natural culture directly from ewe’s raw milk bypassing thermal treatment and selection of pro-technological bacteria can be advantageous in terms of biodiversity preservation, but non-QPS microorganisms can be included in the natural starter and also in cheeses, especially in traditional ones obtained from fermenting raw milk. Following EFSA guidelines, artisanal factories should not be allowed to produce starter cultures by themselves from raw milk, running the risk of including some non-QPS species in their culture, and only selected starters could be used for cheesemaking. A revision of the criteria of QPS guidelines should be necessary. Full article

Cultivation process (CP) modeling and optimization are ambitious tasks due to the nonlinear nature of the models and interdependent parameters. The identification procedures for such models are challenging. Metaheuristic algorithms exhibit promising performance for such complex problems since a near-optimal solution can be found in an acceptable time. The present research explores a new hybrid metaheuristic algorithm built upon the good exploration of the genetic algorithm (GA) and the exploitation of the crow search algorithm (CSA). The efficiency of the proposed GA-CSA hybrid is studied with the model parameter identification procedure of the E. coli BL21(DE3)pPhyt109 fed-batch cultivation process. The results are compared with those of the pure GA and pure CSA applied to the same problem. A comparison with two deterministic algorithms, i.e., sequential quadratic programming (SQP) and the Quasi-Newton (Q-N) method, is also provided. A more accurate model is obtained by the GA-CSA hybrid with fewer computational resources. Although SQP and Q-N find a solution for a smaller number of function evaluations, the resulting models are not as accurate as the models generated by the three metaheuristic algorithms. The InterCriteria analysis, a mathematical approach to revealing certain relations between given criteria, and a series of statistical tests are employed to prove that there is a statistically significant difference between the results of the three stochastic algorithms. The obtained mathematical models are then successfully verified with a different set of experimental data, in which, again, the closest one is the GA-CSA model. The GA-CSA hybrid proposed in this paper is proven to be successful in the collaborative hybridization of GA and CSA with outstanding performance. Full article

Succinic acid (SA), a C4 tricarboxylic acid cycle intermediate, is used as raw material for bulk chemicals and specialty chemicals, such as tetrahydrofuran and 1,4-butanediol, as well as also being used to synthesize the biodegradable biopolymers PBS (polymer poly (butylene succinate)). Actinobacillus succinogenes, which is facultative anaerobic and gram-negative, is one of the most promising natural SA-producing organisms, but genetic engineering of A. succinogenes is rare so far. In this study, a series of engineered strains was constructed using the pLGZ922 expression vector and a cytosine base editor (CBE) based on CRIPSR/Cas9; we found that phosphoenolpyruvate carboxylase (PEPC) was more important for the CO₂ fixation pathway than pyruvate carboxylase (PYC) in A. succinogenes, and the annotated oxaloacetic acid decarboxylase (Asuc_0301 and Asuc_0302) had little correlation with the SA synthesis pathway. The by-product pathway was closely related to cell growth, and overexpression of FDH was beneficial to growth, while the knockout of the ackA gene reduced the growth. For the first time, the hypothetic sugars and SA transporters were mined and identified in A. succinogenes, of which Asuc_0914 was responsible for glucose uptake, and Asuc_0715 and Asuc_0716 constituted SA exporters. This deepens the understanding of SA biosynthesis in A. succinogenes and is also valuable for SA production by fermentation. Full article

A model predictive control (MPC) method was investigated as a route to optimize and control the growth of E. coli BL21 (DE3) and biosynthesis of two different recombinant proteins (nerve growth factor NGF and coat protein of bacteriophage Qβ (Qβ-CP)). To determine the target trajectory for the E. coli cultivation process and estimate the model parameters, the off-line run-to-run optimization method was used. The proven method allowed us to successfully control the growth of microbial biomass, with a deviation of 6–12% from the target trajectory. It was proven that it is possible to obtain a “Golden Batch” profile for the implementation of MPC using datasets from only four to eight fermentation runs. The method showed its robustness when the cultivation of E. coli was carried out with two different titrant supply control systems—volumetric and gravimetric. Furthermore, the MPC method exhibited high adaptability, reliability, and resistance to various types of disturbances. MPC proved to be a reliable and effective method for controlling the cultivation and recombinant protein biosynthesis of fast-growing microorganisms such as E. coli. Full article

Several emerging mosquito control technologies require mass releases of adult male mosquitoes. Previous studies resulted in the generation of a laboratory female-specific larvicidal yeast strain targeting the GGT gene, which facilitated the laboratory sex separation of male Culex quinquefasciatus mosquitoes. Global deployment of this yeast-based sex-separation technology requires engineering second generation yeast strains which can be used in industrial-scale fermentations to support global mosquito control programs. In this study, the RNA-guided Cas-CLOVER system was used in combination with piggyBac transposase to generate robust Saccharomyces cerevisiae strains with multiple integrated copies of the insecticidal GGT shRNA expression cassette. Top expressing Cas-CLOVER strains killed Culex quinquefasciatus female larvae which consumed the yeast, facilitating male sex separation. Scaled fermentation resulted in kilogram-scale production of the yeast, which can be heat-killed and dried for global deployment to mosquito mass-rearing facilities. Full article

Acidic sophorolipids (Acidic SL), congeners of sophorolipid biosurfactants, offer a potential alternative to synthetic sodium lauryl ether sulphate (SLES) in skincare applications. However, major challenges associated with the laboratory-based investigations of the cytotoxic effects of Acidic SL have been the utilisation of impure and/or poorly characterised congeners as well as the use of monolayers of skin cells in in vitro assays. While the former limitation makes glycolipids less attractive for use in academic research and skincare applications, the latter does not provide an accurate representation of the in vivo human skin. The present study, therefore, for the first time, assessed the cytotoxic effects of 96% pure Acidic SL on a 3D in vitro skin model in comparison with SLES, with the aim of investigating a natural alternative to synthetic surfactants for potential use in skincare applications. The 3D in vitro skin model was colonised with Staphylococcus epidermidis for 12 h, and afterwards treated with either Acidic SL or SLES at 100 μg mL⁻¹ for a further 12 h. Subsequently, the cytotoxic effects of Acidic SL in comparison with SLES were assessed using a combination of microbiology, molecular biology techniques, immunoassays, and histological analyses. It was demonstrated that Acidic SL had no deleterious effects on the viability of S. epidermidis, tissue morphology, filaggrin expression, and the production of inflammatory cytokines in comparison to SLES. These findings, in conjunction with the possibility to produce Acidic SL from cheaper renewable natural resources, demonstrate that Acidic SL could offer a potential sustainable alternative to synthetic surfactants. Full article

The yeast Kluyveromyces marxianus has shown the potential to produce $\beta$-fructofuranosidases, which are enzymes capable of hydrolyzing $\beta$-fructofuranosides links of fructans to obtain fructooligosaccharides. The thriving market for fructose syrup and the quality standards imposed by food and pharmaceutical industries have generated an increased search for improved, monitored, and controlled production processes. Monitoring $\beta$-fructofuranosidase activity in a bioprocess requires the use of adequate sensors and the processing of information using efficient software algorithms; nevertheless, currently, such a sensor does not exist for this purpose. In this contribution, a sequential injection analysis system (SIA) developed in our laboratory was adapted to monitor at-line $\beta$-fructofuranosidase activity produced by the yeast K. marxianus. Samples were taken out automatically from the bioreactor and analyzed using 3,5-dinitrosalicylic (DNS). An algorithm was designed to operate the overall components of the lab-made SIA system. The enzymatic activity error obtained with the automatic SIA compared to the off-line laboratory determinations varied from 0.07% at high enzyme concentrations to 20.39% at low $\beta$-fructofuranosidase activity. Further development is required to improve the performance of the lab-made SIA system; nevertheless, such a device must be considered as a potential method for monitoring $\beta$-fructofuranosidase activity in real time. Full article

Understanding the energy metabolism and its regulation is one of the clues to metabolic engineering of stress-resistant lignocellulose-converting microbial strains, also including the promising ethanologen Zymomonas mobilis. Z. mobilis is an obligately fermentative, facultatively anaerobic bacterium, carrying an active respiratory chain with low energy-coupling efficiency. Its respiration does not supply energy to aerobically growing cultures on sugary media, yet oxidative phosphorylation has been demonstrated in non-growing cells with ethanol. Here, we show, for the first time, that in respiring, non-growing Z. mobilis cells receiving regular small amounts of ethanol, oxidative phosphorylation significantly contributes to the maintenance of their viability. No improvement of viability is seen in the NADH dehydrogenase (ndh)-deficient respiratory mutant, which is unable to oxidize ethanol. The ethanol effect is also hampered by the protonophoric uncoupler CCCP, or the inhibitor of ATP synthase, DCCD. At higher concentrations (6% v/v), ethanol causes stress that slows down culture growth. By monitoring the activity of several respiratory gene promoters under ethanol stress with the green fluorescent protein reporter system, we demonstrate downregulation of these promoters, in particular the ndh promoter. We speculate that the decrease in respiratory chain activity in response to stress conditions mitigates the production of reactive oxygen species. Full article

A growing number of science and design scholars and design practitioners have recently embarked on studying fermentation processes to produce alternative materials. The main driver of this trend is the search for a sustainable future by proposing novel alternatives that could substitute or integrate into society’s current production and consumption models. This study presents the development of an open-source bioreactor capable of enhancing and optimizing a symbiotic culture of bacteria and yeast (SCOBY) production process. The bioreactor is part of a greater design-driven project aiming to process edible and non-edible materials. The study presents the experiments and methods that led to the development and refinement of the current bioreactor, and all the information needed to replicate it with tools and equipment currently available under the Creative Commons status. The aim of sharing open-source methods and results to reproduce the bioreactor is to support different interdisciplinary teams of scientists and designers in generating high amounts of SCOBY, accelerating R&D with this auspicious yet underexplored source of bacterial cellulose. Full article

As a life-essential coenzyme, nicotinamide adenine dinucleotide (NAD⁺) has been explored for more than a century. In Saccharomyces, the natural NAD⁺de novo biosynthetic pathway initiating from tryptophan has been well elucidated. To bypass this stringently controlled natural pathway in yeast, an economical C3N pathway that was developed in Escherichia coli previously was constructed in Saccharomyces as a short detour for de novo NAD⁺ biosynthesis. After the functional expressions of the C3N genes were identified in Saccharomyces cerevisiae BY4741 by in vitro enzymatic assays, the C3N module was introduced into an NAD⁺ auxotrophic S. cerevisiae strain BY01, in which the BNA2 gene encoding tryptophan 2,3-dioxygenase was inactivated. The efficient NAD⁺ synthesis via the C3N pathway was confirmed by both plate assays and fermentation analysis. The applicability of the C3N pathway in cofactor engineering was tested by introducing it into S. cerevisiae BY4741, which improved the cellular NAD(H) level considerably. Consequently, this study proved that the de novo NAD⁺ biosynthetic pathway can be replaced by an artificial pathway in yeast, which paves a way to design more promising schemes in eukaryotes for rational manipulation of the cellular NAD(H) levels. Full article

Cocoa pod husks (CPH) and cocoa bean shells (CBS) are the main by-products of the cocoa industry and a source of bioactive compounds. These residues are not completely used and thrown in the fields without any treatment, causing environmental problems. Looking for a holistic valorization, the aim of this work was first to deeply characterize CPH and CBS in their chemical composition, amino acid, and fatty acid profiles, as well as their application as antioxidants. CBS had a high level of protein (17.98% DM) and lipids (16.24% DM) compared with CPH (4.79 and 0.35% DM respectively). Glutamic acid and aspartic acid were the predominant amino acids. The total phenolic compounds (TPC) detected in the ethanolic extracts of CPH and CBS were similar to pyrogallol as the main detected polyphenol (72.57 mg/L). CBS ethanolic extract showed a higher antioxidant activity than CPH. Both extracts increased the oxidation stability of soybean oil by 48% (CPH) and 32% (CBS). In addition, alkaline pretreatment of CPH was found suitable for the release of 15.52 ± 0.78 g glucose/L after subsequent saccharification with the commercial enzyme Cellic^®. CTec2. Alkaline hydrolyzed and saccharified CPH (Ahs-CPH) was assessed for the first time to obtain polyhydroxy alkanoate (PHAs) and bioethanol. Ahs-CPH allowed the growth of both Cupriavidus necator DSM 545 and Saccharomyces cerevisiae Fm17, well-known as PHA- and bioethanol-producing microbes, respectively. The obtained results suggest that such agricultural wastes have interesting characteristics with new potential industrial uses that could be a better alternative for the utilization of biomass generated as million tons of waste annually. Full article

Gasifiers produce a gaseous mixture of CO/CO₂/H₂, also known as synthesis gas (syngas), containing varying compositions and ratios depending on the lignocellulose material types, gasifier design, and gasification conditions. Different physicochemical and thermodynamic properties of each gas type in the various syngas blends can influence syngas fermentation performance for the production of chemicals such as acetate. This study examined the effect of syngas composition (CO, CO/H₂, CO/CO₂/H₂, and CO/H₂) and its corresponding ratio on acetate production using Moorella thermoacetica, a thermophilic homoacetogen as the biocatalyst. We also investigated the effect of yeast extract addition for enhancing acetate production. A syngas fermentation study performed at a total pressure of 19 psig (2.29 atm) demonstrated that syngas fermentation in the absence of CO (30%CO₂/70%H₂) or at low CO proportions (21%CO/24%CO₂/55%H₂) resulted in the highest volumetric productivity of acetate (0.046 ± 0.001 and 0.037 ± 0.001 g/L/h, respectively). Interestingly, syngas fermentation without CO reached the highest Y_P/X of 22.461 ± 0.574 g-acetate/g-biomass, indicating that more acetate was produced compared to cell biomass. Higher biomass production was obtained when the CO proportion was increased up to 75% in CO/H₂ fermentation. However, the cell growth and acetate production dramatically decreased with increasing CO proportion up to 99.5% CO as the sole constituent of the syngas. Even so, acetate production using 99.5% CO could be improved by adding 2 g/L yeast extract. Full article

This work aimed at producing lactic acid (LA) from sugarcane bagasse after steam explosion at 195 °C for 7.5 and 15 min. Enzymatic hydrolysis was carried out with Cellic CTec3 and Cellic HTec3 (Novozymes), whereas fermentation was performed with Bacillus coagulans DSM2314. Water washing of pretreated solids before enzymatic hydrolysis improved both hydrolysis and fermentation yields. The presence of xylo-oligosaccharides (XOS) in substrate hydrolysates reduced hydrolysis efficiency, but their effect on fermentation was negligible. The presence of fermentation inhibitors in C5 streams was circumvented by adsorption on activated carbon powder with no detectable sugar losses. High carbohydrates-to-LA conversions (Y_p/s) of 0.88 g·g⁻¹ were obtained from enzymatic hydrolysates of water-washed steam-exploded materials that were produced at 195 °C, in 7.5 min, and the use of centrifuged-but-never-washed pretreated solids decreased Y_p/s by 16%. However, when the detoxified C5 stream was added at a 10% ratio, Y_p/s was raised to 0.93 g·g⁻¹ for an LA productivity of 2.55 g·L⁻¹·h⁻¹. Doubling the pretreatment time caused a decrease in Y_p/s to 0.78 g·g⁻¹, but LA productivity was the highest (3.20 g·L⁻¹·h⁻¹). For pretreatment at 195 °C for 7.5 min, the elimination of water washing seemed feasible, but the use of longer pretreatment times made it mandatory to eliminate fermentation inhibitors. Full article

The performance of a microbial fermentation on an industrial scale is subjected to the robustness of the strain. Such strains are genetically engineered to optimize the production of desired compounds in minimal time, but they often fail to maintain high productivity levels for many generations, hindering their effective application in industrial conditions. This study focused on assessing the impact of genomic instability in yeasts that were engineered to produce a fluorescent output by incorporating a reporter gene at one or more genomic locations. The fermentation performance of these strains was evaluated over 100 generations in a sequential batch set-up. In order to bridge the gap between strain engineering and industrial implementation, we proposed the use of novel, host-specific parameters to standardize the strain robustness and evaluate potential improvements. It was observed that yeasts carrying multiple copies of the reporter gene exhibited a more pronounced decrease in output, and the genomic integration site significantly influenced the production. By leveraging these new, host-specific parameters, it becomes possible to anticipate strain behavior prior to incurring substantial costs associated with large-scale production. This approach enhances the economic viability of novel microbial fermentation processes and narrows the divide between laboratory findings and industrial applications. Full article

The high loading of lactic acid bacteria (LAB) present in cheese whey still limits its use as hydrogen feedstock. This study aims to investigate the feasibility of producing hydrogen from acid cheese whey via lactate-driven dark fermentation (LD-DF). Mesophilic batch fermentations were performed with delipidated acid cheese whey at a fixed pH of 5.8 and driven by an acidogenic bacterial culture containing LAB and lactate-oxidizing hydrogen producers (LO-HPB). The results obtained indicated that it is technically feasible to produce hydrogen from undiluted cheese whey through lactate oxidation-mediated fermentation. It was elucidated that the acidogenic fermentation of cheese whey followed a two-step lactate-type fermentation, in which fermentable carbohydrates were first converted into lactate, and then lactate was metabolized into hydrogen with the co-production of butyrate. The hydrogen yield and the maximum volumetric hydrogen production rate achieved were 44.5 ± 2.9 NmL/g-COD_fed and 1.9 NL/L-d, respectively. Further microbial community analysis revealed that Lactobacillus, Clostridium, and Klebsiella were the dominant bacterial genera when the hydrogen production rate peaked. It was therefore suggested that the metabolic potential behind the association between LAB and LO-HPB was important in driving the two-step lactate-type fermentation. Overall, the LD-DF can be a strategic hydrogen-producing pathway to be implemented with cheese whey. Full article

Temperature plays a significant role in anaerobic digestion (AD) as it affects the microbial communities and ultimately controls the efficiency of the process. Few studies have looked at temperature-adjusted AD, but it is unclear how the temperature shifts affect biogas production and the dynamics of microorganisms involved in methanogenesis. This study tested two temperature shift scenarios in fed-batch mode using anaerobically digested sewage sludge and glucose-based substrate. The first scenario was acclimatized to upshifting temperatures from 42 °C to 48 °C while the second was acclimatized to downshifting temperatures from 55 °C to 45 °C. Both temperature shift scenarios resulted in a decrease in biogas production, especially at 45 °C. The upshifted scenario experienced a maximum decrease of 83%, and the downshifted scenario experienced a 16–33% decrease in methane production. Next-generation 16S rRNA sequencing revealed the domination of Methanoculleus in the upshifted scenario. However, a low correlation between the number of Methanoculleus and the other hydrogenotrophic methanogens to biogas production indicates inhibition in the hydrogenotrophic pathway. The downshifted scenario showed better biogas production due to the substantial domination of acetoclastic Methanosaeta and the low abundance of sulfate-reducing bacteria. Hence, the temperature shift affects the microbial communities, significantly affecting biogas production performance. Full article

Gas fermentation is an upcoming technology to convert gaseous substrates into value-added products using autotrophic microorganisms. The hydrogen-oxidizing bacteria Cupriavidus necator efficiently uses CO₂ as its sole carbon source, H₂ as electron donor and O₂ as electron acceptor. Surplus CO₂ is stored in microbial storage material poly-(R)-3-hydroxybutyrate. O₂ supply is the most critical parameter for growth and poly-(R)-3-hydroxybutyrate formation. A narrow O₂ optimum between ~0.2 and ~4 mg/L was previously reported. Here, a standard benchtop bioreactor was redesigned for autotrophic growth of C. necator on explosive mixtures of CO₂, H₂ and O₂. The bioreactor was equipped with mass flow control units and O₂ and CO₂ sensors. A controller for automated gas dosage based on a mathematical model including gas mass transfer, gas consumption and sensor response time was developed. Dissolved O₂ concentrations were adjusted with high precision to 1, 2 and 4% O₂ saturation (0.4, 0.8 and 1.5 mg/L dissolved O₂, respectively). In total, up to 15 g/L cell dry weight were produced. Residual biomass formation was 3.6 ± 0.2 g/L under all three O₂ concentrations. However, poly-(R)-3-hydroxybutyrate content was 71, 77 and 58% of the cell dry weight with 1, 2 and 4% dissolved O₂, respectively. Full article

The production of cow manure far exceeds the quantity that can be utilized in primary applications such as fertilizer or for the generation of biogas. As a result, alternative value-added applications are being investigated. The purpose of this study is to evaluate the production of lactic acid, using cow manure as the raw material. The methodology involved the implementation of thermochemical pretreatment for the cow manure, followed by simultaneous saccharification and fermentation for lactic acid production. Response surface methodology based on a central composite design was employed to analyze the simultaneous saccharification and fermentation process. The factorial design of the experiments was carried out with three factors, cow manure concentration, temperature, and enzyme concentration, with 80 g·L⁻¹, 50 °C, and 212.5 IU/gCM_{Dry Matter} as central point values, respectively. Following the addition of Bacillus coagulans DSM2314 inoculum to enzymatically hydrolyzed cow manure at pH 5.0, after a 24 h period the concentration of lactic acid was recorded at 13.65 g·L⁻¹, with a conversion efficiency of 33.1%. Studies were conducted until 48 h to analyze time impact. Characterization studies for native cow manure and that pretreated using acid reagent were conducted. Sugar content and by-product formation were analyzed, resulting in 23.24 g·L⁻¹ of sugar remaining as the maximum after fermentation, while low values of furfural (1.04 g·L⁻¹), 5-hydroxymethylfurfural (1.35 g·L⁻¹), and acetic acid (1.45 g·L⁻¹) were found. Optimal conditions were calculated at 24 and 48 h with R software, obtaining the lactic acid, with yields of 13.4 g·L⁻¹, 36.28% (for 24 h) and 15.27 g·L⁻¹, 32.76% (for 48 h), respectively. Experimental and statistical studies of enzymatic hydrolysis and fermentation stated that cow manure was a feasible substrate for the production of lactic acid. Full article

Type II diabetes is the most important health issue in the whole world. Besides the use of prescribed drugs to control blood glucose level, recently, the development of health supplements is being actively explored. Owing to its high nutritional value, Momordica cochinchinensis Spreng. (Gac) is potentially a good source for developing this supplement. In recent years, the aril of Gac has been utilized as a substrate for developing various forms of supplements, but the pulp has been neglected as a byproduct. However, the pulp contains lots of phytochemicals that could provide health benefits, and the investigation using lactobacilli to ferment the pulp juices to lower blood glucose is not yet to be explored. Therefore, we set out to investigate the potential to develop the pulp-based juices for controlling blood glucose level by selecting an optimal strain of lactobacillus to ferment the pulp juice and measuring the inhibitory action of the fermented juice on α-glucosidase. This enzyme is crucial for controlling postprandial glucose absorbed into the bloodstream because it is the enzyme that hydrolyzes the carbohydrates to release glucose. First, we have successfully isolated a strain of lactobacillus which was capable of fermenting the pulp to produce α-glucosidase-inhibitory activity. Through a 16S rRNA sequence, this lactobacillus was named Lactiplantibacillus plantarum GBI 001. The optimal conditions for its growth in commercial culture medium were found to be 35 °C for 16 h to produce the highest α-glucosidase activity (72.03%). The optimal conditions for the strain to grow in Gac pulp juice were: 20% pulp juices as substrate with an initial pH adjusted to 4.0, growing at 35 °C for 16 h. Under these conditions, the fermented juice exhibited α-glucosidase activity of 24.36%, which is a 2.17-fold increase over the control group (11.23%). From its increase in α-glucosidase potency, using L. plantarum GBI 001 to ferment the pulp juices of Gac as soft drinks has great potential to develop a helpful drink as a food supplement to control postprandial blood glucose in patients with diabetes. Full article

A combined usage of animal and plant proteins-mixture could aid to solve environmental and social problems arising from the use of animal protein alone, while also improving the taste and texture of plant protein. Protein mixtures could be a better protein source due to the high availability of amino acids in the body compared with single proteins. Consuming proteins with probiotics can provide more beneficial health effects by helping to hydrolyze protein and absorb amino acids in the body. In this study, coadministration of an animal and plant protein mixture with a high concentration of probiotics was investigated to increase protein digestibility and amino acids absorbability in a mice model. Lactiplantibacillus plantarum IDCC 3501, which has the maximum ability to hydrolyze a protein mixture, composed of soybean protein and milk protein, was selected, and the changes in mice (C57BL/6J, male, six weeks) were investigated after the coadministration of protein mixture and 5 × 10⁸ or 5 × 10⁹ CFU/mL of L. plantarum for eight weeks. Normal diet, high-protein diet (HPD), and HPD supplementing L. plantarum were separately administered to mice. Food and water consumption of the mice did not differ depending on diet type. Measurements of the serum concentrations of amino acids showed that the absorption of aspartate, glutamate, isoleucine, leucine, valine, and lysine increased when high concentrations of protein and probiotics were administered. Thus, high L. plantarum concentrations could be a protein diet supplementation to improve health by promoting the absorption of amino acids. Full article

Cancer is a significant health burden in the world. Natural product drugs have received widespread attention because of their safety and effectiveness, stable effects and fewer side effects. Some studies have demonstrated that exopolysaccharide (EPS) from lactic acid bacteria (LAB) can inhibit the growth of many types of cancer cells. In this work, the effects of the EPS from Lactiplantibacillus plantarum YT013 on gastric cancer cells were investigated. Its cytotoxicity was evaluated by MTT assay; at the concentration of 1000 μg/mL, the most significant inhibitory effect occurred in AGS cells, followed by SGC-7901, PANC-1 and HCT116, and less inhibited in HepG2 cells. Cell cycle results showed that EPS prevented AGS cells from transitioning from the S phase to the G₂/M phase. In addition, the results of flow cytometry showed that EPS promoted apoptosis in a concentration-dependent manner. Western blotting also indicated that EPS might lead to apoptosis via the endogenous mitochondrial apoptotic pathway. The safety of lyophilized powder, cell-free culture supernatant and EPS from Lactiplantibacillus plantarum YT013 were evaluated by observing tissue organs through H&E staining, and the results showed that the components were safe and effective and could provide a basis for the development of natural anticancer active drugs. Full article

Momordica charantia (MC; commonly known as bitter melon) was fermented with Leuconostoc mesenteroides MKJW, MKSR, and KCTC 3719 (LM), and Leuconosoc citreum KCTC 3526 (LC), and their anti-diabetic, anti-dementia, and antioxidant activities were evaluated. The fermentation was performed for 24 h at 30 °C, and non-fermented MCs (CON1 and CON2) were included for comparison. All fermented MCs produced lactic acids, mannitol, dextran, and oligosaccharides. The highest amount of mannitol (34.76 mg/mL) and lactic acids (10.42 mg/mL) were produced in MKSR-MC, and the highest amount of dextran (22.37%) was produced in MKJW-MC. MKSR-MC showed complete α-glucosidase inhibition (99.91%), but it did not show a significant change in α-amylase inhibition (24.43%) compared to non-fermented MC (20.14%) (p > 0.05). It was also high in acetylcholinesterase inhibition (55.24%) compared to other fermented MCs (31.21–44.46%). Fermentation increased butyrylcholinesterase inhibition, but no significant differences were observed among the groups. Therefore, our results indicated that MKSR-fermented MC might be used as a non-dairy probiotic plant extract to achieve multi-health functional activities such as anti-diabetic, anti-dementia, and antioxidant activities. Full article

This study evaluated the hydrolysis and acidogenesis of food waste at different operating pHs (uncontrolled, 5.5, 6.5, 7.5, 8.5) in a leachate bed reactor (LBR) at room temperature. LBR operation at pH 6.5–8.5 resulted in a hydrolysis yield of 718–729 g SCOD/kg VS_added, which was statistically (p ≤ 0.05) higher than that obtained at pH 5.5 (577 g SCOD/kg VS_added) and the uncontrolled pH (462 g SCOD/kg VS_added). The hydrolysis rate at pH 6.5 was the highest amongst all the pH values. Stabilization at pH at 6.5 also resulted in a high fatty acid (FA) yield of 643 g COD_FA/kg VS_added. Butyrate was the main FA at the pH of 5.5–6.5, while acetate was the main FA at the pH of 7.5–8.5. At the uncontrolled pH, lactate production was the highest, indicating a shift in the microbial community from fatty-acid-producing bacteria to lactate-producing bacteria. The compositions of medium-chain fatty acids, such as caproate, were the highest at pH of 5.5. Full article

In humans and animals, probiotics are widely accepted as crucial for host health and growth. The investigation of the probiotic colonization and expression of probiotics in the host is beneficial for proper usage of probiotics and isolation of indigenous probiotics. In this study, we analyzed commonly used probiotic strains in the intestines/rumen of humans and animals by analyzing metagenomic and paired meta-transcriptomic data from the gut or rumen microbiome of humans (n = 13), pigs (n = 6), chickens (n = 6), cattle (n = 14), sheep (n = 10), and mice (n = 8). First, we generated an expression profile based on 192 selected representative probiotic strains from a published database. A total of 58 probiotic strains were not detected in any samples, while 3 strains were presented and expressed in all individuals. Overall, the probiotic expression of probiotics as detected by meta-transcriptome was significantly higher than the relative abundance of probiotic as detected by metagenomics in cattle, sheep, mice, and humans; however, this difference was not significant in pigs and chickens. In total, 17 (cattle), 21 (sheep), 22 (pig), 14 (chicken), 13 (mouse), and 3 (human) probiotic strains were identified as probiotic strains with significantly higher expression levels [Fold Change (FC) ≥ 2, False Discovery Rate (FDR) ≤ 0.05]. Among them, Clostridium butyricum TOA was found to be significantly expressed in the rumen or gut of all host species. In addition, network analysis based on the expression of probiotics as detected by meta-transcriptomics revealed that several probiotic strains were significantly negatively linked with Salmonella spp., Mycoplasma spp., and Escherichia coli. The results in this study provide a useful reference for developing indigenous probiotics. Full article

Dry fermented sausage innovation trends are linked to consumer preferences for clean label and sodium-reduced foods. This study aims to evaluate the effect of the formulation and production process temperature on the dynamics of bacterial communities in fuet-type dry fermented sausages using metataxonomics. Six fuet batches were manufactured, including formulations without and with the addition of nitrifying salts (replaced or not by pork liver auto-hydrolysate as a colouring agent), processed at 3 to 12 °C, and a partial replacement of NaCl by KCl, processed at 12 °C. Fermentation was performed spontaneously or by a starter culture. Physicochemical characterisation and culture-dependent and independent bacterial analyses were performed at day 0, 4 and 12, at the end of ripening (a_w < 0.90) and after storage. Temperature was the most important factor determining the change in pH, a_w and lactic acid bacteria levels while the presence of a starter culture promoted a pH decrease. Metataxonomic analysis showed that low temperature processes and the absence of nitrifying salts allowed the growth of spoilage-related species, while sausages submitted to a mild temperature containing a starter culture and nitrifying salts showed less bacterial diversity. Liver auto-hydrolysate added putative probiotic species to the product. This study provides valuable information to manufacturers who want to innovate safely. Full article

To improve the nutri-functional quality and, in particular, the antioxidant capacity of corn by-products, fermentation with selected lactic acid bacteria was carried out. To this purpose, white-1 and 2 and yellow corn by-products were fermented and then extracted. In all the samples, the fermentation process shows an improvement in antioxidant activity in comparison to non-fermented by-products. It was observed that the yellow corn by-product extracts have a higher content of total phenols, especially after fermentation with P. pentosaceus, while for white-1 corn by-product extracts, an increment of antioxidant capability was noticed when fermented with L. plantarum. The antioxidant capacity was measured with DPPH and ABTS⁺ assays, showing that yellow corn extracts are more active in comparison with white-1 and white-2 ones. Moreover, L. plantarum and P. pentosaceus provided the best results in increasing the antioxidant activity in all the samples. Analyzing lipid peroxidation in the presence of fermented white-2 corn by-product extracts, we observed an inhibition of the process after treatment with L. citreum compared to the non-fermented control. In all the analyzed samples, through LC-DAD-ESI/MS analysis, the antioxidant dicoumaroyl spermidine (DCS) was detected. The abundance of antioxidant molecules was higher in samples fermented with P. pentosaceus, confirming previous observations. Full article

FODMAPs are fermentable oligo-, di-, monosaccharides, and polyols. The application of homofermentative lactic acid bacteria (LAB) has been investigated as a promising approach for producing low-FODMAP whole-wheat bread. The low-FODMAP diet is recommended to treat irritable bowel syndrome (IBS). Wheat flour is staple to many diets and is a significant source of fructans, which are considered FODMAPs. The reduction of fructans via sourdough fermentation, generally associated with heterofermentative lactic acid bacteria (LAB), often leads to the accumulation of other FODMAPs. A collection of 244 wild-type LAB strains was isolated from different environments and their specific FODMAP utilisation profiles established. Three homofermentative strains were selected for production of whole-wheat sourdough bread. These were Lactiplantibacillus plantarum FST1.7 (FST1.7), Lacticaseibacillus paracasei R3 (R3), and Pediococcus pentosaceus RYE106 (RYE106). Carbohydrate levels in flour, sourdoughs (before and after 48 h fermentation), and resulting breads were analysed via HPAEC-PAD and compared with whole-wheat bread leavened with baker’s yeast. While strain R3 was the most efficient in FODMAP reduction, breads produced with all three test strains had FODMAP content below cut-off levels that would trigger IBS symptoms. Results of this study highlighted the potential of homofermentative LAB in producing low-FODMAP whole-wheat bread. Full article

The use of alternative ingredients in the production of kombucha has seen a recent increase. Our research aimed to characterize the chemical, nutritional, microbial, and aromatic profiles of kombucha beverages prepared with strawberry tree (Arbutus unedo) fruits fermented with three different SCOBYs for 21 days. The analyses showed similar levels of microbiological groups (aerobic mesophilic microorganisms, lactic acid bacteria, acetic acid bacteria, and yeasts)among the SCOBYs used. The beverages studied displayed a decrease in pH value and carbohydrate content, and protein degradation was also observed as fermentation progressed. However, the increase in total phenolic compounds during the first week proved to be a point of interest. A total of 20 volatile organic compounds were detected, giving different sensory qualities to the beverages: higher ethanol, benzaldehyde-4-ethyl, or acetic acid depending on the SCOBY used. The results obtained indicated that strawberry tree kombucha might be an alternative beverage with notable nutritional and aromatic properties, with fermentation time and SCOBY composition being identified as crucial factors. Full article

Non-Saccharomyces wine yeasts are not only proposed to improve the sensory profile of wine but also for several distinctive promising features. Among them, biocontrol action at different steps of the wine production chain could be a suitable strategy to reduce the use of sulfur dioxide. In this work, the activity of a selected strain of Metschnikowia pulcherrima was evaluated as inoculum in cold clarification with the aim to reduce SO₂ and improve the aromatic profile of the wine. Fermentation processes were carried out at the winery level for two consecutive vintages using a pied de cuve as the starter inoculum coming from indigenous Saccharomyces cerevisiae strains. M. pulcherrima revealed an effective bio-protectant action during the pre-fermentative stage even if the timely and appropriate starter inoculum in the two years permitted the effective control of wild yeasts during the fermentation also in the control trials. In general, the main oenological characters did not show differences if compared with an un-inoculated trial, while the inoculum of M. pucherrima in cold clarification determined an enhancement of ethyl hexanoate, isobutanol, acetaldehyde, and geraniol even if they are considered in different amounts for each year. Indeed, the analytical and sensory profiles of wines were also influenced by the vintage and variation pied the cuve population. Nonetheless, the overall results indicated that M. pulcherrima led to biocontrol action and an improvement of the aromatic and sensory profile of the wine. Full article

The popularity of non-alcoholic beers has been increasing over the past few years. Maltose-negative strains of different genera are frequently used to obtain beers of low alcohol content. S. cerevisiae hybrids with other Saccharomyces species offer interesting inherited flavour characteristics; however, their use in non-alcoholic beer production is rare. In this work, we constructed six hybrids of maltose-negative S. cerevisiae parental strains (modified to produce higher amounts of organic acids) and S. mikatae (wild-type). Growth behaviour, osmotolerance and fermentation features of the offspring were compared with parental strains. One hybrid with mitochondrial DNA inherited from both parents was used to produce non-alcoholic beer in which organic metabolites were evaluated by HPLC and HS-SPME-GC-MS. This hybrid produced non-alcoholic beer (≤0.05% (v/v)) with an increased organic acid content, just as its parent S. cerevisiae, but without producing increased amounts of acetic acid. The beer had a neutral aromatic profile with no negative off-flavours, similar to the beer produced by the parent S. mikatae, which was used for the first time to produce non-alcoholic beer. Overall, both parents and hybrid yeast produced non-alcoholic beers with increased amounts of higher alcohols compared with esters. Full article

Amid trends in non-dairy probiotic foods and functional coffees, it is timely to develop a high-count probiotic, fermented coffee beverage. Here, we aimed to enhance the viabilities of different probiotic lactobacilli strains in coffee brews by co-culturing with the probiotic yeast, Saccharomyces boulardii CNCM-I745. The growth, survival, and metabolic activities of Lactiplantibacillus plantarum 299v, Lactobacillus acidophilus NCFM, Limosilactobacillus fermentum PCC, and Lactobacillus gasseri LAC-343 were monitored when cultured individually or co-cultured in coffee brews with S. boulardii CNCM-I745. In co-cultures, all four probiotic lactobacilli maintained viable populations above 5.5 Log CFU/mL for at least 6 months at 4 and 25 °C. In contrast, singly cultured lactobacilli populations generally could not be detected beyond 3 months of storage at either temperature. In co-cultures, vigorous nutrient uptake (glucose, glutamate, and alanine) by the yeast limited lactic acid accumulation by the lactobacilli. Co-culturing also led to accumulations in yeast-derived metabolites (ethanol, 2/3-methylbutanol, 2,3-dimethoxystyrene, and decanoic acid), and lactobacilli-derived metabolites (4-ethylphenol), but the coffee bioactive components (caffeine, trigonelline, and 5-O-caffeoylquinic acid) and antioxidant capacities were maintained. Overall, S. boulardii CNCM-I745 is effective in enhancing the viabilities of probiotic lactobacilli from different species, which may be useful in developing shelf-stable probiotic foods. Full article

By-products from different industries could represent an available source of carbon and nitrogen which could be used for bioethanol production using conventional Saccharomyces cerevisiae yeast. Spent cherry brine and whey are acid food by-products which have a high organic matter content and toxic compounds, and their discharges represent significant environmental and economic challenges. In this study, different combinations of urea, yeast concentrations, and whey as a nutrient source were tested for bioethanol production scale-up using 96-well microplates as well as 7.5 L to 100 L bioreactors. For bioethanol production in vials, the addition of urea allowed increasing the bioethanol yield by about 10%. Bioethanol production in the 7.5 L and 100 L bioreactors was 73.2 g·L⁻¹ and 103.5 g·L⁻¹ with a sugar consumption of 81.5% and 94.8%, respectively, using spent cherry brine diluted into whey (200 g·L⁻¹ of total sugars) supplemented with 0.5 g·L⁻¹ urea and 0.5 g·L⁻¹ yeast at 30 °C and a pH of 5.0 after 96 h of fermentation for both systems. The results allow these by-products to be considered low-economic-value alternatives for fuel- or food-grade bioethanol production. Full article

Fermented foods may confer several benefits to human health and play an important role in a healthy and balanced diet. Vinasse hairtail is a farmhouse-fermented food product with cultural and economic significance to locals in Zhoushan China. It is traditionally produced and subjected to 0–8 days of fermentation. In this study, we aimed to characterize the microbiota and physicochemical profiles of vinasse hairtail across different stages of fermentation. With the increase of fermentation time, pH, total sugar content, reducing sugar content, fat content, salt content, total protein content, myofibrillar protein content, TVB-N, and TBARS index increased, while the peroxide value decreased. The addition of vinasse significantly intensified the lipid and protein oxidation and protein degradation of hairtail, thereby increasing the flavor of its products. The microbial diversity and succession characterization during the fermentation of vinasse hairtail by high-throughput sequencing was measured. Results showed that Firmicutes was the predominant phylum and Lactobacillus was the main genera of bacterial diversity. Ascomycota was the main phylum of fungi and the main fungal genera detected in the samples were Saccharomyces. Additionally, the correlation between microbial community and physicochemical properties was found. Our study revealed that Lactobacillus was the major lactic acid bacteria present throughout the fermentation process. The results may provide a theoretical basis for improving the overall quality of vinasse hairtail. Full article

In this study, the aroma-production profiles of seven different Hanseniaspora strains, namely H. guilliermondii, H. meyeri, H. nectarophila, H. occidentalis, H. opuntiae, H. osmophila and H. uvarum were determined in a simultaneous co-inoculation with the wine yeast Saccharomyces cerevisiae Champagne Epernay Geisenheim (Uvaferm CEG). All co-inoculated fermentations with Hanseniaspora showed a dramatic increase in ethyl acetate levels except the two (H. occidentalis and H. osmophila) that belong to the so-called slow-evolving clade, which had no meaningful difference, compared to the S. cerevisiae control. Other striking observations were the almost complete depletion of lactic acid in mixed-culture fermentations with H. osmophila, the more than 3.7 mg/L production of isoamyl acetate with H. guilliermondii, the significantly lower levels of glycerol with H. occidentalis and the increase in certain terpenols, such as citronellol with H. opuntiae. This work allows for the direct comparison of wines made with different Hanseniapora spp. showcasing their oenological potential, including two (H. meyeri and H. nectarophila) previously unexplored in winemaking experiments. Full article

Talaromyces albobiverticillius 30548, a marine-derived fungus, produces Monascus-like azaphilone red/orange pigments which have the potential for various industrial applications. The objective of this study was to scale up pigment production in a 2 L bioreactor with a working volume of 1.3 L media and to compare its biomass growth and pigment production against small volume (500 mL) shake flasks with 200 mL working volume. Additionally, fungal morphology, pigment intensity, fermentation length and duration of pigment production were also compared. Experiments were carried out at laboratory scale in 200 mL shake flasks without controlling pH and oxygen. In parallel, fermentation was performed in a 2 L bioreactor as an initial scale-up to investigate the influence of dissolved oxygen, agitation speed and controlled pH on pigment production and biomass growth of T. albobiverticillius 30548. The highest orange and red pigment production in bioreactor at 24 °C was noticed after 160 h of fermentation (70% pO₂) with 25.95 AU 470 nm for orange pigments and 22.79 AU 500 nm for red pigments, at pH set point 5.0. Meanwhile, the fermentation using 200 mL shake flasks effectively produced orange pigments with 22.39 AU 470 nm and red pigments with 14.84 AU 500 nm at 192 h under the same experimental conditions (24 °C, pH 5.0, 150 rpm). Regarding fungal morphology, growth of fungus in the bioreactor was in the form of pellets, whereas in the shake flasks it grew in the form of filaments. From the observed differences in shake flasks and closed bioreactor, it is known that the bioprocess was significantly influenced by dissolved oxygen saturation and agitation speed in scale-up. Thus, oxygen transfer appears to be the rate-limiting factor, which highly influences overall growth and production of pigments in Talaromyces albobiverticillius 30548 liquid culture. Full article

Cheese whey management and disposal is a major issue for dairy industries due to its high level of chemical and biochemical oxygen demand. However, it can still represent a source of nutrients (i.e., sugars, proteins and lipids) that can be applied, among other options, as substrate for microbial growth. Yarrowia lipolytica can grow in different environments, consuming both hydrophilic and hydrophobic substrates, and tolerates high salt concentrations. In this work, the lipolytic and proteolytic profile of 20 strains of Y. lipolytica were tested on caseins and butter. Then, their growth potential was evaluated in four types of whey (caciotta, ricotta, squacquerone and their mix). Y. lipolytica showed a very strain-dependent behavior for both hydrolytic profiles and growth capabilities on the different substrates. The best growers for all the types of whey tested were PO1, PO2, and RO2, with the first one reaching up to 8.77 log cfu/mL in caciotta whey after 72 h. The volatile molecule profile of the samples incubated with the best growers were characterized by higher amounts of esters, acids, ketones and alcohols. In this way, cheese whey can become a source of microbial cultures exploitable in the dairy sector. Full article

Laetiporus sulphureus, a wood-decaying basidiomycete, produces yellow-orange pigments in fruiting bodies and, as was recently shown, in submerged cultivated mycelia. Out of four strains, the most potent laetiporic acid producer was identified and its yield compared in different media. The complex Moser b medium was replaced by potato dextrose broth, achieving higher yields at a lower cost. Cultivation was then scaled up from shake flask to a 7 L stirred tank bioreactor. Optimization of parameters led to increased product concentrations up to 1 g L⁻¹, the highest yield reported so far. An in situ product recovery strategy with a biphasic system was established, increasing the yield by 19% on the shake flask scale. A crude ethanolic extract of the biomass was examined for color stability and application trials. In contrast to what has been suggested in the past, the pigment showed limited long-term stability to oxygen and light, but was stable under storage in the dark at 4 °C under nitrogen. The orange extract was successfully incorporated into different matrices like foods, cosmetics and textiles. Laetiporic acid can potentially replace petrochemical based synthetic dyes, and can thus support the development of a circular bioeconomy. Full article

Hanseniaspora vineae and Hanseniaspora opuntiae are apiculate yeasts normally found on the skins of ripe grapes and at the beginning of alcoholic fermentation. Several studies have reported that these species can provide interesting sensory characteristics to wine by contributing high levels of acetate esters and can increase the mouthfeel and body of wines. The present work aims to evaluate the use of these two species sequentially with Saccharomyces cerevisiae to improve the sensory profile of Albillo Mayor white wines. The fermentations were carried out in triplicate in 150 L stainless steel barrels. At the end of the alcoholic fermentation polysaccharides, colour, and an extensive study of the aromatic profiles were measured. Results showed up to 1.55 times higher content of 2-phenylethanol in H. opuntiae wines and up to three times higher concentration of fermentative esters in H. vineae wines than in the controls. Interestingly, it should be noted that the compound safranal was identified only in the H. vineae wines. These results indicated that the species studied are an interesting bio-tool to improve the aromatic profile of Albillo Mayor white wines. A novel non-targeted NMR-based metabolomics approach is proposed as a tool for optimising wine productions with standard and sequential fermentation schemes using apiculate yeast strains due to its discriminant capacity to differentiate fine features between wine samples from the identical geographical origin and grape variety but diverse fermentations or vintages. Full article

To mitigate the various problems caused by using conventional plastics, bioplastic (BP) has emerged as a substitute for plastics. BP wastes after use are commonly treated using composting, causing many environmental problems. Anaerobic digestion (AD) has become prominent as an alternative method of producing renewable energy. The aim of this study was to estimate the methane production yield (MPY) of BPs (polylactic acid (PLA) and polyhydroxyalkanoate (PHA)) with mechanical pretreatment (particle size < 0.5 cm) and investigate the effect of co-digestion of BPs and food waste (FW). Batch experiments were conducted under mesophilic and thermophilic conditions at various mixing ratios (FW/PLA or PHA = 95:5 and 90:10 on a weight basis). During 20 d of digestion at temperatures of 37 and 55 °C, MPYs of PHA were 153.8–172.0 mL CH₄/g chemical oxygen demand (COD), but that of PLA was significantly low (<25.6 mL CH₄/g COD). Higher MPYs were attained at 55 °C than at 37 °C. The synergistic effects of FW addition on BP AD were observed at both temperatures, especially at 55 °C. By comparing theoretical (based on mono-digestion results) and actual (based on co-digestion results) MPYs, the synergistic effect of FW addition on MPY of co-digestion reached 8.5–26.6% and 12.7–25.5% for PLA- and PHA-fed tests, respectively. The biodegradation rates (on a volatile solids (VS) basis) of PLA and PHA were 6.0–13.7% and 49.1–52.3% and increased by 1.8–4.3 and 1.2–1.5 times in the PLA- and PHA-fed co-digestion tests, respectively. Co-digestion of FW might be a feasible treatment option for BPs combined with simple mechanical pretreatment. Full article

Large-scale microalgae cultivation for biofuel production is currently limited by the possibility of maintaining high microalgae yield and high lipid content, concomitantly. In this study, the physiological changes of Chlorella vulgaris NIES 227 during lipid accumulation under nutrient limitation was monitored in parallel with the photosynthetic capacity of the microalgae to fix carbon from the proxy of oxygen productivity. In the exponential growth phase, as the biomass composition did not vary significantly (approx. 53.6 ± 7.8% protein, 6.64 ± 3.73% total lipids, and 26.0 ± 9.2% total carbohydrates of the total biomass dry-weight), the growth capacity of the microalgae was preserved (with net O₂ productivity remaining above (4.44 ± 0.93) × 10⁻⁷ g O₂·µmol PAR⁻¹). Under nutrient limitation, protein content decreased (minimum of approx. 18.6 ± 6.0%), and lipid content increased (lipid content up to 56.0 ± 0.8%). The physiological change of the microalgae was associated with a loss of photosynthetic activity, down to a minimum (1.27 ± 0.26) × 10⁻⁷ g O₂·µmol PAR⁻¹. The decrease in photosynthetic O₂ productivity was evidenced to correlate to the cell internal-protein content (R² = 0.632, p = 2.04 × 10⁻⁶, N = 25). This approach could serve to develop productivity models, with the aim of optimizing industrial processes. Full article

To date, there are very few data regarding new efficient probiotics’ development with their own prebiotic substrate. All commercial products contain prebiotic substrate that was previously purified from external sources and added to the final product. The present study describes Weissella confusa strain fermentations in media with different anthocyanin concentrations from Hibiscus sabdariffa L., in order to increase the exopolysaccharide (EPS) yield, leading to augmented probiotic and prebiotic properties. The extracted and purified EPS were characterized by Gel permeation chromatography, Fourier-transform infrared, and nuclear magnetic resonance spectroscopy; thermal analysis measurements and the whole fermented media’s probiotic properties were evaluated by testing low pH and bile salt resistance, along with hydrophobicity and auto-aggregation capacity. The anthocyanins increased biomass and EPS yields and the high EPS molecular mass improved nutrient access by allowing a good microbial suspension in media. The confirmed dextran structure provides media biocompatibility and very good probiotic properties compared with existing literature. Simultaneously, the anthocyanins in media protected the strain cells against low pH and bile salt compared with the control fermentation. These very good results show that the whole fermented culture media is suitable for further in-vitro and in-vivo studies regarding its probiotic and prebiotic activity. Full article

This study investigated the impacts of different brown seaweed species—Ascophyllum nodosum, Sargassum fulvellum, Ecklonia maxima, Lessonia flavicans, Lessonia nigrescens, and Laminaria japonica—on rumen fermentation and methane (CH₄) mitigation. The current in vitro batch culture study for 24 h at 39 °C evaluated these species in two experimental designs: as feed additive and as feed. The control group for both experimental designs was composed of 500 mg of basal diet (50% grass hay/50% concentrate). For the feed additives experimental design, each seaweed species was evaluated when it was added at 20% of the basal diet, while as a feed, the inclusion level of each species was 20% to partially replace the concentrate in the basal diet as follows (50% hay/30% concentrate/20% seaweed). Chemical analyses showed that the seaweeds were characterized by a high fiber content and high amounts of minerals such as calcium, potassium, and phosphorus, while the protein content ranged within 7 and 13%. When they were applied as feed additives, they increased the production of volatile fatty acids, with L. japonica being the most effective; however, they failed to suppress CH₄ production. In contrast, their inclusion as a feed in the basal diet led to a significant reduction (p < 0.05) in CH₄, especially for E. maxima and L. japonica, by up to 18 and 21%, respectively, but this was associated with general inhibition of the rumen fermentation. Therefore, the tested seaweeds could be used as a source of minerals and as a feed additive to improve rumen fermentation, but without anti-methanogenic potential. Meanwhile, their inclusion as feed at 20% could reduce CH₄ production with an adverse effect on fermentation. Thus, further trials are needed to identify the appropriate inclusion level to achieve effective CH₄ reduction without any detrimental effects on rumen fermentation. Full article

Brewers’ spent grain or BSG is a fiber and protein rich food-grade side stream that has remained underutilized due to its poor technological and sensory characteristics. In this study, BSG was fermented with Weissella confusa A16 in presence of sucrose to induce the synthesis of dextran and maltosyl-isomaltooligosaccharides. Fermented BSG with or without the above polysaccharides was used as ingredient in wheat bread. Digestion of BSG breads was simulated in vitro with Simulator of Human Intestinal Microbial Ecosystem, and levels of fecal metabolites were analyzed. Enrichment of BSG breads with in situ dextran and maltosyl-isomaltooligosaccharides improved the baking quality compared to native BSG. Metabolism of free amino acids and synthesis of short chain fatty acids varied at different stages and parts of colon. The increase in butyric acid was similar in both the proximal and distal colon. In situ dextran and maltosyl-isomaltooligosaccharides, and higher content of proteins and fiber in BSG breads had a positive influence towards gut microbiota functionality. Along with several essential amino acids, an increase in amount of γ-aminobutyric acid was also observed after simulated digestion. BSG breads had a significant effect on the gut metabolome during in vitro digestion, showing increased production of microbial metabolites with potential health benefits. Full article

In recent years, there has been increasing consumer interest and research into plant-based dairy alternatives, due to the increasingly negative impact of animal products on human health, animal welfare, and the environment. The purpose of this study was to investigate the physicochemical and microbiological changes in a Camembert alternative based on the seeds of sweet lupine (Lupinus angustifolius L cv. ‘Boregine’). After heat treatment and homogenization, the seeds were incubated with lactic acid bacteria (LAB) and Penicillium camemberti mold. After fermentation at room temperature, the samples were stored at 12 °C for 14 days, and then ripened until day 49 at 6 °C. Changes in microbial population, acidity, texture, content of polyphenols, flavonoids, reducing sugars, and free amino acids were monitored. In addition, the antioxidant capacity of the samples during ripening was determined. The results showed that LAB and fungi were able to grow well in the lupine matrix. Initially, a decrease in pH was observed, while in the further stages of ripening, alkalization of the product linked with progressive proteolysis associated with an increase in free amino acid content was noted. Hydrolysis of polysaccharides and an increase in antioxidant activity were observed. This indicates the potential of lupine seeds as a raw material for the development of a new group of plant-based ripened cheese alternatives. Full article

Efficient bioconversion of abundant waste glycerol to value-added chemicals calls for a wider range of fermentative workhorses that can catabolize glycerol. In this study, we used quantitative gene expression and solvent profiling, qualitative metabolite analysis, and enzyme activity assays to investigate the factors that limit glycerol utilization as a sole carbon source by Clostridium beijerinckii NCIMB 8052. C. beijerinckii NCIMB 8052 did not produce acetate, acetone and butanol on glycerol. Congruently, the genes encoding the coenzyme A transferase subunits (ctfAB) and bifunctional acetaldehyde-CoA/alcohol dehydrogenase (adhE) were down-regulated up to 135- and 21-fold, respectively, at 12 h in glycerol-grown cells compared to glucose-grown cells. Conversely, NADH-dependent butanol dehydrogenase A (bdhA) was upregulated 2-fold. Glycerol dehydrogenase (gldA) and dihydroxyacetone kinase (subunit dhaK) were upregulated up to 5- and 881-fold, respectively. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) showed mostly similar expression profiles at 12 h on glucose and glycerol. At 24 h, gapdh was downregulated 1.5-fold, while NADP⁺-dependent gapdh was upregulated up to 1.9-fold. Glycerol-grown cells showed higher or similar activity profiles for all solventogenic enzymes studied, compared to glucose-grown cells. Butyraldehyde (3 g/L) supplementation led to the production of ~0.1 g/L butanol, whilst butyrate (3.5 g/L) supplementation produced 0.7 and 0.5 g/L acetone and butanol, respectively, with glycerol. Further, the long chain saturated fatty acids cyclopentaneundecanoic acid, methyl ester and hexadecanoic acid, butyl ester were detected in glucose- but not in glycerol-grown cells. Collectively, growth on glycerol appears to disrupt synthesis of saturated long chain fatty acids, as well as solventogenesis in C. beijerinckii NCIMB 8052. Full article

Intensive horticultural systems for the production of vegetables in greenhouses represent one of the main industries generating organic waste, as those that do not meet the quality standards for the fresh market or the processing industry are discarded. This highlights the importance of using these residues as raw material for other applications, such as bioenergy and bioproducts production, within the framework of a bio-based economy that maximizes the utilization of biomass resources in a sustainable manner. In this work, the microbial oil production from discarded pepper using the oleaginous yeast Cryptococcus curvatus was evaluated. Overall, a total lipid accumulation of 16.8 g/L was achieved with a fatty acid profile suitable to produce biodiesel. The lipid yield obtained was 0.12 g/g sugars. In addition, experimental results were used to assess the techno-economic feasibility of a proposed microbial oil plant using the software Aspen Plus. This plant yields approximately 96 kg of microbial oils/ton dry discarded pepper, with an estimated Minimum Selling Price of 7 €·kg⁻¹. These figures point out the necessity of increasing the yield of microbial oil production and considering the utilization of possible by-products, such as mannitol and cell debris, to improve the economic performance of the process. Full article

The need for alternative energy sources is constantly growing worldwide, while the focus has shifted to the valorization of biomass. The aim of the present study was to determine the optimal pH and hydraulic retention time (HRT) values for treating a mixture of sorghum biomass solution with liquid cow manure (in a ratio 95:5 v/v) through anaerobic digestion, in a two-stage system. Batch tests were initially carried out for the investigation of the pH effect on bio-hydrogen and volatile fatty acids (VFA) production. The highest hydrogen yield of 0.92 mol H₂/mol carbohydrates_consumed was obtained at pH 5.0, whereas the maximum degradation of carbohydrates and VFA productivity was observed at pH 6.0. Further investigation of the effect of HRT on hydrogen and methane production was carried out. The maximum yield of 1.68 mol H₂/mol carbohydrates_consumed was observed at an HRT of 5 d, with H₂ productivity of 0.13 L/L_R·d. On the other hand, the highest CH₄ production rate of 0.44 L/L_R·d was achieved at an HRT of 25 d, with a methane yield of 295.3 mL/g VS_added, whereas at a reduced HRT of 20 d the process exhibited inhibition and/or overload, as indicated by an accumulation of VFAs and decline in CH₄ productivity. Full article

Non-Saccharomyces yeasts represent a very attractive alternative for the production of beers with superior sensory quality since they are able to enhance the flavour of beer. Furthermore, they can produce beers with low ethanol content due to the weak fermentative capacity of a large percentage of non-Saccharomyces species. The objective of this study was to evaluate the ability of 34 non-Saccharomyces yeast strains isolated from Madrilenian agriculture to produce a novel ale beer. The non-Saccharomyces yeast strains were screened at two scales in the laboratory. In the first screening, those with undesirable aromas were discarded and the selected strains were analysed. Thirty-three volatile compounds were analysed by GC, as well as melatonin production by HPLC, for the selected strains. Thirteen strains were then fermented at a higher scale in the laboratory for sensory evaluation. Only yeast strains of the species Schizosaccharomyces pombe and Lachancea thermotolerans were able to complete fermentation. Species such as Torulaspora delbrueckii, Metschnikowia pulcherrima, Wickerhamomyces anomalus, Hanseniaspora vineae, and Hanseniaspora guilliermondii could be used both for production of low ethanol beers and co-fermentation with a Saccharomyces yeast to improve the organoleptic characteristics of the beer. In addition, for these strains, the levels of melatonin obtained were higher than the concentrations found for Saccharomyces strains subjected to the same study conditions. The selected strains can be used in future trials to further determine their viability under different conditions and for different purposes. Full article