Fermentation, Volume 5, Issue 4 (December 2019) – 20 articles

Non-alcoholic beer (NAB) is enjoying growing demand and popularity due to consumer lifestyle trends and improved production methods. In recent years in particular, research into the application of non-Saccharomyces yeasts to produce NAB via limited fermentation has gained momentum. Non-Saccharomyces yeasts are known to produce fruity aromas, owing to a high ester production. This trait could be harnessed to mask the often-criticized wort-like off-flavor of NAB produced via limited fermentation. Six Cyberlindnera strains were characterized and screened in wort extract. Four of the six strains produced a pleasant, fruity aroma while exhibiting low ethanol production. The strain Cyberlindnera subsufficiens C6.1 was chosen for fermentation optimization via response surface methodology (RSM) and a pilot-scale (60 L) brewing trial with subsequent sensory evaluation. A low fermentation temperature and low pitching rate enhanced the fruitiness and overall acceptance of the NAB. The NAB (0.36% ABV) produced on pilot-scale was significantly more fruity and exhibited a significantly reduced wort-like off-flavor compared to two commercial NABs. This study demonstrated the suitability of Cyberlindnera subsufficiens to produce a fruity NAB, which can compete with commercial NABs. The outcome strengthens the position of non-Saccharomyces yeasts as a serious and applicable alternative to established methods in NAB brewing. Full article

Eighty-seven yeasts were isolated from Bombino bianco, a white grape variety from Apulian Region (Southern Italy). The isolates were characterized for the splitting of arbutin, the hydrolysis of pectins, sulphite production, the resistance to acetic acid, SO₂, and ethanol. An enhanced arbutin splitting (β-glucosidase) and a moderate pectolytic activity were found. Concerning ethanol resistance, the most of yeast population showed a low-to-moderate resistance, but some isolates, identified as Saccharomyces cerevisiae, were able to grow in presence of 15% v/v of ethanol. Four isolates were selected (coded as 43D, 44D, 45D, and 46D), studied for their ability to decarboxylate amino acids and used in small-scale fermentation trial; for this last experiment a reference strain was used (S. cerevisiae EC1118). This experiment suggested the existence of an isolate (S. cerevisiae 46D) with interesting traits and performances, which could be potentially proposed as a starter for Bombino bianco. Full article

Non-Saccharomyces yeasts have aroused interest in brewing science as an innovative and seminal way of creating new beer flavors. A screening system for potential brewing strains of non-Saccharomyces yeasts was set up to investigate the yeast’s utilization of wort sugars and to examine the effect of hop acids as well as ethanol on the growth of different yeast strains. Additionally, phenolic off-flavor (POF) and sensory odor tests of fermented wort samples were performed. The promising strains were further investigated for their propagation ability and for following fermentation trials. The produced beers were analyzed for secondary metabolites, ethanol content and judged by trained panelists. Subsequently to the screening, it was discovered that among the 110 screened yeast strains, approx. 10 strains of the species Saccharomycopsis fibuligera, Schizosaccharomyces pombe and Zygosaccharomyces rouxii generate promising fruity flavors during fermentation and were able to metabolize maltose and maltotriose as a prerequisite for the production of alcoholic beers. Consequently, the screening method described in this study makes it possible to investigate a tremendous number of different non-Saccharomyces yeasts and to test their brewing ability in a relatively short period of time. Full article

We isolated several strains of human and animal origin, focusing on the genera Lactobacillus and Bifidobacterium. Samples of cow colostrum, feces and saliva of calves and piglets, and infant feces were acquired, bacterial strains were isolated and identified, and twenty strains of lactobacilli and bifidobacteria were shortlisted for further in vitro analysis of adhesion capacity to human cells, surface hydrophobicity, and auto-aggregation. Adhesion capacity was evaluated using a mixture of Caco-2 and HT-29-MTX cells and hydrophobicity was measured using the microbial adhesion to the hydrocarbon method. Lactobacillus reuteri was the most frequently isolated species, followed by L. casei subsp. paracasei and L. gasseri; all strains were isolated from infant feces. Bifidobacteria were represented by the species B. longum (infant feces), and B. thermophilum and B. pseudolongum (calves, piglets). All twenty strains showed good adhesion capacities to the mixed cell culture (17.7–37.2%), particularly, L. reuteri isolates K7 and K14 (37.2% and 35.5%, respectively). In hydrophobicity and auto-aggregation assays, strain-specific differences irrespective of the origin or taxonomic group were observed. Hydrophobicity values varied considerably (from 6.1% to 87.4%), whereas auto-aggregation ability ranged from 21.7% to 69.7%. No relation was observed between hydrophobicity and adhesion capacity; instead, auto-aggregation was apparently related with adhesion. Full article

The rapid growth of aquaculture and scarcity of conventional fish feed supplements has prompted the introduction of new sustainable supplementation sources. In this study, the potential of five strains of fungal biomass of Ascomycetes and Zygomycetes edible filamentous fungi, Aspergillus oryzae, Neurospora intermedia, Rizhopus oryzae, Monascus purpureus, and Fusarium venenatum, cultivated on vinasse, a by-product of the bioethanol industry, as alternative protein sources for fishmeal in the fish diet was evaluated. It was observed that 5% vinasse with an initial pH of 5–6.5 can support fungal biomass yields of 34.3 ± 2.4–118.5 ± 3.9 g DM/L for A. Oryzae, N. intermedia, and R. oryzae. High protein contents of about 44.7%, 57.6%, and 50.9% (w/w), and fat contents of 7.0%, 3.5%, and 5.5% (w/w) were obtained for A. oryzae, N. intermedia, and R. oryzae, respectively. The latter three fungi species contained noticeable amino acid contents, including promising profiles of amino acids that are highly compatible with those of fishmeal. These findings provide evidence that fungal biomasses, with their relatively high protein content, good amino acid profiles, and other essential nutrients, are a promising supplementation alternative that can be produced from low-value by-products and organic-rich waste streams like vinasse to meet the dietary protein requirements in fish feed. Full article

Food waste disposal and transportation of commodity chemicals to the point-of-need are substantial challenges in military environments. Here, we propose addressing these challenges via the design of a microbial consortium for the fermentation of food waste to hydrogen. First, we simulated the exchange metabolic fluxes of monocultures and pairwise co-cultures using genome-scale metabolic models on a food waste proxy. We identified that one of the top hydrogen producing co-cultures comprised Clostridium beijerinckii NCIMB 8052 and Yokenella regensburgei ATCC 43003. A consortium of these two strains produced a similar amount of hydrogen gas and increased butyrate compared to the C. beijerinckii monoculture, when grown on an artificial garbage slurry. Increased butyrate production in the consortium can be attributed to cross-feeding of lactate produced by Y. regensburgei. Moreover, exogenous lactate promotes the growth of C. beijerinckii with or without a limited amount of glucose. Increasing the scale of the consortium fermentation proved challenging, as two distinct attempts to scale-up the enhanced butyrate production resulted in different metabolic profiles than observed in smaller scale fermentations. Though the genome-scale metabolic model simulations provided a useful starting point for the design of microbial consortia to generate value-added products from waste materials, further model refinements based on experimental results are required for more robust predictions. Full article

In this study, we explored the diversity of yeasts and lactic acid bacteria (LAB) associated with six spontaneous sourdough fermentations from the northern part of the Apulian region (Italy). Bacterial and yeast isolates from sourdough were investigated by amplified ribosomal DNA restriction analysis (ARDRA) and restriction fragment length polymorphism (RFLP) analysis, respectively. The identification of the isolates was confirmed by sequencing bacterial 16S gene and yeast ITS1–5.8S–ITS2 rRNA gene amplicons. Microbiological analysis of all sourdough samples revealed that LAB and yeast counts ranged between 1.7 × 10⁵ and 6.5 × 10⁸ cfu/g, and 7.7 × 10⁵ and 2.5 × 10⁷ cfu/g, respectively. The molecular identification at species level revealed the occurrence of Lactobacillus plantarum as the dominant LAB and Saccharomyces cerevisiae as the dominant yeast species in all different sourdough samples. Then, the ability of all isolated strains to inhibit and/or reduce the growth of several selected fungi was valued through the overlay method. In light of their antifungal performances, ten LAB strains were inoculated, singularly and in combination, in subsequent bread-making trials. Overall, we confirmed the potential of LAB to extend the shelf life of bread through spoilage inhibition and, for the first time, we observed a synergistic effect due to the combination of several isolated LAB on the inhibition behavior against selected fungal spoilage strains. Our findings suggest the exploration of a LAB-based approach in order to extend the shelf life of bread, reducing, at the same time, the use of chemical agents for food preservation. Full article

With rapidly ageing populations, the world is experiencing unsustainable healthcare from chronic diseases such as metabolic, cardiovascular, neurodegenerative, and cancer disorders. Healthy diet and lifestyle might contribute to prevent these diseases and potentially enhance health outcomes in patients during and after therapy. Fermented dairy foods (FDFs) found their origin concurrently with human civilization for increasing milk shelf-life and enhancing sensorial attributes. Although the probiotic concept has been developed more recently, FDFs, such as milks and yoghurt, have been unconsciously associated with health-promoting effects since ancient times. These health benefits rely not only on the occurrence of fermentation-associated live microbes (mainly lactic acid bacteria; LAB), but also on the pro-health molecules (PHMs) mostly derived from microbial conversion of food compounds. Therefore, there is a renaissance of interest toward traditional fermented food as a reservoir of novel microbes producing PHMs, and “hyperfoods” can be tailored to deliver these healthy molecules to humans. In FDFs, the main PHMs are bioactive peptides (BPs) released from milk proteins by microbial proteolysis. BPs display a pattern of biofunctions such as anti-hypertensive, antioxidant, immuno-modulatory, and anti-microbial activities. Here, we summarized the BPs most frequently encountered in dairy food and their biological activities; we reviewed the main studies exploring the potential of dairy microbiota to release BPs; and delineated the main effectors of the proteolytic LAB systems responsible for BPs release. Full article

This study demonstrates that the consortium, which consists of the microbial flora of methane production (MFMP) and Clostridium cellulovorans grown with cellulose, can perform the direct conversion of cellulosic biomass to methane. The MFMP was taken from a commercial methane fermentation tank and was extremely complicated. Therefore, C. cellulovorans grown with cellobiose could not perform high degradation ability on cellulosic biomass due to competition by various microorganisms in MFMP. Focusing on the fact that C. cellulovorans was cultivated with cellulose, which is armed with cellulosome, so that it is now armed C. cellulovorans; the direct conversion was carried out by the consortium which consisted of MFMP and the armed C. cellulovorans. As a result, the consortium of C. cellulovorans grown with cellobiose and MFMP (CCeM) could not degrade the purified cellulose and mandarin orange peel. However, MFMP and the armed C. cellulovorans reduced 78.4% of the total sugar of the purified cellulose such as MN301, and produced 6.89 mL of methane simultaneously. Furthermore, the consortium consisted of MFMP and the armed C. cellulovorans degraded mandarin orange peel without any pretreatments and produced methane that was accounting for 66.2% of the total produced gas. Full article

Yeasts during alcoholic fermentation form a vast number of volatile compounds that significantly influence wine character and quality. It is well known that the capacity to form aromatic compounds is dependent on the yeast strain. Thus, the use of native yeast strains, besides promoting biodiversity, encourages the conservation of regional sensory properties. In this work, we studied the volatile profile of Malvar wines fermented with 102 Saccharomyces cerevisiae yeast strains, isolated from vineyards and cellars belonging to the D.O. “Vinos de Madrid”. The wines elaborated with different S. cerevisiae showed a good classification by cellar of origin. Additionally, seven sensory descriptors have helped to classify the wines depending on their predominant aromatic character. Twenty-nine Saccharomyces strains, belonging to five of six cellars in the study, were characterized by producing wines with a fruity/sweet character. Floral, solvent, and herbaceous descriptors are more related to wines elaborated with Saccharomyces strains from organic cellars A, E, and F. Based on these findings, winemakers may use their best native S. cerevisiae strains, which add personality to their wine. Therefore, this study contributes to promoting the use of native Saccharomyces yeasts in winemaking. Full article

The high concentration of sugars in Icewine juice causes formidable stress for the fermenting Saccharomyces cerevisiae, causing cells to lose water and shrink in size. Yeast can combat this stress by increasing the internal concentration of glycerol by activating the high osmolarity glycerol response to synthesize glycerol and by actively transporting glycerol into the cell from the environment. The H⁺/glycerol symporter, Stl1p, has been previously characterized as being glucose repressed and inactivated, despite osmotic stress induction. To further investigate the role of Stl1p in Icewine fermentations, we developed a rapid single plasmid CRISPR-Cas9-based genome editing method to construct a strain of the common Icewine yeast, S. cerevisiae K1-V1116, that lacks STL1. In an Icewine fermentation, the ∆STL1 strain had reduced fermentation performance, and elevated glycerol and acetic acid production compared to the parent. These results demonstrate that glycerol uptake by Stl1p has a significant role during osmotically challenging Icewine fermentations in K1-V1116 despite potential glucose downregulation. Full article

Nattokinase is a serine protease in the subtilisin family which is produced by Bacillus subtilis subsp. natto and exhibits vigorous fibrinolytic activity that has been suggested to be able to prevent and treat thromboembolic diseases. In this study, WTC016, a spore-forming and rod-shaped bacterium with fibrinolytic activity was successfully isolated from soil, which was identified as Bacillus subtilis subsp. natto based on morphological and physiological tests, and phylogenetic analysis of 16S rRNA and gyrA. According to the growth curve of WTC016, the nattokinase production reached the highest amount in the stationary phase. To optimize the liquid fermentation condition for nattokinase yield of WTC016, further optimal tests of four factors, including the temperature, pH, inoculum size, and loading volume, followed by orthogonal test of all these factors, was performed. The optimal fermentation conditions were determined as 30 °C, 7.0 pH, 2% inoculum size, and 60 mL of loading volume in 250 mL conical flask, which indicates the highest nattokinase production of 3284 ± 58 IU/mL while fermented for 26 h. This work laid the foundation for producing nattokinase using Bacillus subtilis subsp. natto WTC016. Full article

(1) Background: Commercial production of fuel ethanol currently uses sugarcane and corn as feedstocks. Attempts to develop other renewable feedstocks that are more abundant have led to lignocellulosic biomass, which requires pretreatment prior to enzymatic hydrolysis to generate fermentable sugars. One of the largest cost components of pretreatment is chemical cost. Ethanol fermentation also produces large quantities of CO₂ as a co-product contributing to global warming. (2) Methods: Sweet sorghum has emerged as a potential new feedstock for ethanol production. In the present study, the CO₂ produced in sweet sorghum juice (SSJ) fermentation was captured by absorption in 5 M NaOH. The resultant Na₂CO₃ solution was used for pretreatment of sweet sorghum bagasse (SSB), which is the solid residue in SSJ extraction. The pretreated SSB was fermented in SSJ to produce additional ethanol. (3) Results: CO₂ absorption efficiency of 92.0% was observed. Pretreatment of SSB by the obtained Na₂CO₃ solution resulted in no loss of glucan and only 8.1 wt% loss of xylan. Ethanol yield from glucan in the pretreated SSB was 81.7% theoretical. (4) Conclusions: CO₂ from SSJ fermentation captured as Na₂CO₃ could be used for efficient SSB pretreatment. Further study focusing on pretreatment process optimization is needed. Full article

Anhydrous ethanol is a promising alternative to gasoline in fuel engines. However, since ethanol forms an azeotrope with water, high-energy-consumption separation techniques such as azeotropic distillation, extractive distillation, and molecular sieves are needed to produce anhydrous ethanol. This work discusses the potential development of an integrated process for bioethanol production using ionic liquids and Ceratonia siliqua as a carbohydrate source for further fermentation of the aqueous extracts. A four-stage counter-current system was designed to improve the sugar extraction yield to values close to 99%. The alcoholic fermentation of the extracts showed ethanol concentrations of 95 g/L using the microorganism Saccharomyces cerevisae. The production of anhydrous ethanol through extractive distillation with ethylene glycol was simulated using CHEMCAD software, with an energy consumption of 13.23 MJ/Kg of anhydrous ethanol. Finally, several ionic liquids were analyzed and are proposed as potential solvents for the recovery of bioethanol for the design of an integrated extraction–fermentation–separation process, according to their ability to extract ethanol from aqueous solutions and their biocompatibility with the microorganism used in this study. Full article

This paper aimed to investigate the influence of certain wheat and wheat malt quality indicators on limit of attenuation of wort (LAT). The experiment was conducted using wheats that have been proven to display the best malting properties with heightened total and soluble N and very good viscosity. Standard micromalting and brewing processes and analysis were applied. The obtained results showed that the quality of analyzed malts was satisfying. Statistical analysis determined no significant correlation between the limit of attenuation of wort and any of the other analyzed quality indicators. The lack of close correlations between indicators is probably due to the extremely complex intertwine of factors influencing the LAT, pointing to the fact that this particular indicator should be observed as separate and mainly variety-dependent. Full article

The efficiency of anaerobic digestion relies upon activity of the inoculum converting organic substrate into biogas. Often, metabolic capacity of the inoculum needs to be augmented with new capabilities to accommodate changes in the substrate feed composition. However, bioaugmentation is not a widely used strategy possibly due to the lack of studies demonstrating successful applications. This study describes the bioaugmentation of granular anaerobic sludge digesting mixed algal biomass in batch-scale reactors. The addition of an algalytic bacterial mixture to the granular consortium increased methane yield by 11%. This study also investigated changes in the microbial 16SrRNA composition of the augmented and non-augmented granular inoculum, which demonstrates a significant change in the hydrolytic microbial community. Overall, the studies’ results aim to provide a feasible checklist to assess the success rates of bioaugmentation of anaerobic digestion applications. Full article

Wine production by inoculated fermentation with commercial Saccharomyces cerevisiae strains is an ordinary practice in modern winemaking in order to assure the final quality of wine, although this procedure results in the production of highly homogeneous wines. The use of indigenous selected starters represents a useful tool to control alcoholic grape must fermentation, safeguarding the typical sensory characteristics of wine produced from specific regions. In this study, we selected three indigenous S. cerevisiae strains among 16 indigenous strains previously isolated from the spontaneous fermentation of Primitivo grapes, which were collected from the vineyards of three different cellars. The three selected starters (one for each cellar) were tested during fermentations at pilot scale by performing in each cellar two trials: one with an indigenous starter (specific for the winery), and one with the commercial starter AWRI796 (common to all the cellars). Starter dominance ability and influence on aromatic quality of the wine were used as criteria to test the suitability of these indigenous starters to be used at the cellar scale. The results obtained in this study showed that the indigenous strains were characterized by very high dominance ability, and the aromatic quality of wine was strongly influenced both by the inoculated strain and the interaction strain/grape must. Full article

Saccharomyces cerevisiae populations occurring in spontaneous wine fermentations display a high polymorphism, although few strains are generally able to dominate the fermentative process. Recent studies have suggested that these indigenous S. cerevisiae strains are representative of a specific oenological ecosystem, being associated to a given wine-producing area or a single winery. In contrast, according to other ecological studies, no correlation between genotypic and phenotypic groups of the native S. cerevisiae strains and their origin was found. In this work, several S. cerevisiae strains were isolated in consecutive years from spontaneous fermentations carried out in the same wineries located in different oenological areas in Tuscany, and their persistence was assessed by molecular methods. Some predominant S. cerevisiae strains persisted in different fermentations in the same winery from one year to another and they seemed to be representative of a single winery rather than of an oenological area. Therefore, data suggested the idea of the “winery effect” or a microbial terroir at a smaller scale. The use of these typical strains as starter yeasts could provide wines with the distinctive characteristics of a particular winery or sub-zone. Full article

Climate change threatens food systems, with huge repercussions on food security and on the safety and quality of final products. We reviewed the potential of food microbiology as a source of biotechnological solutions to design climate-smart food systems, using wine as a model productive sector. Climate change entails considerable problems for the sustainability of oenology in several geographical regions, also placing at risk the wine typicity. The main weaknesses identified are: (i) The increased undesired microbial proliferation; (ii) the improved sugars and, consequently, ethanol content; (iii) the reduced acidity and increased pH; (iv) the imbalanced perceived sensory properties (e.g., colour, flavour); and (v) the intensified safety issues (e.g., mycotoxins, biogenic amines). In this paper, we offer an overview of the potential microbial-based strategies suitable to cope with the five challenges listed above. In terms of microbial diversity, our principal focus was on microorganisms isolated from grapes/musts/wines and on microbes belonging to the main categories with a recognized positive role in oenological processes, namely Saccharomyces spp. (e.g., Saccharomyces cerevisiae), non-Saccharomyces yeasts (e.g., Metschnikowia pulcherrima, Torulaspora delbrueckii, Lachancea thermotolerans, and Starmerella bacillaris), and malolactic bacteria (e.g., Oenococcus oeni, Lactobacillus plantarum). Full article

Developing integrated biorefineries requires the generation of high-value co-products produced alongside cellulosic ethanol. Most industrial yeast strains produce ethanol at high titers, but the small profit margins for generating ethanol require that additional high-value chemicals be generated to improve revenue. The aim of this research was to boost xylose utilization and conversion to high-value co-products that can be generated in an integrated biorefinery. Pretreated sweet sorghum bagasse (SSB) was hydrolyzed in sweet sorghum juice (SSJ) followed by ethanol fermentation. Ethanol was removed from the fermentation broth by evaporation to generate a stillage media enriched in xylose. Candida mogii NRRL Y-17032 could easily grow in non-detoxified stillage media, but a high xylitol yield of 0.55 g xylitol/g xylose consumed was achieved after recovered cells were resuspended in synthetic media containing supplemented xylose. Phaffia rhodozyma ATCC 74219 could be cultivated in non-detoxified stillage media, but astaxanthin generation was increased 4-fold (from 17.5 to 71.7 mg/L) in detoxified media. Future processing strategies to boost product output should focus on a two-step process where the stillage media is used as the growth stage, and a synthetic media for the production stage utilizing xylose generated from SSB through selective hemicellulase enzymes. Full article