Characterization of Two Zymomonas mobilis Wild Strains and Analysis of Populations Dynamics during Their Leavening of Bread-like Doughs.

Two Zymomonas mobilis wild strains (UMB478 and 479) isolated from water kefir were characterized for their biomass production levels and leavening performance when used as the inoculum of a real bread-like dough formulation. The obtained baked product would be consumable by people with adverse responses to Saccharomyces cerevisiae. In liquid cultures, the two strains reached similar biomass concentration (0.7 g CDW/L). UMB479 showed an interesting resistance to NaCl (MBC 30 g/L), that may be useful in the bakery sector. When inoculated in doughs, UMB479 produced the maximum dough volume (650 mL) after 5 h, glucose was almost consumed and 1 g/100 g of ethanol produced, +200% respective to UMB478. Using S. cerevisiae for comparison purposes, the dough doubled its volume fast, in only 2 h, but reached a final level of 575 mL, lower than that achieved by Z. mobilis. The analysis of bacterial and fungal population dynamics during dough leavening was performed through the Automated Ribosomal Intergenic Spacer Analysis (ARISA); doughs leavened by UMB479 showed an interesting decrease in fungal richness after leavening. S. cerevisiae, instead, created a more complex fungal community, similar before and after leavening. Results will pave the way for the use of Z. mobilis UMB479 in commercial yeast-free leavened products.

Mild Pretreatments to Increase Fructose Consumption in Saccharomyces cerevisiae Wine Yeast Strains.

The present research investigates the effect of different pretreatments on glucose and fructose consumption and ethanol production by four Saccharomyces cerevisiae wine strains, three isolated and identified from different wine regions in Turkey and one reference strain. A mild stress temperature (45 °C, 1 h) and the presence of ethanol (14% v/v) were selected as pretreatments applied to cell cultures prior to the fermentation step in synthetic must. The goodness fit of the mathematical models was estimated: linear, exponential decay function and sigmoidal model were evaluated with the model parameters R2 (regression coefficient), RMSE (root mean square error), MBE (mean bias error) and χ2 (reduced Chi-square). Sigmoidal function was determined as the most suitable model with the highest R2 and lower RMSE values. Temperature pretreatment allowed for an increase in fructose consumption rate by two strains, evidenced by a t90 value 10% lower than the control. One of the indigenous strains showed particular promise for mild temperature treatment (45 °C, 1 h) prior to the fermentation step to reduce residual glucose and fructose in wine. The described procedure may be effective for indigenous yeasts in preventing undesirable sweetness in wines.

Assessment of anammox, microalgae and white-rot fungi-based processes for the treatment of textile wastewater.

The treatability of seven wastewater samples generated by a textile digital printing industry was evaluated by employing 1) anammox-based processes for nitrogen removal 2) microalgae (Chlorella vulgaris) for nutrient uptake and biomass production 3) white-rot fungi (Pleurotus ostreatus and Phanerochaete chrysosporium) for decolorization and laccase activity. The biodegradative potential of each type of organism was determined in batch tests and correlated with the main characteristics of the textile wastewaters through statistical analyses. The maximum specific anammox activity ranged between 0.1 and 0.2 g N g VSS-1 d-1 depending on the sample of wastewater; the photosynthetic efficiency of the microalgae decreased up to 50% during the first 24 hours of contact with the textile wastewaters, but it improved from then on; Pleurotus ostreatus synthetized laccases and removed between 20-62% of the colour after 14 days, while the enzymatic activity of Phanerochaete chrysosporium was inhibited. Overall, the findings suggest that all microbes have great potential for the treatment and valorisation of textile wastewater after tailored adaptation phases. Yet, the depurative efficiency can be probably enhanced by combining the different processes in sequence.

From cheese whey permeate to Sakacin-A/bacterial cellulose nanocrystal conjugates for antimicrobial food packaging applications: a circular economy case study.

Applying a circular economy approach, this research explores the use of cheese whey permeate (CWP), by-product of whey ultrafiltration, as cheap substrate for the production of bacterial cellulose (BC) and Sakacin-A, to be used in an antimicrobial packaging material. BC from the acetic acid bacterium Komagataeibacter xylinus was boosted up to 6.77 g/L by supplementing CWP with ß-galactosidase. BC was then reduced to nanocrystals (BCNCs, 70% conversion yield), which were then conjugated with Sakacin-A, an anti-Listeria bacteriocin produced by Lactobacillus sakei in a CWP based broth. Active conjugates (75 Activity Units (AU)/mg), an innovative solution for bacteriocin delivery, were then included in a coating mixture applied onto paper sheets at 25 AU/cm2. The obtained antimicrobial food package was found effective in reducing Listeria population in storage trials carried out on a fresh Italian soft cheese (named "stracchino") intentionally inoculated with Listeria. Production costs of the active material have been mainly found to be associated (90%) to the purification steps. Setting a maximum prudential 50% cost reduction during process up-scaling, conjugates coating formulation would cost around 0.89 €/A4 sheet. Results represent a practical example of a circular economy production procedure by using a food industry by-product to produce antimicrobials for food preservation.

Yeast-Free Doughs by Zymomonas mobilis: Evaluation of Technological and Fermentation Performances by Using a Metabolomic Approach.

This research focuses on the leavening performances and development of volatile compounds of three strains of Zymomonas mobilis in the production of yeast-free doughs. Z. mobilis DSM 3580, 424, and 473 were used in doughs supplemented with glucose and with or without NaCl. Z. mobilis produced about 10 mg ethanol/g dough, with maximum dough volumes (640-680 mL) being reached after 2 h leavening. NaCl addition postponed this parameter up to 6 h. Among organic acids, hexanoic acid resulted the highest produced compound; DSM 424 and 473 formed more propanoic, butanoic and pentanoic acid, being both negatively affected by NaCl. Esters were mainly discriminated on NaCl addition, with octanoic acid (DSM 3580), butanoic acid (DSM 424), and propanoic acid (DSM 473) ethyl esters as main components. DSM 3580 specifically produced 2-heptanal, DSM 424 2-hexadecenal, (E) and DSM 473 octanal, while DSM 424 and DSM 473 produced 2-butanone-4-hydroxy better than DSM 3580. Z. mobilis unique signatures were the production of nonanoic and undecanoic acids, 2-hexadecenal, (E), L(+)-tartaric acid diethyl ester and 3-decen-5-one, 4-methyl, (E). This outcome can pave the way for using Z. mobilis in baking goods, providing innovation possibilities in the area of yeast-free leavened products.

From Cheese Whey Permeate to Sakacin A: A Circular Economy Approach for the Food-Grade Biotechnological Production of an Anti-Listeria Bacteriocin.

Cheese Whey Permeate (CWP) is the by-product of whey ultrafiltration for protein recovery. It is highly perishable with substantial disposal costs and has serious environmental impact. The aim of the present study was to develop a novel and cheap CWP-based culture medium for Lactobacillus sakei to produce the food-grade sakacin A, a bacteriocin exhibiting a specific antilisterial activity. Growth conditions, nutrient supplementation and bacteriocin yield were optimized through an experimental design in which the standard medium de Man, Rogosa and Sharpe (MRS) was taken as benchmark. The most convenient formulation was liquid CWP supplemented with meat extract (4 g/L) and yeast extract (8 g/L). Although, arginine (0.5 g/L) among free amino acids was depleted in all conditions, its supplementation did not increase process yield. The results demonstrate the feasibility of producing sakacin A from CWP. Cost of the novel medium was 1.53 €/L and that of obtaining sakacin A 5.67 €/106 AU, with a significant 70% reduction compared to the corresponding costs with MRS (5.40 €/L, 18.00 €/106 AU). Taking into account that the limited use of bacteriocins for food application is mainly due to the high production cost, the obtained reduction may contribute to widening the range of applications of sakacin A as antilisterial agent.

Zymomonas mobilis in Bread Dough: Characterization of Dough Leavening Performance in Presence of Sucrose.

Zymomonas mobilis, because of its fermentative metabolism, has potential food applications in the development of leavened baked goods consumable by people with adverse responses to Saccharomyces cerevisiae. Since Z. mobilis is not able to utilize maltose present in flour, the effect of sucrose addition (2.5 g/100 g flour) on bread dough leavening properties was studied. For comparison purposes, leavening performances of S. cerevisiae with and without sucrose were also investigated. Doughs leavened by Z. mobilis without sucrose addition showed the lowest height development (14.95 ± 0.21 mm) and CO2 production (855 ± 136 mL). When sucrose was added, fermentative performances of Z. mobilis significantly (p < 0.05) improved (+80% and +85% of gas production and retention, respectively), with a dough maximum height 2.6 times higher, results indicating that Z. mobilis with sucrose can be leavened in shorter time with respect to the sample without addition. S. cerevisiae did not benefit the sucrose addition in terms of CO2 production and retention, even if lag leavening time was significantly (p < 0.05) shorter (about the half) and time of porosity appearance significantly (p < 0.05) longer (about 26%) with respect to S. cerevisiae alone. Results demonstrate that in the presence of sucrose, Z. mobilis can efficiently leaven a bread dough, thus providing innovation possibilities in the area of yeast-free leavened products.

Cellulose nanofiber (CNF)-sakacin-A active material: production, characterization and application in storage trials of smoked salmon.

BACKGROUND: Sakacin-A due to its specific antimicrobial activity may represent a good candidate to develop active packaging solutions for food items supporting Listeria growth. In the present study a protein extract containing the bacteriocin sakacin-A, produced by Lactobacillus sakei Lb 706 in a low-cost culture medium containing deproteinized cheese whey, was adsorbed onto cellulose nanofibers (CNFs) to obtain an active material to be used as a mat (or a separator) in direct contact with foods. RESULTS: The applied fermentation conditions allowed 4.51 g L-1 of freeze-dried protein extract to be obtained, characterized by an antimicrobial activity of near 16 700 AU g-1 , that was used for the preparation of the active material by casting. The active material was then characterized by infrared spectra and thermogravimetric analyses. Antimicrobial trials were carried out in vitro using Listeria innocua as indicator strain; results were also confirmed in vivo, employing smoked salmon fillets intentionally inoculated with Listeria innocua: its final population was reduced to about 2.5-3 Log cycles after 28 days of storage at 6 °C in presence of sakacin-A, compared with negative control mats produced without the bacteriocin extract. CONCLUSION: This study demonstrates the possibility of producing an antimicrobial active material containing sakacin-A absorbed onto CNFs to decrease Listeria population in smoked salmon, a ready-to eat-food product. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Can Zymomonas mobilis Substitute Saccharomyces cerevisiae in Cereal Dough Leavening?

Baker’s yeast intolerance is rising among Western populations, where Saccharomyces cerevisiae is spread in fermented food and food components. Zymomonas mobilis is a bacterium commonly used in tropical areas to produce alcoholic beverages, and it has only rarely been considered for dough leavening probably because it only ferments glucose, fructose and sucrose, which are scarcely present in flour. However, through alcoholic fermentation, similarly to S. cerevisiae, it provides an equimolar mixture of ethanol and CO2 that can rise a dough. Here, we propose Z. mobilis as a new leavening agent, as an alternative to S. cerevisiae, overcoming its technological limit with different strategies: (1) adding glucose to the dough formulation; and (2) exploiting the maltose hydrolytic activity of Lactobacillus sanfranciscensis associated with Z. mobilis. CO2 production, dough volume increase, pH value, microbial counts, sugars consumption and ethanol production were monitored. Results suggest that glucose addition to the dough lets Z. mobilis efficiently leaven a dough, while glucose released by L. sanfranciscensis is not so well fermented by Z. mobilis, probably due to the strong acidification. Nevertheless, the use of Z. mobilis as a leavening agent could contribute to increasing the variety of baked goods alternative to those leavened by S. cerevisiae.

Use of solid digestate for lignocellulolytic enzymes production through submerged fungal fermentation.

Studies were performed on the use of the solid fraction of digestate (D) for the production of lignocellulolytic enzymes (endo- and exo-glucanase, xylanase, ß-glucosidase and laccase) by fungi, in comparison with wheat straw (benchmark) (W). To date, this is the first report on the use of such an inexpensive substrate in a liquid environment. Submerged instead of solid state fermentation was applied to overcome pH inhibition and increase surface accessibility. A total of 21 fungal strains were tested: the most performing ones were Irpex lacteus DSM1183 for both ß-glucosidase (52 IU/g with D, + 400% compared to W) and endo-glucanase (236 IU/g with D, + 470% compared to W), Schizophyllum commune CBS30132 for xylanase (715 IU/g with W, + 145% compared to D) and Pleurotus ostreatus ATCC96997 for laccase (124 IU/g with D, +230% compared to D). Cultures from S. commune and P. ostreatus were analyzed at the beginning and at the end of the growth test to determine soluble COD, total (TS) and volatile (VS) solids. COD was always lower at the end of the test suggesting a faster uptake than hydrolysis. P. ostreatus evidenced a higher VS reduction (-11% rather than -32%), suggesting a more effective growth of this strain on D. Results may open up new avenues for the utilization of solid digestate, an inexpensive agricultural by-product, for the production of value-added products as well as to increase biodegradation of lignocellulosic materials.

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thin-cut meat: preliminary assessment.

BACKGROUND: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio). RESULTS: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm-2 ) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 °C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g-1 ). CONCLUSION: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Unconventional bacterial association for dough leavening.

The purpose of the research was to obtain innovative yeast-free doughs leavened by Zymomonas mobilis and Lactobacillus sanfranciscensis. Z. mobilis, as well as Saccharomyces cerevisiae, produces an equimolar mixture of ethanol and CO2 through glucose, fructose or sucrose fermentation. In the present work, the inability of Z. mobilis to metabolize maltose has been circumvented by the addition of L. sanfranciscensis in the formulation. Indeed, L. sanfranciscensis, a heterofermentative lactic acid bacterium (LAB) typical of sourdough environment, hydrolyzes maltose releasing glucose which can be used by Z. mobilis for its metabolism. Biomass samples of Z. mobilis subs. mobilis DSM 424 and L. sanfranciscensis DSM 20663 were grown separately in liquid media and then associated in a model dough. Leavening trials set up by using three different microbial combinations (Lactobacillus:Zymomonas 80+80mg, 15+145mg and 145+15mg biomass, i.e. 1:1, 1:10 and 10:1 respectively on cell dry weight basis) evidenced CO2 production levels (mL) higher than the mathematical sum of CO2 produced by the single bacteria. In particular, when the biomass combination of L. sanfranciscensis and Z. mobilis was 1:1 (80+80mg cdw) and 10:1 (145+15mg cdw) a CO2 production of 46.3-41.4mL versus 26.7-28.5mL was achieved. The calculated productivity gain showed positive performances of the microbial combination up to 180-240min leavening. The subsequent efficiency loss may be due several factors, above all glucose shortage for Z. mobilis, as well as decrease of dough pH that can negatively affect both Lactobacillus and Zymomonas metabolism. As in traditional sourdoughs, L. sanfranciscensis was responsible for the souring activity with positive effects on both dough tasting and reduction of spoilage microbiota; Z. mobilis was instead responsible for most of the CO2 production. A bakery product leavened with the unconventional association Z. mobilis - L. sanfranciscensis will be addressed to people having adverse responses to the ingestion of bakery goods, thus providing innovation in the area of yeast-free leavened food.

Comparative performance of enzymatic and combined alkaline-enzymatic pretreatments on methane production from ensiled sorghum forage.

This study investigated the effect of enzymatic and combined alkaline-enzymatic pretreatments on chemical composition and methane production from ensiled sorghum forage. Four commercial enzymatic preparations were tested and the two yielding the highest sugars release were added to evaluate any hydrolytic effect on both untreated and alkaline pretreated samples. In the combined alkaline-enzymatic pretreatment trials, the highest sugar release was found with Primafast and BGL preparations (added at a final concentration 0.12 and 0.20 mL/g TS, respectively), with a total monomeric content of 12 and 6.5 g/L. Fibre composition analysis confirmed that the combined alkaline-enzymatic pretreatment led to cellulose (up to 32 %) and hemicelluloses (up to 56 %) solubilisation, compared to the enzymatic pretreatment alone. BMP tests were performed on both untreated and pretreated samples, and time courses of methane production were fitted. Both enzymatic and combined alkaline-enzymatic pretreatment led to a methane production increase (304 and 362 mL CH4/g VS), compared to that of untreated sorghum (265 mL CH4/g VS), as  +15 and  +37 %, respectively. Moreover, higher specific methane production rates, compared to that of untreated sorghum (20.31 mL CH4/g VS/d), were obtained by applying the enzymatic and combined alkaline-enzymatic pretreatment (33.94 and 31.65 mL CH4/g VS/d), respectively.

Post-fermentative production of glutathione by baker's yeast (S. cerevisiae) in compressed and dried forms.

The study was aimed at investigating the best biotransformation conditions to increase intracellular glutathione (GSH) levels in samples of baker's yeast (Saccharomyces cerevisiae) employing either the commercially available compressed and dried forms. Glucose, GSH precursors amino acids, as well as other cofactors, were dissolved in a biotransformation solution and yeast cells were added (5%dcw). Two response surface central composite designs (RSCCDs) were performed in sequence: in the first step the influence of amino acid composition (cysteine, glycine, glutamic acid and serine) on GSH accumulation was investigated; once their formulation was set up, the influence of other components was studied. Initial GSH content was found 0.53 and 0.47%dcw for compressed and dried forms. GSH accumulation ability of baker's yeast in compressed form was higher at the beginning of shelf life, that is, in the first week, and a maximum of 2.04%dcw was obtained. Performance of yeast in dried form was not found satisfactory, as the maximum GSH level was 1.18%dcw. When cysteine lacks from the reaction solution, yeast cells do not accumulate GSH. With dried yeast, the highest GSH yields occurred when cysteine was set at 3 g/L, glycine and glutamic acid at least at 4 g/L, without serine. Employing compressed yeast, the highest GSH yields occurred when cysteine and glutamic acid were set at 2-3 g/L, while glycine and serine higher than 2 g/L. Results allowed to set up an optimal and feasible procedure to obtain GSH-enriched yeast biomass, with up to threefold increase with respect to initial content.