Biorefinery of brewery spent grain to obtain bioproducts with high value-added in the market.

The brewery industry is under economic and environmental pressure to minimize residual management costs, particularly brewery spent grain (BSG), which accounts for 80-85% (w/w) of the total by-products generated. BSG is a lignocellulosic material primarily composed of carbohydrates, proteins and lipids. Developing a biorefinery model for conversion of BSG into value-added products is a plausible idea. Previous work optimized the pretreatment of BSG with the ionic liquid [N1112OH][Gly] and further release of fermentable sugar-containing solutions by enzymatic hydrolysis, using an enzymatic cocktail obtained by solid-state fermentation of BSG with Aspergillus brasiliensis CECT 2700 and Trichoderma reesei CECT 2414. The current work ends the biorefinery process, studying the fermentation of these culture media with two LAB strains, Lactobacillus pentosus CECT 4023 and Lactobacillus plantarum CECT 221, from which the production of organic acids, bacteriocins, and microbial biosurfactants (mBS) was obtained. In addition to the bacteriocin activity observed, a mass balance of the whole biorefinery process quantified the production of 106.4 g lactic acid and 6.76 g mBS with L. plantarum and 116.1 g lactic acid and 4.65 g mBS with L. pentosus from 1 kg of dry BSG. Thus, BSG shows a great potential for waste valorization, playing a major role in the implementation of biomass biorefineries in circular bioeconomy.

Biorefinery of Brewery Spent Grain by Solid-State Fermentation and Ionic Liquids.

Novel environmentally friendly pretreatments have been developed in recent years to improve biomass fractionation. Solid-state fermentation (SSF) and treatment with ionic liquids show low environmental impact and can be used in biorefinery of biomass. In this work, these processes were assessed with brewery spent grain (BSG). First, BSG was used as a substrate to produce cellulases and xylanases by SSF with the fungi Aspergillus brasiliensis CECT 2700 and Trichoderma reesei CECT 2414. Then, BSG was pretreated with the ionic liquid [N1112OH][Gly] and hydrolyzed with the crude enzymatic extracts. Results showed that SSF of BSG with A. brasiliensis achieved the highest enzyme production; meanwhile, the pretreatment with ionic liquids allowed glucan and xylan fractions to increase and reduce the lignin content. In addition, a mixture of the extracts from both fungi in a ratio of 2.5:0.5 Aspergillus/Trichoderma (v/v) efficiently hydrolyzed the BSG previously treated with the ionic liquid [N1112OH][Gly], reaching saccharification percentages of 80.68%, 54.29%, and 19.58% for glucan, xylan, and arabinan, respectively. In conclusion, the results demonstrated that the BSG biorefinery process developed in this work is an effective way to obtain fermentable sugar-containing solutions, which can be used to produce value-added products.

Influence of Casein Hydrolysates and Yeast on the Rheological Properties of Wheat Dough.

The influence of casein hydrolysates (CHs) and yeast on the viscoelasticity of wheat dough at 25 °C were analysed. Three wheat doughs were studied: the unyeasted dough (UYD), the unyeasted dough with CHs (UYD-C) and the yeasted dough (YD). The characteristic parameters in the linear viscoelastic range (LVER) were analysed by stress sweep at 6.3 rad/s: UYD-C dough exhibited higher values of stress (σmax) and strain (γmax) amplitudes, and softer gel network (lower complex modulus, G*) comparing with UYD dough. The oscillatory data suggest that CHs would work as (energy and time) stabilising-agents based on the greatest reticular energy (E parameter) and the lowest frequency dependence of phase angle (δ) at the low frequency range. The rotatory tests show that CHs may act as shear thinning agents in the gluten-starch network, facilitating the solid-fluid transition at the yield point (UYD-C dough). The yeasted dough (YD) exhibited a more shear sensitive structure, evidenced in the highest influence of frequency on the elastic (G') and viscous (G″) parameters, and gel to sol transition at 0.23 rad/s was observed.

The Chemical, Microbiological and Volatile Composition of Kefir-like Beverages Produced from Red Table Grape Juice in Repeated 24-h Fed-Batch Subcultures.

The aim of this work was to study the production of kefir-like beverages via the fed-batch fermentation of red table grape juice at initial pHs of 3.99 (fermentation A) and 5.99 (fermentation B) with kefir grains during 4 repeated 24-h fed-batch subcultures. All kefir-like beverages (KLB) were characterized by low alcoholic grade (≤3.6%, v/v) and lactic and acetic acid concentrations. The beverages obtained from fermentation B had lower concentrations of sugars and higher microbial counts than the KLB obtained in fermentation A. Additionally, the KLB samples from fermentation B were the most aromatic and had the highest contents of alcohols, esters, aldehydes and organic acids, in contrast with the nonfermented juice and KLB from fermentation A. These results indicate the possibility of obtaining red table grape KLB with their own distinctive aromatic characteristics and high content in probiotic viable cells, contributing to the valorization of this fruit.

Production of a Potentially Probiotic Product for Animal Feed and Evaluation of Some of Its Probiotic Properties.

Nowadays, probiotics have been proposed for substituting antibiotics in animal feed since the European Union banned the latter compounds in 2006 to avoid serious side effects on human health. Therefore, this work aimed to produce a probiotic product for use in animal feed by fed-batch fermentation of whey with a combination of kefir grains, AGK1, and the fermented whole milk used to activate these kefir grains. The probiotic culture obtained was characterized by high levels of biomass (8.03 g/L), total viability (3.6 × 108 CFU/mL) and antibacterial activity (28.26 Activity Units/mL). Some probiotic properties of the probiotic culture were investigated in vitro, including its survival at low pH values, under simulated gastrointestinal conditions, after freezing in skim milk at -20 °C, and in the commercial feed during storage at room temperature. The viable cells of lactic and acetic acid bacteria and yeasts exhibited higher tolerance to acidic pH and simulated gastrointestinal conditions when the cells were protected with skim milk and piglet feed, compared with washed cells. The results indicated the feasibility of producing a probiotic product at a low cost with a potential application in animal feed.

Enhancing the saccharification of pretreated chestnut burrs to produce bacteriocins.

The present study aims to valorize chestnut burrs, an important lignocellulosic waste, through a biorefinery concept. A solid residue rich in glucan (41.36 ± 0.59 %) and lignin (39.06 ± 0.01 %) obtained from a previous process of pre-hydrolysis was subjected to four treatments with water or NaOH to enhance enzymatic hydrolysis. Saccharification was performed using different ratios of commercial cellulases and ß-glucosidases and at controlled pH 4.8 or 6.0 (with citrate buffer) or uncontrolled pH. Carbohydrate-rich solutions with or without nutrients were used to produce bacteriocins by Lactobacillus plantarum CECT 211. The use of NaOH at high temperatures (120 and 130 °C) was the most suitable treatment to improve saccharification. Regarding the production of bacteriocins, the best result was obtained using the enzymatic solution obtained at controlled pH 6.0, supplemented with MRS broth nutrients (except glucose). Thus, the concentrations of bacteriocins obtained in this culture medium (9.21 BU/mL) was 1.22 and 1.98 times higher than those obtained in the nutrient supplemented medium buffered at pH 4.8 (7.56 BU/mL) and in the commercial MRS broth (4.65 BU/mL), respectively. These results highlight the feasibility of the technology developed in this work.

Enzymatic hydrolysis of brewer's spent grain to obtain fermentable sugars.

Lignocellulosic biomass is a feedstock with the potential to be converted into value-added bioproducts. The use of enzymatic hydrolysis allows the cleavage of lignocellulose into their monomeric units, but there are some drawbacks that make its use in industrial biocatalysis unfeasible. In the present study, we describe the hydrolysis of brewer's spent grain (BSG) with an enzymatic cocktail produced by Aspergillus niger CECT 2700 and its comparison with commercial enzymes. In addition, it was determined whether pretreating the BSG (non-pressurized alkaline hydrolysis or treatment with cholinium glycinate ionic liquid) is necessary. Results show that both pretreatments enhanced xylose release (10.55 ±â€¯0.07 g/L and 8.14 ±â€¯0.13 g/L respectively), meanwhile the hydrolysis of raw BSG with the enzymatic cocktail produced solutions containing high levels of glucose (18.45 ±â€¯1.66 g/L) and xylose (6.38 ±â€¯0.26 g/L).

Production of a highly concentrated probiotic culture of Lactococcus lactis CECT 539 containing high amounts of nisin.

In this study, probiotic biomass and nisin productions by Lactococcus lactis CECT 539 were followed in two realkalized fed-batch cultures in diluted whey (DW) supplemented with KH2PO4 up to a total phosphorus concentration of 0.459 g/L. Increased biomass (5.12 g/L, 2.18 × 1010 CFU/mL) and nisin (235.23 BU/mL) concentrations were obtained in the culture fed concentrated whey and concentrated mussel processing waste (CMPW) medium supplemented with glucose up to a concentration of 400 g/L (CMPW + G medium) compared with similar fed-batch fermentations in DW medium. In the second fed-batch fermentation, the feeding medium CMPW + G was supplemented with KH2PO4 up to a TP concentration of 3.21 g/L. With this approach, increased production of biomass (5.49 g/L, 2.33 × 1010 CFU/mL) and nisin (257.59 BU/mL) was obtained. Considering the substantial availability of these wastes at very low prices from local dairy and mussel processing plants in Galicia, their use as culture media could offer an attractive alternative for a low-cost production of probiotic biomass and nisin at a high scale.

Simultaneous production of amylases and proteases by Bacillus subtilis in brewery wastes.

The simultaneous production of amylase (AA) and protease (PA) activity by Bacillus subtilis UO-01 in brewery wastes was studied by combining the response surface methodology with the kinetic study of the process. The optimum conditions (T=36.0°C and pH=6.8) for high biomass production (0.92g/L) were similar to the conditions (T=36.8°C and pH=6.6) for high AA synthesis (9.26EU/mL). However, the maximum PA level (9.77EU/mL) was obtained at pH 7.1 and 37.8°C. Under these conditions, a considerably high reduction (between 69.9 and 77.8%) of the initial chemical oxygen demand of the waste was achieved. In verification experiments under the optimized conditions for production of each enzyme, the AA and PA obtained after 15h of incubation were, respectively, 9.35 and 9.87EU/mL. By using the Luedeking and Piret model, both enzymes were classified as growth-associated metabolites. Protease production delay seemed to be related to the consumption of non-protein and protein nitrogen. These results indicate that the brewery waste could be successfully used for a high scale production of amylases and proteases at a low cost.

Nisin production in realkalized fed-batch cultures in whey with feeding with lactose- or glucose-containing substrates.

Nisin production by Lactococcus lactis CECT 539 was followed in batch cultures in whey supplemented with different concentrations of glucose and in two realkalized fed-batch fermentations in unsupplemented whey, which were fed, respectively, with concentrated solutions of lactose and glucose. In the batch fermentations, supplementation of whey with glucose inhibited both the growth and bacteriocin production. However, fed-batch cultures were characterized with high productions of biomass (1.34 and 1.51 g l(-1)) and nisin (50.6 and 60.3 BU ml(-1)) in comparison to the batch fermentations in unsupplemented whey (0.48 g l(-1) and 22.5 BU ml(-1)) and MRS broth (1.59 g l(-1) and 50.0 BU ml(-1)). In the two realkalized fed-batch fermentations, the increase in bacteriocin production parallels both the biomass production and pH drop generated in each realkalization and feeding cycle, suggesting that nisin was synthesized as a pH-dependent primary metabolite. A shift from homolactic to heterolactic fermentation was observed at the 108 h of incubation, and other metabolites (acetic acid and butane-2,3-diol) in addition to lactic acid accumulated in the medium. On the other hand, the feeding with glucose improved the efficiencies in glucose, nitrogen, and phosphorus consumption as compared to the batch cultures. The realkalized fed-batch fermentations showed to be an effective strategy to enhance nisin production in whey by using an appropriate feeding strategy to avoid the substrate inhibition.

Lipases and esterases from extremophiles: overview and case example of the production and purification of an esterase from Thermus thermophilus HB27.

Extremophiles are organisms that have evolved to exist in a variety of extreme environments. They fall into a number of different classes that include thermophiles, halophiles, acidophiles, alkalophiles, psychrophiles, and barophiles (piezophiles). Extremophiles have the potential to produce uniquely valuable biocatalysts that function under conditions in which usually the enzymes of their nonextremophilic counterparts could not. Among novel enzymes isolated from extremophilic microorganisms, hydrolases, and particularly lipases and esterases are experiencing a growing demand. Lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) catalyze the cleavage of ester bounds in aqueous media and the reverse reaction in organic solvents. Both lipolytic enzymes have relevant applications in food, dairy, detergent, biofuel, and pharmaceutical industries. Here, we summarize the properties of lipases and esterases from the main extremophile groups: thermophiles and hyperthermophiles, psychrophiles, halophiles, alkalophiles/acidophiles, and solvent-resistant microorganisms.We report the biomass and lipolytic activity production by Thermus thermophilus HB27 in 5-L stirred-tank bioreactor at 70°C. Suitability of thermal spring water for culture media formulation is shown. In addition, a protocol to isolate and purify a cell-bound esterase from this microorganism is described.

Production and characterization of distilled alcoholic beverages obtained by solid-state fermentation of black mulberry (Morus nigra L.) and black currant (Ribes nigrum L.).

The present study was conducted to appraise the potential of black mulberry and black currant to be used as fermentation substrates for producing alcoholic beverages obtained by distillation of the fruits previously fermented with Sacchromyces cerevisiae IFI83. In the two distillates obtained, the volatile compounds that can pose health hazards are within the limits of acceptability fixed by the European Council (Regulation 110/2008) for fruit spirits. However, the amount of volatile substances in the black currant distillate (121.1 g/hL absolute alcohol (aa)) was lower than the minimum limit (200 g/hL aa) fixed by the aforementioned regulation. The mean volatile composition of both distillates was different from other alcoholic beverages such as four commercial Galician orujo spirits, Portuguese bagaceiras, and two distillates obtained from fermented whey and blackberry. The results obtained showed the feasibility for obtaining distillates from fermented black mulberry and black currant, which have their own distinctive characteristics.

Performance and intestinal coliform counts in weaned piglets fed a probiotic culture (Lactobacillus casei subsp. casei CECT 4043) or an antibiotic.

The production of biomass and antibacterial extracellular products by Lactobacillus casei subsp. casei CECT 4043 was followed in both batch and in realkalized fed-batch cultures. Enhanced concentrations of biomass and antibacterial extracellular products were obtained with the use of the latter fermentation technique in comparison with the batch mode. The culture obtained by fed-batch fermentation was mixed with skim milk and used to prepare a probiotic feed for weaned piglets. To test the effect of the potentially probiotic culture of L. casei on body weight gain, feed intake, feed conversion efficiency, and on fecal coliform counts of piglets, two groups of animals received either feed supplemented with the probiotic preparation or avilamycin for 28 days. The control group was fed nonsupplemented feed. At the end of the administration period (day 28), the groups receiving probiotic and avilamycin exhibited the highest average body weight gain values, although the mean feed intake and feed conversion efficiency values were not different among the groups (P > 0.05). For the entire experimental period (42 days), the control group exhibited the lowest feed intake value, the probiotic group exhibited the highest feed conversion efficiency value, and the antibiotic group exhibited the highest body weight gain (P < 0.05). Interestingly, no significant difference in body weight gain was observed between the probiotic and the control groups by day 42 (P > 0.05). Fecal coliform values decreased (although not significantly) by day 28 in the three groups. However, the mean counts returned to pretreatment levels by day 42 in all groups.

Fed-batch pediocin production by Pediococcus acidilactici NRRL B-5627 on whey.

Cell growth and pediocin production by Pediococcus acidilactici NRRL B-5627 on whey were compared by using batch fermentation and re-alkalized fed-batch fermentation. The batch fermentations were performed on DWG [DW (diluted whey) supplemented with 1% (w/v) glucose], DWYE [DW supplemented with 2% (w/v) yeast extract] and DWGYE (DW supplemented with 1% glucose plus 2% yeast extract) media. The fed-batch culture on DWYE medium was fed with a mixture of concentrated whey (48 g of total sugars/l) supplemented with 2% yeast extract and 400 g/l concentrated glucose. The re-alkalized fed-batch culture was characterized by higher biomass (6.57 g/l) and pediocin [517.6 BU (bacteriocin activity units)/ml] concentrations compared with the batch processes on MRS (de Man, Rogosa and Sharpe) broth (1.76 g/l and 493.2 BU/ml), DW (0.17 g/l and 57.7 BU/ml), DWG (0.14 g/l and 53.6 BU/ml), DWYE (1.43 g/l and 187.6 BU/ml) and DWGYE (1.28 g/l and 167.3 BU/ml) media. A mixed acid fermentation was observed during the growth of P. acidilactici NRRL B-5627 in the fed-batch culture on DWYE medium, and other products (acetic acid and ethanol) in addition to lactic acid accumulated in the medium. Mathematical models were set up to describe fed-batch production of biomass and pediocin by P. acidilactici. The models developed offer a better fit and a more realistic description of the experimental biomass and pediocin production data when compared with the logistic and Luedeking and Piret model.

Production of bacteriocins from Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 using mussel-processing wastes.

The growth and bacteriocin production by Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 were investigated on mussel-processing wastes. Both bacteriocin productions were satisfactorily modelled using a modified form of the Luedeking and Piret expression, which includes a term for the influence of the pH reduction rate. Experimental data from cultures buffered at different initial concentrations (0, 0.03, 0.10 and 0.25 M) of both bacteria were used to fit and verify the model. The influence of total sugars, nitrogen, phosphorus and buffer concentration on nisin and pediocin production was also studied using response-surface methodology and empirical modelling. Enhanced nisin production (33 BU/ml) was achieved in media buffered with 0.10 M potassium hydrogen phthalate/NaOH. However, the highest levels of pediocin (368 BU/ml) were obtained in the non-buffered media.