Bioavailable Phenolic Compounds from Olive Pomace Present Anti-Neuroinflammatory Potential on Microglia Cells.

The neuroinflammatory process is considered one of the main characteristics of central nervous system diseases, where a pro-inflammatory response results in oxidative stress through the generation of reactive oxygen and nitrogen species (ROS and RNS). Olive (Olea europaea L.) pomace is a by-product of olive oil production that is rich in phenolic compounds (PCs), known for their antioxidant and anti-inflammatory properties. This work looked at the antioxidant and anti-neuroinflammatory effects of the bioavailable PC from olive pomace in cell-free models and microglia cells. The bioavailable PC of olive pomace was obtained through the process of in vitro gastrointestinal digestion of fractionated olive pomace (OPF, particles size < 2 mm) and micronized olive pomace (OPM, particles size < 20 µm). The profile of the PC that is present in the bioavailable fraction as well as its in vitro antioxidant capacity were determined. The anti-neuroinflammatory capacity of the bioavailable PC from olive pomace (0.03-3 mg L-1) was evaluated in BV-2 cells activated by lipopolysaccharide (LPS) for 24 h. The total bioavailable PC concentration and antioxidant activity against peroxyl radical were higher in the OPM than those observed in the OPF sample. The activation of BV-2 cells by LPS resulted in increased levels of ROS and nitric oxide (NO). The bioavailable PCs from both OPF and OPM, at their lowest concentrations, were able to reduce the ROS generation in activated BV-2 cells. In contrast, the highest PC concentration of OPF and OPM was able to reduce the NO levels in activated microglial cells. Our results demonstrate that bioavailable PCs from olive pomace can act as anti-neuroinflammatory agents in vitro, independent of particle size. Moreover, studies approaching ways to increase the bioavailability of PCs from olive pomace, as well as any possible toxic effects, are needed before a final statement on its nutritional use is made.

Understanding the effect of fermentation time on physicochemical characteristics, sensory attributes, and volatile compounds in green tea kombucha.

Kombuchas are a trend in the fermented beverage field and the effect of fermentation time on their characteristics is necessary to better understand the process, mainly concerning volatile compounds, which are scarce information in the current literature. Thus, the present work aimed to evaluate the features of green tea kombucha during fermentation, monitoring the changes in pH, acidity, turbidity, polyphenols, ethanol, acetic acid, volatile compounds, and sensory profile and acceptance up to 14 days of fermentation. Kombuchas' pH and acidity decreased through time as expected, but after 4 days of fermentation, the beverage exceeded the Brazilian legal limits of acidity (130 mEq/L) and produced more than 0.5% AVB, which labels the beverage as alcoholic. Total polyphenols and condensed tannins content enhanced until the seventh day of fermentation and remained constant. Fermentation highly impacted the aroma of the infusion with a high formation of volatile acids, such as alcohols, esters, and ketones. Aldehydes were degraded during the bioprocess. Sensory characterization of kombucha showed that fermentation of 4 days increased perceived turbidity; vinegar, citric fruit, acid, and alcoholic aroma; and produced the beverage with sour, bitter, and vinegar flavor. Thus, the fermentation time of kombuchas must be controlled as they rapidly change and impact on the physicochemical parameters and sensory profile of the beverage can be negative.

Biosynthesis of 1,3-propanodiol and 2,3-butanodiol from residual glycerol in continuous cell-immobilized Klebsiella pneumoniae bioreactors.

In recent years, residual glycerol from biodiesel synthesis made this chemical a cheap, readily available carbon source to bioprocess, which is also a form to reduce costs in the fuel industry. We propose and describe a bioprocess using fluidized and packed-bed continuous bioreactors to convert this residual glycerol into value-added products such as 1,3-propanediol (1,3-PD) and 2,3-butanediol (2,3-BD), largely used in the chemical industry. The bacterium Klebsiella pneumoniae BLh-1, strain isolated by us, was immobilized in the permeable support of polyvinyl alcohol (LentiKats®). After testing different dilution rates (D) for all bioreactor configurations, the best obtained productivities of 1,3-PD was 8.69 g L-1  h-1 at a D = 0.45 h-1 , and 2.99 g L-1  h-1 at a D = 0.30 h-1 for 2,3-BD, both in the packed-bed configuration. In the fluidized-bed reactor, the highest productivity values achieved were 4.48 and 1.16 g L-1  h-1 for 1,3-PD and 2,3-BD, respectively, both at D = 0.33 h-1 . These results show the potential of setting up a bioprocess based on continuous cultures using immobilized K. pneumoniae BLh-1 in PVA matrices in order to efficiently convert the abundant surplus of glycerol into commercially important chemicals such as 1,3-PD and 2,3-BD.

Prebiotic effect of sorghum biomass xylooligosaccharides employing immobilized endoxylanase from Thermomyces lanuginosus PC7S1T.

Purified endoxylanase from Thermomyces lanuginosus PC7S1T was immobilized in calcium alginate, resulting in a yield of 78.5% and a reusability for 11 cycles. The stability of the immobilized enzyme was given for a pH range of 4 to 9 for 96 h. Endoxylanase immobilized in calcium alginate at 65 °C exhibited thermal stability equal to the soluble enzyme for 5 h, and at high temperatures of 75 °C and 85 °C showed half-lives of 4 and 3 h, respectively. Both soluble endoxylanase and immobilized forms were able to hydrolyze hemicellulose, obtained from low-lignin sorghum biomass pretreated with 5% H2O2 and 2% NaOH, after 1 h of incubation at 65 °C, releasing a mixture of short-chain xylooligosaccharides (X2-X6). The highest amounts of XOS generated were those for X5 (24 to 40%), X4 (33 to 39%), and X3 (11 to 22%). These XOS acted as prebiotics, promoting the growth of the probiotic L. acidophilus, similar to glucose in the MRS broth. These results show the potential of low-lignin sorghum to generate XOS with prebiotic activity, suggesting the application of these compounds in the food industry.

High Cell Density Culture of Dairy Propionibacterium sp. and Acidipropionibacterium sp.: A Review for Food Industry Applications.

The dairy bacteria Propionibacterium sp. and Acidipropionibacterium sp. are versatile and potentially probiotic microorganisms showing outstanding functionalities for the food industry, such as the production of propionic acid and vitamin B12 biosynthesis. They are the only food grade microorganisms able to produce vitamin B12. However, the fermentation batch process using these bacteria present some bioprocess limitations due to strong end-product inhibition, cells slow-growing rates, low product titer, yields and productivities, which reduces the bioprocess prospects for industrial applications. The high cell density culture (HCDC) bioprocess system is known as an efficient approach to overcome most of those problems. The main techniques applied to achieve HCDC of dairy Propionibacterium are the fed-batch cultivation, cell recycling, perfusion, extractive fermentation, and immobilization. In this review, the techniques available and reported to achieve HCDC of Propionibacterium sp. and Acidipropionibacterium sp. are discussed, and the advantages and drawbacks of this system of cultivation in relation to biomass formation, vitamin B12 biosynthesis, and propionic acid production are evaluated.

Effect of freeze-dried kombucha culture on microbial composition and assessment of metabolic dynamics during fermentation.

Kombucha is a traditional fermented beverage gaining popularity around the world. So far, few studies have investigated its microbiome using next-generation DNA sequencing, whereas the correlation between the microbial community and metabolites evolution along fermentation is still unclear. In this study, we explore this correlation in a traditionally produced kombucha by evaluating its microbial community and the main metabolites produced. We also investigated the effects of starter cultures processed in three different ways (control, starter culture without liquid suspension (CSC), and a freeze-dried starter culture (FDSC)) to evaluate changes in kombucha composition, such as antioxidant activity and sensory analysis. We identified seven genera of bacteria, including Komagataeibacter, Gluconacetobacter, Gluconobacter, Acetobacter, Liquorilactobacillus, Ligilactobacillus, and Zymomonas, and three genera of yeasts, Dekkera/Brettanomyces, Hanseniaspora, and Saccharomyces. Although there were no statistically significant differences in the acceptance test in sensory analysis, different starter cultures resulted in products showing different microbial and biochemical compositions. FDSC decreased Zymomonas and Acetobacter populations, allowing for Gluconobacter predominance, whereas in the control and CSC kombuchas the first two were the predominant genera. Results suggest that the freeze-drying cultures could be implemented to standardize the process and, despite it changes the microbial community, a lower alcohol content could be obtained.

Performance of xylose-fermenting yeasts in oat and soybean hulls hydrolysate and improvement of ethanol production using immobilized cell systems.

We investigated the fermentation of a mixture of oat and soybean hulls (1:1) subjected to acid (AH) or enzymatic (EH) hydrolyses, with both showing high osmotic pressures (> 1200 Osm kg-1) for the production of ethanol. Yeasts of genera Spathaspora, Scheffersomyces, Sugiymaella, and Candida, most of them biodiverse Brazilian isolates and previously untested in bioprocesses, were cultivated in these hydrolysates. Spathaspora passalidarum UFMG-CM-469 showed the best ethanol production kinetics in suspended cells cultures in acid hydrolysate, under microaerobic and anaerobic conditions. This strain was immobilized in LentiKats® (polyvinyl alcohol) and cultured in AH and EH. Supplementation of hydrolysates with crude yeast extract and peptone was also performed. The highest ethanol production was obtained using hydrolysates supplemented with crude yeast extract (AH-CYE and EH-CYE) showing yields of 0.40 and 0.44 g g-1, and productivities of 0.39 and 0.29 g (L h)-1, respectively. The reuse of the immobilized cells was tested in sequential fermentations of AH-CYE, EH-CYE, and a mixture of acid and enzymatic hydrolysates (AEH-CYE) operated under batch fluidized bed, with ethanol yields ranging from 0.31 to 0.40 g g-1 and productivities from 0.14 to 0.23 g (L h)-1. These results warrant further research using Spathaspora yeasts for second-generation ethanol production.

Expression of Bacillus amyloliquefaciens transglutaminase in recombinant E. coli under the control of a bicistronic plasmid system in DO-stat fed-batch bioreactor cultivations.

We studied the expression of Bacillus amyloliquefaciens transglutaminase cloned in Escherichia coli BL21(DE3)pLysS harboring the plasmid pBAD/3C/bTGase, a bicistronic expression system, in bioreactor cultivation. Batch and fed-batch controlled as DO-stat strategies were employed for the production of the recombinant enzyme. In 30 h-batch cultivations using Terrific broth (TB), 6 g/L of biomass and 3.12 U/mgprotein of transglutaminase activity were obtained. DO-stat fed-batch cultivations under the control of oxygen concentration (DO-stat) using TB as medium but fed with glucose allowed the increment in biomass formation (17.5 g/L) and enzyme activity (6.43 U/mgprotein). DO-stat fed-batch using mineral medium (M9) and fed with glucose under the same conditions produced even higher enzymatic activity (9.14 U/mgprotein). The pH effect was investigated, and the best enzymatic activity could be observed at pH 8. In all cultivations, the bicistronic system remained stable, with 100% of plasmid-bearing cells. These results show that E. coli bearing bicistronic plasmid constructs to express recombinant TGase could be cultivated in bioreactors under DO-stat fed-batch using mineral medium and it is a promising strategy in future optimizations to produce this important enzyme.

Construction of Recombinant Klebsiella pneumoniae to Increase Ethanol Production on Residual Glycerol Fed-Batch Cultivations.

K. pneumoniae BLh-1 strain was genetically modified aiming at obtaining high ethanol productivity in cultivations using residual glycerol from biodiesel synthesis as substrate. The recombinant strain K. pneumoniae Kp17 was obtained by inserting the multicopy plasmid pTOPOBL17 containing the AdhE gene, and its own promoter, from K. pneumoniae BLh-1. Influence of Fe2+ supplementation and initial glycerol concentration on culture conditions were analyzed, both in rotatory shaker and in batch bioreactors. In the bioreactor cultures, K. pneumoniae Kp17 strain produced 4.5 g L-1 of ethanol (productivity of 0.50 g L-1 h-1 and yields of 0.15 g g-1) after 24-h cultivation, corresponding to an increase of approximately 40% in ethanol concentration compared to wild strain, K. pneumoniae BLh-1. Best conditions were then applied in exponential fed-batch bioreactors, with final ethanol concentration of 17.30 g L-1 (productivity of 0.59 g L-1 h-1 and yields of 0.16 g g-1) after 30 h of feeding, representing 11.5% of increment in titer of ethanol compared to the wild strain. Mutant cells kept 92.5% of the plasmids under batch in 24 h, and 71.9% under fed-batch after 27 h of exponential feeding. The findings in this work show the possibility of using a simple approach to genetically modify K. pneumoniae to be employed this versatile bacterium for the bioconversion of residual glycerol into ethanol.

Bioreactor production of 2,3-butanediol by Pantoea agglomerans using soybean hull acid hydrolysate as substrate.

Production of 2,3-butanediol (2,3-BD) by Pantoea agglomerans strain BL1 was investigated using soybean hull hydrolysate as substrate in batch reactors. The cultivation media consisted of a mixture of xylose, arabinose, and glucose, obtained from the hemicellulosic fraction of the soybean hull biomass. We evaluated the influence of oxygen supply, pH control, and media supplementation on the growth kinetics of the microorganism and on 2,3-BD production. P. agglomerans BL1 was able to simultaneously metabolize all three monosaccharides present in the broth, with average conversions of 75% after 48 h of cultivation. The influence of aeration conditions employed demonstrated the mixed acid pathway of 2,3-BD formation by enterobacteria. Under fully aerated conditions (2 vvm of air), up to 14.02 g L-1 of 2.3-BD in 12 h of cultivation were produced, corresponding to yields of 0.53 g g-1 and a productivity of 1.17 g L-1 h-1, the best results achieved. These results suggest the production potential of 2,3-BD by P. agglomerans BL1, which has been recently isolated from an environmental consortium. The present work proposes a solution for the usage of the hemicellulosic fraction of agroindustry biomasses, carbohydrates whose utilization are not commonly addressed in bioprocess.

Production of volatile compounds by yeasts using hydrolysed grape seed oil obtained by immobilized lipases in continuous packed-bed reactors.

Lipases CAL-B, TLL, and RML were used in the synthesis of free fatty acids of grape seed oil as heterogeneous substrate. The best enzyme was used to optimize the reaction variables temperature, enzyme content, and molar ratio of water:oil in batch reactions using experimental planning. The ideal conditions to produce free fatty acids using pure RML were 45 °C, 12:1 substrate molar ratio, and 15% enzyme, resulting in 66% of oil hydrolysis and a productivity of 0.54 mol L-1 min-1 in 4 h of reaction at 180 rpm. Repeated batches of reaction were performed testing the operational stability of RML, results showing that this enzyme could be used for at least 20 cycles keeping more than 80% of its initial activity, suggesting its potential use in industrial processes. The synthesis of free fatty acids was then evaluated in continuous reactions using packed-bed reactor (PBR). The highest productivity in the continuous process was 6.85 mol L-1 min-1, using only RML, showing an operational stability higher than 80% of its initial conversion capacity after 11 days of operation, at a flow rate of 0.13 mL min-1 at 45 °C. We evaluated the use of this hydrolyzed oil as substrate for lactone bioproduction using Galactomyces geotrichum UFMG-CM-Y3276, G. geotrichum UFMG-CM-Y3558, and Geotrichum klebahnii UFMG-CM-Y3014 screened for their oil-hydrolysis ability. Volatile compounds were qualitatively identified in GC-MS as γ-octalactone and γ-nonalactone.

Cloning and expression of the Bacillus amyloliquefaciens transglutaminase gene in E. coli using a bicistronic vector construction.

Transglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxicity, we constructed a bicistronic plasmid containing the B. amyloliquefaciens TGase gene fused to the inhibitory Streptomyces caniferus prodomain. To make the enzyme active and avoid the need of prodomain removal in vitro, we also cloned the 3C protease gene into the same plasmid. After a fast single-step purification protocol, we obtained a partially purified recombinant TGase with 37 mU/mg protein activity, that crosslinked bovine serum albumin (BSA). This is the first report on the expression of B. amyloliquefaciens TGase in E. coli in its mature and active form.

Transglutaminases: part I-origins, sources, and biotechnological characteristics.

The transglutaminases form a large family of intracellular and extracellular enzymes that catalyze cross-links between protein molecules. Transglutaminases crosslinking properties are widely applied to various industrial processes, to improve the firmness, viscosity, elasticity, and water-holding capacity of products in the food and pharmaceutical industries. However, the extremely high costs of obtaining transglutaminases from animal sources have prompted scientists to search for new sources of these enzymes. Therefore, research has been focused on producing transglutaminases by microorganisms, which may present wider scope of use, based on enzyme-specific characteristics. In this review, we present an overview of the literature addressing the origins, types, reactions, and general characterizations of this important enzyme family. A second review will deal with transglutaminases applications in the area of food industry, medicine, pharmaceuticals and biomaterials, as well as applications in the textile and leather industries.

Review transglutaminases: part II-industrial applications in food, biotechnology, textiles and leather products.

Because of their protein cross-linking properties, transglutaminases are widely used in several industrial processes, including the food and pharmaceutical industries. Transglutaminases obtained from animal tissues and organs, the first sources of this enzyme, are being replaced by microbial sources, which are cheaper and easier to produce and purify. Since the discovery of microbial transglutaminase (mTGase), the enzyme has been produced for industrial applications by traditional fermentation process using the bacterium Streptomyces mobaraensis. Several studies have been carried out in this field to increase the enzyme industrial productivity. Researches on gene expression encoding transglutaminase biosynthesis were performed in Streptomyces lividans, Escherichia coli, Corynebacterium glutamicum, Yarrowia lipolytica, and Pichia pastoris. In the first part of this review, we presented an overview of the literature on the origins, types, mediated reactions, and general characterizations of these important enzymes, as well as the studies on recombinant microbial transglutaminases. In this second part, we focus on the application versatility of mTGase in three broad areas: food, pharmacological, and biotechnological industries. The use of mTGase is presented for several food groups, showing possibilities of applications and challenges to further improve the quality of the end-products. Some applications in the textile and leather industries are also reviewed, as well as special applications in the PEGylation reaction, in the production of antibody drug conjugates, and in regenerative medicine.

Fermentation of hexoses and pentoses from sugarcane bagasse hydrolysates into ethanol by Spathaspora hagerdaliae.

The present study evaluated 13 strains of yeast for ethanol and xylitol production from xylose. Among them, Spathaspora hagerdaliae UFMG-CM-Y303 produced ethanol yields (YP/S) of 0.25 g g- 1 and 0.39 g g- 1 under aerobic and microaerophilic conditions, respectively, from a mixture of glucose and xylose in flasks. A pH of 5.0 and an inoculum of 3.0 × 108 cells mL- 1r resulted in the highest ethanol yields. These conditions were tested in a bioreactor for fermenting a medium containing an enzymatic hydrolysate of sugarcane bagasse with 15.5 g L- 1 of glucose and 3 g L- 1 of xylose, and achieved a YP/S of 0.47 g g- 1, in relation to total available sugar. These results suggest that S. hagerdaliae UFMG-CM-Y303 has potential for use in second-generation ethanol studies.

Transesterification of Waste Frying Oil and Soybean Oil by Combi-lipases Under Ultrasound-Assisted Reactions.

This work describes the use of an ultrasound system for the enzymatic transesterification of oils using combi-lipases as biocatalyst. The reactions were carried out evaluating the individual use of waste oil and fresh soybean oil, and the immobilized lipases CALB, TLL, and RML were used as biocatalysts. It was performed in a mixture design of three factors to obtain the ideal mixture of lipases according to the composition of fatty acids present in each oil, and the main reaction variables were optimized. After 18 h of reaction, ultrasound provided a biodiesel yield of about 90% when using soybean oil and 70% using the waste oil. The results showed that ultrasound technology, in combination with the application of enzyme mixtures, known as combi-lipases, and the use of waste oil, could be a promising route to reduce the overall process costs of enzymatic production of biodiesel.

Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates.

An industrial ethanol-producing Saccharomyces cerevisiae strain with genes of fungal oxido-reductive pathway needed for xylose fermentation integrated into its genome (YRH1415) was used to obtain haploids and diploid isogenic strains. The isogenic strains were more effective in metabolizing xylose than YRH1415 strain and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting from the hydrolysis of lignocellulosic biomass (switchgrass). The rate of xylose consumption did not appear to be affected by the ploidy of strains or the presence of two copies of the xylose fermentation genes but by heterozygosity of alleles for xylose metabolism in YRH1415. Furthermore, inhibitor tolerance was influenced by the heterozygous genome of the industrial strain, which also showed a marked influenced on tolerance to increasing concentrations of toxic compounds, such as furfural. In this work, selection of haploid derivatives was found to be a useful strategy to develop efficient xylose-fermenting industrial yeast strains.

Screening of filamentous fungi to produce xylanase and xylooligosaccharides in submerged and solid-state cultivations on rice husk, soybean hull, and spent malt as substrates.

We investigated the enzymatic complex produced by selected fungi strains isolated from the environment using the agro-industrial residues rice husk, soybean hull, and spent malt as substrates. Microbial growth was carried out in solid-state cultivation (SSC) and in submerged cultivations (SC) and the enzymatic activities of xylanase, cellulase, ß-xylosidase, and ß-glucosidase were determined. All substrates were effective in inducing enzymatic activities, with one strain of Aspergillus brasiliensis BLf1 showing maximum activities for all enzymes, except for cellulases. Using this fungus, the enzymatic activities of xylanase, cellulase, and ß-glucosidase were generally higher in SSC compared to SC, producing maxima activities of 120.5, 25.3 and 47.4 U g-1 of dry substrate, respectively. ß-xylosidase activity of 28.1 U g-1 of dry substrate was highest in SC. Experimental design was carried out to optimize xylanase activity by A. brasiliensis BLf1 in SSC using rice husk as substrate, producing maximum xylanase activity 183.5 U g-1 dry substrate, and xylooligosaccharides were produced and characterized. These results suggest A. brasiliensis BLf1 can be used to produce important lytic enzymes to be applied in the preparation of xylooligosaccharides.

Lactobacillus plantarum BL011 cultivation in industrial isolated soybean protein acid residue

Abstract In this study, physiological aspects of Lactobacillus plantarum BL011 growing in a new, all-animal free medium in bioreactors were evaluated aiming at the production of this important lactic acid bacterium. Cultivations were performed in submerged batch bioreactors using the Plackett-Burman methodology to evaluate the influence of temperature, aeration rate and stirring speed as well as the concentrations of liquid acid protein residue of soybean, soy peptone, corn steep liquor, and raw yeast extract. The results showed that all variables, except for corn steep liquor, significantly influenced biomass production. The best condition was applied to bioreactor cultures, which produced a maximal biomass of 17.87 g L-1, whereas lactic acid, the most important lactic acid bacteria metabolite, peaked at 37.59 g L-1, corresponding to a productivity of 1.46 g L-1 h-1. This is the first report on the use of liquid acid protein residue of soybean medium for L. plantarum growth. These results support the industrial use of this system as an alternative to produce probiotics without animal-derived ingredients to obtain high biomass concentrations in batch bioreactors.

Lactobacillus plantarum BL011 cultivation in industrial isolated soybean protein acid residue.

In this study, physiological aspects of Lactobacillus plantarum BL011 growing in a new, all-animal free medium in bioreactors were evaluated aiming at the production of this important lactic acid bacterium. Cultivations were performed in submerged batch bioreactors using the Plackett-Burman methodology to evaluate the influence of temperature, aeration rate and stirring speed as well as the concentrations of liquid acid protein residue of soybean, soy peptone, corn steep liquor, and raw yeast extract. The results showed that all variables, except for corn steep liquor, significantly influenced biomass production. The best condition was applied to bioreactor cultures, which produced a maximal biomass of 17.87gL-1, whereas lactic acid, the most important lactic acid bacteria metabolite, peaked at 37.59gL-1, corresponding to a productivity of 1.46gL-1h-1. This is the first report on the use of liquid acid protein residue of soybean medium for L. plantarum growth. These results support the industrial use of this system as an alternative to produce probiotics without animal-derived ingredients to obtain high biomass concentrations in batch bioreactors.