Yeast Fermentation Broth Research Articles

Development of a rapid, efficient, and reusable magnetic bead-based immunocapture system for recombinant human procollagen type II isolation from yeast fermentation broth

Recombinant collagen production, especially using yeasts as expression systems, could represent a promising alternative over traditional extractive methods from animal sources, offering controllable, scalable, and high-quality products. Monitoring the efficiency and efficacy of procollagen/collagen expression, especially in the initial fermentation phases, can be difficult and time consuming, as biological matrices necessitate purification and commonly used analytical methods are only partially informative. We propose a straightforward, efficient, and reusable immunocapture system able to specifically isolate human procollagen type II from fermentation broths and to release it in few experimental steps. A recovered sample allows for a detailed characterization providing information on structural identity and integrity, which can strongly support the monitoring of fermentation processes. The immunocapture system relies on the use of protein A–coated magnetic beads which have been functionalized and cross-linked with a human anti-procollagen II antibody (average immobilization yield of 97.7%) to create a stable and reusable support for the specific procollagen fishing. We set up the binding and release conditions ensuring specific and reproducible binding with a synthetic procollagen antigen. The absence of non-specific interaction with the support and binding specificity was demonstrated, and the latter was also confirmed by a peptide mapping epitope study in reversed-phase liquid chromatography high-resolution mass spectrometry (RP-LC-HRMS). The bio-activated support proved to be reusable and stable over 21 days from the initial use. Finally, the system was successfully tested on a raw yeast fermentation sample to provide a proof of concept of the applicability within recombinant collagen production.Graphical

Two Laminaria japonica Fermentation Broths Alleviate Oxidative Stress and Inflammatory Response Caused by UVB Damage: Photoprotective and Reparative Effects.

UVB radiation can induce oxidative stress and inflammatory response in human epidermal cells. We establish a UVB-induced damage model of human immortalized epidermal keratinocytes (HaCaT) to explore the protective and reparative effects of Laminaria japonica on UVB-damaged epidermal inflammation after fermentation by white Ganoderma lucidum (Curtis) P. Karst and Saccharomyces cerevisiae. Compared with unfermented Laminaria japonica, fermented Laminaria japonica possesses stronger in vitro free radical scavenging ability. Laminaria japonica white Ganoderma lucidum fermentation broth (LJ-G) and Laminaria japonica rice wine yeast fermentation broth (LJ-Y) can more effectively remove excess reactive oxygen species (ROS) in cells and increase the content of the intracellular antioxidant enzymes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO-1). In addition, fermented Laminaria japonica effectively reduces the content of pro-inflammatory factors ILs, TNF-α and MMP-9 secreted by cells. The molecular research results show that fermented Laminaria japonica activates the Nrf2 signaling pathway, increases the synthesis of antioxidant enzymes, inhibits the gene expression levels of pro-inflammatory factors, and alleviates cellular oxidative stress and inflammatory response caused by UVB radiation. Based on the above results, we conclude that fermented Laminaria japonica has stronger antioxidant and anti-inflammatory activity than unfermented Laminaria japonica, possesses good safety, and can be developed and used as a functional inflammation reliever. Fermented Laminaria japonica polysaccharide has a more slender morphological structure and more rockulose, with better moisturizing and rheological properties.

The superposition anti-viral activity of porcine tri-subtype interferon expressed by Saccharomyces cerevisiae

Interferon (IFN)-mediated antiviral responses are central to host defense against viral infection. Porcine viral infection has emerged as a serious hazard for the pig industry. The construction of an engineered Saccharomyces cerevisiae strain that efficiently produces porcine IFN has demonstrated several advantages. It can be easily fed to pigs, which helps in reducing antibiotic residues in pork and improve meat quality. In this study, the stable expression of several porcine IFN molecules (pIFN-α1, pIFN-β, pIFN-λ1, pIFN-λ1-β, pIFN-λ1-β-α1) were determined using an engineered S. cerevisiae system. With the YeastFab assembly method, the complete transcriptional units containing promoter (GPD), secretory peptide (α-mating factor), target gene (IFN) and terminator (ADH1) were successfully constructed using the characteristics of type II restriction endonuclease, and then integrated into the chromosomes Ⅳ and XVI of ST1814 yeast host strain, respectively. The expression kinetics of recombinant pIFNs were further analyzed. Synergism in the expression level of IFN receptor, antiviral protein, and viral loading was observed in viral-cell infection model treated with different porcine IFN subtypes. The porcine reproductive and respiratory syndrome viral load and antibody titer in serum decreased significantly after oral administration of IFN expression yeast fermentation broth. These findings indicate the potential efficacy of multi-valent pIFNs expressing S. cerevisiae as a potent feed material to prevent viral infections of pigs.

An Alternative Hard Capsule Prepared with the High Molecular Weight Pullulan and Gellan: Processing, Characterization, and In Vitro Drug Release

Albeit gelatin hard capsules were predominantly applied, their disadvantages call for endeavours on non-gelatin capsules in healthcare aspects. Herein, high molecular weight (Mw = 1 × 106) pullulan was purified from yeast fermentation broth. This high Mw pullulan and commercial low Mw pullulan were respectively co-blended with gellan to prepare the optimal solutions for capsule production. Further investigations, including loss-on-drying, brittleness and tightness, showed that the obtained pullulan-gellan capsules were of low water content, brittleness, leakage, and of high tightness. These capsules exhibited an extended release of amoxicillin over 60 min in simulated gastric fluid. Although the use of high and low Mw pullulan produced composite capsules with similar properties, the required concentration of high Mw pullulan was nearly half of that for low Mw pullulan, suggesting a cost-saving characteristic of using high Mw pullulan. This work proposes a potential substitution of gelatin with pullulan-gellan composites for preparing hard capsules.

Comparison of antimicrobial peptide purification via free-flow electrophoresis and gel filtration chromatography.

Antimicrobial peptides (AMPs) are usually small and cationic biomolecules with broad-spectrum antimicrobial activities against pathogens. Purifying them from complex samples is essential to study their physiochemical properties. In this work, free-flow zone electrophoresis (FFZE) was utilized to purify AMPs from yeast fermentation broth. Meanwhile, gel filtration chromatography (GFC) was conducted for comparison. The separation efficiency was evaluated by SDS-PAGE analysis of the fractions from both methods. Our results demonstrated as follows: (i) FFZE had more than 30-fold higher processing capacity as compared with GFC; (ii) FFZE could achieve 87% purity and 89% recovery rate while in GFC these parameters were about 93 and 82%, respectively; (iii) the former had ∼2-fold dilution but the latter had ∼13-fold dilution. Furthermore, Tricine-SDS-PAGE, Native-PAGE, and gel IEF were carried out to characterize the purified AMPs. We found that two peptides existed as a pair with the molecularmass of ∼5.5and 7.0kDa, while the same pI 7.8. These two peptides were proved to have the antimicrobial activity through the standardized agar diffusion method. Therefore, FFZE could be used to continuously purify AMPs with high bioactivity, which will lead to its wide application in the clinical and pharmaceutical fields.

Efficient Ethanol Recovery from Yeast Fermentation Broth with Integrated Distillation–Membrane Process

A hybrid process integrating vapor stripping with vapor compression and vapor permeation membrane separation, termed Membrane Assisted Vapor Stripping (MAVS), was evaluated for recovery and dehydration of ethanol from aqueous solution as an alternative to conventional distillation–molecular sieve processes. Ethanol removal/drying performance of the MAVS system with binary ethanol–water mixtures and a yeast fermentation broth were evaluated and the fate of secondary fermentation products in the system was assessed. Simple alcohols, esters, and organic acids displayed varying degrees of recovery in the vapor stripping based on the relative vapor–liquid partitioning of the compounds. All volatilized organic compounds were concentrated to the same degree in the membrane step. Membrane permeance, permselectivity, and overall energy usage of the hybrid process were the same with the fermentation broth as with binary ethanol–water solutions. The MAVS system required less than half the energy of a distillation–molecular sieve system.

Spray Drying of Extracts from Red Yeast Fermentation Broth

Red yeast rice is a pigmented material that is traditionally used in Asia as a food colorant. In addition to food applications, red yeast rice is known in traditional Chinese medicine for its therapeutic actions. The aim of this work was to study the quality interactions during spray drying of extracts from the Monascus ruber van Tiegham fermentation broth. The quality indicators used for the dry powder properties were the levels of monacolin K, ratio of red to yellow pigments, as well as their antioxidant activity. The experiments followed a Box-Behnken design to study the effects of the adjuvant/drug ratio, adjuvant incorporation time, and oulet drying temperature on the pharmacotechnical, chemical, and biological properties of the dry extract. The influences of these factors on the characteristics of the dry powder were evaluated by the bulk density, tapped density, Carr index, Hausner factor, residual moisture content, water activity, antioxidant activity, monacolin K, yellow-to-red pigment ratio, and antioxidant activity. The analysis of variance (ANOVA) on experimental data revealed that an increase in drying temperature significantly increased the dry powder yield and caused an improvement in powder flow properties, which may be related to lower moisture contents. The drying temperature did not affect the monacolin K content in dry powder but showed a complex influence on its antioxidant activity. The increase in drying adjuvant-to-drug ratio affected the yield and also indicated a protective effect on the monacolin K content. The duration of drying adjuvant incorporation had little or negligible effect on powder properties. The dry extracts of red yeast rice showed adequate properties and the process proposed herein can be used to prepare nutraceutical products.

Factors affecting alcohol–water pervaporation performance of hydrophobic zeolite–silicone rubber mixed matrix membranes

Mixed matrix membranes (MMMs) consisting of ZSM-5 zeolite particles dispersed in silicone rubber exhibited ethanol–water pervaporation permselectivities up to five times that of silicone rubber alone and three times higher than simple vapor–liquid equilibrium (VLE). A number of conditions resulted in the deterioration in pervaporation performance of these MMMs. Long-term exposure to simple binary ethanol–water mixtures resulted in slow declines in ethanol permeability, water permeability, and selectivity. Treating the particles by quenching them in linear short-chain alcohols immediately after calcination protected the MMM against this performance decline. In addition, rapid and dramatic reductions in both ethanol permeability and selectivity occurred when the MMMs were exposed to a centrifuged yeast fermentation broth. Exposure to a clarified acetone/n-butanol/ethanol (ABE) fermentation broth also resulted in marked reductions in performance, albeit less severe and not as rapid as with the yeast broth. The effect of the broths was not easily reversed. The most effective regeneration scheme was soaking the MMM in 100% n-butanol, which fully recovered the original performance. Zeolite pore blockage due to adsorption of minor constituents in the broths, including organic acids, esters, alcohols, and esterification reaction products was identified as the likely cause of performance changes.

Liquid–Liquid Extraction of GSH Using DEHAP/Octanol Reverse Micelles

In this study, di-(2-ethylhexyl) ammonium phosphate (DEHAP)/octanol reverse micellar extraction was investigated and demonstrated to be an effective method to separate GSH from yeast fermentation broth. The effect of several important factors i.e., pH, DEHAP concentration, and type & concentration of cations on both extraction and strip-extraction was investigated. The optimum operating conditions for both extraction and strip-extraction were obtained by two groups of orthogonal experiments. At the optimum operating conditions, the yield of GSH can reach 68%. In the lyophilized product, the purity of GSH was higher than 75%, all the proteins can be separated, and only a small portion of amino acids were left.

Glutathione extraction and mass transfer in di-(2-ethylhexyl) ammonium phosphate/octanol reverse micelles

Di-(2-ethylhexyl) ammonium phosphate (DEHAP)/octanol reverse micellar extraction was demonstrated to be an effective method to separate glutathione (GSH) from yeast fermentation broth. The effect of several important factors including pH, DEHAP concentration, and types and concentration of cations was investigated, and the optimum operating conditions were obtained by orthogonal experiments, for forward extraction: pH 2.5, DEHAP concentration = 0.16 mol/L, W 0 = 10, phase ratio (organic/aqueous, v/v) = 2:1, K + concentration = 0.1 mol/L, and extraction time = 15 min. For back extraction: pH 10.5, Ca 2+ concentration = 0.6 mol/L, phase ratio (aqueous/organic, v/v) = 3:1, back extraction time = 45 min. Under the optimum operating conditions, the yield of GSH can reach 65% after one forward and three back extractions. No protein and only a small portion of amino acids were detected in the lyophilized product of GSH. The study on the extraction kinetics showed that the volumetric mass transfer rates of the forward and back extraction are around 0.209 s −1 and 0.054 s −1, respectively, which is very close to the literature results of electrostatic force-based reverse micellar extraction of protein.

Isolation of ATP from a yeast fermentation broth using a cryogel column at high flow velocities

This communication presents an effective method for isolating adenosine triphosphate (ATP) from a yeast fermentation broth using an anion-exchange supermacroporous cryogel column at high flow velocities. The breakthrough and elution behaviors of pure ATP in the cryogel bed were investigated at flow velocities of 2, 5, and 10 cm/min and the ATP binding capacities were determined. Then the ATP-containing yeast fermentation broth was employed as the test feedstock and various chromatographic runs were conducted to isolate ATP by the cryogel at different high flow velocities. The ATP samples obtained were analyzed quantitatively by HPLC. The results showed that even at a flow velocity of 5 or 10 cm/min, a product purity of 97.4 or 98.0% can be achieved, illustrating the potential of the present method for separation of high-purity ATP directly from fermentation feedstock at high flow velocities.

A New Integrated Membrane Filtration and Chromatographic Device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.

Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth

Recovery of therapeutic protein from high cell density yeast fermentations at commercial scale is a challenging task. In this study, we investigate and compare three different harvest approaches, namely centrifugation followed by depth filtration, centrifugation followed by filter-aid enhanced depth filtration, and microfiltration. This is achieved by presenting a case study involving recovery of a therapeutic protein from Pichia pastoris fermentation broth. The focus of this study is on performance of the depth filtration and the microfiltration steps. The experimental data has been fitted to the conventional models for cake filtration to evaluate specific cake resistance and cake compressibility. In the case of microfiltration, the experimental data agrees well with flux predicted by shear induced diffusion model. It is shown that, under optimal conditions, all three options can deliver the desired product recovery ( >80%), harvest time ( <15 h including sequential concentration/diafiltration step), and clarification ( <6 NTU). However, the three options differ in terms of process development time required, capital cost, consumable cost, ease of scale-ability and process robustness. It is recommended that these be kept under consideration when making a final decision on a harvesting approach.

Dehydration of light oil by pervaporation

The pervaporation recovery of ethanol from yeast fermentation broth was investigated using poly[1-(trimethylsilyl)-1-propyne]. The deterioration of membrane performance in the presence of fermentation broth was observed. The fouled membrane was characterized by gas permeation and density measurements, and sorption of pure components of the fermentation broth in PTMSP was studied to clarify the fouling mechanism. It was concluded that properties of the membrane deteriorate due to internal contamination of the PTMSP free volume with non-volatile by-products of the fermentation. The PTMSP film did not show appreciable deterioration of membrane properties in the pervaporation of aqueous solution of organic compounds with high volatility.

Effect of yeast fermentation by-products on poly[1-(trimethylsilyl)-1-propyne] pervaporative performance

The pervaporation recovery of ethanol from yeast fermentation broth was investigated using poly[1-(trimethylsilyl)-1-propyne]. The deterioration of membrane performance in the presence of fermentation broth was observed. The fouled membrane was characterized by gas permeation and density measurements, and sorption of pure components of the fermentation broth in PTMSP was studied to clarify the fouling mechanism. It was concluded that properties of the membrane deteriorate due to internal contamination of the PTMSP free volume with non-volatile by-products of the fermentation. The PTMSP film did not show appreciable deterioration of membrane properties in the pervaporation of aqueous solution of organic compounds with high volatility.

Mass transfer characteristics of yeast fermentation broth in the presence of pluronic F-68

A nonionic surfactant, Pluronic F-68, was used to investigate mass transfer characteristics of a fermentation medium in a 1 l bench-top bioreactor. At low flow rate (0.08 vvm), k L a did not change significantly with increasing agitation speed. In all air flow rates studied, pluronic caused a remarkable reduction in k L a. When an oxygen carrier such as perfluorocarbon (PFC) was added to the medium, k L a increased with increasing PFC concentration. Moreover, PFC emulsified with pluronic F-68 resulted in a significant increase in k L a values. The inclusion of 4 g l −1 pluronic in yeast fermentation medium resulted in a longer lag phase, while the final biomass concentration was almost unaffected.

Mass transfer characteristics of a fermentation broth in a reactor: co-current downflow contacting reactor

Gas holdups, dispersion height, volumetric mass transfer coefficients and kLa of water and a yeast fermentation broth were studied in a co-current downflow contacting reactor. kLa and gas holdup increased with increasing superficial gas velocity and there was a parallelism between kLa and gas holdup. The values of kLa gas holdup, and dispersion height measured in the air/fermentation broth system were all lower than those in air/water system.

Phospholipid coatings for the prevention of membrane fouling.

The aim of the present work was the development of phosphorylcholine-based treatments for biofiltration membranes and the demonstration that such treatments prevent or inhibit protein fouling. Microfiltration membranes of cellulose triacetate, polyether sulphone and polyvinylidene fluoride were etched with oxygen in a plasma chamber to generate surface hydroxyl groups and were then treated with the monomer 2-methacryloyloxyethyl phosphorylcholine. These membranes were evaluated with water, buffer, bovine serum albumin (BSA), yeast fermentation broth, beer and orange juice. The treatment of cellulose triacetate membranes reduced both the initial flux and the extent of water fouling. In terms of the integrated flux, these factors tended to cancel each other out. For protein, the membranes gave similar or higher fluxes but worse fouling. The cellular feed (yeast) reacted more favourably to the coating than the BSA. The polyether sulphone was scarcely affected by the coating; fouling remaining high with most 'real' feeds. There was lower initial flux but less flux decline with water and beer. Washing with water and cleaning with Tergazyme did not restore the initial flux. Polyvinylidene fluoride membranes gave the most positive results. In most cases, the coating both increased initial flux and decreased the rate of fouling. The coating was particularly effective for BSA and for beer and orange juice, where fouling is probably caused by a polysaccharide rather than by a protein. Electron microscopy showed, nonetheless, that fouling by proteins was accompanied by protein adsorption primarily on the upper surface of the membrane and that coated membranes showed less deposition and in different places than did untreated membranes.

Biofuel Cell Utilizing Saccharomyces cerevisiae - Modelling of the Process

Biofuel cell is a device generating electrical energy by using microorganism. The driving force of a biofuel cell is the redox reaction of substrate such as glucose by using microorganism as the catalyst In this paper a biofuel cell device is presented by using Saccharomyces cerevisiae baker’s yeast as microorganism. A fairly large power output (120 μW/cm3) was obtained by using nickel net packing with graphite particles as the anode electrode. The cell can be run in batch, fed-batch, or continuous mode. Experiments indicate that yeast fermentation broth includes large amount of electroactive substances which can be utilized in the anode without the aid of mediators. An electro-biological model is proposed from the results of theoretical and experimental analysis.

Characterization of human and rat intestinal trefoil factor produced in yeast.

Intestinal trefoil factor (ITF) from human (hITF) and rat (rITF) have been produced in Saccharomyces cerevisiae. The DNA encoding the two peptides were cloned by polymerase chain reactions (PCR) from a human normal colon library and a rat small intestinal epithelial cell library. Recombinant plasmids were constructed to encode a fusion protein consisting of a hybrid leader sequence and the rat and human ITF sequences, respectively. The leader sequence used serves to direct the fusion protein into the secretory (and processing) pathway of the cell. The secreted recombinant hITF was found in a monomer and a dimer form, whereas the rITF was only secreted as a dimer. The secreted peptides were purified by a combination of ionic exchange chromatography and preparative HPLC. From 8 L of yeast fermentation broth, 256 mg of hITF (monomer) and 133 mg of hITF (dimer) were isolated, and from 8.7 L of fermentation broth, 236 mg of rITF (dimer) was isolated. The structure of hITF (monomer), hITF (dimer), and rITF (dimer) was determined by amino acid analyses, peptide mapping, sequence analyses, and electrospray mass spectrometry analyses. In hITF (monomer) six of the seven cysteines are disulfide-linked to form 3 disulfide bridges. Mass analysis indicated that the last cysteine residue (Cys-57) did not exist as free (-SH) cysteine, but have reacted with cysteine to form an S-S linked cystine. Sequence and mass spectrometry analyses as well as peptide mapping showed that the dimer form of both hITF and rITF is mediated by a disulfide bridge between Cys-57 residues of two monomers.