Increased Protein Yield Research Articles

Impact of genetic variants on the separation efficiency of β-lactoglobulin during microfiltration of skim milk

Genetic variants of milk proteins are often overlooked by dairy processors as a parameter that can effect process efficiency. This study assessed the impact of the A and B genetic variants of β-lactoglobulin on the isolation of whey proteins from skim milk by microfiltration. The A and B genetic variants of β-lactoglobulin differ by two amino acids at positions 64 (A = aspartic acid, B = glycine) and 118 (A = valine, B = alanine), with the A variant thus having a higher negative charge. Mass balance calculations indicated that 46.7 % more whey proteins permeated during microfiltration for the A compared to the B variant, with the A also having significantly (P < 0.05) more total and reversible fouling accumulation than the B variant. These findings could be of importance to the dairy industry to improve microfiltration efficiency and increase protein yield during manufacture of native whey protein ingredients.

An efficient approach for overproduction of DNA polymerase from Pyrococcus furiosus using an optimized autoinduction system in Escherichia coli.

High fidelity DNA polymerase from Pyrococcus furiosus (Pfupol) is an attractive alternative to the highly popular DNA polymerase from Thermus aquaticus. Because this enzyme is in great demand for biotechnological applications, optimizing Pfupol production is essential to supplying the industry's expanding demand. T7-induced promoter expression in Escherichia coli expression systems is used to express recombinant Pfupol; however, this method is not cost-effective. Here, we have effectively developed an optimized process for the autoinduction approach of Pfupol expression in a defined medium. To better examine Pfupol's activities, its purified fraction was used. A 71mg/L of pure Pfupol was effectively produced, resulting in a 2.6-fold increase in protein yield when glucose, glycerol, and lactose were added in a defined medium at concentrations of 0.05%, 1%, and 0.6%, respectively, and the condition for production in a 5 L bioreactor was as follow: 200rpm, 3 vvm, and 10% inoculant. Furthermore, the protein exhibited 1445 U/mg of specific activity when synthesized in its active state. This work presents a high level of Pfupol production, which makes it an economically viable and practically useful approach.

Soluble and insoluble expression of recombinant human interleukin-2 protein using pET expression vector in Escherichia coli

Interleukin-2 has emerged as a potent protein-based drug to treat various cancers, AIDS, and autoimmune diseases. Despite its immense requirement, the production procedures are inefficient to meet the demand. Therefore, efficient production procedures must be adopted to improve protein yield and decrease procedural loss. This study analyzed cytoplasmic and periplasmic IL-2 expression for increased protein yield and significant biological activity. The study is focused on cloning IL-2 into a pET-SUMO and pET-28a vector that expresses IL-2 in soluble form and inclusion bodies, respectively. Both constructs were expressed into different E. coli expression strains, but the periplasmic and cytoplasmic expression of IL-2 was highest in overnight culture in Rosetta 2 (DE3). Therefore, E. coli Rosetta 2 (DE3) was selected for large-scale production and purification. Purified IL-2 was characterized by SDS-PAGE and western blotting, while its biological activity was determined using MTT bioassay. The results depict that the periplasmic and cytoplasmic IL-2 achieved adequate purification, yielding 0.86 and 0.51 mg/mL, respectively, with significant cytotoxic activity of periplasmic and cytoplasmic IL-2. Periplasmic IL-2 has shown better yield and significant biological activity in vitro which describes its attainment of native protein structure and function.

Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level.

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, abbreviated as SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets (e.g. nuclear proteins such as cbp1 and mis16, and the centromere-specific histone protein cnp1). The SExY protocol optimizations increasing protein yield could be beneficial for many studies, when targeting low abundance proteins, or for studies that rely on genetic labelling for various reasons (e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality).

Effect of pre-sowing treatment of soybean seeds with inoculant and microelements for yield and quality in the conditions of south-east of Kazakhstan

Soybean is a high-protein and at the same time an oilseed crop. Its cultivation brings profit from the sale of seeds and enriches the soil through the activity of nitrogen-fixing plants. The effectiveness of pre-sowing treatment of soybean seeds with a nitrogen-fixing preparation (HiStick© inoculant) in combination with molybdenum and cobalt salts was evaluated at field level from 2019 to 2021 in the conditions of south-east Kazakhstan. The profitability of the crop could be increased by the use of pre-sowing treatment with these micronutrients which are involved in the processes of photosynthesis and nitrogen fixation. The most indicative characteristics for assessing the impact of pre-sowing seed treatment are the accumulation of protein and oil per hectare, since grain yield increased with pre-sowing treatment without changing quality characteristics (expressed as concentrations). Our research showed an increase in protein yield per hectare with pre-sowing treatment. When seeds of the early ripening Ivushka variety were treated with molybdenum and cobalt without HiStick©, protein yield per hectare increased by 8%. When using the HiStick© preparation together with microelements, the protein yield increased by 4.8%, 8.7% and 12.8% for Zhansaya, Lastochka (late-maturing) and Birlik CV varieties respectively. The increase in oil yield after pre-sowing seed treatment for the early-ripening Ivushka variety was 8.8% (complete treatment) and 6.8% for the late-ripening Lastochka variety (with micronutrients only). Therefore the increase of grain protein and oil yields improves the profitability of this crop for a range of soybean varieties.

The Causes for Genomic Instability and How to Try and Reduce Them Through Rational Design of Synthetic DNA.

Genetic engineering has revolutionized our ability to manipulate DNA and engineer organisms for various applications. However, this approach can lead to genomic instability, which can result in unwanted effects such as toxicity, mutagenesis, and reduced productivity. To overcome these challenges, smart design of synthetic DNA has emerged as a promising solution. By taking into consideration the intricate relationships between gene expression and cellular metabolism, researchers can design synthetic constructs that minimize metabolic stress on the host cell, reduce mutagenesis, and increase protein yield. In this chapter, we summarize the main challenges of genomic instability in genetic engineering and address the dangers of unknowingly incorporating genomically unstable sequences in synthetic DNA. We also demonstrate the instability of those sequences by the fact that they are selected against conserved sequences in nature. We highlight the benefits of using ESO, a tool for the rational design of DNA for avoiding genetically unstable sequences, and also summarize the main principles and working parameters of the software that allow maximizing its benefits and impact.

Determination of process parameters and precipitation methods for potential large-scale production of sugar beet leaf protein concentrate.

Sugar beet is one of the most produced industrial plants in the world, and during manufacturing it produces a large quantity of leaf waste. Because this waste is rich in protein, this study aimed to identify an efficient method for producing large-scale protein concentrate from sugar beet leaves. Results showed that protein extraction from fresh leaves was more effective than from dried leaves. Maximum protein extraction was achieved at pH 9, compared with pH 7 or 8. Blanching as a pretreatment reduced protein yield during isoelectric precipitation, with a yield of 2.31% compared to 20.20% without blanching. Consequently, blanching was excluded from the extraction process. After extraction, isoelectric precipitation, heat coagulation, and isoelectric-ammonium sulfate precipitation were compared. Although the latter resulted in the highest protein yield, Fourier transform infrared analysis revealed that excessive salt was not removed during dialysis, making it unsuitable for scale-up due to its additional cost and complexity. Therefore, isoelectric precipitation was selected as the appropriate method for protein precipitation from sugar beet leaves. To increase yield, extractions were assisted by ultrasound or enzyme addition. Ultrasound-assisted extraction resulted in an increased protein yield from 20.20% to 28.60%, while Pectinex Ultra SP-L-assisted extraction was the most effective, increasing protein yield from 20.20% to 38.09%. Proteins were extracted from fresh sugar beet leaves using optimum conditions (50 °C, 30 min, pH 9) and precipitated at isoelectric point, with enzymatic-assisted extraction yielding the maximum protein recovery. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Unlocking nutritional and functional insights: exploring the impact of germination time on bean protein isolate

SummaryThe study aims to investigate the effect of germination time on kidney bean protein isolate (KBPI), enhancing its nutritional and functional qualities to fill a knowledge gap. Using an alkaline extraction‐isoelectric precipitation strategy, KBPI was extracted with different germination periods (0–72 h). Germination up to 48 h significantly increased GKBPI (germinated kidney bean protein isolate) yield to 44.01%–50.01%, with G48H at 81.20% showing the highest protein recovery at 45 °C. Essential amino acids like valine and threonine were notably retained up to 48 h at 45 °C, preserving GKBPI's nutritional profile. Gelling capacity, protein solubility, foaming and emulsification properties were favourable at 35 °C with 48‐h germinated samples, making GKBPI versatile for food applications. GKBPI also displayed antioxidant activity, with DPPH% values ranging from 20.19% to 24.47%. After 72 h of germination, phytic acid levels decreased by 47.94%, leading to higher in vitro protein digestibility (IVDP) in G48R (germinated kidney bean for 48 h at 35 °C alkali extraction temperature; 82.82%) and G48H (germinated kidney bean for 48 h at 45 °C alkali extraction temperature; 85.55%) due to reduced anti‐nutrients. The study delves into the novelty of germination time's impact on KBPI. Extended germination, up to 48 h, increases protein yield, recovery, IVDP, antioxidant activity and enhances functional properties and reduced anti‐nutrients which indicates that GKBPI has significant potential as an ingredient for various food applications. However, further research is needed for in vitro data, toxins profile and a comprehensive understanding of its practical, economic and health‐related implications.

Enhancing silkworm protein yield, extraction efficiency, structure, functionality, and antioxidant activity using ultrasound-, microwave-, and freeze-thaw-assisted methods.

Silkworm protein applications are limited in the food industry because of their low emulsifying and foaming properties. This study investigated the effect of ultrasound-assisted extraction (UAE) for 15 and 30 min, microwave-assisted extraction (MAE) for 1 and 2 min, and freeze-thaw-assisted extraction (FTAE) for one and three cycles on the yield, extraction efficiency, functional properties, and antioxidant activities of proteins from silkworm pupae. Relationships of protein structure and functionality were also examined. UAE for 15 and 30 min and MAE for 1 and 2 min significantly increased protein yield and extraction efficiency compared to the control. Both UAE and MAE processes, especially MAE for 2 min, greatly improved the emulsifying and foaming properties of extracted proteins. FTAE one and three cycles did not increase the protein yield and extraction efficiency but showed enhanced functional properties, especially foaming. All samples showed changes in protein structure, such as increased exposed sulfhydryl (SH) contents, denaturation temperatures, and enthalpy. Only MAE samples had low-molecular-weight proteins based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. UAE and FTAE samples had significantly higher antioxidant activities, while the MAE process showed the opposite. UAE and MAE processes improved the yield and functionality of extracted silkworm proteins, while MAE negatively impacted protein antioxidant activities. © 2023 Society of Chemical Industry.

Isoelectric point (pI)-based phase separation (pI-BPS) purification of elastin-like polypeptides (ELPs) containing charged, biologically active fusion proteins (ELP-FPs).

Elastin-like polypeptides (ELPs) are peptide-based biomaterials with residue sequence (VPGXG)n where X is any residue except proline. ELPs are a useful modality for delivering biologically active proteins (growth factors, protease inhibitors, anti-inflammatory peptides, etc.) as fusion proteins (ELP-FP). ELP-FPs are particularly cost-effective because they can be rapidly purified using Inverse Temperature Cycling (ITC) via the reversible formation and precipitation of entropically driven aggregates above a transition temperature (Tt ). When ELP fusion proteins (ELP-FPs) contain significant charge density at physiological pH, electrostatic repulsion between them severely inhibits aggregate formation. The literature does not currently describe methods for purifying ELP-FPs containing charged proteins on either side of the ELP sequence as fusion partners without organic solvents. Here, the isoelectric point (pI) of ELP-FPs is discussed as a means of neutralizing surface charges on ELP-FPs and increasing ITC yield to dramatically high levels. We use pI-based phase separation (pI-BPS) to purify ELP-FPs containing cationic and anionic fusion proteins. We report a dramatic increase in protein yield when using pI-BPS for purification of ELP-FPs. Proteins purified by this method also retain the functional activity of the protein present in the ELP-FP. Techniques developed here enable significant diversification of possible fusion proteins delivered by ELPs as ELP-FPs by allowing them to be produced and purified at higher quantities and yields.

Structural and computational design of a SARS-CoV-2 spike antigen with improved expression and immunogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern challenge the efficacy of approved vaccines, emphasizing the need for updated spike antigens. Here, we use an evolutionary-based design aimed at boosting protein expression levels of S-2P and improving immunogenic outcomes in mice. Thirty-six prototype antigens were generated in silico and 15 were produced for biochemical analysis. S2D14, which contains 20 computationally designed mutations within the S2 domain and a rationally engineered D614G mutation in the SD2 domain, has an ~11-fold increase in protein yield and retains RBD antigenicity. Cryo-electron microscopy structures reveal a mixture of populations in various RBD conformational states. Vaccination of mice with adjuvanted S2D14 elicited higher cross-neutralizing antibody titers than adjuvanted S-2P against the SARS-CoV-2 Wuhan strain and four variants of concern. S2D14 may be a useful scaffold or tool for the design of future coronavirus vaccines, and the approaches used for the design of S2D14 may be broadly applicable to streamline vaccine discovery.

Extraction and modification of protein from sesame oil cake by the application of emerging technologies

Present work evaluated the extraction yield and quality of protein from defatted sesame meal (DSM) using ultrasound-assisted extraction (UAE), low-temperature microwave-assisted extraction (MAE), and moderate electric field-assisted extraction (MEFAE). It was observed that the application of emerging technologies as pre-treatments significantly increased protein yield from 26.4% (without pre-treatments) to 38.9% (with UAE 450 W–20 min), 36.05% for MAE (with 500 W–20 min) and 32.75% for MEFAE (with 100 V–30 min) and improved the functional properties of Sesame protein isolate (SPI). FTIR revealed Amide I to III bands in SPI, with characteristic changes in protein secondary structure. The treated sample showed higher thermal stability evident from the denaturation temperature increase from 203.41 to 221.40 °C. The protein's microstructure in this study was transformed using various extraction techniques into a compact structure with a wrinkled surface. MAE-SPI showed maximum Invitro protein digestibility as 94.44%. The molecular weight profile revealed that the SPI samples' protein bands had molecular weights between 6.5 and 200 kDa. SDS-PAGE results revealed a change in the intensity of protein bands, and all of the main allergen protein bands were degraded.

An Investigation into the Mechanism of Alkaline Extraction-Isoelectric Point Precipitation (AE-IEP) of High-Thiol Plant Proteins

Hempseed protein isolate (HPI) has drawn significant attention as a promising source of plant-based protein due to its high nutritional value. The poor functionality (e.g., solubility and emulsifying properties) of HPI has impeded its food application for years. This study provides important new information on hempseed protein extraction, which may provide further insights into the extraction of other high-thiol-based plant proteins to make valuable plant-based products with improved functional properties. In this study, HPI was produced from hempseed meals using the AE-IEP method. The underlying mechanisms and extraction kinetics were investigated under different experimental conditions (pH 9.0–12.0, temperature 24–70 °C, and time 0–120 min). The results suggested that disulphide bond formation is an inevitable side reaction during hempseed protein extraction and that the protein yield and the free -SH content can be influenced by different extraction conditions. A high solution pH and temperature, and long extraction time result in increased protein yield but incur the formation of more intermolecular disulphide bonds, which might be the reason for the poor functionality of the HPI. For instance, it was particularly observable that the protein solubility of HPI products reduced when the extraction pH was increased. The emulsifying properties and surface tension data demonstrated that the functionality of the extracted hempseed protein was significantly reduced at longer extraction times. A response surface methodology (RSM) optimization model was used to determine the conditions that could maximise HPI functionality. However, a three-fold reduction in protein yield must be sacrificed to obtain the protein with this high functionality.

Modifications to functional and biological properties of proteins of cowpea pulse crop by ultrasound-assisted extraction

Natural resource depletion, negative environmental effects and the challenge to secure global food security led to the establishment of the Sustainable Development Goals (SDGs). In need to explore underutilized sustainable protein sources, this study aims at isolating protein from cowpea by ultrasound-assisted extraction (UAE), where the techno-functional characteristics of the protein isolates were studied at different sonication conditions i.e., 100 W and 200 W at processing times ranging from 5 to 20 min. The US at 200 W-10 min produced the optimal results for all properties. In this process combination, there was an increase in protein yield, solubility, water-holding capacity, foaming capacity and stability, emulsion activity and stability, zeta-potential, and in-vitro protein digestibility from 31.78% to 58.96%, 57.26% to 68.85%, 3.06 g/g to 3.68 g/g 70.64% to 83.74%, 30.76% to 60.01%, 47.48% to 64.26%, 56.59% to 87.71%, –32.9 mV to −44.2 mV and 88.27% to 89.99%, respectively and particle size dropped from 763 nm to 559 nm in comparison to control. The microstructure and secondary-structure alterations of proteins caused by sonication were validated by SEM images, SDS-PAGE, and FTIR analyses. Sonication leads to acoustic cavitation and penetrate the cell walls, improving extraction from the solid to liquid phase. After sonication, the hydrophobic protein groups were exposed and proteins were partially denatured which increased its functionality. The findings demonstrated that UAE of cowpea protein improved yield, modify characteristics to fit the needs of the food industry, and contribute to achieving SDGs 2, 3, 7, 12, and 13.

Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway

Pichia pastoris is a commonly used microbial host for recombinant protein production. It is mostly cultivated in fed-batch mode, in which the environment of the cell is continuously changing. Hence, it is vital to understand the influence of feeding strategy parameters on the intracellular reaction network to fine-tune bioreactor performance. This study used dynamic flux balance analysis (DFBA) integrated with transcriptomics data to simulate the recombinant P. pastoris (Muts ) growth during the induction phase for three fed-batch strategies, conducted at constant specific growth rates (μ-stat). The induction phase was split into equal time intervals, and the correlated reactions with protein yield were identified in the three fed-batch strategies using the Pearson correlation coefficient. Subsequently, principal component analysis (PCA) was applied to cluster induction phase time intervals and identify the role of correlated reactions on metabolic differentiation of time intervals. It was found that increasing fluxes through the methanol dissimilation pathway increased protein yield. By adding a methanol assimilation pathway inhibitor (HgCl2 ) to the shake flask medium growing on glycerol: methanol mixture (10%: 90%, v/v), the protein titre increased by 60%. As per DFBA, the higher the methanol to biomass flux ratio (Rmeoh/Δx ), the higher the protein yield. Finally, a novel feeding strategy was developed to increase the amount of Rmeoh/Δx compared to the three feeding strategies. The concentration of recombinant human growth hormone (rhGH), used as the model protein, increased by 16% compared to the optimal culture result obtained previously (800 mg L-1 to 928 mg L-1 ), while production yield improved by 85% (24.8 mg gDCW -1 to 46 mg gDCW -1 ).

Optimization and evaluation of tear protein elution from Schirmer's strips in dry eye disease.

Extraction of tear protein from Schirmer's strip is a prerequisite for the proper identification and screening of biomarkers in dry eye disease. The study compares different methods of extraction of tear proteins from the Schirmer's strip. Reflex tear was collected from healthy controls (HC; n = 12), Stevens-Johnson syndrome (SJS; n = 3) and dry eye disease (DED; n = 3) patients using capillary tube. This tear was used to measure the volume absorbed by Schirmer's strip per microliter. Different buffers (6) were used to compare the protein yield from the Schirmer's strip in four different conditions. The tear proteins extracted using the highest protein yield buffer were analyzed by mass spectrometry. A linear relationship between the tear volume and wetting length was observed (r = 0.0.997, n = 6). The highest yield was observed after incubation of the Schirmer's strip in 100 mM ammonium bicarbonate (ABC) with 0.25% Nonidet P-40(NP-40) at 4°C for an hour (P < 0.00005). The in-solution digestion of tear eluted in the above condition 100 Mm ABC + 0.25% NP-40 with one-hour incubation yielded a total of 2119 proteins in HC, SJS, and DED. The unique protein observed in SJS and DED was 0.6% and 17.9%, respectively. The significantly expressed proteins are associated with innate immune response, proteolysis, wound healing, and defense response. A method for extraction of protein from Schirmer's strip was optimized for increase in protein yield from the tear sample. SJS and DED tear samples have unique protein signature. The study will aid in better design of tear protein-based experimental study.

Effects of nitrogen fertilizer on protein synthesis, accumulation, and physicochemical properties in common buckwheat

Nitrogen (N) fertilization affects grain quality in common buckwheat (Fagopyrum esculentum Moench). But the effects of N fertilizer on various buckwheat protein parameters are not fully understood. This study aimed to investigate the synthesis, accumulation, and quality of buckwheat protein under four N application rates in the Loess Plateau, China. Optimal N application (180 kg N ha−1) improved yield, agronomic traits, and N transport and increased protein yield and protein component accumulation. Prolamin and glutelin accumulation first increased and then decreased with increasing N application. The relationships between the contents of protein components and the amount of applied N generally followed quadratic functions. Nitrate reductase and glutamine synthetase activities first increased and then decreased with increasing N levels. Optimal N fertilizer increased the waterholding capacity and thermal stability of buckwheat protein and reduced its emulsification capacity, but negligibly changed its oil-absorption capacity. Hydrophobic amino acids and glutelin content were the main factors affecting protein quality.

Effects of feeding rumen-protected methionine pre- and postpartum in multiparous Holstein cows: Health disorders and interactions with production and reproduction

Study objectives were to evaluate the effects of feeding rumen-protected Met (RPM) in pre- and postpartum total mixed rations (TMR) on health disorders and the interactions of health disorders with lactation and reproductive performance. Multiparous Holstein cows [470; 235 cows at University of Wisconsin (UW) and 235 cows at Cornell University (CU)] were enrolled at approximately 4 wk before parturition and housed in close-up dry cow (n = 6) and replicated lactation pens (n = 16). Pens were randomly assigned to treatment diets (pre- and postpartum, respectively): (1) control (CON): basal diet = 2.30% and 2.09% Met as % of metabolizable protein (MP) (UW) or 2.22% and 2.19% Met as % of MP (CU); (2) RPM: basal diet fed with RPM with 2.83% and 2.58% Met (Smartamine M, Adisseo Inc.; 12 g prepartum and 27 g postpartum), as % of MP (UW) or 2.85% and 2.65% Met (Smartamine M; 13 g prepartum and 28 g postpartum), as % of MP (CU). Total serum Ca was evaluated at the time of parturition and on d 3 ± 1 postpartum. Daily rumination was monitored from 7 d before parturition until 28 d postpartum. Health disorders were recorded during the experimental period until the time of first pregnancy diagnosis (32 d after timed artificial insemination; 112 ± 3 d in milk). Uterine health was evaluated on d 35 ± 3 postpartum. Time to pregnancy and herd exit were evaluated up to 350 d in milk. Treatment had no effect on the incidence of most health disorders and did not alter daily rumination. Cows fed RPM had reduced subclinical hypocalcemia (13.6 vs. 22%; UW only) on day of parturition relative to CON. Percentage of cows culled (13.1 vs. 19.3%) and hazard of herd exit due to culling [hazard ratio = 0.65, 95% confidence interval (CI): 0.42-1.02] tended to be reduced for cows fed RPM compared with CON. Moreover, cows fed RPM had greater milk protein concentration and protein yield overall, although retrospective analysis indicated that RPM only significantly increased protein yield in the group of cows with one or more health disorders (1.47 vs. 1.40 kg/d), not in cows without health disorders (1.49 vs. 1.46 kg/d) compared with CON. Overall, treatment had no effect on pregnancy per timed artificial insemination; however, among cows with health disorders, those fed RPM had reduced time to pregnancy compared with CON (hazard ratio = 0.71, 95% CI: 0.53-0.96). Thus, except for subclinical hypocalcemia on the day of parturition, feeding RPM in pre- and postpartum TMR did not reduce the incidence of health disorders, but our retrospective analysis indicated that it lessened the negative effects of health disorders on milk protein production and time to pregnancy.

Cultivation of microbial cultures at high cell density in microbioreactor fermentations to increase protein yield.

Microbioreactors have been proven to be a useful tool in high-throughput applications, such as clone screening, synthetic library testing, and media optimization. Most were designed for low cell density applications, where the optical density of the cultures typically does not exceed an OD600 of 10. In microbial applications, where protein is to be expressed, such a scale is not sufficient to produce material for extensive target molecule testing. Here, we present a method for growing high-cell density Escherichia coli cultures in milliliter-scale bioreactors, to produce milligram quantities of target protein. We used a micro-Matrix system with a starting volume of 3ml per culture. A combination of defined medium, a fed-batch feeding strategy at low temperature, and an advanced self-adapting control algorithm achieved up to 0.7g of wet cell weight (WCW) in a 5.7ml final culture volume, which corresponds to 123g/L WCW. This translates to an estimated protein yield of 1150mg of target protein per liter final volume.

Limited enzymatic hydrolysis of green coffee protein as a technique for preparing new functional food components

Valorisation of side-streams in food production has become an important booster for increased sustainability in food production. The objective of this work was to study and improve the functional properties of green coffee (GC) protein. Extraction of defatted GC meal by using PVPP slightly increased protein yield and significantly decreased the amount of covalently and non-covalently bound CQA, therefore decreasing the antioxidant activity of the meal. Peptic hydrolysis at pH 1.5 led to a significantly higher degree of hydrolysis (DH) than at pH 3. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weights of peptides of GC protein hydrolysates were in the range of 11–60 kDa, while peptides were in the range of 500–5000 Da using matrix-assisted laser desorption/ionization-time of flight mass spectroscopy (MALDI-TOF MS). Additionally, the enzymatic hydrolysis significantly improved the antioxidant activity of the GC protein. Finally, the results suggest that enzymatic hydrolysis with pepsin is an effective technique to provide bioactive compounds. The works presented in our manuscript may help in further exploiting the potential use of green coffee beans for food, cosmetic or pharmaceutical industry.