Maximum Protein Yield Research Articles

Optimization of extraction for efficient recovery of kenaf seed protein isolates: evaluation of physicochemical and techno-functional characteristics.

Kenaf seeds are a rich source of protein; however, finding the best extraction method is crucial to obtaining high-quality protein from these underutilized seeds. This research devised an optimized extraction process for best recovery of kenaf seeds protein using response surface methodology. The key parameters affecting the yield and protein content were optimized, including extraction pH (2-11), seed:water ratio (5:1-50:1), temperature (30-90 °C), and duration (20-360 min). The physicochemical and techno-functional properties of kenaf seed protein isolates (KSPIs) were examined. A maximum protein yield of 12.05 g/100 g with purity level 91.94 g/100 g was obtained using an optimized extraction with pH 11.0, seed:water ratio 50:1, 360 min duration, and temperature 50 °C. The oil and water retention capacities of KSPI were 1.14 mL g-1 and 1.37 mL g-1 respectively. After 30 min at pH 7, KSPIs demonstrated remarkable emulsion capacity (83.12%) and stability (75.63%), along with high foaming capacity (106%) and stability (18.3%). As per high-performance liquid chromatography analysis, arginine, glutamic acid, leucine, phenylalanine, and lysine were the most abundant amino acids detected in KPSIs. The KSPIs' globular protein structure was successfully verified using analytical approaches, including Fourier transform infrared spectroscopy, protein fraction ratios, and differential scanning calorimetry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that KPSI has a molecular weight distribution ranging from 10 kDa to 50 kDa. The results of this study support the application of the proposed response-surface-methodology-optimized extraction method for efficient recovery of high-quality kenaf seed proteins that meet the necessary physicochemical and techno-functional requirements. © 2024 Society of Chemical Industry.

Response surface optimization of protein extraction from cold-pressed terebinth (Pistacia terebinthus L.) oil byproducts: Physicochemical and functional characteristics.

The current study focused on optimizing the extraction parameters of terebinth seed proteins from cold-pressed terebinth oil byproducts to maximize protein purity and protein yield. The isolated proteins were characterized to evaluate their properties; thus revealing the valorization potential of these byproducts. Response surface methodology was used to detect the effect of three extraction parameters (pH, temperature, and time). The protein isolates were studied for their physicochemical and functional characteristics. The results indicated that an extraction pH of 8, a temperature of 50°C, and an extraction period of 60min are optimum conditions for obtaining protein isolates with the highest purity. On the other hand, it was demonstrated that an extraction pH of 12, a temperature of 46.4°C, and an extraction duration of 102.4min were optimum conditions for the maximum protein yield. The proteins produced under these two sets of conditions, referred to as TRP (terebinth protein with maximum purity)and TRY (terebinth protein with maximum yield), respectively, exhibited comparable oil absorption capacity (OAC), foaming, emulsifying capabilities, and stability. Both proteins showed the highest solubility at pH 11, and their zeta potentials approached zero at pH 4, indicating proximity to their isoelectric points. However, FRAP and DPPH assays showed that TRP and TRY offered low antioxidative capacity. The high β-sheet content in TRP and TRY suggests enhanced thermal stability but reduced digestibility of these proteins. Therefore, in addition to protein enrichment, TRP and TRY protein isolates can be utilized in muffins and other food applications thanks to their favorable oil absorption, foaming and emulsifying capacities, and thermal stabilities.

The effect of cultivation technologies on weed infestation and productivity of peas

The current paper has presented the study results obtained in 2020–2023, in the stationary field trial of the FSBSI “KurskFARC”. The purpose of the study was to identify and evaluate the impact of technologies with different levels of soil tillage minimization on weed infestation, the formation of yield structure elements and pea productivity in the conditions of the CBR. There have been studied four pea cultivation technologies, such as traditional (plowing at 20–22 cm, main application of N15P40K40 + top dressing with N34); differentiated (chiseling by 20–22 cm + disking by 8–10 cm, main application N15P40K40 + top dressing with N34); minimal (discing up to 8 cm, main application N15P40K40 + top dressing with N34); direct sowing (no-tillage, main application N5P14K14 + pre-sowing N10P26K26 + top dressing with N34). There has been established the advantage of traditional technology in reducing weed infestation, and direct sowing in the formation of yield structure elements, indicators of grain quality and pea productivity. The traditional pea cultivation technology reduced the total number of weeds by 1.9–2.0 times and their dry weight by 1.2–1.8 times during the period critical for the growth and development of the crop, and by 2.1–2.5 and 1.3–2.0 times before harvesting. The highest indicators of plant density for harvesting, the mean number of grains per plant and unit area, and protein percentage in grain were identified with direct sowing, which made it possible to obtain the maximum pea productivity (2.31 t/ha) and protein yield with the harvest (460.6 kg/ha). The use of traditional, differentiated, and minimal technologies contributed to a reduction of plant density before harvesting by 5.4–9.3 %, the mean number of grains per 1 m2 by 4.2–22.6 % and per plant by 0.6–18. 6 %, protein percentage in grain by 0.25–0.90 %, which led to a productivity decrease by 4.3–10.8 %, protein yield per hectare by 5.3–12.6 %.

COMPARATIVE ASSESSMENT OF THE PRODUCTIVITY OF VARIOUS GRAIN CROPS

COMPARATIVE ASSESSMENT OF THE PRODUCTIVITY OF VARIOUS GRAIN CROPS

Development of Cell-Free Transcription-Translation Systems in Three Soil Pseudomonads.

In vitro transcription-translation (TX-TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX-TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth: Pseudomonas synxantha, Pseudomonas chlororaphis, and Pseudomonas aureofaciens. All three species demonstrated multiple sonication, runoff, and salt conditions producing detectable protein synthesis. One of these new TX-TL systems, P. synxantha, demonstrated a maximum protein yield of 2.5 μM at 125 proteins per DNA template, a maximum protein synthesis rate of 20 nM/min, and a range of DNA concentrations with a linear correspondence with the resulting protein synthesis. A set of different constitutive promoters driving mNeonGreen expression were tested in TX-TL and integrated into the genome, showing similar normalized strengths for in vivo and in vitro fluorescence. This correspondence between the TX-TL-derived promoter strength and the in vivo promoter strength indicates that these lysate-based cell-free systems can be used to characterize and engineer biological parts without genomic integration, enabling a faster design-build-test cycle.

Influence of weather conditions on yield and protein content in winter wheat grain

The results of three-year studies (2020–2022) conducted in the forest-steppe zone of the Samara region in the agrocenosis of winter wheat are presented. The purpose of the work was to study the influence of natural and climatic conditions of the research area on the formation of yield indicators, protein content in winter wheat grain. The material was 10 varieties from the Vavilov All-Russian Institute of Plant Genetic Resources (VIR) collection and 5 varieties of selection of the Povolzhsky Scientific Research Institute of Selection and Seed Growing named after P.N. Konstantinov. Yield, grain protein content and protein yield per hectare were determined. Varieties characterized by high yield and high amount of protein in grain, showing maximum protein yield in different weather conditions of the years were identified. Over the years of research, the year 2022 was the most favorable for the formation of high yields (between 6 and 8.5 t/ha were harvested in the indicated year), where a large amount of precipitation was observed in May – the first ten-day period of June. The protein content in grain was the highest in 2021 (14–19%), when high temperatures were observed during the grain filling period in the absence of precipitation. For each variety, the correlation dependence of the sum of active temperatures and the amount of precipitation during the spring-summer vegetation period with the studied indicators was established. There was a negative correlation between the sum of active temperatures and yield (from –0.553 to –0.981) and a positive correlation with the protein content indicator (from 0.605 to 0.984) depending on the variety. The amount of precipitation was in a positive correlation with the yield index, the dependence on the protein content in the grain was ambiguous and depended on the variety. The studied indicators were influenced not only by natural and climatic conditions, but also by the genotypic features of the studied varieties, which explains the diverse correlation dependence.

In vitro α-Amylase and α-Glucosidase Inhibitory Activity of Dioscorin from Dioscorea alata

Dioscorea alata is a species of yam found in many countries in Southeast Asia. It contains many bioactive compounds consisting of steroidal saponins, phenolic compounds flavonoids, polysaccharides, and proteins. Eighty to eighty-five of the soluble proteins in the tuber of D. alata are dioscorin. Dioscorin (31 kDa) is the major storage protein of yam tuber, exhibiting biochemical and immunomodulatory properties, such as antioxidant activity, reducing blood pressure and angiotensin I converting enzyme (ACE) inhibitors activity. The aims of this research focused on extraction of the total soluble protein from D. alata tuber, purification of dioscorin, and study of in vitro inhibitory activity of α-amylase and α-glucosidase of dioscorin extracts. The results showed that a maximum yield of total soluble protein was obtained from the buffers extraction using Tris-HCl buffer at pH 9.0 resulting in the protein concentration of 15.10 mg/g with a protein extraction yield of 31.56 ± 2.36%. The crude protein was further recovered using ammonium sulfate precipitation and dialysis. SDS-PAGE analysis showed that dioscorin from crude extracts was precipitated at the concentration of ammonium sulfate of 60% saturation. The purified dioscorin was obtained from anion exchange chromatography (DEAE-Sephacel) and its molecular weight of 31 kDa was estimated on SDS-PAGE. Furthermore, the α-amylase inhibitory activities of crude extract (1 mg/mL), purified dioscorin obtain from gastric digestion and purified dioscorin obtained from intestinal digestion were at 14.44% 2.21% and 78.13%, respectively. Moreover, the results showed that the α-glucosidase inhibitory activity of the crude extract, and purified dioscorin obtained from gastric digestion were at 10.56% and 2.61%, respectively.

Optimization of Soybean Protein Extraction with Ammonium Hydroxide (NH4OH) Using Response Surface Methodology.

Plants have been recognized as renewable and sustainable sources of proteins. However, plant protein extraction is challenged by the plant's recalcitrant cell wall. The conventional extraction methods make use of non-reusable strong alkali chemicals in protein-denaturing extraction conditions. In this study, soy protein was extracted using NH4OH, a weak, recoverable, and reusable alkali. The extraction conditions were optimized using response surface methodology (RSM). A central composite design (CCD) with four independent variables: temperature (25, 40, 55, 70, and 85 °C); NH4OH concentration (0.5, 1, and 1.5%); extraction time (6, 12, 18, and 24 h) and solvent ratio (1:5, 1:10, 1:15 and 1:20 w/v) were used to study the response variables (protein yield and amine concentration). Amine concentration indicates the extent of protein hydrolysis. The RSM model equation for the independent and response variables was computed and used to create the contour plots. A predicted yield of 64.89% protein and 0.19 mM amine revealed a multiple R-squared value of 0.83 and 0.78, respectively. The optimum conditions to obtain the maximum protein yield (65.66%) with the least amine concentration (0.14 Mm) were obtained with 0.5% NH4OH concentration, 12 h extraction time, and a 1:10 (w/v) solvent ratio at 52.5 °C. The findings suggest that NH4OH is suitable to extract soybean protein with little or no impact on protein denaturation.

Ultrasound-assisted alkaline extraction of protein from Tenebrio molitor larvae: Extraction kinetics, physiochemical, and functional traits

Currently, as a promising alternative protein source, the interest of edible insect protein has been continuously increased. However, the extraction processing had distinct effects on the physicochemical properties and functionalities of this novel and sustainable protein. In this study, Tenebrio molitor larvae protein (TMLP) was extracted via ultrasound (US)-assisted alkaline extraction. The changes of extraction kinetics, physicochemical characteristics, and functional properties of TMLP as a function of US time (10, 20, 30, 40, 50 min) were investigated. The results showed that 30 min US treatment rendered the maximum protein yield (60.04 %) (P < 0.05). Meanwhile, Peleg's model was considered a suitable model to represent the extraction kinetics of TMLP, with a correlation coefficient of 0.9942. Moreover, the protein secondary structure, particle size, and amino acid profiles of TMLP were changed under the US-assisted alkaline extraction process. Additionally, a significant improvement of the functional properties of TMLP extracted with this method was observed compared to traditional alkaline extraction. In conclusion, the present work suggests that US-assisted alkaline extraction could be considered as a potential method to improve the protein yield, quality profiles, and functional properties of TMLP.

Ultrasound-Assisted Extraction of Protein from Moringa oleifera Seeds and Its Impact on Techno-Functional Properties.

Plant proteins can be an important alternative to animal proteins subject to minor modification to address sustainability issues. The impact of ultrasound application on the yield, techno-functional properties, and molecular characteristics of protein extracted from Moringa oleifera seeds was studied. For this purpose, a central composite design (CCD) was applied to optimize ultrasound-assisted extraction (UAE) parameters such as amplitude (25-75%), solute-to-solvent ratio (1:10-1:30), and pH (9-13) for obtaining the maximum protein yield. At the optimized conditions of 75% amplitude, 1:20 solute-to-solvent ratio, and 11 pH, a protein yield of 39.12% was obtained in the UAE process. Moreover, the best sonication time at optimized conditions was 20 min, which resulted in about 150% more extraction yield in comparison to conventional extraction (CE). The techno-functional properties, for instance, solubility, water (WHC)- and oil-holding capacity (OHC), and emulsifying and foaming properties of the protein obtained from UAE and CE were also compared. The functional properties revealed high solubility, good WHC and OHC, and improved emulsifying properties for protein obtained from UAE. Although protein from UAE provided higher foam formation, foaming stability was significantly lower.

Optimization of Keratin Hydrolysate Extraction from Tannery Sheep Hair Waste

Tannery hair wastes are becoming a challenge for tanners regarding environmental pollution control and human health. In this study, an experiment had been designed to hydrolyse sheep hair in an alkaline medium, and the operational condition for the alkaline extraction of KH has been modeled and optimized. The structure, morphology, functional groups, particle size, and molecular mass of the KH extracts were evaluated experimentally by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), particle size analysis, and SDS-PAGE analysis, respectively. FTIR analysis of the extract confirmed the presence of carboxylic, amide, and aldehyde functional groups and alkyl side chains of amino acids. The molecular weight of the extracted keratin ranges between 3–15 kDa, and X-ray diffraction (XRD) analysis showed an amorphous form of structure with two peaks at 2 theta of 9.36° and 21.16° due to α -helix and β - sheet structure in keratin. Response surface methodology (RSM) coupled with BOX-Behnken design was applied as a statistical tool to investigate the effect of extraction time, the concentration of the hydrolysing agent, and temperature on the response variable (yield of keratin protein). The concentration of the hydrolysing agent was found to be the most significant factor affecting the speed of extraction, but its gradual increase tends to affect the protein content of the extract. Optimum parameters of 0.5 N, 80°C, and 3.5 hr were obtained for the concentration of NaOH, temperature, and extraction time, respectively, with a maximum average protein yield of 91.5% and a percentage total nitrogen content of 14.6% using the Kjeldahl method and 86.57% using the biuret test method.

Effect of foliar application of salicylic acid and mycorrhiza on quantitative and qualitative traits of maize.

An experiment was performed to investigate the effect of mycorrhizal symbiosis and foliar application of salicylic acid on quantitative and qualitative traits of maize during 2018 and 2019 in the research farm of Islamic Azad University, Chalous Branch. Split plot in a randomized complete block design with three replications was used. Experimental factors included mycorrhiza species of (G. mosseae), (G. geosporum) and (G. intraradices) at two levels (no consumption and consumption of mycorrhiza) and salicylic acid at two levels (no consumption and consumption of 1 mμ of salicylic acid). Results of interaction effects of mycorrhiza and salicylic acid on the measured traits revealed that the maximum 1000-grain weight, grain yield, biological yield, phosphorus, potassium, nitrogen percentage and yield of maize grain protein were observed in G. mosseae treatment under foliar application of salicylic acid. Foliar application of salicylic acid increases the root length and provides the necessary conditions for increasing water and nutrient uptake alongwith increase in photosynthesis and thus allocates more photosynthetic substance for development of reproductive organs. Hence, it increases maize grain weight and accordingly grain yield. In general, the results revealed that mycorrhiza and foliar application of salicylic acid increase growth indicators, yield and yield components. It also improved the quality traits of the maize plant. Based on results, the interaction effect of G. mosseae treatment and foliar application of salicylic acid yielded better results than other treatments. Mycorrhiza increases the number of grain in the ear, the number of rows in the ear, increases the plant's ability to absorb phosphorus, and the increase of mycorrhiza along with salicylic acid shows the maximum grain yield in maize. Finally, it can be concluded that the use of mycorrhiza and salicylic acid can be effective in increasing grain in the plant.

Dot-Blotting: A Quick Method for Expression Analysis of Recombinant Proteins.

Expressing recombinant proteins in heterologous host cells is a prerequisite for purification and other downstream processes. Cell cultures require a protein expression test to optimize incubation time, temperature, and additives (like chemical inducers) to identify the best growth conditions with maximum recombinant protein yield. However, running SDS-PAGE followed by western blotting is cumbersome and results are not quick. Here, I describe a simple protocol to quickly check the presence of recombinant protein in cell cultures using a dot-blot experiment. The cells can be rapidly lysed and directly spotted on the nitrocellulose membrane. Then, the membrane is incubated with a horseradish peroxidase (HRP) conjugated antibody raised against the affinity tag present on the recombinant protein to confirm the protein expression by chemiluminescence. It takes less than an hour to get results. This method rapidly investigates recombinant protein expression in different cell lines and tests other variables. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Protein expression analysis for eukaryotic systems Basic Protocol 2: Protein expression analysis for bacterial systems.

The effect of long-and short-stemmed oat in vetch-oat intercropping on weed infestation, agronomic performance, and grain quality in low input systems

As protein-rich crops, grain legumes are of great importance in organic farming. Common vetch (Vicia sativa L.) has not only a higher protein content than faba bean (Vicia faba L.) and pea (Pisum sativum L.) but also has lower requirements for soil and climatic conditions. The phenology of common vetch with the formation of high biomass requires cultivation in an intercropping system with a strong partner ensuring a stable crop stand for machine harvest of the grains. In this study, low- and high-growing common vetch cultivars were intercropped with short- and long-stemmed oats in different seed ratios to find the best combination of both. Therefore, a field trial was conducted at the Thünen Institute of Organic Farming in Northern Germany in a randomised block design with four replications over three years. The agronomic performance showed a slight inhibition in pod formation of vetches in long-stemmed oat mixtures. Furthermore, the high-growing vetch suppressed the short-stemmed oat more than the long-stemmed oat. The two different growth characters of oat had no effect on weed suppression. Moreover, the high-growing vetch showed better weed suppression over all seed ratios in comparison to the low-growing vetch due to its vigorous growth and higher crop height. The regression models showed a strong influence of the year on total and partial grain and protein yields. Long-stemmed oat mixtures had a higher total grain yield than short-stemmed oat mixtures. A comparison of the common vetch cultivars in long-stemmed oat-mixtures demonstrates a total grain yield advantage for mixtures of high-growing vetch (5.3 Mg ha−1 dry matter (d. m.) (2016)) than for low-growing vetch (5.0 Mg ha−1 d. m. (2016)) in seed mixtures with a 51.9 % and a 44.6 % share of vetch. In short-stemmed oat mixtures, total grain yields of 4.2 Mg ha−1 d. m. (2016) were estimated for the low-growing vetch as well as for the high-growing vetch in seed mixtures with a 48.8 % and a 33.6 % share of vetch, respectively. The total grain yields of intercropped common vetches were higher than the respective sole-cropped cultivars. According to the regression model, a maximum total protein yield of 1.0 Mg ha−1 d. m. (2014 and 2015) was estimated for the high-growing vetch in a seed mixture with 79.8 % (2014) and 59.6 % (2015) share of vetch in short- and long-stemmed oat, respectively. The low-growing vetch had the highest total protein yields of 0.8 Mg ha−1 d. m. (2016) in short-stemmed oat mixtures with a 61.9 % share of vetch in the seed mixture. When intercropped with long-stemmed oat, the total protein yield of low-growing vetch-mixtures was lower with 0.7 Mg ha−1 d. m. (2016) in a seed mixture with a 59.6 % share of vetch. Mixtures showed a total grain yield advantage for high-growing vetches in both oat cultivars. Seed mixtures with a 34–64 % share of vetch reached the highest total grain yields. High total protein yields were reached in seed mixtures with higher vetch shares of 58–83 % for the high-growing and 60–95 % for the low-growing vetch. However, the risk of collapse of crop stand increased with high precipitation and high shares of vetch in the seed mixture which makes a machine harvest for grain yield challenging. Therefore, a seed mixture with a 50 % share of vetch is recommended to reach high levels of grain and protein yield but also to prevent an overgrowth of the cereal.

Radial discharge high shear homogenization and ultrasonication assisted pH-shift processing of herring co-products with antioxidant-rich materials for maximum protein yield and functionality

Cross-processing herring co-products with antioxidant-rich helpers including lingonberry-press-cake, shrimp-shells and seaweed was reported to mitigate lipid oxidation but reduce protein yield. Here, four strategies were used to counteract such yield-reduction; optimizing solubilization/precipitation pH, increasing raw-material-to-water-ratio, replacing single-stage-toothed- by radial-discharge- high-shear-mechanical-homogenization (RD-HSMH) and ultrasonication (US). The effects of RD-HSMH and US on lipid oxidation, protein structural and functional properties were studied. Combining four strategies improved total protein yield by 5–12 %, depending on helper type. More than the confirmed antioxidant effects, cross-processing also improved protein water solubility and emulsification activity but reduced gelation properties. RD-HSMH generally improved protein emulsifying and gelation properties but reduced protein water solubility. US reduced protein water solubility and gelation properties. Altogether, it was recommended for all helpers to increase solubilization pH to 12 and raw-material-to-water-ratio to 1:6 followed by RD-HSMH at 8000 rpm for 90 s, aiming for maximum protein yield and emulsifying and gelation properties.

Interaction Effect of Phosphorus and Boron on Yield Components, Productivity Parameters and Quality Traits of Lentil (Lens culinaris L.)

A field trial was conducted on sandy loam soil having low status of organic carbon and accessible nitrogen, medium in accessible phosphorous and high in accessible potassium at pot house of department of Soil Science and Agricultural Chemistry of C.S.A.U.A&amp;T, Kanpur (campus) under Indo-Gangetic Plain zone of Uttar Pradesh, amid Rabi season of 2021-22. The experiment comprised of 16 treatment combinations in Factorial randomized block design with three replications. Lentil variety Lentil Shekhar 2 (KLB-303) was grown with the recommended agronomic practices. On the premise of the comes about exuded from the present investigation, it might be concluded that application of 60 kg P2O5 ha-1+ 2.5 kg Boron ha-1 significantly recorded maximum yield components viz. no. of pod plant -1 (71), Seed pod -1 (1.84) and Seed Index (3.23 g). Similarly, among the productivity parameters such as grain yield (15.67 q ha-1), straw yield (28.22 q ha-1), biological yield (43.89 q ha-1) and harvest index (35.70%) was also associated with the application of 60 kg P2O5 ha-1+ 2.5 kg Boron ha-1. Among the quality traits maximum protein content (22.40%) and protein yield (351.00 kg ha-1) was also associated with the application of 60 kg P2O5 ha-1+ 2.5 kg Boron ha-1. The present experiment clearly points out the significance of balanced use of Phosphorus and Boron nutrients in Lentil for improving the yield components, yield and quality of Lentil crop.

Extraction optimization, characterization and anti- inflammatory activity of functional protein from Mangifera indica seeds kernel

Mango seed kernel (MSK) was observed to be a likely source of bioactive protein. Box-Behnken Design (BBD) was employed to optimize the protein extraction conditions. The optimum extraction conditions for maximum protein yield (68.336%) were corresponding to extraction temperature 54.53 °C, buffer-to-sample values 41.79 mL/g and extraction time 120 min with desirability value of 0.471%. The results of electrophoresis patterns of MSKP revealed two types of proteins; low molecular weight between 6 to 98 kDa explained the presence of globulin with type (2S) and another principal protein band of 27.6 kDa explained the presence of lectins. The extracted proteins under optimized conditions showed inhibition activity of nitric oxide (NO) release and pro-inflammatory inducers like (IL-6), (IL-1r3) and (TNF-a) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. These findings revealed that MSK could be used as a promising nutraceutical of functional and health-promoting proteins.

Highly Expressed Soluble Recombinant Anti-GFP VHHs in Escherichia coli via Optimized Signal Peptides, Strains, and Inducers.

Antigen-binding variable domains of the H chain of heavy-chain antibodies (VHHs), also known as nanobodies (Nbs), are of great interest in imaging technique, disease prevention, diagnosis, and therapy. High-level expression of soluble Nbs is very important for its industrial production. In this study, we optimized the expression system of anti-green fluorescent protein (GFP) VHHs with three different signal peptides (SPs), outer-membrane protein A (OmpA), pectate lyase B (PelB), and L-asparaginase II SP (L-AsPsII), in different Escherichia coli strains via isopropyl β-D-thiogalactoside (IPTG) induction and auto-induction, respectively. The solubility of recombinant anti-GFP VHHs with PelB or OmpA was significantly enhanced to the same extent by IPTG induction and auto-induction in BL21 (DE3) E. coli strain and the maximum yield of target protein reached approximately 0.4 mg/l in a shake flask. The binding activity of recombinant anti-GFP VHHs was also confirmed to be retained by native-polyacrylamide gel electrophoresis (PAGE). These results suggest that SPs like OmpA and PelB could efficiently improve the recombinant anti-GFP VHH solubility without changing its bioactivity, providing a novel strategy to optimize the E. coli expression system of soluble VHHs, and lay the foundation for the industrial production of soluble recombinant anti-GFP VHHs and the research of other VHHs in the future.

Cottage cheese from blends of fresh green peas (Pisum sativum L.) and dairy milk (pEaneer): Preparation, characterization, and sensory evaluation

Traditional tofu from soybeans provides a bunch of health benefits which makes it a staple option in high-protein diets. Concerns on allergenicity/hypercholesterolemia from animal foods and lactose intolerance from dairy based ones have surged the demand for alternative and sustainable plant-based proteins. In this study, a tofu-like coagulated product named pEaneer was prepared using a blend of fresh green peas and dairy milk. Initially, fresh peas were ground with water in 1:2 (w/v) ratio and used for protein extraction by alkali solubilisation followed by isoelectric precipitation. The process parameters such as pH (6-12), time (40–120 min) and temperature (30–90 °C) were optimized for maximum protein yield using full factorial design. The maximum protein yield of 53.99% w/w was obtained at pH 12/60 °C/40min. The pea protein extract (PPE) obtained under optimized conditions was used to obtain pEaneer with and without dairy milk by blending them in various proportions. Efforts to achieve a desirable appearance and texture for pEaneer using D-optimal design indicated 0.3% (w/v) gellan to be effective. The best pEaneer obtained with 80:20 (w/w) of PPE and dairy milk had 58.01% protein and 16.16% fat vis-à-vis 42.07% protein and 48.78% fat in dairy paneer.

Exploiting genetic variability in the trajectory of lactation yield and somatic cell score with each progressing parity

The inclusion of reproductive performance in dairy cow breeding schemes has resulted in a cumulative improvement in genetic merit for reproductive performance; this improvement should manifest in longer productive lives through a reduced requirement for involuntary culling. Nonetheless, the average length of dairy cow productive life has not changed in most populations, suggesting that risk factors for culling, especially in older cows, are possibly more associated with lower yield or high somatic cell score (SCS) than compromised reproductive performance. The objective of the present study was to understand the dynamics of lactation yields and SCS in dairy cows across parities and, in doing so, quantify the potential to alter this trajectory through breeding. After edits, 3,470,520 305-d milk, fat, and protein yields, as well as milk fat and protein percentage and somatic cell count records from 1,162,473 dairy cows were available for analysis. Random regression animal models were used to identify the parity in which individual cows reached their maximum lactation yields, and highest average milk composition and SCS; also estimated from these models were the (co)variance components for yield, composition, and SCS per parity across parities. Estimated breeding values for all traits per parity were calculated for cows reaching ≥fifth parity. Of the cows included in the analyses, 91.0%, 92.2%, and 83.4% reached maximum milk, fat, and protein yield in fifth parity, respectively. Conversely, 95.9% of cows reached their highest average fat percentage in first parity and 62.9% of cows reached their highest average protein percentage in third parity. In contrast to both milk yield and composition traits, 98.4% of cows reached their highest average SCS in eighth parity. Individual parity estimates of heritability for milk yield traits, milk composition, and SCS ranged from 0.28 to 0.44, 0.47 to 0.69, and 0.13 to 0.23, respectively. The strength of the genetic correlations per trait among parities was inversely related to the interval between the parities compared; the weakest genetic correlation was 0.67 (standard error = 0.02) between milk yield in parities 1 and 8. Eigenvalues and eigenfunctions of the additive genetic covariance matrices for all investigated traits revealed potential to alter the trajectory of parity profiles for milk yield, milk composition, and SCS. This was further demonstrated when evaluating the trajectories of animal estimated breeding values per parity.