Cell wall as a barrier for protein extraction from tomato leaves: A biochemical study.

Solanum lycopersicum (Tomato) leaves and stems are considered waste. Valorization of this waste can be achieved by for example the extraction of proteins. This prospect is promising but currently not feasible, since protein extraction yields from tomato leaves are low, amongst other due to the (physical) barrier formed by the plant cell walls. However, the molecular aspects of the relationship between cell wall properties and protein extractability from tomato leaves are currently not clear and thus objective of this study. To fill this knowledge gap the biochemical composition of plant cell walls was measured and related to protein extraction yields at different plant ages, leaf positions, and across different tomato accessions, including two Solanum lycopersicum cultivars and the wildtype species S. pimpinellifolium and S. pennellii. For all genotypes, protein extraction yields from tomato leaves were the highest in young tissues, with a decreasing trend towards older plant material. This decrease of protein extraction yield was accompanied by a significant increase of arabinose and galacturonic acid content and a decrease of galactose content in the cell walls of old-vs-young tissues. This resulted in strong negative correlations between protein extraction yield and the content of arabinose and galacturonic acid in the cell wall, and a positive correlation between the content of galactose and protein extraction yield. Overall, these results point to the importance of the pectin network on protein extractability, making pectin a potential breeding target for enhancing protein extractability from tomato leaves.

Oilseed meal proteins: From novel extraction methods to nanocarriers of bioactive compounds.

The global demand for animal proteins is predicted to increase twofold by 2050. This has led to growing environmental and health apprehensions, thereby prompting the appraisal of alternative protein sources. Oilseed meals present a promising alternative due to their abundance in global production and inherent dietary protein content. The alkaline extraction remains the preferred technique for protein extraction from oilseed meals in commercial processes. However, the combination of innovative techniques has proven to be more effective in the recovery and functional modification of oilseed meal proteins (OMPs), resulting in improved protein quality and reduced allergenicity and environmental hazards. This manuscript explores the extraction of valuable proteins from sustainable sources, specifically by-products from the oil processing industry, using emerging technologies. Chemical structure, nutritional value, and functional properties of the main OMPs are evaluated with a particular focus on their potential application as nanocarriers for bioactive compounds.

Alternative methods for RuBisCO extraction from sugar beet waste: A comparative approach of ultrasound and high voltage electrical discharge.

Ultrasound (US) and high voltage electric discharge (HVED) with water as a green solvent represent promising novel non-thermal techniques for protein extraction from sugar beet (Beta vulgaris subsp. vulgaris var. altissima) leaves. Compared to HVED, US proved to be a better alternative method for total soluble protein extraction with the aim of obtaining high yield of ribulose-1,5-bisphosphate carboxylase-oxygenase enzyme (RuBisCO). Regardless of the solvent temperature, the highest protein yields were observed at 100% amplitude and 9 min treatment time (84.60 ± 3.98 mg/gd.m. with cold and 96.75 ± 4.30 mg/gd.m. with room temperature deionized water). US treatments at 75% amplitude and 9 min treatment time showed the highest abundance of RuBisCO obtained by immunoblotting assay. The highest protein yields recorded among HVED-treated samples were observed at a voltage of 20 kV and a treatment time of 3 min, disregarding the used gas (33.33 ± 1.06 mg/gd.m. with argon and 34.89 ± 1.59 mg/gd.m. with nitrogen as injected gas), while the highest abundance of the RuBisCO among HVED-treated samples was noticed at 25 kV voltage and 3 min treatment time. By optimizing the US and HVED parameters, it is possible to affect the solubility and improve the isolation of RuBisCO, which could then be purified and implemented into new or already existing functional products.

Plant leaf proteins for food applications: Opportunities and challenges.

Plant-based proteins are gaining a lot of attention for their health benefits and are considered as an alternative to animal proteins for developing sustainable food systems. Against the backdrop, ensuring a healthy diet supplemented with good quality protein will be a massive responsibility of governments across the globe. Increasing the yield of food crops has its limitations, including low acceptance of genetically modified crops, land availability for cultivation, and the need for large quantities of agrochemicals. It necessitates the sensible use of existing resources and farm output to derive the proteins. On average, the protein content of plant leaves is similar to that of milk, which can be efficiently tapped for food applications across the globe. There has been limited research on utilizing plant leaf proteins for food product development over the years, which has not been fruitful. However, the current global food production scenario has pushed some leading economies to reconsider the scope of plant leaf proteins with dedicated efforts. It is evident from installing pilot-scale demonstration plants for protein extraction from agro-food residues to cater to the protein demand with product formulation. The present study thoroughly reviews the opportunities and challenges linked to the production of plant leaf proteins, including its nutritional aspects, extraction and purification strategies, anti-nutritional factors, functional and sensory properties in food product development, and finally, its impact on the environment. Practical Application: Plant leaf proteins are one of the sustainable and alternative source of proteins. It can be produced in most of the agroclimatic conditions without requiring much agricultural inputs. It's functional properties are unique and finds application in novel food product formulations.

The Health-promoting Potential of Edible Mushroom Proteins.

Edible mushrooms have been classified as "next-generation food" due to their high nutritional value coupled with their biological and functional potential. The most extensively studied and reported mushroom macromolecules are polysaccharides. However, macrofungi proteins and peptides are also a representative and significant bioactive group. Several factors such as species, substrate composition and harvest time significantly impact the mushroom protein content, typically ranging between 19 and 35% on a dry weight basis. Proteins work based on their shape and structure. Numerous extraction methods, including chemical and non-conventional, and their implications on protein yield and stability will be discussed. Beyond their biological potential, a great advantage of mushroom proteins is their uniqueness, as they often differ from animal, vegetable, and microbial proteins. According to recently published reports, the most relevant mushroom bioactive proteins and peptides include lectins, fungal immunomodulatory proteins, ubiquitin-like proteins, and proteins possessing enzymatic activity such as ribonucleases laccases, and other enzymes and ergothioneine. These are reported as antioxidant, antiviral, antifungal, antibacterial, antihypertensive, immunomodulatory, antitumour, antihypercholesterolemic or antihyperlipidemic, antidiabetic and anti-inflammatory properties, which improved proteins and peptides research interest and contributed to the increase of mushroom market value. This review provides an overview of the most relevant biochemical and biological properties of the main protein groups in edible mushrooms, explicitly focusing on their biomedical potential. Although mushrooms are a rich source of various proteins, many of these molecules have yet to be identified and characterised. Accordingly, it is crucial to identify and characterise new macromolecules of macrofungi origin, which opens an opportunity for further investigation to identify new bioactives for food, nutraceutical, or medicinal applications.

Protein extraction from cold-pressed hempseed press cake: From laboratory to pilot scale.

During the production of industrial hempseed oil, a press cake is formed as a byproduct, which is often used as animal feed although it contains a high amount of protein that could be used for human consumption. Extracting this valuable protein would reduce food waste and increase the availability of plant-based protein. A protein extraction process based on the pH-shift method was adapted to improve the protein extraction yield from industrial hempseed press cake (HPC). Parameters such as alkali extraction pH, time, and temperature, as well as isoelectric precipitation pH, were investigated in laboratory scale and were thereafter carried out in a pilot trial to explore the suitability for future scale up. The phytic acid content of the extracted protein isolate was also analyzed to investigate any potential inhibitory effect on mineral absorption. A final protein yield of 60.6%, with a precipitated protein content of 90.3% (dw), was obtained using a constant alkali extraction pH of 10.5 for 1 h at room temperature, followed by precipitation at pH 5.5. The pilot trial showed promising results for the future production of industrial hemp protein precipitate on a larger scale, showing a protein yield of 57.0% and protein content of 90.8% (dw). The amount of phytic acid in the protein isolate produced in the optimal laboratory experiment and in the pilot trial was 0.595 and 0.557 g phytic acid/100 g dw, respectively, which is 83%-88% less than in the HPC. This is in the range of other plant-based protein sources (tofu, kidney beans, peas, etc.). PRACTICAL APPLICATION: Industrial hempseed press cake is a byproduct in the production of industrial hempseed oil, which is mostly used as animal feed, but has the potential to become an additional source of plant-based protein for human consumption with a suitable protein extraction method. The extracted hemp protein could be used to develop new plant-based dairy or meat analog products.

Honey and Its Antimicrobial Properties: A Function of a Single Component, or the Sum of Its Parts?

INTRODUCTION: Honey is known for exhibiting antibacterial properties, indicating its use as part of traditional medicine since the early ages. With the advent of antibiotic-resistant bacteria, the need for alternative antimicrobials has outpaced the actual development of novel, broad-spectrum antibiotics. Previous research has revolved around the sugar content of honey because its sweetness makes it an attractive food source. However, research assessing the protein and lipid components of honey is lagging behind that of its sugar counterpart. The goal of this investigation was to examine the antimicrobial properties of honey and to identify any distinct proteins or lipids. METHODS: In order to isolate individual peptides and lipids, the different samples of local and foreign-sourced honeys were dialyzed, and the resulting dialysate proteins were screened via gel electrophoresis (sodium dodecyl-sulfate polyacrylamide gel electrophoresis [SDS-PAGE]) with Coomassie blue and silver stain, while lipids were examined using thin layer chromatography (TLC). To assess antimicrobial potency, a series of Kirby-Bauer disc diffusion assays was performed on Mueller-Hinton agar using different types of raw honey with Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis. The process was then repeated using the peptide extracts from the dialyzed fractions of the honeys. RESULTS: The SDS-PAGE trials revealed repetitive promising protein bands across several gels below 75kDa with both Coomassie blue and silver staining. The TLC analysis of varying raw honey samples consistently demonstrated the presence of medium and long-chain fatty acids, likely in the range of C12-C14. In the disc diffusion assays, the greatest amount of inhibition was seen when the honeys were tested as a whole instead of its constituent parts. CONCLUSION: Instead of an individual component acting as the key to honey's action against bacteria, it appears there is a synergistic relationship amongst the sugars, proteins, and lipids that make each honey unique.

Physico-chemical and colloidal properties of protein extracted from black soldier fly (Hermetia illucens) larvae.

The black soldier fly larvae (BSFL), Hermetia illucens (Linnaeus), has been largely utilized for animal feed. Due to its interesting composition, BSFL has great potential to be further implemented in the human diet. Herein we compared the flour and protein extract composition based on their moisture, ash, amino acids, mineral, and protein content. To have wide knowledge on protein profile and behavior, SDS-page electrophoresis, Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) were used to give information about protein structure and thermal stability, respectively. The flour and protein extract contained respectively 37.3% and 61.1% of protein. DSC graph reported a glass transition temperature around 30 °C, recognizable by a shift in the curve, and an endothermic peak for solid melting at around 200 °C. FTIR analysis showed the main amide bands (A, B, I, II, III) for the flour and protein extract. The foam properties of BSFL protein extract were explored under different temperatures treatment, and the best foam stability was reached at 85 °C with 15 min of treatment. The data highlight the promising techno-functional properties of BSFL protein extract, and that the nutritional composition might be suitable for further use of BSFL as food fortification system.

Extraction and identification of biologically important proteins from the medicinal plant God's crown (Phaleria macrocarpa).

The fruit and leaf of God's crown (Phaleria macrocarpa) have been traditionally used to treat a wide variety of diseases. However, the proteins of this tropical plant are still heavily understudied. Three protein extraction methods; phenol (Phe), trichloroacetic acid (TCA)-acetone-phenol (TCA-A-Phe), and ultrasonic (Ult) were compared on the fruit and leaf of P. macrocarpa. The Phe extraction method showed the highest percentage of recovered protein after the resolubilization process for both leaf (12.24%) and fruit (30.41%) based on protein yields of the leaf (6.15 mg/g) and fruit (36.98 mg/g). Phe and TCA-A-Phe extraction methods gave well-resolved bands over a wide range of molecular weights through sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following liquid chromatography-tandem mass spectrometry analysis, proteins identified through the Phe extraction method were 30%-35% enzymatic proteins, including oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases that possess various biological functions. PRACTICAL APPLICATIONS: Every part of God's crown plant is traditionally consumed to treat various illnesses. While plant's benefits are well known and have led to a plethora of health products, the proteome remains mostly unknown. This study compares three protein extraction methods for the leaf and fruit of P. macrocarpa and identifies their proteins thru LC-MS/MS coupled with PEAKS. These method comparisons can be a guide for works on other plants as well. In addition, the proteomics data from this study may shed light on the functional properties of these plant parts and their products.

Rapid extraction of watermelon seed proteins using microwave and its functional properties.

Solid food industry waste like watermelon seed is an excellent source of value-added components such as proteins, oil, and carbohydrate. In the present study, protein extraction was carried out using microwave energy from defatted watermelon seeds (DWS), containing 50% of proteins. Microwave-assisted extraction (MAE) was optimized with different parameters, namely, solid to solvent ratio (1:10-1:40), pH (7-10), microwave power (30 W, 50 W, 70 W), extraction time (30 s-8 min) and moisture content or pre-leaching effect. Maximum protein recovery was achieved with 50 W microwave power, solid to solvent ration of 1:30, and pH 10 in 2 minutes of microwave irradiation time. MAE gave higher yield in less time compared to conventional extraction. SDS-PAGE confirmed the molecular weight of watermelon seed proteins (WSP) in the range of 25-250 kDa. A comparative study showed 90% protein recovery with MAE in 2 min with 1:30 (w/v) solid to solvent ratio, whereas ultrasound gave 87% in 9 min with 1:50 (w/v) ratio and batch 72% in 25 min with 1:70 (w/v) ratio. Watermelon seed proteins obtained from MAE method possess excellent functional properties with reference to conventional extraction method indicating its application in food products.

Optimization of extraction and deamidation of edible protein from evening primrose (Oenothera biennis L.) oil processing by-products and its effect on structural and techno-functional properties.

The optimization of ultrasound-assisted alkaline extraction and enzymatic deamidation by protein-glutaminase (PG) on evening primrose seed cake (EPSC) protein and its effect on structure (amino acid composition, secondary structure and electrophoresis pattern) and techno-functional properties (water-holding and oil-binding capacities, solubility, emulsifying and foaming properties) of EPSC protein were evaluated. The optimum conditions of the both processes were measured using response surface methodology (RSM). The maximum yield (26.4%) and protein content (86.1%) were reached at the optimized extraction conditions. Optimal conditions of PG deamidation based on reaching a high degree of deamidation (DD) with a simultaneously low degree of hydrolysis (DH). Under these conditions, DD and DH were 39.40 and 2.11%, respectively. Ultrasound-assisted alkaline extraction and enzymatic deamidation by PG have great potential to produce edible EPSC protein with modified techno-functional characteristics that can be used for several aims in the food and pharmaceutical applications.

Optimization of protein extraction for proteomic analyses of fresh and frozen "Musang King" durian pulps.

Four different methods were evaluated to extract proteins from "Musang King" durian pulps and subsequently proteins with different abundance between fresh and long term frozen storage were identified using two-dimensional polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analyses. The acetone-phenol method was found to produce good protein yields and gave the highest gel resolution and reproducibility. Differential protein analyses of the durian pulp revealed that 15 proteins were down-regulated and three other proteins were up-regulated after a year of frozen storage. Isoflavone reductase-like protein, S-adenosyl methionine synthase, and cysteine synthase isoform were up-regulated during frozen storage. The down-regulation of proteins in frozen durian pulps indicated that frozen storage has affected proteins in many ways, especially in their functions related to carbohydrate and energy metabolisms, cellular components, and transport processes. This study will enable future detailed investigations of proteins associated with quality attributes of durians to be studied.

Effect of Ultrasound Application on Protein Yield and Fate of Alkaloids during Lupin Alkaline Extraction Process.

The objective of this work is to elucidate the fate of quinolizidine alkaloids (QA) during the lupin protein extraction process assisted with ultrasound and the evaluation of the nutritional and functional properties of the protein fraction. Proximal characterization, concentration of anti-nutritional compounds, amino acid profile and protein solubility profile of flours from three lupin species were (L. albus, L. angustifolius and L. mutabilis) assessed. The result showed a significant difference (p < 0.05) in protein concentration, fat, total alkaloids and particle size between the three species flours. Based on these parameters, the most different Lupinus species (L. mutabilis and L. angustifolius) were chosen to study the behavior of the protein fraction in terms of functionality, composition and resistance to thermal treatments. The results obtained for L. mutabilis described the ultrasound effect as beneficial for protein yield (14% more than control), QA reduction from bagasse (81% less than control) and protein isolate production (50% less than control). On the other hand, L. angustifolius was more resistant to the ultrasound effect with no significant difference between treatments (10 and 15 min) and control but with the lower toxicity and better amino acid score. These results will be useful to design processes to assist in the objective of meeting the future protein demand of the population.

Appropriate Activity Assays Are Crucial for the Specific Determination of Proline Dehydrogenase and Pyrroline-5-Carboxylate Reductase Activities.

Accumulation of proline is a widespread plant response to a broad range of environmental stress conditions including salt and osmotic stress. Proline accumulation is achieved mainly by upregulation of proline biosynthesis in the cytosol and by inhibition of proline degradation in mitochondria. Changes in gene expression or activity levels of the two enzymes catalyzing the first reactions in these two pathways, namely pyrroline-5-carboxylate (P5C) synthetase and proline dehydrogenase (ProDH), are often used to assess the stress response of plants. The difficulty to isolate ProDH in active form has led several researchers to erroneously report proline-dependent NAD+ reduction at pH 10 as ProDH activity. We demonstrate that this activity is due to P5C reductase (P5CR), the second and last enzyme in proline biosynthesis, which works in the reverse direction at unphysiologically high pH. ProDH does not use NAD+ as electron acceptor but can be assayed with the artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP) after detergent-mediated solubilization or enrichment of mitochondria. Seemingly counter-intuitive results from previous publications can be explained in this way and our data highlight the importance of appropriate and specific assays for the detection of ProDH and P5CR activities in crude plant extracts.

An efficient and rapid method to extract and purify protein - Liquid Triphasic Flotation system.

Microalgae are rich in valuable biomolecules and grow on non-arable land with rapid growth rate, which has a host of new possibility as alternative protein sources. In the present study, extraction of proteins from Chlorella vulgaris via an efficient technique, Liquid Triphasic Flotation (LTF) system, was studied. The optimized conditions in LTF system were 70% v/v of t-butanol, 40% w/v of salt solution, 0.5% w/v of biomass, pH 5.54, 1:1 of salt to t-butanol solution, and 10 min of air flotation time to attain 87.23% of protein recovery and 56.72% of separation efficiency. Besides, the study on recycling t-butanol has demonstrated that only one run was sufficient to maintain the performance of system. The efficiency of LTF in extracting protein has performed better than just Three Phase Partitioning (TPP) system. LTF system is hence an effective protein extraction and purification method with minimum operation unit and processing time.

Sesame bran as an unexploited by-product: Effect of enzyme and ultrasound-assisted extraction on the recovery of protein and antioxidant compounds.

Significant amount of bran is discarded from sesame processing plants and yet it is seen as waste or animal feed. This study for the first time designed to recover protein and antioxidant compounds from sesame bran. In this respect, effectiveness of four different techniques i.e. viscozyme L, alcalase, ultrasound and ultrasound-assisted enzymatic extractions were tested and compared with standard alkaline method. RSM was used to investigate the effects of extraction parameters and to determine optimum process conditions. All of the independent parameters (enzyme concentrations, pH, ultrasound power, temperature and time) had significant effects on all of the responses. Alcalase exhibited higher recovery efficiency than viscozyme L. The highest protein yield, total phenolic compound and antioxidant capacities were found in ultrasound-assisted enzymatic extraction at 836 W ultrasound power, 43 °C, 98 min, 9.8 pH value and 1.248 AU/100 g enzyme concentration. SDS-PAGE and SEM analyses were also carried out to compare extraction techniques.

Evaluation of the Use of TRIzol-Based Protein Extraction Approach for Gel-Based Proteomic Analysis of Dried Seafood Products and Chinese Tonic Foods.

Although the emergence of gel-free approaches has greatly enhanced proteomic studies, two-dimensional gel electrophoresis (2-DE) remains one of the most widely used proteomic techniques for its high resolving power, relatively low cost, robustness, and high resolution. Preparation of high-quality protein samples remains the key in high-quality 2-DE for proteomic analysis. Samples with high endogenous levels of interfering molecules, such as salts, nucleic acids, lipids, and polysaccharides, would yield a low-quality 2-DE gel and hinder the analysis. Recently, a TRIzol-based protein extraction method has gained prominence and has attracted attention due to its promising performance in high-quality 2-DE. The authors evaluate the use of this approach for four valuable dried food products, namely two dried seafood products (abalone slices and whelk slices) and two traditional Chinese tonic foods (ganoderma and caterpillar fungus). The results indicate that 2-DE gels obtained through the TRIzol-based method are of high-quality and are comparable to those obtained through the trichloroacetic acid⁻acetone method in terms of spot number, spot intensity, and resolution. The TRIzol-based method is generally applicable to dried food samples and is simple and fast, which greatly streamlines the protein extraction procedure. Additionally, it enables the concurrent extraction and analysis of RNA, DNA, and protein from the same sample.

Quantification of Photosynthetic Enzymes in Leaf Extracts by Immunoblotting.

In this chapter, we describe a method to extract and quantify photosynthetic enzymes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The method is particularly suitable for characterizing altered protein amounts in leaves of plants produced from genetic engineering or gene-editing approaches. We focus on RuBisCO and RuBisCO activase, a molecular chaperone required to sustain the activity of RuBisCO and CO2 fixation, yet the method can be easily adapted to investigate other leaf proteins of interest.

Extraction of RuBisCO to Determine Catalytic Constants.

RuBisCO enables net carbon fixation through the carboxylation of RuBP during photosynthesis. Its complex biochemistry and catalytic diversity found among different plants make characterization of RuBisCO properties useful for investigations aimed at improving photosynthetic performance. This chapter reports methods for rapid extraction of soluble proteins to examine RuBisCO catalytic properties, and for large-scale purification of RuBisCO from leaves to measure the specificity of the enzyme toward its gaseous substrates.

Analyzing AMPK Function in the Hypothalamus.

Hypothalamic AMPK plays a key role in the control of energy homeostasis by regulating energy intake and energy expenditure, particularly modulating brown adipose tissue (BAT) thermogenesis. The function of AMPK can be assayed by analyzing its phosphorylated protein levels in tissues, since AMPK is activated when it is phosphorylated at Thr-172. Here, we describe a method to obtain hypothalamic (nuclei-specific) protein extracts and the suitable conditions to assay AMPK phosphorylation by Western blotting.