It is necessary to develop and deploy novel protein production to allow the establishment of a sustainable supply for both humans and animals, given the ongoing expansion of protein demand to meet the future needs of the increased world population and high living standards. In addition to plant seeds, green biomass from dedicated crops or green agricultural waste is also available as an alternative source to fulfill the protein and nutrient needs of humans and animals. The development of extraction and precipitation methods (such as microwave coagulation) for chloroplast and cytoplasmic proteins, which constitute the bulk of leaf protein, will allow the production of leaf protein concentrates (LPC) and protein isolates (LPI). Obtained LPC serves as a sustainable alternative source of animal-based protein besides being an important source of many vital phytochemicals, including vitamins and substances with nutritional and pharmacological effects. Along with it, the production of LPC, directly or indirectly, supports sustainability and circular economy concepts. However, the quantity and quality of LPC largely depend on several factors, including plant species, extraction and precipitation techniques, harvest time, and growing season. This paper provides an overview of the history of green biomass-derived protein from the early green fodder mill concept by Károly Ereky to the state-of-art of green-based protein utilization. It highlights potential approaches for enhancing LPC production, including dedicated plant species, associated extraction methods, selection of optimal technologies, and best combination approaches for improving leaf protein isolation.

Jerusalem artichoke (JA) is widely known to have inulin-rich tubers. However, its fresh aerial biomass produces significant levels of leaf protein and economic bioactive phytochemicals. We have characterized leaf protein concentrate (JAPC) isolated from green biomass of three Jerusalem artichoke clones, Alba, Fuseau, and Kalevala, and its nutritional value for the human diet or animal feeding. The JAPC yield varied from 28.6 to 31.2 g DM kg⁻¹ green biomass with an average total protein content of 33.3% on a dry mass basis. The qualitative analysis of the phytochemical composition of JAPC was performed by ultra-high performance liquid chromatography-electrospray ionization-Orbitrap/mass spectrometry analysis (UHPLC-ESI-ORBITRAP-MS/MS). Fifty-three phytochemicals were successfully identified in JAPC. In addition to the phenolic acids (especially mono- and di-hydroxycinnamic acid esters of quinic acids) several medically important hydroxylated methoxyflavones, i.e., dimethoxy-tetrahydroxyflavone, dihydroxy-methoxyflavone, hymenoxin, and nevadensin, were detected in the JAPC for the first time. Liquiritigenin, an estrogenic-like flavanone, was measured in the JAPC as well as butein and kukulkanin B, as chalcones. The results also showed high contents of the essential amino acids and polyunsaturated fatty acids (PUFAs; 66-68%) in JAPC. Linolenic acid represented 39–43% of the total lipid content; moreover, the ratio between ω-6 and ω-3 fatty acids in the JAPC was ~0.6:1. Comparing the JA clones, no major differences in phytochemicals, fatty acid, or amino acid compositions were observed. This paper confirms the economic and nutritional value of JAPC as it is not only an alternative plant protein source but also as a good source of biological valuable phytochemicals.

The main objective of this study was to increase the economic value of broccoli green agro-waste using three wet fractionation methods in the shadow of green biorefinery and the circular economy. Product candidates were obtained directly by using a mechanical press, and indirectly by using microwave coagulation or via lactic acid fermentation of green juice. The leaf protein concentrates (LPC) fractions displayed significantly higher dry matter content and crude protein content (34–39 m/m% on average) than the green juice fraction (27.4 m/m% on average), without considerable changes in the amino acids composition ratio. UHPLC-ESI-ORBITRAP-MS/MS analysis showed that kaemferol and quercetin are the most abundant flavonols, forming complexes with glycosides and hydroxycinnamic acids in green juice. Lacto-ermentation induced a considerable increase in the quantity of quercetin (48.75 μg·g⁻¹ dry weight) and kaempferol aglycons (895.26 μg·g⁻¹ dry weight) of LPC. In contrast, chlorogenic acid isomers and sulforaphane disappeared from LPC after lactic acid fermentation, while microwave treatment did not cause significant differences. These results confirm that both microwave treatment and lacto-fermentation coagulate and concentrate most of the soluble proteins. Also, these two processes affect the amount of valuable phytochemicals differently, so it should be considered when setting the goals.

A pot experiment, under greenhouse conditions, was carried out aiming at investigating the agronomic biofortification of alfalfa (Medicago sativa L.) with Se and monitoring the Se uptake and accumulation dynamics within four consecutive harvests within the same growing season. Two ionic Se forms, i.e., sodium selenate (Se (VI)) and sodium selenite (Se (IV)), were applied once at a rate of 1, 10, and 50 mg kg⁻¹ (added on Se basis), while 10 and 50 mg L⁻¹ of a red elemental Se (red Se⁰) were used; all Se treatments were added as soil application. Application of Se (VI) at the rate of 50 mg kg⁻¹ was toxic to alfalfa plants. The effect of Se forms on Se accumulation in alfalfa tissues, regardless of the applied Se concentration, follows: Se (VI) > Se (IV) > red Se⁰. The leaf, in general, possessed higher total Se content than the stem in all the treatments. The accumulation of Se in stem and leaf tissues showed a gradual decline between the harvests, especially for plants treated with either Se (VI) or Se (IV); however, the chemically synthesized red Se⁰ showed different results. The treatment of 10 mg kg⁻¹ Se (VI) resulted in the highest total Se content in stem (202.5 and 98.0 µg g⁻¹) and leaf (643.4 and 284.5 µg g⁻¹) in the 1st and 2nd harvests, respectively. Similar tendency is reported for the Se (IV)-treated plants. Otherwise, the application of red Se⁰ resulted in a lower Se uptake; however, less fluctuation in total Se content between the four harvests was noticed compared to the ionic Se forms. The Se forms in stem and leaf of alfalfa extracted by water and subsequently by protease XIV enzyme were measured by strong anion exchange (SAX) HPLC-ICP-MS. The major Se forms in our samples were selenomethionine (SeMet) and Se (VI), while neither selenocysteine (SeCys) nor Se (IV) was detected. In water extract, however, Se (VI) was the major Se form, while SeMet was the predominant form in the enzyme extract. Yet, Se (VI) and SeMet contents declined within the harvests, except in stem of plants treated with 50 mg L⁻¹ red Se⁰. The highest stem or leaf SeMet yield %, in all harvests, corresponded to the treatment of 50 mg L⁻¹ red Se⁰. For instance, 63.6% (in stem) and 38.0% (in leaf) were calculated for SeMet yield % in the 4th harvest of plants treated with 50 mg L⁻¹ red Se⁰. Our results provide information about uptake and accumulation dynamics of different ionic Se forms in case of multiple-harvested alfalfa, which, besides being a good model plant, is an important target plant species in green biorefining.

Plant-based foods offer a sustainable alternative to meet the growing protein demand. Legumes are the most promising of these, as they contain relatively high concentrations of protein, low digestible starch, and dietary fiber, as well as them possibly featuring low levels of fat. Enzymatically modified legume proteins provide us with tempting perspectives in terms of enhancing foods’ biological values. However, their bioavailability and digestibility are generally less sufficient than that of proteins of animal origin, which may be improved by well-tailored enzyme modification. In this study, the efficacy of three plant-based proteases (bromelain, ficin, and papain) were evaluated at two distinct concentrations (2.5% and 10%) and three hydrolysis durations (1, 2, and 12 h) when transforming chickpea and lentil proteins. The degree of hydrolysis (DH), peptide profiles, and free amino acid content were analyzed to determine the efficiency of each enzyme. Results showed significant variations in DH, which was influenced by enzyme type, concentration, and hydrolysis duration. Papain exhibited the highest DH, particularly at a 10% concentration, reaching 27.8% efficiency in chickpea and 34.8% in lentils after 12 h. Bromelain and ficin were proven to be less effective, with ficin showing the least hydrolytic activity. SDS-PAGE analysis revealed substantial protein degradation, especially subsequent to papain treatment, pointing out that most proteins were cleaved into smaller peptides. SEC-HPLC indicated a predominant release of peptides within the 200–1000 Da range, suggesting enhanced bioavailability. Papain and bromelain treatments resulted in a significant release of oligopeptides and dipeptides. UHPLC analysis highlighted a marked post-hydrolysis increase in total free amino acids, with arginine, leucine, and lysine being the most abundant ones. Notably, tryptophan, being undetectable in untreated samples, was released in measurable amounts post-hydrolysis. These findings demonstrate papain’s superior performance in protein hydrolysis and its potential in producing bioactive peptides, highlighting its applicability in food processing and the development of both nutraceuticals and functional foods.

Background and Aims: Wet processed alfalfa-based products can provide high-quality concentrated protein, also contain nutrients and phytochemicals. Agronomic fortification can increase selenium (Se) incorporation into green biomass. For this reason, the aim was to investigate how different forms of Se are incorporated into the green biomass of multiple-harvest alfalfa and how they affect the chemical quality of the processed product candidates. Methods: In this research, alfalfa was enriched with three forms of selenium at different concentrations [selenate (Se(VI)); selenite (Se(IV)); and red elemental Se (Se0)]. The fortified green biomass was fractionated into leaf protein concentrate (LPC), fiber and phytoserum, and changes in selenium content and speciation, protein and phytochemical composition were determined. Results: Total Se content in alfalfa fractions drastically reduced within the four consecutive harvests, particularly for Se(VI) and Se(IV) forms, and significantly varied according to the Se treatments. Selenomethionine (SeMet) was the predominant organic Se species in LPC (35.7–246.0 µg g −¹ DW) and fiber (9.0- 47.7 µg g −¹ DW) fractions. Se-fortification induced considerable changes in the crude protein of LPC, which varied between 29—45 (m/m)%. Phytochemical composition markedly varied among Se-treatments. Besides several flavonoids, aglycones and glycosyl derivatives, apigenin glucuronide, and apigenin were the most abundant with a concentration up to 170.85 µg g −¹ DW. Medicagenic acid concentration was below 1.86 µg g −¹ DW in LPC. Conclusions: Se-enhanced alfalfa green biomass could provide functional products, such as LPC, for human and animal consumption with enhanced nutritional value, including Se, protein, and phytochemical contents.