Potential of Leucaena leucocephala and Leucaena esculenta Seeds in Human Nutrition: Composition, Techno-functional Properties, Toxicology and Pretreatment Technologies

BSTRACT The consumption of legume seeds has benefits for human nutrition and health. Seeds of Leucaena leucocephala and Leucaena esculenta are fast-growing legumes that are used for a variety of purposes. In general, they are used to preserve/restore the soil; the foliage can be used as fodder for livestock and the seeds for human consumption in rural populations. Leucaena seeds are a source of nutrition, since they contain proteins, carbohydrates and lipids (including omega 3, 6 and 9 unsaturated acids). The protein fraction has shown pharmaceutical effects of interest (analgesic, lipolytic, emollient, anticancer, anti-inflammatory, anti-coagulant and anti-diabetic) and some techno-functional properties for the food industry. However, the main limitation to the human consumption of leucaena seeds is the presence of mimosine, an antinutrient that produces side effects such as alopecia, growth retardation, cataracts and infertility in animals. In this sense, some technological processes that could contribute to its decrease. This work was carried out at the Nutraceuticals Department of the Interdisciplinary Research Center for Integral Regional Development, Oaxaca Unit, Mexico. A comprehensive and integrative review of research work carried out in different parts of the world was conducted. The bibliographic search was carried out from January 2020 to January 2022. Several databases such as Scopus, Latindex, Dialnet, Redalyc.org, FAO and Academic Google were explored. A total of 81 documents were used to prepare this review document. This review integrates aspects related to the potential use of leucaena seeds

The family Leguminosae include thousands of species distributed in about 750 genera (Hutchinson, 1964).These species are creeping, shrubby or arboreal growth species and their life cycle can be annual or perennial (Sprent and Parsons, 2000;Bianco and Cenzano, 2018).Around 150 legumes have been listed by the Codex alimentarius as safe for human consumption.Soybeans (Glycine max), common beans (Phaseolus vulgaris), lentils (Lens culinaris), beans (Vicia faba), peas (Pisum sativum) and chickpeas (Cicer arientinum) are the most important legumes due to their nutritional characteristics and their low ecological impact, except for soybeans (World Health Organization, 2007;Delgado-Andrade et al., 2016).
The legumes for human consumption contain a high concentration of carbohydrates and proteins as the main nutritional compounds of interest for the human nutrition (Haytowitz et al., 2011).In addition, they contain several compounds of biological activity classified as undesirable or anti-nutritional such as phytates, galactosesoligosaccharides, protease inhibitors, spooning and etcetera (Messina, 1999).The biological effect of these anti-nutritional molecules directly affects the digestibility of proteins and carbohydrates.
The leucaenas (also called guajes in Spanish) are arboreal legumes distributed throughout many ecosystems, from semi-arid areas, tropical deciduous forest, medium subevergreen forest and mangroves.They are widely distributed in Mexico, especially in the southeastern center of the country (Zárate, 1994).In Mexico, the seeds of various Potential of Leucaena leucocephala and Leucaena esculenta Seeds in Human Nutrition: Composition, Techno-functional... species are consumed, especially Leucaena esculenta and Leucaena leucocephala.Protein concentration of these species varies from 26 to 31%, while the carbohydrate concentration range is 57 to 61% (Román-Cortés et al., 2014).Additionally, the seeds contain some non-polar vitamins, as well as compounds of phenolic origin that can exert a biological function such as nutraceuticals or probiotics (Román-Cortés et al., 2014).
The tannins, trypsin inhibitors, phytic acid, tannins, oligosaccharides and mimosine have been reported as unwanted/anti-nutritive compounds in leucaena seeds.The biological effect of those anti-nutritive molecules directly affects the digestibility of protein and carbohydrates (Román-Cortés et al., 2014;Tran et al., 2016).The non-protein amino acid called mimosine, acid (S)-α-Amino-β-[1-(3-hydroxy-4oxopyridin), inhibits some intracellular processes (Tran et al., 2016;Chaichun et al., 2019) and can be used as an immunosuppressive, antiarrhythmic and hypocholesterolemic agent (Syamsudin et al., 2010).The use of leucaena seeds as an ingredient in livestock feed has been widely evaluated (Barác et al., 2015), but their use in human food has not been formally studied.However, they have been consumed ancestrally in the south of Mexico (Almeida-Costa et al., 2006).
This review evaluates, from de human nutrition point of view, the published data on the nutritional composition, pharmacological and techno-functional properties and toxicity of Leucaena leucocephala and Leucaena esculenta seeds and compares them with other important edible legumes.

Protein content
Legume flours, concentrates and protein isolates are used in several food matrices (Barác et al., 2015).The average crude protein content in leucaena seeds ranges from 31-33% (Table 1) and these values are higher than those reported for legumes of widespread consumption such as Glycine max (soy) or Phaseolus vulgaris (bean), where the protein content ranges between 18-25% (Almeida-Costa et al., 2006).On the other hand, protein values of Leucaena seeds are lower than that reported for other legumes of lower commercial use (e.g.several species of Lupinus spp and Inga paterno) where the protein content ranges from 32-56% (Carvajal-Larenas et al., 2016;Sánchez-Mendoza et al., 2017).
In legumes, the protein fraction is constituted by different sub fractions: globulins, the highest concentration protein sub fraction and albumins, glutelins and prolamins, the lowest concentration protein sub fractions (Desphande, 1990).W ithin these sub fractions, the biologically active polypeptides are the lectins and the enzyme inhibitors (Hall et al., 2017).In cereals, the glutelins and prolamins are the main protein sub fractions.In leucaena seeds, the globulins constitute the main protein sub fraction, 43.5% of proteins (Chandrasekhara-Rao et al., 1984;Sethi and Kulkarni, 1995).The concentration of albumins is 28.4%, glutelins 25% of the protein fraction; and the prolamins <4% (Sethi and Kulkarni, 1995).Similar distributions have been reported for other legumes of widespread consumption such as Glycine max and/or Phaseolus vulgaris, where the fraction of the globulins represent the 54-79% and 85-95 % of the protein fraction, while in Lupinus album the concentrations range was reported of 80-90% of the protein fraction (Almeida-Costa et al., 2006;Carvajal-Larenas et al., 2016).
In the legume seeds, their nutrition/biological potential must be evaluated beyond the quantification of the protein concentration and the protein amino acid residues constitution.Although the correlation between a higher concentration of protein and a better attributes from the nutritional point of view is not correct, these polypeptides may be enzymes with unwanted biological functions, such as trypsin inhibitors and lectins (Sánchez-Mendoza et al., 2017).

Amino acids
Deficiency of methionine and cysteine residues is characteristic in all legume protein fractions; therefore, when legumes are used in human nutrition it must be enriched with protein fractions from cereals (Desphande, 1990).Leucaena seeds are particularly rich in glutamic acid, arginine and leucine (Table 2).
In Glycine max seeds, the amino acids with the highest concentrations are glutamic acid, aspartic acid and proline (Colina et al., 2017;Duke, 2022) while in the seeds of Phaseolus vulgaris, the amino acids of higher concentration correspond to glutamic acid, leucine and serine (Menchú and Méndez, 2012).In other legumes such as lupine (Lupinus spp); glutamic acid, arginine and aspartic acid are the amino acids with the highest concentration (Carvajal-Larenas et al., 2016).
The quality evaluation of leucaena protein by the DIASS criterion, referred to the quality of the protein of the whole milk powder (UN-FAO, 2013) is 8.73% for infants, 17.45% for child and 22.48% for adolescents and adults.Based in Potential of Leucaena leucocephala and Leucaena esculenta Seeds in Human Nutrition: Composition, Techno-functional... the DIAAS quantity criteria, the protein fraction must be catalogued as a high protein source, but based in the DIAAS quantity and quality criteria, the protein faction must be catalogued as a low-quality protein source for the human nutrition; then, its use must be part of a food matrix constituted by a mix with a cereal/animal proteins (UN-FAO, 2013).

Carbohydrates
In legumes, the carbohydrates are considered the molecules of highest concentration.They make up 50% of the dry weight of the seed (Ter Meulen et al., 1979;Middelbos and Fahey, 2008;Ahmed and Abdelati, 2009).In leucaena seeds, the carbohydrate concentration ranges from 35-60%, Table 1 (Sethi and Kulkarni, 1995;Román-Cortés et al., 2014) and the metabolic conversion rate in animal models range from 10.7 to 12.4 kilojoule per gram on a dry basis, kj g db -1 . These values are lower than those reported by Glycine max, 20.95 kj g db -1 (Middelbos and Fahey, 2008;Hall et al., 2017) and Lupinus spp.20 kj g db -1 (Carvajal-Larenas et al., 2016).There is no information regarding the complete profile and concentration of oligosaccharides present in leucaena seeds.Sethi and Kulkarni (1995) and Román-Cortes et al. (2014) suggest that the carbohydrate fraction is composed mainly of non-structural polysaccharides.Plouvier (1962) and Beveridge et al. (1977) report the presence of oligosaccharides such as raffinose, glucose, sucrose, stachyose, D-pinal and Myo-inositol.
NC Non considered values for the recommended intake by UN-FAO.
Potential of Leucaena leucocephala and Leucaena esculenta Seeds in Human Nutrition: Composition, Techno-functional...

Vitamins
Little information is available about the vitamin content in leucaena seeds, where the tocopherols (especially the αtocopherol) and carotenoids are the vitamin family of the higher concentration (Table 4).The lipophilic molecules from grains and legumes, such as carotenoids and tocopherols, have been cited as anti-cardiovascular and eye pathologies molecules (Boschin and Arnoldi, 2011;Monge-Rojas, 2013).
The recommended intake of tocopherol (vitamin E as αtocopherol) is of 5-6 mg d -1 for kids and 9-12 mg d -1 for adult females/males (Institute of Medicine, 2007).In humans, the tocopherols (especially the α-tocopherol, the most abundant and the -tocopherol, with the highest antioxidant activity) and their physiological role depends on their ability to quench free radicals in cell membranes and other lipid environment, preventing the autoxidation of polyunsaturated fatty acids (Bramley et al., 2000).The recommended intake of vitamin A (as retinol) is 0.76-0.87mg d -1 for kids and 0.66-0.76mg d -1 for adult females/males (Institute of Medicine, 2007).They are considered as an antioxidant molecules with therapeutic valu e in p hotosensitivity disorders and leukoplakia pathologies (Olson, 1999).The recommended intake of vitamin K is 6-7 mg d -1 for kids and 11-15 mg d -1 for adult females/males (Institute of Medicine, 2007).In humans, the vitamin K is a cofactor for the function of the enzyme glutamyl carboxylase, necessary for the activation of multiple vitamin K dependent-proteins (VKDPs).The VKDPs proteins have been correlated with bone, vascular and reproduction health as well as in cancer progression (Fusaro et al., 2019).

Minerals
Regarding minerals, phosphorus and potassium have the highest concentration (Table 5).Information about the mineral profile and concentration of leucaena seeds is limited.As other legumes, an intra/inter specific variations have been reported due to agronomic aspects (sowing/ harvest variables), the extraction/pre-treatments and genetic flow, among other reasons.Potential of Leucaena leucocephala and Leucaena esculenta Seeds in Human Nutrition: Composition, Techno-functional...

Anti-nutritive compounds
Leucaena leucocephala seeds contain mimosine as its main ant nutrient (Table 6).Mimosine [3-N-(3-hydroxy-4-pyridone)ao-aminopropionic acid], present in the Leucaena family, is considered one of the most important anti-nutritional factors, given side effects (alopecia, growth retardation, cataracts and infertility in animals) that occur when the permitted intake dose is exceeded (Sethi and Kulkarni, 1995).
In human diet, the intake of unprocessed legumes has been related to human pathologies and nutritional disorders.These pathologies are caused by molecules called antinutrients (De W reede and W ayman, 1970; Sethi and Kulkarni, 1995).For this reason, it is essential to perform a thermal, chemical or physical pretreatment, in order to ensure that the side effects are not harmful to human health.In processed legumes, the remaining anti nutrients concentration after the pre-treatments (e.g.germination, cooking or soaking) is lower than in unprocessed legumes (Elizalde et al., 2009).Tran et al. (2016) reported the absence of significant adverse effects in the consumption of tea made from dried leaves of Leucaena leucocephala.Lim (2012) indicated that the ethno pharmacological use of roasted seeds of Leucaena leucocephala as an emollient in humans has no side effects.Villaseñor et al. (1997) evaluated the effects of the compound of the non-polar extract of Leucaena leucocephala seeds, alkaloids, observing analgesic effects comparable to those of mefenamic acid -depressive effects on the central nervous system-and anti-mutagenic effects in murine models.The same extracts showed vermifuge effects against Ascaris suum.

Pharmacological uses
Gamal -Eldeen et al. (2007) reported chemo-preventive and/or anticancer effect of some polysaccharide-protein complexes isolated from Leucaena leucocephala seeds that inhibited/modulated the action of cytochrome P450 1A (CYP1A) and the transduction of glutathione-S-transferases (GST).Souza-Pinto et al. (1995), reported that trypsin 1 (LLTI-1) and trypsin 2 (LLTI-2), inhibitors isolated from the seed protein fraction of Leucaena leucocephala, showed an anti-inflammatory effect in murine models.Oliva et al. (2000) found that other serine proteinase inhibitor isolated from Leucaena leucocephala seeds, modified the activity of plasmin, plasma trypsin and human chymotrypsin.Syamasudin et al. (2010) reported that galactose as well as other several glycose compounds extracted from Leucaena leucocephala seeds, showed a hypoglycemic effect in mouse models where a diabetic pathology was previously induced.In vitro studies showed some effects on mitotic frequency and incorporation of thiamine in healthy human cells, as well as in cell division and proliferation in human carcinoma without significant effects on the mechanisms of protein synthesis and DNA synthesis (Tsai and Ling, 1971;Krude, 1999;Kubota et al., 2014).Kuppusamy et al. (2014) reported that aqueous extracts of leucaena seeds could be used as complementary therapy in the treatment of type 2 diabetes due to its hypoglycemic  effect as well as in weight control treatments due to its adipogenesis and lipolytic properties.Pendyala et al. (2010) reported that Leucaena leucocephala gum (galactomanase gums) can serve as a drug-carrying suspensions and emulsions.In addition, these gums can act as antitumor, hemo binder and anticoagulant with similar actions to those reported from soybeans (Deodhar et al., 1998).

Techno-functional properties
The water holding capacity (WHC) and oil holding capacity (OHC) of the protein fraction in commercial legumes are in the range from 1.8-6.8 and 3.5-6.8grams per gram of protein (g g -1 ), respectively.The legume protein fractions exhibit good foaming formation, emulsion capacity and solubility (Hassan et al., 2009;Pendyala et al., 2010).These properties were preserved in a wide range of process/variable values (e.g.extreme pH, 2.0 and 10.0) (Pendyala et al., 2010) .
Leucaena seed flour has a good oil absorption capacity, 2.2±0.2g of oil/g of flour, an important quality in the formulation of fried foods, bakery and confectionery products.This attribute also helps to reduce the development of oxidative rancidity and consequently increases the shelf life of the products (Bravo-Delgado et al., 2019).Sethi and Kulkarni (1994) evaluated the functional and organoleptic properties of Leucaena leucocephala seed protein isolate.They found an oil and water absorption capacity very similar to soybean protein isolate and that these values are higher than those presented by other edible legumes such as bean, pea, chickpea and fava bean.They also carried out successful organoleptic tests of cake and mayonnaise supplemented with protein isolate as a replacement for egg protein.Deodhar et al. (1998) and Pendyala et al. (2010) analyzed the techno-functional properties of gums from leucaena seeds and proposed other possible industrial applications (e.g. as pharmaceutical tablet formulation, as a carrier in modified release dosage forms and as a suspending agent and emulsifying agent owing to its pseudo plastic and thixotropic properties).

Processes used to reduce the toxicity of leucaena seed
Various methodologies are used to reduce the antinutritional molecules concentration in leucaena seeds, most of them being widely used in other legumes, e.g.Phaseolus vulgaris.These are based on two fundamental principles, the high solubility in polar solutions (water) and the thermolabile behavior of those molecules.
The human protease inhibitors from legume seeds exhibit thermo-sensitive properties (Hegarty et al., 1964), while other compounds such as those of phenolic origin (phenols, flavonoids, tannins and anthocyanins) and alkaloids exhibit good solubility in polar solvents.
Regarding mimosine, several authors have evaluated the use of extraction processes (by aqueous extractions, methane sulfonate and ethanol) and biological processes (germination) as a practical and effective process to reduce its concentration (Tangendjaja et al., 1984;Sethi and Kulkarni, 1995;Alabi and Alausa, 2006;Mohamed et al., 2014).
A complete degradation of mimosine was reported by Tangeudjaja et al. (1984) in an aqueous slurry at pH 8.0 and 45C in 10 minutes.Heating the intact leaf at 70C resulted in 90% reduction of mimosine in 15 minutes (Labadan, 1969;Ter Meulen et al., 1979;Padmavathy and Shobha 1987).Washing with water and soaking, the leaves and seeds had a significant effect in lowering their mimosine contents.Prolonged soaking (48 hours) in 30C water was most effective in reducing virtually all the mimosine in the leaves (Wee and Wang, 1987).One of the most effective reagents for extracting 95% mimosine is 0.05 N sodium acetate (Tawada, 1988;Tawata, 1990).Studies were carried out by treating L. leucocephala seeds with several reagents.Urea and sodium hydrogen carbonate completely removed mimosine.The protein content of the mimosine-free seed mass was reduced to 80% of the original after treatment with urea and 88% after treatment with sodium hydrogen carbonate solution (Hossain et al., 1991).In the livestock industry, the reduction of mimosine toxicity of Leucaena leucocephala has been reached in leaf and seed feed by adding iron and zinc salts (Chang, 1987).The use of bacteria that produce lactic acid (Inafuku and Pongo, 1992), bacteria of the species Rhizobium sp., Soedarjo et al. (1994) as well as native microorganisms in the rumen of animals.Allison et al. (1992) and Domínguez-Bello and Stewart (2020) have been proven alternatives that can significantly decrease the effect of mimosine in ruminant animal feed.Its mechanism of action consists of the degradation of mimosine to 3-hydroxy-4(4H)-pyridone (Arjona-Alcocer et al., 2012;Contreras-Hernández et al., 2013).It has been determined that 3-hydroxy-4(4H)-pyridone is also toxic in ruminant animals, but with biological effects of lesser magnitude than those caused by mimosine (Ruiz-Ruiz, 2013).Sethi and Kulkarni (1994) found that derived from the preparation process of leucaena protein isolates, the mimosine content decreased by 93%.The resulting isolates were used as egg replacers in the preparation of cake and mayonnaise.

Future trends
The UN-FAO estimates that for 2050 will be necessary to increase food production from animal and vegetable sources by nearly 26% from 2014 levels (Mulhollen 2017).In the same way, the agency estimates a rapid increase in the demand of protein for human food of animal origin, mainly, associated with the expected increase in the human population.This means an increase in the consumption of cereals and legumes for livestock production, altering the balance between the consumption of legumes and cereals by humans and agricultural production systems.
Some economic analyses indicate a consistent increase in the demand for plant-based protein, estimating a global market of USD 75.8 billion in 2025 (Hexa Research, 2019).The industry is then concerned about how introduce a large variety of legume-based foods.Marr (2022) suggests that plant-based meat alternatives could represent 10 per cent of the global meat industry by 2029.
Potential of Leucaena leucocephala and Leucaena esculenta Seeds in Human Nutrition: Composition, Techno-functional... Nowadays, the market is dominated by soy protein derived food, but multiples factors limit their production (mainly the environmental impact of the production) and acceptance (associated health risks in human population exposed to the glyphosate and chemical additives of the acrylamide type).Other vegetable proteins such as Pisum sativum, Vicia faba or Phaseolus vulgaris exhibit a minor market penetration, although the presence of anti-nutrient compounds and their effect on the human nutrition/health does not differ substantially from those found in soybeans.Probably, the less penetration of these legumes is due to various socio-cultural factors (e.g. the decrease in its consumption, cultivated area and a stagnation in production of P. vulgaris).

CONCLUSION
The uses of non-traditional legumes seeds, as leucaena or prosopis, in human nutrition are limited, mainly due to their potential being unknown.Therefore, research and development should focus on the following aspects:  Genetic/agronomic selection of varieties with desirable nutritional potential. Genetic improvement: breeding of wild spices/varieties with desirable nutritional potential. Establishment of the maximum daily intake of mimosine referenced to specific nutritional requirements associated with sex and age. The biological effects of prolonged mimosine intake below the determined maximum level. Research and development of industrial anti nutrient extraction process and compounds with no desirable biological activity. Techno-functional properties of proteins and carbohydrate fractions (starches) present in seeds to improve food matrices. Evaluation of polypeptides with desirable biological activity.

Table 1 :
Comparative nutrient composition of some legume seeds.
a Lupinus spp refers to the average value reported for the species of Lupinus mutabilis, Lupinus albus, Lupinus angustifolia, Lupinus termis and Lupinus mutabilis.

Table 2 :
Comparative amino acid concentration of legume seeds.
a Lupinus spp refers to the average value reported for the species of Lupinus mutabilis, Lupinus albus, Lupinus angustifolia, Lupinus termis and Lupinus mutabilis.b Recommended intake for older child (>6 years), adolescents and adults.

Table 4 :
Comparison of vitamins from legume seeds lipid fraction.
ND a Lupinus spp refers to the average value reported for the species of Lupinus mutabilis, Lupinus albus, Lupinus angustifolia, Lupinus termis and Lupinus mutabilis.ND No reported values.PR Qualitative presence.

Table 3 :
Comparison of fatty acid profile from legume seeds.
a Lupinus spp refers to the average value reported for the species of Lupinus mutabilis, Lupinus albus, Lupinus angustifolia, Lupinus termis and Lupinus mutabilis.ND No reported values.
ND No reported values.

Table 5 :
Mineral composition of legume seeds.
a Lupinus spp refers to the average value reported for the species of Lupinus mutabilis, Lupinus albus, Lupinus angustifolia, Lupinus termis and Lupinus mutabilis.ND No reported values.