Leaf protein concentrate (LPC) refers to the proteinaceous mass extracted from leaves. It can be a lucrative source of low-cost and sustainable protein for food as well as feed applications. Although the proteinaceous extracts from leaves have been described as early as 1773 by Rouelle, [1] large scale extraction and production of LPC was pioneered post the World War II. In fact, many innovations and advances made with regards to LPC production occurred in parallel to the Green Revolution. [2] In some respects, these two technologies were complimentary in that the Green Revolution sought to increase agrarian productivity through increased crop yields via fertiliser use, mechanisation and genetically modified crops, while LPC offered the means to better utilise available agrarian resources through efficient protein extraction. [3]
Over the years, numerous sources have been experimented. Pirie [4] and Telek [5] described LPC production using a combination of pulping and heat coagulation. Leaves are typically sourced from shrubs or agricultural wastes given their ease of access and relative abundance. Trees are generally considered a poor source of leaf mass for the production of LPC given restrictions on the ease of access. Fallen leaves / leaf litter have negligible protein-content and are of no extractive value. [6]
Plants belonging to the Fabaceae family such as clover, peas and legumes have also been prime candidates for LPC production. [7] While most plants have a mean leaf protein content of 4 to 6% w/v. Fabaceae plants tend to have nearly double that value at 8 to 10% v/w, depending on the protein estimation method employed. Other non-traditional sources include agricultural wastes such as pea (Pisum sativum) pods, cauliflower (Brassica oleracea) leaves, as well as invasive plants such as Gorse (Ulex europeaus), Broom (Cytisus scoparius), and Bracken (Pteridium aquilinum). [8]
LPC production processes are two-staged, with the first focusing on the expression of leaf juice or production of a leaf extract, and the second being the purification or protein recovery stage that recovers protein from the solution.
The most commonly employed method of leaf protein extraction is pulping / juicing. [9] [10] Other assisted extraction methods have also been reported such as alkali treatment, [11] pressurised extraction, and enzyme treatment [12] Each method comes with its own advantages although pulping produces the most “native” protein composition and does not require significant investment in complex machinery.
Alkali extraction has been employed with some success [13] although it significantly affects lysine and threonine residues in the protein. Pressurised extraction have limited success. Enzyme treatment is another well reported method which targets the plant cell wall to aid the release of bound proteins. However, enzymes are generally more expensive compared to physical or chemical methods of protein extraction.
Recovering the protein from the extract however is most critical to the nutritive value of the LPC. Commonly reported methods were heat coagulation, [14] acid precipitation, [15] ultrafiltration, solvent precipitation [8] and chromatography.
Heat coagulation is the easiest and the oldest method of protein recovery, albeit the least preferred as most of the nutritive value of the LPC is lost. Acid precipitation is the most commonly employed method of protein recovery although it results in the loss of methionine and tryptophan in the LPC. Ultrafiltration is the most hardware demanding option for protein recovery although it serves more as a protein concentration step rather than complete recovery. Chromatographic methods may be used in tandem with ultrafiltration to help increase solute mass and subsequent recovery. Solvent precipitation is not often reported although it produces the highest protein recovery among other methods and preserves the nutritional integrity of the LPC. The extraction and purification methods are largely inter-compatible and may be employed depending on local facilities. Interestingly, the purity of the final LPC was influenced by the protein content in the initial leaf mass rather than the purification method employed. Furthermore, the amino acid composition of the LPC was dependent on the extraction method employed. [8]
In laboratory conditions, protein fractions of 96% purity could be produced with a recovery of 56% w/w and an overall yield of 5.5%. [12] Telek on the other hand experimented with numerous tropical plants at a large scale using a combination of pulping and heat coagulation. Yields were around 3% with protein recoveries <50%. [16]
Depending on the purity of the recovered protein, they are either called leaf protein extract (<60% w/w), leaf protein concentrate (>60% w/w), or leaf protein isolate (>90% w/w), [17] although publications use these terms interchangeably.
Whole leaf protein concentrate is a dark green substance with a texture similar to cheese. Approximately 60% of this is water, while the remaining dry matter is 9-11% nitrogen, 20-25% lipid, 5-10% starch and a variable amount of ash. It is a mixture of many individual proteins. Its flavour has been compared to spinach or tea. [18]
Because the colour and taste may make it unpalatable for humans, LPC can instead be separated into green and white fractions. The green fraction has proteins mainly originating from the chloroplasts, while the white fraction has proteins mainly originating from the cytoplasm. [19]
LPC was first suggested as a human food in the early 20th century, but it has not achieved much success, despite early promise. Norman Pirie, the Copley Medal winner from the UK, studied LPC and promoted its use for human consumption. He and his team developed machines for extraction of LPC, including low-maintenance "village units" intended for poor rural communities. These were installed in places such as villages in south India. [20] The non profit organization, Leaf for Life, maintains a list of human edible leaves and provides recommendations for the top choices of plants. [21]
There has recently been an interest in using LPCs as an alternative food (or resilient food) during times of catastrophe or food shortages. [22] Such resilient food LPCs would be derived from widely geogrphically dispersed tree leaves from forests [23] or agricultural waste. [24]
LPC have been evaluated for infant weaning foods. [25]
The increasing reliance on feedlot based animal rearing to satisfy human appetites for meat has increased demand for cheaper vegetable protein sources. This has recently led to renewed interest in LPC to reduce the use of human-edible vegetable protein sources in animal feed. [26]
Leaf protein has had successful trials as a substitute for soy feed for chickens and pigs. [27]
LPC from alfalfa can be included in feed for tilapia as a partial replacement for fish meal. [28]
The amino acid composition of the LPC:
Ref | Purity | Ala | Arg | Asp | Glu | Gly | His | Ile | Leu | Lys | Met | Phe | Pro | Ser | Thr | Tyr | Val |
[29] | 68.6 | 6.4 | 8.3 | 10.3 | 12.6 | 5.7 | 3.4 | 5.3 | 9.7 | 6.9 | 2.6 | 6.6 | 4.8 | 2.8 | 5.7 | 5.4 | 6.9 |
[30] | 69.8 | 6.7 | 7.8 | 10.8 | 13.2 | 5.9 | 3.3 | 5.7 | 10.2 | 6.3 | 2.6 | 7.0 | 4.3 | 3.6 | 5.9 | 5.6 | 7.2 |
[31] | 88.5 | 5.5 | 12.7 | 14.2 | 26.3 | 5.6 | 3.0 | 4.4 | 7.8 | 2.8 | 2.3 | 5.8 | 5.1 | 5.2 | 3.4 | 4.0 | 6.1 |
[31] | 94.9 | 5.4 | 12.3 | 14.9 | 28.1 | 6.0 | 3.1 | 4.6 | 8.0 | 2.3 | 2.3 | 5.9 | 4.9 | 5.3 | 3.6 | 4.1 | 6.4 |
[32] | 50.0 | 5.2 | 5.4 | 12.0 | 10.0 | 4.5 | 2.0 | 4.0 | 7.8 | 5.5 | 1.8 | 5.3 | 3.3 | 4.7 | 4.2 | 4.1 | 5.2 |
[33] | 68.8 | 4.8 | 10.0 | 9.3 | 15.9 | 4.7 | 2.9 | 4.8 | 7.3 | 3.5 | 3.7 | 4.8 | 3.5 | 3.8 | 4.2 | 4.2 | 6.0 |
[34] | 56.0 | 4.1 | 7.1 | 11.5 | 21.2 | 4.2 | 2.3 | 5.4 | 7.6 | 5.8 | 1.8 | 5.5 | 5.1 | 5.1 | 3.4 | 3.9 | 5.2 |
[35] | 50.0 | 4.5 | 4.6 | 7.9 | 11.0 | 4.4 | 1.7 | 4.1 | 8.4 | 5.7 | 1.8 | 6.1 | 3.0 | 3.6 | 3.7 | 3.3 | 5.8 |
[12] | 96.9 | 7.8 | 6.0 | 12.7 | 12.8 | 7.6 | 1.7 | 2.4 | 8.6 | 6.8 | 2.0 | 4.9 | 5.3 | 6.7 | 4.0 | 4.6 | 3.4 |
[36] | 57.2 | 6.1 | 6.6 | 10.5 | 11.6 | 5.5 | 2.6 | 6.0 | 9.7 | 6.6 | 1.8 | 6.4 | 4.7 | 4.7 | 5.3 | 4.8 | 7.2 |
[36] | 60.1 | 6.9 | 6.5 | 9.4 | 11.0 | 6.0 | 2.3 | 6.0 | 10.1 | 6.8 | 2.3 | 6.8 | 5.9 | 4.7 | 5.0 | 4.6 | 6.8 |
[36] | 53.6 | 6.8 | 6.4 | 9.4 | 11.2 | 5.9 | 2.6 | 6.2 | 9.9 | 6.1 | 2.2 | 7.0 | 4.7 | 4.5 | 5.6 | 4.7 | 7.0 |
[36] | 63.1 | 6.1 | 6.6 | 10.1 | 11.1 | 5.4 | 2.6 | 6.2 | 10.0 | 6.2 | 2.1 | 6.6 | 6.0 | 4.4 | 5.3 | 4.7 | 6.8 |
[37] | 34.0 | 3.3 | 3.1 | 4.5 | 5.0 | 2.9 | 1.1 | 2.5 | 4.5 | 2.6 | 1.1 | 3.0 | 2.3 | 1.8 | 2.3 | 1.9 | 3.1 |
[38] | 60.7 | 3.3 | 3.8 | 5.8 | 6.3 | 3.1 | 1.5 | 2.6 | 5.2 | 3.9 | 1.1 | 3.4 | 3.0 | 2.8 | 3.1 | 2.8 | 3.4 |
[39] | 76.4 | 5.5 | 14.2 | 12.5 | 11.2 | 5.1 | 3.5 | 4.5 | 7.1 | 3.0 | 1.7 | 5.4 | 5.5 | 5.2 | 3.6 | 3.2 | 5.2 |
[40] | 59.8 | 5.6 | 7.2 | 9.8 | 12.9 | 4.7 | 2.9 | 4.4 | 9.7 | 7.6 | 2.4 | 6.3 | 5.3 | 3.7 | 5.5 | 5.7 | 6.3 |
[41] | 80.0 | 10.9 | 4.9 | 6.5 | 23.8 | 2.2 | 2.1 | 4.6 | 13.1 | 3.4 | 2.7 | 7.7 | 5.1 | 5.2 | 2.8 | 2.9 | 5.6 |
[41] | 75.6 | 9.3 | 4.8 | 7.7 | 22 | 2.9 | 2.1 | 4.6 | 13.6 | 3.9 | 3.1 | 6.3 | 5.5 | 4.6 | 3.7 | 2.4 | 5.8 |
[42] | 83.4 | 3.7 | 8.5 | 12.3 | 6.4 | 3.4 | 2.7 | 3.8 | 5.0 | 8.3 | 1.7 | 5.8 | 3.3 | 4.0 | 4.5 | 4.0 | 5.6 |
[43] | 95.5 | 4.0 | 11.5 | 7.0 | 14.5 | 3.9 | 2.5 | 4.0 | 6.7 | 2.4 | 3.2 | 4.8 | 5.9 | 4.3 | 3.6 | 3.9 | 4.9 |
[43] | 97.0 | 4.2 | 11.2 | 8.0 | 13.5 | 4.0 | 2.6 | 4.1 | 6.6 | 2.2 | 2.1 | 4.6 | 5.7 | 4.1 | 3.7 | 3.6 | 5.2 |
[44] | 46.8 | 8.3 | 6.9 | 13.9 | 15.9 | 7.4 | 2.3 | 7.0 | 13.2 | 8.8 | 2.9 | 7.7 | 6.9 | 3.7 | 5.9 | 6.1 | 9.3 |
[45] | 58.4 | 6.3 | 6.2 | 9.7 | 11.3 | 5.7 | 3.0 | 4.6 | 9.1 | 6.3 | 1.1 | 5.9 | 4.0 | 4.3 | 4.9 | 5.0 | 5.6 |
[46] | 55.4 | 3.4 | 3.6 | 5.1 | 5.9 | 3.0 | 1.1 | 2.3 | 5.1 | 2.7 | 1.3 | 3.4 | 2.7 | 2.6 | 2.6 | 2.2 | 2.8 |
[47] | 46.1 | 6.0 | 3.4 | 11.6 | 12.4 | 5.9 | 1.9 | 6.3 | 9.0 | 2.8 | 1.9 | 4.4 | 4.8 | 4.3 | 3.1 | 4.3 | 5.5 |
Leaf protein is a good source of amino acids, with methionine being a limiting factor. [48] It is nutritionally better than seed proteins and comparable to animal proteins (other than those in egg and milk). [18]
In terms of digestibility, whole LPC has digestibility in the range 65–90%. The green fraction has a much lower digestibility that may be <50%, while the white fraction has digestibility >90%. [19]
The challenges that have to be overcome using lucerne and cassava, two high density monoculture crops, include the high fiber content and other antinutritional factors, such as phytate, cyanide, and tannins. [48]
Lablab beans, Moringa oleifera , tree collards and bush clover may also be used. Flavors of different species vary greatly. [27]
For testing new leaf species for use as LPCs a non-targeted approach has been developed that uses an ultra-high-resolution hybrid ion trap orbitrap mass spectrometer with electrospray ionization coupled to an ultra-high pressure two-dimensional liquid chromatograph system. [49] An open source software toolchain was also developed for automated non‐targeted screening of toxic compounds for LPCs. [50] The process uses three tools 1) mass spectrometry analysis with MZmine 2, [51] [52] 2) formula assignment with MFAssignR, [53] [54] and 3) data filtering with ToxAssign. [55] Studies have looked at the potential for deciduous trees [49] and coniferous tree leaves. [56] The latter showed yields for LPC extraction from 1% to 7.5% and toxicity screenings confirm that coniferous trees may contain toxins that can be consumed in small amounts, and additional studies including measuring the quantity of each toxin are needed. [56]
Tannins are a class of astringent, polyphenolic biomolecules that bind to and precipitate proteins and various other organic compounds including amino acids and alkaloids.
Polyphenols are a large family of naturally occurring phenols. They are abundant in plants and structurally diverse. Polyphenols include flavonoids, tannic acid, and ellagitannin, some of which have been used historically as dyes and for tanning garments.
The cowpea is an annual herbaceous legume from the genus Vigna. Its tolerance for sandy soil and low rainfall have made it an important crop in the semiarid regions across Africa and Asia. It requires very few inputs, as the plant's root nodules are able to fix atmospheric nitrogen, making it a valuable crop for resource-poor farmers and well-suited to intercropping with other crops. The whole plant is used as forage for animals, with its use as cattle feed likely responsible for its name.
Phytic acid is a six-fold dihydrogenphosphate ester of inositol, also called inositol hexaphosphate, inositol hexakisphosphate (IP6) or inositol polyphosphate. At physiological pH, the phosphates are partially ionized, resulting in the phytate anion.
Whey protein is a mixture of proteins isolated from whey, the liquid material created as a by-product of cheese production. The proteins consist of α-lactalbumin, β-lactoglobulin, serum albumin and immunoglobulins. Glycomacropeptide also makes up the third largest component but is not a protein. Whey protein is commonly marketed as a protein supplement, and various health claims have been attributed to it. A review published in 2010 in the European Food Safety Authority Journal concluded that the provided literature did not adequately support the proposed claims. For muscle growth, whey protein has been shown to be slightly better compared to other types of protein, such as casein or soy.
Myrcene, or β-myrcene, is a monoterpene. A colorless oil, it occurs widely in essential oils. It is produced mainly semi-synthetically from Myrcia, from which it gets its name. It is an intermediate in the production of several fragrances. α-Myrcene is the name for the isomer 2-methyl-6-methylene-1,7-octadiene, which has not been found in nature.
Norman Wingate Pirie FRS, was a British biochemist and virologist who, along with Frederick Bawden, discovered that a virus can be crystallized by isolating tomato bushy stunt virus in 1936. This was an important milestone in understanding DNA and RNA.
Moringa oleifera is a fast-growing, drought-resistant tree of the family Moringaceae, native to the Indian subcontinent and used extensively in South and Southeast Asia. Common names include moringa, drumstick tree, horseradish tree, or malunggay.
Proanthocyanidins are a class of polyphenols found in many plants, such as cranberry, blueberry, and grape seeds. Chemically, they are oligomeric flavonoids. Many are oligomers of catechin and epicatechin and their gallic acid esters. More complex polyphenols, having the same polymeric building block, form the group of tannins.
Soy protein is a protein that is isolated from soybean. It is made from soybean meal that has been dehulled and defatted. Dehulled and defatted soybeans are processed into three kinds of high protein commercial products: soy flour, concentrates, and isolates. Soy protein isolate has been used since 1959 in foods for its functional properties.
Procyanidins are members of the proanthocyanidin class of flavonoids. They are oligomeric compounds, formed from catechin and epicatechin molecules. They yield cyanidin when depolymerized under oxidative conditions.
Olive leaf is the leaf of the olive tree. Although olive oil is well known for its flavor and possible health benefits, the leaf and its extracts remain under preliminary research with unknown effects on human health.
Cleome gynandra is a species of Cleome that is used as a green vegetable. It is known by many common names including Shona cabbage, African cabbage, spiderwisp, cat's whiskers, and stinkweed. It is an annual wildflower native to Africa but has become widespread in many tropical and sub-tropical parts of the world.
Chrysanthemin is an anthocyanin. It is the 3-glucoside of cyanidin.
Condensed tannins are polymers formed by the condensation of flavans. They do not contain sugar residues.
Pea protein is a food product and protein supplement derived and extracted from yellow and green split peas, Pisum sativum. It can be used as a dietary supplement to increase an individual's protein or other nutrient intake, or as a substitute for other food products. As a powder, it is used as an ingredient in food manufacturing, such as a thickener, foaming agent, or an emulsifier.
Fish protein powder (FPP) describes a food grade powder product designated primarily for human consumption applications. It differs significantly from fish meal products which are designated for animal feed applications. Fish protein powders have various sanitary processing, purity and functional characteristics which establish them as human food ingredients. Production plants registered for the USA market are located in Peru and France.
Protein quality is the digestibility and quantity of essential amino acids for providing the proteins in correct ratios for human consumption. There are various methods that rank the quality of different types of protein, some of which are outdated and no longer in use, or not considered as useful as they once were thought to be. The Protein Digestibility Corrected Amino Acid Score (PDCAAS), which was recommended by the Food and Agriculture Organization of the United Nations (FAO), became the industry standard in 1993. FAO has recently recommended the newer Digestible Indispensable Amino Acid Score (DIAAS) to supersede PDCAAS. The dairy industry is in favor of this, because while PDCAAS truncates all protein types that exceed the essential amino acid (EAA) requirements to 1.0, DIAAS allows a higher than 1.0 ranking: while for example both soy protein isolate and whey isolate are ranked 1.0 according to PDCAAS, in the DIAAS system, whey has a higher score than soy.
Digestible Indispensable Amino Acid Score (DIAAS) is a protein quality method proposed in March 2013 by the Food and Agriculture Organization to replace the current protein ranking standard, the Protein Digestibility Corrected Amino Acid Score (PDCAAS).
Instant tea is a powdered mix in which water is added, in order to reconstitute it into a cup of tea. The earliest form of instant tea was developed in the United Kingdom in 1885. A patent was granted for a paste made of concentrated tea extract, sugar, and evaporated milk, which became tea when hot water was added. However, no notable developments were made until spray drying technology allowed for drying the tea concentrates at a temperature which did not damage the flavors of the product.
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