**2.7 Transgenic soybean**

"Genetically modified plants are typically created by the addition or deletion of existing innate genes in the plant's own genome or transferring external non-host genome through DNA splicing" [38]. Genetic approaches were applied for creating a prototype soybean that synthetize and accumulate a n-3 long chain polyunsaturated fatty acid (*i.e.* eicosapentaenoic acid, EPA) and a carotenoid (*i.e.* astaxanthin) in the seed [15]. Soybean contains very low levels of lutein (10 μg/g seed), but the expression of the phytoene synthase gene in transgenic soybean increases the accumulation of ß-carotene up to 800 μg/g seed and significantly reduces lutein content (29%). The expression of fatty acid elongases and Δ5 desaturase in transgenic soybean increase the synthesis of EPA up to 5%, but EPA content need to be improve in transgenic soybean to better reflect FO fatty acid profile [15]. It should be mentioned that the annual sales of astaxanthin reaches to over USD 200 million and inclusion of such trait in transgenic soybean can impressively enhance the attractiveness of such product especially for incorporating in aquafeeds for salmon, trout and shrimp [39, 40].

## **2.8 Pea protein**

Pea (*Pisum sativum*) is another promising APPS for aquaculture species with highly digestible protein and energy levels and it is a good source of digestible starch (40-50%) [41, 42]. This legume contains low levels of ANF (*e.g.* tannins) and does not have trypsin inhibitors, but contain high levels of saponins. The protein (21-25%) and methionine contents in peas also lower compared to soybean. Pea

protein concentrate (PPC) is a pea derived product that produced by fine grinding dehulled peas and air processing to remove fiber and carbohydrates. The PPC contains higher protein and lower ANF compared to unprocessed pea meal, thus it is more suitable APPS for aquafeeds. Like other plant protein derived products, extrusion and micronizing processes improve protein and energy digestibility of pea meals [41].

#### **2.9 Peanut**

Peanut (*Arachis hypogaea*) is the fourth largest oilseed crop in the world and peanut pulp that remain after the oil extraction can be used as an APPS in aquafeeds [43, 44]. Peanut meal (PNM) is a residue after solvent extraction of whole shelled peanuts and considered as a great APPS due to its higher protein content (47.8%) than SBM, higher palatability and the same cost as SBM [45, 46]. However, the protein quality of PNM is inferior compared to SBM and it contains lower levels of lysine and methionine than SBM, but a higher level of arginine [43, 45]. Lysine and methionine deficiencies in PNM can be met by using crystalline amino acids.

#### **2.10 Lupines**

Lupines include many legumes species with a considerable protein ( 35%) but low lipid (8–10%) levels [21, 47]. About 80% of lupines species the can be used as feed ingredients, particularly *Lupinus angustifolius,* is produced in Australia. Among different lupines, Andean lupin (*L. mutabilis*) seed contain 50% protein (dry matter) and its derivatives such as dehulled, deoiled and lupine protein concentrate contain higher protein content (61%) [48]. Although lupines have low levels of lysine and sulfur amino acids, they contain more arginine content than soybean [49]. Four species of lupines including *L. angustifolius, L. albus, L. luteus and L. mutabilis* named as "sweet lupins" as they contains low levels of alkaloids and because of their high protein contents they have great potential as APPS [50]. However, using lupins in aquafeeds are still limited because of their low protein digestibility and the presence of various ANF [51].

#### **2.11 Faba bean**

Faba bean, (*Vicia faba* L.) is a legume with high amount of protein ( 20 to 41%), carbohydrate (51% to 68%), B-vitamins and minerals depending on its variety [52, 53]. Its protein composition is mainly consisted of albumins (20%) and globulins (80%) and rich in glutamic and aspartic acids. But, the levels of sulfur amino acids and tryptophan residues are low [54]. The main carbohydrates in faba been are starch (41–53%), low molecular weight carbohydrates (*e.g.* raffinose , stachyose, and verbascose), and fiber mainly hemicellulose [52, 55]. Faba bean contains some ANF such as trypsin inhibitors and lectins, condensed tannin, phytic acid, vicine and convicine [56]. Processing of faba bean protein does provide ingredients with higher protein contents such as faba bean protein concentrate (55% crude protein) and faba bean isolate (80% crude protein) that contain lower levels of ANF [57–59].

#### **2.12 Other protein sources**

Carob seed (*Ceratonia siliqua*) germ does have a high protein content (45–50% crude protein) and it is cheaper than SBM [60]. Carob seed germ meal is produced

*Legumes, Sustainable Alternative Protein Sources for Aquafeeds DOI: http://dx.doi.org/10.5772/intechopen.99778*

from the germ of the carob seed after the separation of the gums and the fibrous [61–63]. However, it contains high levels of tannins [64].

Alfalfa (*Medicago sativa*) protein concentrate is another APPS that produced by pressing fresh alfalfa foliage (mainly leaves and stems) to make a protein-rich juice which is centrifuged and heated to fractionate proteins from the juice [65]. This byproduct contains reasonable protein level (52% crude protein) with high amounts of lysine, threonine, and methionine. It also contains high levels of vitamins and antioxidants such as carotenoids, but low content of fiber and ANF (*e.g.* phytic acid or lectins) [65, 66].

## **3. Anti-nutritional factors in legumes**

The ANF are defined as compounds that disturb feed utilization and can affect the health condition and production of livestock [67]. Legumes contain various ANF such as saponins, tannins, phytic acid, gossypol, lectins, protease inhibitors, amylase inhibitors, antivitamin factors, metal binding ingredients, goitrogens, etc. (**Table 1**) that combine with nutrients and reduce bioavailability of them in aquafeeds [8]. Some ANF such as protease inhibitors and phytates abate digestibility of proteins and energy as well as reduce mineral absorption that consequently results in malnutrition and microelements deficiencies.

The ANF can be divided into four classes [66]:


#### **4. Improvement of legumes efficiency in aquafeeds**

Digestibility of an ingredient is a pivotal parameter for determining its potential for use in the aquafeeds [68]. In order to validate the nutritional quality of a feedstuff, determination of apparent digestibility coefficient (ADC) of its dry matter and nutrients is necessary. As previously mentioned, generally legumes contain high amounts of starch and NSP and ANF [66] that negatively affect ADC in most fish and shrimp species. Carnivorous fish species are more susceptible to legumes. The ADC of crude protein of legumes are generally over 0.80, indicating high quality of protein provided by these APS. However, the ADC of gross energy in these research showed great fluctuations from 0.5 to 0.7 [69]. It has been reported that the ADC of legumes in diet mainly depends on fish species. Thus, ADC of legumes in omnivorous species such as Nile tilapia is higher than carnivorous fish such as rainbow trout [69].

Several strategies were applied for improving nutrients digestibility in legumes such as processing techniques (*e.g.* dehulling, soaking, extrusion cooking, fermentation etc.), using novel and new variety of plant protein sources (*e.g.* transgenic legumes), nutritional programming and selective breeding of fish to be more

adapted to legumes in aquafeeds, modulation of gut microbiota (*e.g.* probiotics and short chain fatty acids), and inclusion of additives (*e.g.* acidifiers, CAA, phospholipids etc.) in aquafeeds [53, 70]. Here the most efficient strategies for reducing ANF in APPS were described:
