**1. Introduction**

190 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

Wright, K. N. (1968). Soybean meal processing and quality control. *Journal of the American* 

Yoshiki, Y.; Kudou, S. & Okubo, K. (1998). Relationship between chemical structures and

Zhenyu, G. ; Jianrong, L. ; Ping, Y. ; Xinle, L. & Peilin, C. (2000). Removal of Goitrogen in

*Biotechnology and Biochemistry,* vol. 62, pp. 2291–2299, ISSN : 1347-6947 Zanella, I.; Sakomura, N. K.; Silverside, F. G. & (1999). Effect of enzyme supplementation of

biological activities of triterpenoid saponins from soybean (Review). *Bioscience* 

broiler diets based on corn and soybeans. *Poultry Science*., v. 78, pp.561–568, ISSN

Soybean. Journal of the Chinese Cereals and Oils Association., vol. 01, ISSN : 1003-

*Oil Chemists' Society*, Vol. 58, No. 3, pp. 294-300, ISSN: 0003-021X

0032-5791

0174

Since 2001 the European Commission banned the use of meat and bone meal and its byproducts in diets for livestock animals (EC directive 999/2001) in order to assure consumer safety on animal products. Consequently, soybean meal became the most utilised protein source in the intensive livestock systems.

Moreover, the proteins of this source are low degradable in the rumen and well proportioned to the non structural carbohydrates (NSC).

Soybean meal solvent extract (s.e.) is a by-product of oil industry, where soybean seeds are treated with organic solvents (e.g. hexane) and subsequently with high temperature. For this reason soybean meal has been banned in the organic livestock (EC directive 2092/1991; EC directive 834/2007).

Even if in Europe the high part of soybean is imported, soybean solvent extract represents the less expensive protein source for its high crude protein content (44-50 % as fed). However soybean meal costs and availability are strongly related with the price development of agricultural commodities on the world market (Jezierny et al., 2010). Factors which may influence world market prices include variations in population and economic growth, changes in consumer's product preferences, but world market prices are also dependent on weather conditions (Gill, 1997; Trostle, 2008).

Finally, another factor has to be evaluated that is the genetically modification (GM) of soybean. Indeed, public concerns are increasing in GM food consumption due to the fact, even if for several years no direct evidence that it may represent a possible danger for health has been reported, recently, a number of papers have been published with controversial results.

Thus, the search for alternative protein sources has led to an increasing interest in the use of grain legumes, as they supply the important source of plant protein.

The botanical family of grain legumes is known as *Fabaceae*, also referred to as *Leguminosae*. Grain legumes are cultivated primarily for their seeds which are harvested at maturity, and which are rich in protein and energy. The mature dry seeds of grain legumes are used either as animal feed ingredient or for human consumption (Singh et al., 2007). Beans, lentils and chickpeas are utilised exclusively for human nutrition, while the other grains are used in animal feeding too.

In Italy grain legumes cultivation is progressively increased due the presence of new cultivars more hardy and productive. These new cultivars were selected principally in

Protein Sources in Ruminant Nutrition 193

Soybean meal s.e. proteins, due to the heat treatment, are medium to low degradable in the rumen (Chaubility et al., 1991; Infascelli et al., 1995). In addition, soybean meal shows an

Legume grains are characterised by high energy density allowed to the high protein, starch

The proteins of legume grain are highly degradable in the rumen and digestible in the intestine. Notwithstanding, large part of legume grain shows anti-nutritional factors (i.e. lecithin, trypsin inhibitors, tannins, saponin, phytase), that are inactivated by the enzymes produced by the bacteria present in the rumen. Within the grain legumes, lupins have higher amounts of crude protein (324–381 g/kg dry matter), compared to faba beans (301 g/kg dry matter) and peas (246 g/kg dry matter) (Degussa, 2006). Jezierny et al. (2007) reported similar contents of crude protein in different batches of lupins, faba beans and peas averaging 387, 308 and 249 g/kg dry matter, respectively. In comparison to soybean meal, faba beans and peas contain between 45 to 55% and lupins (L. albus) even up to 70% of its

The ether extract content in peas and faba beans is generally rather low compared to lupins; crude fat contents of faba beans and peas range from 15 to 20 g/kg dry matter, thus being in a similar range as values for soybean meal (15–28 g/kg dry matter) (DLG, 1999; Jezierny et al., 2007). In lupins, the crude fat content varies between cultivars, with values of about 57 g/kg dry matter (*L. luteus*, *L. angustifolius*) to 88 g/kg dry matter (*L. albus*) (DLG, 1999). The carbohydrate fraction includes the low molecular-weight sugars, starch and various non-starch-polysaccharides (NSP) (Bach Knudsen, 1997). The NSP and lignin are the principal components of cell walls and are commonly referred to as dietary fibre (Theander et al., 1989; Canibe and Bach Knudsen, 2002). Generally, faba beans and peas are rich in starch (422–451 and 478–534 g/kg dry matter, respectively) (DLG, 1999; Jezierny, 2009), whereas lupins have comparatively low levels of starch (42–101 g/kg dry matter) (DLG, 1999; Jezierny, 2010). However, it needs to be emphasized that the determination of starch in

grain legumes may be confounded by the analytical method used (Hall et al., 2000).

Faba beans and peas contain rather low amounts of fibre fractions in comparison to lupins (Bach Knudsen, 1997; Jezierny, 2009), and, with regard to lignin content, faba beans and *L. angustifolius* have similar amounts of lignin (1 to 7 and 6 to 9 g/kg dry matter, respectively), whereas the lignin content in peas is of minor importance (0.4–3 g/kg dry matter) (Salgado

The NSP fraction of faba beans consists mainly of cellulose (89–115 g/kg dry matter), with lower levels of hemicellulose (21–57 g/kg dry matter) (Salgado et al., 2002a,b; Jezierny,

Hemicellulose contents in peas range from 23 to 95 g/kg dry matter and cellulose contents

Lupins contain high levels of NSP, with contents of cellulose generally being higher than hemicellulose (131 to 199 vs. 40 to 66 g/kg dry matter) (Bach Knudsen, 1997; Salgado et al., 2002a,b; Jezierny, 2009), and they also have considerable amounts of oligosaccharides (Bach

Comparing the different batches in each species, lupin show lower variability than faba bean and peas, probably because the genetic selection in this species was addressed principally on the reduction of secondary plant metabolites (Colombini, 2004) than to the

range from 52 to 77 g/kg dry matter (Salgado et al., 2002a,b; Jezierny, 2009).

and/or fat concentrations, as more than sufficient is their calcium concentration.

elevated ratio of protein/non structural carbohydrates.

crude protein content (Degussa, 2006).

et al., 2002a; Jezierny, 2010).

Knudsen, 1997; Salgado et al., 2002a).

improving of chemical characteristics.

2009).

France and are characterised by lower water requirements, higher production and higher resistance to the parasitic infestations and to the adverse environmental conditions.

Generally, legumes are characterised by their ability to use atmospheric nitrogen as a nutrient due to the symbiosis with nitrogen-fixing bacteria from the *Rhizobium* species (Sprent and Thomas, 1984; Zahran, 1999). Therefore, unlike other cultivated plants, legume crops need less nitrogen fertiliser for optimal growth, and the use of legumes in crop rotation systems reduces the need of nitrogen fertiliser in subsequent crops (López-Bellido et al., 2005). Nitrogen benefits in legume-cereal rotation systems have been attributed not only to the transfer of biologically fixed nitrogen (Díaz-Ambrona and Mínguez, 2001; Evans et al., 2001), but also to lower immobilisation of nitrate in the soil during the decomposition of legumes compared to cereal residues (Green and Blackmer, 1995), also termed as the nitrogen-sparing effect. Thus, nitrogen benefits may result from a combination of legume nitrogen sparing effects and the bacterial nitrogen fixation (Chalk et al., 1993; Herridge et al., 1995). In addition, crop rotation and intercropping with legumes may provide successful strategies for weed suppression (Liebman and Dyck, 1993; Bulson et al., 1997). Weed growth and development may be disrupted due to varying cultivation conditions prevailing for the different crops used (e.g. fertiliser requirements, planting or maturation dates), thereby preventing domination of only a few weed species (Froud-Williams, 1988; Liebman and Janke, 1990). Due to these crop effects, cultivation of grain legumes is an important part of crop rotation, particularly in organic farming (Badgley et al., 2007).

In animal nutrition, grain legumes are mainly used as protein supplements, but also as a valuable energy source, due to their partly high contents of starch (faba bean, peas) and lipids (lupins) (Gatel, 1994; Bach Knudsen, 1997; Salgado et al., 2002a).

However, the use of grain legumes in animal nutrition has been hampered due to partially high concentrations of secondary plant metabolites, also referred as antinutritional factors (ANFs), including condensed tannins, protease inhibitors, alkaloids, lectins, pyrimidine glycosides and saponins. Possible negative effects of these secondary plant metabolites include, for example, feed refusals (tannins, alkaloids), reduced nutrient digestibility (tannins, protease inhibitors, lectins) or even toxic effects (alkaloids) (Rubio and Brenes, 1995; Lallès and Jansman, 1998; Huisman and Tolman, 2001).

The objectives of the following chapters are the comparison of chemical composition as well as the nutritive value of soybean, soybean meal solvent extract and several legume grain (e.g.: peas, lupine, faba bean). In addition, in order to evaluate the opportunity of soybean replacement with grain, the results of *in vitro* studies are described and the influence of protein sources on meat quality are discussed.

#### **2. Nutritional characteristics of soybean meal solvent extract and legume grains**

The chemical composition of some grain legumes in comparison to soybean and soybean meals s.e are pictured in Table 1.

Soybeans (*Soja hispida*) are characterized by high protein (380 g/kg dry matter) and lipid (200 g/kg dry matter) concentrations, which provide high energy density (1.1-1.2 UFL/kg dry matter). In ruminant feeding, soybean integral seeds could represent the only source of protein supplementation for cattle fattening. However, in dairy cows it is preferable not to exceed the dry matter administration of soybean seeds as it may modify fatty acid profile of milk fat and worse butter consistency and conservation.

France and are characterised by lower water requirements, higher production and higher

Generally, legumes are characterised by their ability to use atmospheric nitrogen as a nutrient due to the symbiosis with nitrogen-fixing bacteria from the *Rhizobium* species (Sprent and Thomas, 1984; Zahran, 1999). Therefore, unlike other cultivated plants, legume crops need less nitrogen fertiliser for optimal growth, and the use of legumes in crop rotation systems reduces the need of nitrogen fertiliser in subsequent crops (López-Bellido et al., 2005). Nitrogen benefits in legume-cereal rotation systems have been attributed not only to the transfer of biologically fixed nitrogen (Díaz-Ambrona and Mínguez, 2001; Evans et al., 2001), but also to lower immobilisation of nitrate in the soil during the decomposition of legumes compared to cereal residues (Green and Blackmer, 1995), also termed as the nitrogen-sparing effect. Thus, nitrogen benefits may result from a combination of legume nitrogen sparing effects and the bacterial nitrogen fixation (Chalk et al., 1993; Herridge et al., 1995). In addition, crop rotation and intercropping with legumes may provide successful strategies for weed suppression (Liebman and Dyck, 1993; Bulson et al., 1997). Weed growth and development may be disrupted due to varying cultivation conditions prevailing for the different crops used (e.g. fertiliser requirements, planting or maturation dates), thereby preventing domination of only a few weed species (Froud-Williams, 1988; Liebman and Janke, 1990). Due to these crop effects, cultivation of grain legumes is an important part of

In animal nutrition, grain legumes are mainly used as protein supplements, but also as a valuable energy source, due to their partly high contents of starch (faba bean, peas) and

However, the use of grain legumes in animal nutrition has been hampered due to partially high concentrations of secondary plant metabolites, also referred as antinutritional factors (ANFs), including condensed tannins, protease inhibitors, alkaloids, lectins, pyrimidine glycosides and saponins. Possible negative effects of these secondary plant metabolites include, for example, feed refusals (tannins, alkaloids), reduced nutrient digestibility (tannins, protease inhibitors, lectins) or even toxic effects (alkaloids) (Rubio and Brenes,

The objectives of the following chapters are the comparison of chemical composition as well as the nutritive value of soybean, soybean meal solvent extract and several legume grain (e.g.: peas, lupine, faba bean). In addition, in order to evaluate the opportunity of soybean replacement with grain, the results of *in vitro* studies are described and the influence of

**2. Nutritional characteristics of soybean meal solvent extract and legume** 

The chemical composition of some grain legumes in comparison to soybean and soybean

Soybeans (*Soja hispida*) are characterized by high protein (380 g/kg dry matter) and lipid (200 g/kg dry matter) concentrations, which provide high energy density (1.1-1.2 UFL/kg dry matter). In ruminant feeding, soybean integral seeds could represent the only source of protein supplementation for cattle fattening. However, in dairy cows it is preferable not to exceed the dry matter administration of soybean seeds as it may modify fatty acid profile of

resistance to the parasitic infestations and to the adverse environmental conditions.

crop rotation, particularly in organic farming (Badgley et al., 2007).

lipids (lupins) (Gatel, 1994; Bach Knudsen, 1997; Salgado et al., 2002a).

1995; Lallès and Jansman, 1998; Huisman and Tolman, 2001).

protein sources on meat quality are discussed.

milk fat and worse butter consistency and conservation.

meals s.e are pictured in Table 1.

**grains** 

Soybean meal s.e. proteins, due to the heat treatment, are medium to low degradable in the rumen (Chaubility et al., 1991; Infascelli et al., 1995). In addition, soybean meal shows an elevated ratio of protein/non structural carbohydrates.

Legume grains are characterised by high energy density allowed to the high protein, starch and/or fat concentrations, as more than sufficient is their calcium concentration.

The proteins of legume grain are highly degradable in the rumen and digestible in the intestine. Notwithstanding, large part of legume grain shows anti-nutritional factors (i.e. lecithin, trypsin inhibitors, tannins, saponin, phytase), that are inactivated by the enzymes produced by the bacteria present in the rumen. Within the grain legumes, lupins have higher amounts of crude protein (324–381 g/kg dry matter), compared to faba beans (301 g/kg dry matter) and peas (246 g/kg dry matter) (Degussa, 2006). Jezierny et al. (2007) reported similar contents of crude protein in different batches of lupins, faba beans and peas averaging 387, 308 and 249 g/kg dry matter, respectively. In comparison to soybean meal, faba beans and peas contain between 45 to 55% and lupins (L. albus) even up to 70% of its crude protein content (Degussa, 2006).

The ether extract content in peas and faba beans is generally rather low compared to lupins; crude fat contents of faba beans and peas range from 15 to 20 g/kg dry matter, thus being in a similar range as values for soybean meal (15–28 g/kg dry matter) (DLG, 1999; Jezierny et al., 2007). In lupins, the crude fat content varies between cultivars, with values of about 57 g/kg dry matter (*L. luteus*, *L. angustifolius*) to 88 g/kg dry matter (*L. albus*) (DLG, 1999).

The carbohydrate fraction includes the low molecular-weight sugars, starch and various non-starch-polysaccharides (NSP) (Bach Knudsen, 1997). The NSP and lignin are the principal components of cell walls and are commonly referred to as dietary fibre (Theander et al., 1989; Canibe and Bach Knudsen, 2002). Generally, faba beans and peas are rich in starch (422–451 and 478–534 g/kg dry matter, respectively) (DLG, 1999; Jezierny, 2009), whereas lupins have comparatively low levels of starch (42–101 g/kg dry matter) (DLG, 1999; Jezierny, 2010). However, it needs to be emphasized that the determination of starch in grain legumes may be confounded by the analytical method used (Hall et al., 2000).

Faba beans and peas contain rather low amounts of fibre fractions in comparison to lupins (Bach Knudsen, 1997; Jezierny, 2009), and, with regard to lignin content, faba beans and *L. angustifolius* have similar amounts of lignin (1 to 7 and 6 to 9 g/kg dry matter, respectively), whereas the lignin content in peas is of minor importance (0.4–3 g/kg dry matter) (Salgado et al., 2002a; Jezierny, 2010).

The NSP fraction of faba beans consists mainly of cellulose (89–115 g/kg dry matter), with lower levels of hemicellulose (21–57 g/kg dry matter) (Salgado et al., 2002a,b; Jezierny, 2009).

Hemicellulose contents in peas range from 23 to 95 g/kg dry matter and cellulose contents range from 52 to 77 g/kg dry matter (Salgado et al., 2002a,b; Jezierny, 2009).

Lupins contain high levels of NSP, with contents of cellulose generally being higher than hemicellulose (131 to 199 vs. 40 to 66 g/kg dry matter) (Bach Knudsen, 1997; Salgado et al., 2002a,b; Jezierny, 2009), and they also have considerable amounts of oligosaccharides (Bach Knudsen, 1997; Salgado et al., 2002a).

Comparing the different batches in each species, lupin show lower variability than faba bean and peas, probably because the genetic selection in this species was addressed principally on the reduction of secondary plant metabolites (Colombini, 2004) than to the improving of chemical characteristics.

Protein Sources in Ruminant Nutrition 195

contain less than 50% of these AA in comparison to soybean meal (Table 2), thus

CP 301 246 381 324 361 541 Indispensable AA Arginine 26.4 21.0 39.3 33.5 38.0 39.7 Histidine 7.8 6.1 9.3 8.8 9.7 14.4 Isoleucine 11.8 10 15.3 12.7 14.2 24.3 Leucine 21.4 17.4 27.5 21.5 24.1 40.9 Lysine 18.4 17.3 18.2 15 16.3 33.1 Methionine 2.2 2.2 2.5 2.0 2.0 7.3

alanine 12.6 11.7 14.9 12.5 13.6 27.2 Threonine 10.5 9.1 13.3 10.9 11.9 21.3 Tryptophan 2.6 2.2 3.0 2.6 3.0 7.4 Valine 13.3 11.4 14.5 12.5 13.6 25.5 Dispensable AA Alanine 11.9 10.5 12.5 10.9 11.8 23.3

acid 31.6 28.2 38.5 31.5 35.1 62.0 Cystine 3.5 3.5 6.7 4.3 4.8 8.0

acid 46.9 40.0 79.3 65.6 72.5 97.6 Glycine 12.2 10.6 15.0 13.4 14.3 23.0 Proline 11.8 10.2 15.3 13.5 14.3 27.5 Serine 14.1 11.5 19.0 15.3 17.0 27.3 Table 2. Amino acid contents of grain legumes compared to soybean meal (g/kg dry matter)

As concerns the fatty acid profile, the rather high proportion of essential unsaturated fatty acids of some grain legumes, e.g. some *Vicia* species (Akpinar et al., 2001) or *L. albus* (Erbas¸ et al., 2005) may be attractive both from the human and animal nutrition perspective (Bézard et al., 1994), while adverse effects of unsaturated fatty acids on meat quality should be taken into account (Wood et al., 2003). For example, in faba beans a ratio of saturated to unsaturated fatty acids of 40–60 has been reported (Akpinar et al., 2001), whereas in *L*. *albus*, a ratio of saturated, monounsaturated and polyunsaturated fatty acids of 13.5 to 55.4 to 31.1

As concerns the mineral composition of soybean and grain legumes, only few data are

The calcium concentration ranges between 1.0 g/kg (faba beans) and 1.9 g/kg (*L. angustifolius*). Phosphorus concentration varies between 4.2 g/kg (*L. angustifolius*) and 7.6 g/kg (*L. luteus*). No extreme differences in trace mineral concentrations occurred except for the manganese concentration of *L. albus*, which containes approximately 10 times more

(Jezierny et al., 2010) SBM= soybean meal; CP= crude protein; AA= amino acids.

*angustifolius* 

*Lupinus* 

*luteus* SBM

constraining the use of grain legumes as sole protein source in pig diets.

Phenyl-

Aspartic

Glutamic

has been established (Erbas¸ et al., 2005).

manganese than the other legume grains (Brand et al., 2004).

reported in literature.

*Vicia faba Pisum sativum Lupinus albus Lupinus* 

The protein of faba beans and peas contains similar or even higher proportions of lysine (70 and 80 g/kg crude protein, respectively), when compared to protein from soybean meal s.e. (69 g/kg crude protein) or lupins (51 to 54 g/kg crude protein) (Degussa, 2006).

Comparing the different batches in each species, lupin show lower variability than faba bean and peas, probably because the genetic selection in this species was addressed principally on the reduction of secondary plant metabolites (Colombini, 2004) than to the improving of chemical characteristics.

Comparing the different batches in each species, lupin show lower variability than faba bean and peas, probably because the genetic selection in this species was addressed principally on the reduction of secondary plant metabolites (Colombini, 2004) than to the improving of chemical characteristics.


Table 1. Mean values of chemical composition (%dry matter) of different protein sources (Calabrò et al., 2001; Calabrò et al., 2009; Calabrò et al., 2010).

Comparing the different batches in each species, lupin show lower variability than faba bean and peas, probably because the genetic selection in this species was addressed principally on the reduction of secondary plant metabolites (Colombini, 2004) than to the improving of chemical characteristics.

The protein of faba beans and peas contains similar or even higher proportions of lysine (70 and 80 g/kg crude protein, respectively), when compared to protein from soybean meal (69 g/kg crude protein) or lupins (51 to 54 g/kg crude protein) (Degussa, 2006).

The proportion of threonine in grain legume protein (38 to 42 g/kg crude protein) is similar to that in soybean meal (45 g/kg soybean meal) (Degussa, 2006), however, there is a severe deficiency in the sulphur containing AA methionine + cystine, while tryptophan is marginally deficient to fulfill nutrient requirements for pigs (20 to 50 kg body weight) (NRC, 1998; Degussa, 2006). In fact, apart from *L. albus*, the seeds of faba beans, peas and lupins

The protein of faba beans and peas contains similar or even higher proportions of lysine (70 and 80 g/kg crude protein, respectively), when compared to protein from soybean meal s.e.

Comparing the different batches in each species, lupin show lower variability than faba bean and peas, probably because the genetic selection in this species was addressed principally on the reduction of secondary plant metabolites (Colombini, 2004) than to the

Comparing the different batches in each species, lupin show lower variability than faba bean and peas, probably because the genetic selection in this species was addressed principally on the reduction of secondary plant metabolites (Colombini, 2004) than to the

Soybean 93.30 33.2 25.0 5.57 14.5 Soybean meal s.e. 89.80 41.3 1.36 6.00 22.2 Lupin Lublanc 93.26 36.92 6.78 4.33 4.33 Lupin Lutteur 94.31 35.30 5.73 4.19 4.19 Lupin Multitalia 93.97 36.67 9.54 3.92 3.92 Faba bean Irena 89.22 25.62 1.05 3.99 20.94 Faba bean Lady 88.48 25.17 1.02 4.21 25.82 Faba bean Scuro di Torre Lama 90.55 26.91 0.90 4.32 21.75 Faba bean Chiaro di Torre Lama 90.80 24.69 1.01 4.19 21.23 Faba bean ProtHABAT69 90.60 28.69 1.10 4.51 18.21 Faba bean Sicania 90.29 26.52 0.95 3.89 21.43 Peas Alembo 88.49 31.27 0.73 3.94 21.60 Peas Alliance 89.20 28.47 0.56 3.82 20.40 Peas Attika 89.68 25.04 0.81 4.01 18.09 Peas Corallo 88.80 28.50 0.80 3.69 21.82 Peas Iceberg 90.08 27.30 0.78 4.12 22.75 Peas Ideal 89.94 28.28 0.88 4.17 18.37 Peas Spirale 93.00 28.74 0.55 4.30 19.05 Table 1. Mean values of chemical composition (%dry matter) of different protein sources

Comparing the different batches in each species, lupin show lower variability than faba bean and peas, probably because the genetic selection in this species was addressed principally on the reduction of secondary plant metabolites (Colombini, 2004) than to the

The protein of faba beans and peas contains similar or even higher proportions of lysine (70 and 80 g/kg crude protein, respectively), when compared to protein from soybean meal (69

The proportion of threonine in grain legume protein (38 to 42 g/kg crude protein) is similar to that in soybean meal (45 g/kg soybean meal) (Degussa, 2006), however, there is a severe deficiency in the sulphur containing AA methionine + cystine, while tryptophan is marginally deficient to fulfill nutrient requirements for pigs (20 to 50 kg body weight) (NRC, 1998; Degussa, 2006). In fact, apart from *L. albus*, the seeds of faba beans, peas and lupins

g/kg crude protein) or lupins (51 to 54 g/kg crude protein) (Degussa, 2006).

**Protein sources DM CP EE Ash NDF** 

(69 g/kg crude protein) or lupins (51 to 54 g/kg crude protein) (Degussa, 2006).

improving of chemical characteristics.

improving of chemical characteristics.

(Calabrò et al., 2001; Calabrò et al., 2009; Calabrò et al., 2010).

improving of chemical characteristics.


contain less than 50% of these AA in comparison to soybean meal (Table 2), thus constraining the use of grain legumes as sole protein source in pig diets.

Table 2. Amino acid contents of grain legumes compared to soybean meal (g/kg dry matter) (Jezierny et al., 2010) SBM= soybean meal; CP= crude protein; AA= amino acids.

As concerns the fatty acid profile, the rather high proportion of essential unsaturated fatty acids of some grain legumes, e.g. some *Vicia* species (Akpinar et al., 2001) or *L. albus* (Erbas¸ et al., 2005) may be attractive both from the human and animal nutrition perspective (Bézard et al., 1994), while adverse effects of unsaturated fatty acids on meat quality should be taken into account (Wood et al., 2003). For example, in faba beans a ratio of saturated to unsaturated fatty acids of 40–60 has been reported (Akpinar et al., 2001), whereas in *L*. *albus*, a ratio of saturated, monounsaturated and polyunsaturated fatty acids of 13.5 to 55.4 to 31.1 has been established (Erbas¸ et al., 2005).

As concerns the mineral composition of soybean and grain legumes, only few data are reported in literature.

The calcium concentration ranges between 1.0 g/kg (faba beans) and 1.9 g/kg (*L. angustifolius*). Phosphorus concentration varies between 4.2 g/kg (*L. angustifolius*) and 7.6 g/kg (*L. luteus*). No extreme differences in trace mineral concentrations occurred except for the manganese concentration of *L. albus*, which containes approximately 10 times more manganese than the other legume grains (Brand et al., 2004).

Protein Sources in Ruminant Nutrition 197

prediction. However, the tables supply mean values, which cannot be used for individual lots, and all the *in vivo* techniques are very expensive and time consuming while accuracy

The *in vitro* gas production technique (IVGPT, Theodorou et al., 1994) has proved to be a potentially useful technique for ruminant feed evaluation (Herrero et al., 1996; Getachew et al., 2004), as it is capable of measuring rate and extent of nutrient degradation (Groot et al.,

To evaluate a feedstuff by IVGPT, it is incubated at 39°C and under anaerobiosis condition with buffered rumen fluid and gas produced is measured as an indirect indicator of fermentation kinetics. During the incubation the feedstuff is first degraded and the degraded fraction may either be fermented to produce gas (CO2 and methane) and

The IVGPT is considered the most complete *in vitro* technique, because it allows to estimate the fermentation kinetics and contemporary gives information on the fermentation products

The IVGPT has been used by our research group for many years in order to investigate feed

In particular, in this chapter we report some results obtained incubating different protein sources (i.e. soybean and legumes grains) in order to compare their fermentation kinetics (Calabrò et al., 2001a), to assess the effect of some technological treatments (i.e. crushing and flaking) on the carbohydrates fermentation kinetics (Calabrò et al., 2001b) and to test

Calabrò et al. (2001a), in order to study the *in vitro* fermentation characteristics and kinetics proposed the following protocol: the samples (about 1.00 g), ground to pass a 1 mm screen, were incubated in triplicate at 39°C in 120 ml serum bottles under anaerobic conditions. Rumen liquor for the *inoculum* was collected from four buffaloes fed a standard diet, and

The gas measurements was made at 2-24 time intervals using a manual a pressure

The cumulative gas produced at each time was fitted to the Groot et al. (1996) model which estimates the asymptotic value (A, ml/g), the time after incubation at which A/2 is formed (B, h), the time to reach the maximum rate (tmax, h) and the maximum rate (Rmax, ml/h). At the end of incubation, the degraded organic matter (dOM, %) was calculated as a difference between incubated and residual OM (filtering the bottle content through preweighed glass crucibles and burning at 550°C for 3 hours), and pH and volatile fatty acid concentration (VFA, mM/g) were determined, using a pH-meter and a gas chromatography,

Several concentrate ingredients such as cereals and grain legumes, used in ruminant diets in order to increase production levels, were evaluated. In particular barley, maize, hard wheat,

Carbohydrates fractionation was carried out according to Cornell Net Carbohydrate and

The Cornell Net Carbohydrate and Protein System was developed to predict requirements, feed utilization, animal performance and nutrient excretion for dairy and beef cattle and sheep, using accumulated knowledge about feed composition, digestion, and metabolism in

immediately transported to the laboratory where it was homogenised and filtered.

fermentation acids, or incorporated into microbial biomass.

(degradability of dry matter, volatile fatty acids).

different legumes grain cultivars (Calabrò et al., 2009).

soft wheat, oats, faba bean and pea were used as test substrates.

Protein System (CNCPS, Sniffen *et al.,* 1992).

supplying nutrients to meet requirements.

may be low.

1996; Cone et al., 1996).

fermentation kinetics.

transducer (figure 1).

respectively.

Concerning the trace elements, Cabrera et al (2003) reported that in legumes their levels ranged from 1.5–5.0 μg Cu/g, 0.05–0.60 μg Cr/g, 18.8–82.4 μg Fe/g, 32.6–70.2 μg Zn/g, 2.7– 45.8 μg Al/g, 0.02–0.35 μg Ni/g, 0.32–0.70 μg Pb/g and not detectable—0.018 μg Cd/g. In nuts, the levels ranged from 4.0–25.6 μg Cu/g, 0.25–1.05 μg Cr/g, 7.3–75.6 μg Fe/g, 25.6– 69.0 μg Zn/g, 1.2–20.1 μg Al/g, 0.10–0.64 μg Ni/g, 0.14–0.39 μg Pb/g, and not detectable— 0.018 μg Cd/g. The authors found a direct statistical correlation between Cu–Cr, Zn–Al and Cr–Ni (P<0.05), and Al–Pb (P<0.001).

Sankara Rao and Deosthale (2006) comparing for their total ash, calcium, phosphorus, iron, magnesium, zinc, manganese, copper, and chromium contents five grain legumes (indian legumes, chick pea, pigeon pea, green gram, and black gram), found significant varietal differences only for chromium content in black gram. The cotyledons of these legumes were significantly lower in calcium content as compared to the whole grains. The authors concluded that for human nutrition, differences in mineral composition of whole grain and cotyledons were marginal except for calcium. These legumes as whole grain and cotyledons, appeared to be significant contributors to the daily requirements of magnesium, manganese, and copper in the diet.

As concerns the mineral composition of soybean and grain legumes, only few data are reported in literature.

The calcium concentration ranges between 1.0 g/kg (faba beans) and 1.9 g/kg (*L. angustifolius*). Phosphorus concentration varies between 4.2 g/kg (*L. angustifolius*) and 7.6 g/kg (*L. luteus*). No extreme differences in trace mineral concentrations occurred except for the manganese concentration of *L. albus*, which containes approximately 10 times more manganese than the other legume grains (Brand et al., 2004).

Concerning the trace elements, Cabrera et al (2003) reported that in legumes their levels ranged from 1.5–5.0 μg Cu/g, 0.05–0.60 μg Cr/g, 18.8–82.4 μg Fe/g, 32.6–70.2 μg Zn/g, 2.7– 45.8 μg Al/g, 0.02–0.35 μg Ni/g, 0.32–0.70 μg Pb/g and not detectable—0.018 μg Cd/g. In nuts, the levels ranged from 4.0–25.6 μg Cu/g, 0.25–1.05 μg Cr/g, 7.3–75.6 μg Fe/g, 25.6– 69.0 μg Zn/g, 1.2–20.1 μg Al/g, 0.10–0.64 μg Ni/g, 0.14–0.39 μg Pb/g, and not detectable— 0.018 μg Cd/g. The authors found a direct statistical correlation between Cu–Cr, Zn–Al and Cr–Ni (P<0.05), and Al–Pb (P<0.001).

Sankara Rao and Deosthale (2006) comparing for their total ash, calcium, phosphorus, iron, magnesium, zinc, manganese, copper, and chromium contents five grain legumes (indian legumes, chick pea, pigeon pea, green gram, and black gram), found significant varietal differences only for chromium content in black gram. The cotyledons of these legumes were significantly lower in calcium content as compared to the whole grains. The authors concluded that for human nutrition, differences in mineral composition of whole grain and cotyledons were marginal except for calcium. These legumes as whole grain and cotyledons, appeared to be significant contributors to the daily requirements of magnesium, manganese, and copper in the diet.

#### **3.** *In vitro* **evaluation of soybean and legume grains**

The nutritive value of a feed is assessed by its chemical composition, digestibility and level of voluntary intake. Feed evaluation methods are use to express nutritive value of feed. It is basically description of feeds interns that allow for a prediction of the performance of animals offered the feeds (Medsen et al., 1997).

Several methods are used in feed evaluation such as chemical analysis, rumen degradability measurement using the nylon bag technique, digestibility measurement and feed intake

Concerning the trace elements, Cabrera et al (2003) reported that in legumes their levels ranged from 1.5–5.0 μg Cu/g, 0.05–0.60 μg Cr/g, 18.8–82.4 μg Fe/g, 32.6–70.2 μg Zn/g, 2.7– 45.8 μg Al/g, 0.02–0.35 μg Ni/g, 0.32–0.70 μg Pb/g and not detectable—0.018 μg Cd/g. In nuts, the levels ranged from 4.0–25.6 μg Cu/g, 0.25–1.05 μg Cr/g, 7.3–75.6 μg Fe/g, 25.6– 69.0 μg Zn/g, 1.2–20.1 μg Al/g, 0.10–0.64 μg Ni/g, 0.14–0.39 μg Pb/g, and not detectable— 0.018 μg Cd/g. The authors found a direct statistical correlation between Cu–Cr, Zn–Al and

Sankara Rao and Deosthale (2006) comparing for their total ash, calcium, phosphorus, iron, magnesium, zinc, manganese, copper, and chromium contents five grain legumes (indian legumes, chick pea, pigeon pea, green gram, and black gram), found significant varietal differences only for chromium content in black gram. The cotyledons of these legumes were significantly lower in calcium content as compared to the whole grains. The authors concluded that for human nutrition, differences in mineral composition of whole grain and cotyledons were marginal except for calcium. These legumes as whole grain and cotyledons, appeared to be significant contributors to the daily requirements of magnesium, manganese,

As concerns the mineral composition of soybean and grain legumes, only few data are

The calcium concentration ranges between 1.0 g/kg (faba beans) and 1.9 g/kg (*L. angustifolius*). Phosphorus concentration varies between 4.2 g/kg (*L. angustifolius*) and 7.6 g/kg (*L. luteus*). No extreme differences in trace mineral concentrations occurred except for the manganese concentration of *L. albus*, which containes approximately 10 times more

Concerning the trace elements, Cabrera et al (2003) reported that in legumes their levels ranged from 1.5–5.0 μg Cu/g, 0.05–0.60 μg Cr/g, 18.8–82.4 μg Fe/g, 32.6–70.2 μg Zn/g, 2.7– 45.8 μg Al/g, 0.02–0.35 μg Ni/g, 0.32–0.70 μg Pb/g and not detectable—0.018 μg Cd/g. In nuts, the levels ranged from 4.0–25.6 μg Cu/g, 0.25–1.05 μg Cr/g, 7.3–75.6 μg Fe/g, 25.6– 69.0 μg Zn/g, 1.2–20.1 μg Al/g, 0.10–0.64 μg Ni/g, 0.14–0.39 μg Pb/g, and not detectable— 0.018 μg Cd/g. The authors found a direct statistical correlation between Cu–Cr, Zn–Al and

Sankara Rao and Deosthale (2006) comparing for their total ash, calcium, phosphorus, iron, magnesium, zinc, manganese, copper, and chromium contents five grain legumes (indian legumes, chick pea, pigeon pea, green gram, and black gram), found significant varietal differences only for chromium content in black gram. The cotyledons of these legumes were significantly lower in calcium content as compared to the whole grains. The authors concluded that for human nutrition, differences in mineral composition of whole grain and cotyledons were marginal except for calcium. These legumes as whole grain and cotyledons, appeared to be significant contributors to the daily requirements of magnesium, manganese,

The nutritive value of a feed is assessed by its chemical composition, digestibility and level of voluntary intake. Feed evaluation methods are use to express nutritive value of feed. It is basically description of feeds interns that allow for a prediction of the performance of

Several methods are used in feed evaluation such as chemical analysis, rumen degradability measurement using the nylon bag technique, digestibility measurement and feed intake

manganese than the other legume grains (Brand et al., 2004).

**3.** *In vitro* **evaluation of soybean and legume grains** 

animals offered the feeds (Medsen et al., 1997).

Cr–Ni (P<0.05), and Al–Pb (P<0.001).

Cr–Ni (P<0.05), and Al–Pb (P<0.001).

and copper in the diet.

reported in literature.

and copper in the diet.

prediction. However, the tables supply mean values, which cannot be used for individual lots, and all the *in vivo* techniques are very expensive and time consuming while accuracy may be low.

The *in vitro* gas production technique (IVGPT, Theodorou et al., 1994) has proved to be a potentially useful technique for ruminant feed evaluation (Herrero et al., 1996; Getachew et al., 2004), as it is capable of measuring rate and extent of nutrient degradation (Groot et al., 1996; Cone et al., 1996).

To evaluate a feedstuff by IVGPT, it is incubated at 39°C and under anaerobiosis condition with buffered rumen fluid and gas produced is measured as an indirect indicator of fermentation kinetics. During the incubation the feedstuff is first degraded and the degraded fraction may either be fermented to produce gas (CO2 and methane) and fermentation acids, or incorporated into microbial biomass.

The IVGPT is considered the most complete *in vitro* technique, because it allows to estimate the fermentation kinetics and contemporary gives information on the fermentation products (degradability of dry matter, volatile fatty acids).

The IVGPT has been used by our research group for many years in order to investigate feed fermentation kinetics.

In particular, in this chapter we report some results obtained incubating different protein sources (i.e. soybean and legumes grains) in order to compare their fermentation kinetics (Calabrò et al., 2001a), to assess the effect of some technological treatments (i.e. crushing and flaking) on the carbohydrates fermentation kinetics (Calabrò et al., 2001b) and to test different legumes grain cultivars (Calabrò et al., 2009).

Calabrò et al. (2001a), in order to study the *in vitro* fermentation characteristics and kinetics proposed the following protocol: the samples (about 1.00 g), ground to pass a 1 mm screen, were incubated in triplicate at 39°C in 120 ml serum bottles under anaerobic conditions. Rumen liquor for the *inoculum* was collected from four buffaloes fed a standard diet, and immediately transported to the laboratory where it was homogenised and filtered.

The gas measurements was made at 2-24 time intervals using a manual a pressure transducer (figure 1).

The cumulative gas produced at each time was fitted to the Groot et al. (1996) model which estimates the asymptotic value (A, ml/g), the time after incubation at which A/2 is formed (B, h), the time to reach the maximum rate (tmax, h) and the maximum rate (Rmax, ml/h).

At the end of incubation, the degraded organic matter (dOM, %) was calculated as a difference between incubated and residual OM (filtering the bottle content through preweighed glass crucibles and burning at 550°C for 3 hours), and pH and volatile fatty acid concentration (VFA, mM/g) were determined, using a pH-meter and a gas chromatography, respectively.

Several concentrate ingredients such as cereals and grain legumes, used in ruminant diets in order to increase production levels, were evaluated. In particular barley, maize, hard wheat, soft wheat, oats, faba bean and pea were used as test substrates.

Carbohydrates fractionation was carried out according to Cornell Net Carbohydrate and Protein System (CNCPS, Sniffen *et al.,* 1992).

The Cornell Net Carbohydrate and Protein System was developed to predict requirements, feed utilization, animal performance and nutrient excretion for dairy and beef cattle and sheep, using accumulated knowledge about feed composition, digestion, and metabolism in supplying nutrients to meet requirements.

Protein Sources in Ruminant Nutrition 199

(% DM) 11.0 8.79 12.2 14.4 13.5 29.8 23.9 A 3.80 0.49 12.6 2.40 5.00 6.00 18.0 B1 66.4 75.4 45.6 67.6 65.7 43.6 40.8 B2 11.4 6.14 16.0 9.86 9.64 9.99 7.92 C 3.60 2.57 6.77 2.64 2.16 6.41 4.18

Legumes were characterised by high gas production alongside a rather slow fermentative process. However, interpretation of faba bean and pea data was complicated by their high protein content, whose degradation can interact with gas production (Schofield*.,* 2000).

Oats reached the maximum rate before all the other grains (figure 2) and remained the highest until 12 incubation hours, subsequently decreasing below the soft wheat level. Until 6 hours of incubation barley was faster than the two wheat, later becoming slower until the end; up to this time the three sharp curves were practically overlapping, suggesting that the fermentation trend was similar for the three grains. Maize, faba bean and pea always showed the slowest fermentation rate with overlapping trends: fermentation slowly reaches its maximum rate and slowly decreases. The results of this investigation evidenced the

The range of rates obtained with the GPT (oats>wheat>barley>maize>legumes) approximately reflects that reported by other authors cited by Sniffen *et al.* (1992):

validity of the IVGPT in describing the kinetics fermentation of the examined grains.

wheat

Soft

wheat Faba bean Pea

Barley Maize Oats Hard

Table 3. Chemical composition and CNCPS fraction of the tested grains.

At 72 h the pH always remains good to guarantee microbial activity.

Fig. 2. Gas production fractional rate plots of different grains.

Organic matter degradability for all tested grains was always higher than 95%.

CP

wheat>oats>barley>maize.

Fig. 1. Pressure transducer.

The CNCPS partitions crude protein into fractions A, B, and C, depending on their rate and extent of degradability in the rumen (NRC, 2001). Fraction A represents the non-protein N (NPN) (ammonia, peptides, amino acids) and is considered to be completely soluble; fraction B, subdivided into B1, B2, and B3, consists of true protein with progressively declining ruminal degradability. Fraction C is unavailable true protein. Broadly, these crude protein fractions are categorized into rumen degradable protein (RDP) and rumen undegradable protein (RUP). The rumen degradable protein meets protein requirements for ruminal microbial growth and protein synthesis.

Once reaching the rumen, feed and protein degradation is a function of microbial activity. Rumen microbial activity, growth and protein synthesis is primarily limited by the rate and extent of carbohydrate fermentation in the rumen. Consequently, dietary fiber fractions in the forage determine the animal response to feed.

Microbial protein and rumen undegradable protein reaching the small intestine are absorbed to meet the ruminant's protein requirement. When rumen degradable protein exceeds the capacity of the rumen microbes to assimilate it, ammonia builds up in the rumen. This is followed by absorption of ammonia into the blood, conversion into urea by the liver, and excretion in the urine. The conversion of ammonia to urea costs the dairy cow energy that could otherwise be used for milk production. This loss of dietary crude protein and energy reduces the utilization efficiency of rumen degradable protein and therefore, reduced ruminant production (NRC, 2001). It also causes a negative energy balance that leads to a reduced fertility.

In the study of Calabrò et al. (2001a), the CNCPS carbohydrate fractions (table 3) was consistent with the values reported by Sniffen *et al.* (1992).

Oats (table 4) had the lowest potential gas production (A: 251 *ml/g,* P<0.0) and a very fast fermentation process (evidenced by low B and high RM). This result was probably due to its high soluble sugars contents (A fraction). Interestingly, oat values were very high compared to *in situ* observations. As reported by Van Soest *et al., (*1992), maize proved to be the slowest because of its high starch content (B1 fraction), which degraded slowly. Hard and soft wheat showed very similar fermentation characteristics according to their chemical composition. Barley has fermentation kinetics between those of wheat and maize. Results related to these last three grains were similar to the results obtained *in vitro* using the IGPT (Mould *et al.,* 2005).

The CNCPS partitions crude protein into fractions A, B, and C, depending on their rate and extent of degradability in the rumen (NRC, 2001). Fraction A represents the non-protein N (NPN) (ammonia, peptides, amino acids) and is considered to be completely soluble; fraction B, subdivided into B1, B2, and B3, consists of true protein with progressively declining ruminal degradability. Fraction C is unavailable true protein. Broadly, these crude protein fractions are categorized into rumen degradable protein (RDP) and rumen undegradable protein (RUP). The rumen degradable protein meets protein requirements for

Once reaching the rumen, feed and protein degradation is a function of microbial activity. Rumen microbial activity, growth and protein synthesis is primarily limited by the rate and extent of carbohydrate fermentation in the rumen. Consequently, dietary fiber fractions in

Microbial protein and rumen undegradable protein reaching the small intestine are absorbed to meet the ruminant's protein requirement. When rumen degradable protein exceeds the capacity of the rumen microbes to assimilate it, ammonia builds up in the rumen. This is followed by absorption of ammonia into the blood, conversion into urea by the liver, and excretion in the urine. The conversion of ammonia to urea costs the dairy cow energy that could otherwise be used for milk production. This loss of dietary crude protein and energy reduces the utilization efficiency of rumen degradable protein and therefore, reduced ruminant production (NRC, 2001). It also causes a negative energy balance that

In the study of Calabrò et al. (2001a), the CNCPS carbohydrate fractions (table 3) was

Oats (table 4) had the lowest potential gas production (A: 251 *ml/g,* P<0.0) and a very fast fermentation process (evidenced by low B and high RM). This result was probably due to its high soluble sugars contents (A fraction). Interestingly, oat values were very high compared to *in situ* observations. As reported by Van Soest *et al., (*1992), maize proved to be the slowest because of its high starch content (B1 fraction), which degraded slowly. Hard and soft wheat showed very similar fermentation characteristics according to their chemical composition. Barley has fermentation kinetics between those of wheat and maize. Results related to these last three grains were similar to the results obtained *in vitro* using the IGPT

Fig. 1. Pressure transducer.

leads to a reduced fertility.

(Mould *et al.,* 2005).

ruminal microbial growth and protein synthesis.

the forage determine the animal response to feed.

consistent with the values reported by Sniffen *et al.* (1992).


Table 3. Chemical composition and CNCPS fraction of the tested grains.

Legumes were characterised by high gas production alongside a rather slow fermentative process. However, interpretation of faba bean and pea data was complicated by their high protein content, whose degradation can interact with gas production (Schofield*.,* 2000).

Organic matter degradability for all tested grains was always higher than 95%.

At 72 h the pH always remains good to guarantee microbial activity.

Oats reached the maximum rate before all the other grains (figure 2) and remained the highest until 12 incubation hours, subsequently decreasing below the soft wheat level. Until 6 hours of incubation barley was faster than the two wheat, later becoming slower until the end; up to this time the three sharp curves were practically overlapping, suggesting that the fermentation trend was similar for the three grains. Maize, faba bean and pea always showed the slowest fermentation rate with overlapping trends: fermentation slowly reaches its maximum rate and slowly decreases. The results of this investigation evidenced the validity of the IVGPT in describing the kinetics fermentation of the examined grains.

The range of rates obtained with the GPT (oats>wheat>barley>maize>legumes) approximately reflects that reported by other authors cited by Sniffen *et al.* (1992): wheat>oats>barley>maize.

Fig. 2. Gas production fractional rate plots of different grains.

Protein Sources in Ruminant Nutrition 201

Fermentation characteristics (Table 4 and 5) were affected by the grain type and treatment;

*ml/g H h-1 % ml/g mM/g*

whole grain 321 334 10.6 0.107 87.8 362 90.6 crushed grain 272 311 10.3 0.106 86.9 332 90.5 whole +crushed 296B 322B 10.4C 0.0106Aa 87.3aCD 347B 90.5AB

whole grain 335 360 14.4 0.071 96.7 344 94.3 crushed grain 325 367 14.8 0.062 89.9 359 79.3 whole +crushed 330A 364A 14.6A 0.066B 93.3B 352B 86.8AbB

whole grain 324 348 12.2 0.099 97.6 331 90.6 crushed grain 327 350 13.4 0.092 96.7 338 100 whole +crushed 326Ab 349A 12.8B 0.096AbC 97.2A 335B 95.5aA

whole grain 310 327 8.68 0.138 81.5 398 77.3 crushed grain 370 386 9.59 0.128 90.1 395 91.7 whole +crushed 340Aa 357A 9.13D 0.133D 85.8bD 397A 84.5B

**Main effect**  Grain \*\*\* \*\*\* \*\*\* \*\*\* \*\*\* \*\*\* \*\*\* Treatment n.s. n.s. \* n.s. n.s. n.s. n.s. **Interaction**  gran x treat \*\*\* \*\*\* n.s. n.s. \*\*\* \*\*\* n.s. Var. er 204 398 0.73 1•10-1 1.85 437 48.19

*OMCV, cumulative gas production related to incubated OM at 120 h; A, potential gas production; B, time at which A/2 is produced; Rmax, maximum fermentation rate; Y, yield related to the degraded OM; VFA, volatile* 

Flaking increased the potential gas production (A) for soybean and barley (P< 0.01), and slowed their fermentation kinetics (increased t/1/2 and decreased RM values). By contrast, maize flakes showed lower gas production (P<0.05) and the fermentation process was faster

Organic matter degradability decreased after the treatment for soybean (P<0.01), while it increased for barley (P<0.01) and, not significantly, for maize. The slower fermentative process for barley flakes (increased t/1/2and decreased RM values) agrees with the results of other authors on treated cereals for both *in vitro* (Bittante et al., 1989) and *in situ*

Comparing each grain with the respective flakes, OM degradability did not strictly follow the trend of potential gas production, confirming that not all the degraded OM is fermented. This result holds mainly for soybean flakes, which had a higher A (P<0.01) and a lower

Such decreased OM degradability was probably due to the thermal treatment causing a

Overall, the two cereals showed higher gas and VFA production, as well as a faster

**<sup>V</sup> A B Rmax dOM Y VFA**

in addition, the two factors interacted with each other.

Oats

Faba bean

Maize

Barley

*fatty acids related to degraded OM.* 

(P<0.01), compared to whole grain.

experiments (Arieli et al., 1995).

fermentative process.

dOM (P<0.01) compared to the whole grain.

decrease in the rapidly degradable fraction (Sarubbi, 1999).

 *a,b,c: P<0.05; A,B: P<0.001ns, not significant;\*\*\*: p < 0.001.* 

Table 5. *In vitro* fermentation characteristics of whole and crushed.

**OMC**

In the second investigation Calabrò et al. (2001b) evaluated the effects of flaking and crushing on some cereal and legume grains, using the *in vitro* cumulative gas production technique (Theodorou *et al.*, 1994).

The grains used in animal nutrition are commonly subjected to different technological treatments (i.e.: grounding, crushing, flaking, extrusion, cooking, micronization). Flaking is a hydrothermal-mechanical process which causes starch gelatinization and increases the enzymatic hydrolysis rate of the polysaccharide, which in turn favours the rumen microbial attack. Crushing differs from flaking in the shorter exposure to vapour. Also in this case, the obtained product presents a larger surface area compared to the primary grain, and a modified starch, which promotes faster fermentation kinetics.

Several investigations to evaluate the effect of flaking on organic matter degradability in the rumen, indicated the considerable influence of the primary grain (Arieli *et al*., 1995; Bittante *et al*., 1989); by contrast, little was known about the effects of crushing.


*OMCV, cumulative gas production related to incubated OM at 120 h; A, potential gas production; B, time at which A/2 is produced; Rmax, maximum fermentation rate; Y, yield related to the degraded OM; VFA, volatile fatty acids related to degraded OM.* 

 *a,b,c: P<0.05; A,B: P<0.001ns, not significant;\*\*\*: p < 0.001.* 

Table 4. *In vitro* fermentation characteristics of whole and flaked grains.

In this experiment three whole and flaked grains (barley, maize, soybean) and four whole and crushed grains (barley, oats, maize, faba bean) were used as test substrates.

In the second investigation Calabrò et al. (2001b) evaluated the effects of flaking and crushing on some cereal and legume grains, using the *in vitro* cumulative gas production

The grains used in animal nutrition are commonly subjected to different technological treatments (i.e.: grounding, crushing, flaking, extrusion, cooking, micronization). Flaking is a hydrothermal-mechanical process which causes starch gelatinization and increases the enzymatic hydrolysis rate of the polysaccharide, which in turn favours the rumen microbial attack. Crushing differs from flaking in the shorter exposure to vapour. Also in this case, the obtained product presents a larger surface area compared to the primary grain, and a

Several investigations to evaluate the effect of flaking on organic matter degradability in the rumen, indicated the considerable influence of the primary grain (Arieli *et al*., 1995; Bittante

*ml/g ml/g h h-1 % ml/g mM/g*

wholegrain 340 362 14.9 0.078 92.9 363 99.1 flaked grain 338 347 13.2 0.096 96.6 343 83.3 whole + flaked 339A 355A 14.1B 0.087B 94.8 353A 91.2A

wholegrain 304 323 10.2 0.125 90.6 341 92.4 flaked grain 346 369 11.7 0.104 97.9 359 94.8 whole + flaked 325B 346A 11.0C 0.115A 92.6 350A 93.6A

wholegrain 204 236 15.5 0.051 96.7 209 71.6 flaked grain 225 280 19.8 0.039 86.8 257 76.0 whole + flaked 215C 258B 17.6A 0.045C 91.8 233B 73.8B

**Main effect** grain \*\*\* \*\*\* \*\*\* \*\*\* n.s. \*\*\* \*\* treatment \*\*\* \*\* \*\* n.s. n.s. \*\* n.s.

**Interaction** grain. x treat. \*\*\* \*\*\* \*\*\* \*\*\* \*\*\* \*\*\* n.s. Var.er. 87.9 135 1.21 0.00009 10.5 206 103

*OMCV, cumulative gas production related to incubated OM at 120 h; A, potential gas production; B, time at which A/2 is produced; Rmax, maximum fermentation rate; Y, yield related to the degraded OM; VFA, volatile* 

In this experiment three whole and flaked grains (barley, maize, soybean) and four whole

Table 4. *In vitro* fermentation characteristics of whole and flaked grains.

and crushed grains (barley, oats, maize, faba bean) were used as test substrates.

 **VCSOI A B RM dOM Y VFA**

technique (Theodorou *et al.*, 1994).

Maize

Barley

Soybe an

*fatty acids related to degraded OM.* 

 *a,b,c: P<0.05; A,B: P<0.001ns, not significant;\*\*\*: p < 0.001.* 

modified starch, which promotes faster fermentation kinetics.

*et al*., 1989); by contrast, little was known about the effects of crushing.


Fermentation characteristics (Table 4 and 5) were affected by the grain type and treatment; in addition, the two factors interacted with each other.

*OMCV, cumulative gas production related to incubated OM at 120 h; A, potential gas production; B, time at which A/2 is produced; Rmax, maximum fermentation rate; Y, yield related to the degraded OM; VFA, volatile fatty acids related to degraded OM.* 

 *a,b,c: P<0.05; A,B: P<0.001ns, not significant;\*\*\*: p < 0.001.* 

Table 5. *In vitro* fermentation characteristics of whole and crushed.

Flaking increased the potential gas production (A) for soybean and barley (P< 0.01), and slowed their fermentation kinetics (increased t/1/2 and decreased RM values). By contrast, maize flakes showed lower gas production (P<0.05) and the fermentation process was faster (P<0.01), compared to whole grain.

Organic matter degradability decreased after the treatment for soybean (P<0.01), while it increased for barley (P<0.01) and, not significantly, for maize. The slower fermentative process for barley flakes (increased t/1/2and decreased RM values) agrees with the results of other authors on treated cereals for both *in vitro* (Bittante et al., 1989) and *in situ* experiments (Arieli et al., 1995).

Comparing each grain with the respective flakes, OM degradability did not strictly follow the trend of potential gas production, confirming that not all the degraded OM is fermented. This result holds mainly for soybean flakes, which had a higher A (P<0.01) and a lower dOM (P<0.01) compared to the whole grain.

Such decreased OM degradability was probably due to the thermal treatment causing a decrease in the rapidly degradable fraction (Sarubbi, 1999).

Overall, the two cereals showed higher gas and VFA production, as well as a faster fermentative process.

Protein Sources in Ruminant Nutrition 203

**Cultivar pH dOM OMCV Yield A B tmax Rmax**   *% ml/g ml/g ml/g h h ml/h*  **Faba bean**  Irene 6.46 92.9 370 397 328 22.9 12.42 9.14 Lady 6.35 93.3 354 363 333 24.5 15.67 9.26

Lama 6.49 87.8 308 351 269 22.0 15.39 8.84

Lama 6.41 91.8 348 379 310 23.1 13.20 8.69 ProtHABAT69 6.47 93.8 359 383 303 20.2 12.49 10.02 Sicania 6.40 92.9 324 349 299 21.0 12.83 9.71 MSD 0.135 3.19 60.1 49.3 60.1 3.90 3.65 1.89 **Lupine**  Lublanc 6.63 93.4 256 279 283 26.1 10.25 6.73 Luteur 6.69 92.4 275 298 309 25.6 5.58 7.83 Multitalia 6.72 91.2 273 297 303 27.0 8.48 7.08 MSD 0.219 5.40 26.6 91.5 13.9 45.8 15.6 4.74 **Peas**  Alembo 6.57 99.0 406 410 361 20.6 12.52 11.73 Alliance 6.49 99.3 397 396 358 20.1 11.99 11.72 Attika 6.57 98.4 397 404 360 20.5 11.82 11.46 Corallo 6.53 98.9 393 394 365 22.3 11.42 10.38 Iceberg 6.55 98.8 381 385 347 21.0 12.45 10.86 Ideal 6.58 97.0 371 383 336 20.7 13.24 11.06 Spirale 6.58 98.8 344 343 310 17.1 10.66 12.14 MSD 0.188 2.81 52.5 53.5 3.68 74.1 5.22 2.47

Faba bean1 6.52Ab 90.9b 368B 405A 321a 21.1ab 12.4 10.0Ab Lupine1 6.64B 91.8ab 284C 309B 293b 24.4a 9.03 7.42B Peas1 6.60a 95.1a 394A 413A 336a 18.2b 11.4 12.6Aa Soybean meal2 6.73 96.5 295 306 323 18.7 6.01 10.67 MSE 0.001 2.31 56.2 30.5 101 4.40 2.99 0.56

*MSD: Minimum Significant Differences for P<0.01. MSE: Mean Square Error.,In the column A,B,C: P<0.01; a,b,c: P<0.05. 1Data obtained from the grain legumes incubated in vitro as a pool. 2Data not statistically assessed.*  Table 6. Fermentation characteristics of the different grain legume cultivars and soybean

Several researches have been carried out in order to compare the nutritional characteristics of soybean solvent extract and legume grains, as faba bean, peas and lupine, for ruminant

Di Francia et al. (2007) evaluated the effect of partial replacement of soybean cake with extruded peas in the diet of lactating buffalo cows on milk yield and quality over the first 100 days of lactation. Their results showed that peas could represent an attractive GMO free

**4. Effect of different protein sources on animal performance** 

Scuro di Torre

Chiaro di Torre

meal.

feeding.

Crushing showed slighter effects compared to flaking, and also the results of the grain x treatment interaction were less important.

The different technological condition of the two treatments (less drastic for crushing) may well have contributed to this trend. In particular, crushed barley presents, with respect to the whole grain, the same behaviour as the flakes (higher A and dOM, P<0.001; higher *t/1/2*  and lower RM, not significant).

Interestingly, there was a decrease in faba bean degradability due to the treatment (P<0.01), which agrees with the results of a contemporaneous *in situ* trial (Sarubbi, 1999). Besides, faba bean also showed a lower VFA concentration, which was probably also caused by the considerable decrease in their protein degradability (P<0.001) as found *in situ* (Sarubbi, 1999). As usually observed *in vivo* in young bulls fed a cereal-rich diet, pH values were always quite low.

In the third study Calabrò *et al*. (2009) evaluated the fermentation characteristics of different cultivars of grain legumes using IVGPT.

Three grain legumes were tested: lupine (*Lupinus* spp.) (Lublanc, Luteur, Multitalia), faba bean (*Vicia faba* L.) (Chiaro di Torre Lama, Irena, Lady, ProtHABAT69, Scuro di Torre Lama, Sicania) and peas (*Pisum sativum*) (Alembo, Alliance, Attika, Corallo, Iceberg, Ideal, Spirale).

The fermentation characteristics are reported in table 6.

The values of pH ranged between 6.35 and 6.72, indicating a normal pattern of fermentation, and were consistent to the crude protein content.

As regards faba bean, the cultivar "Scuro di Torre Lama" showed significantly (P<0.01) lower values of dOM and OMCV than the other 5 cultivars.

In the case of lupine the cultivar "Lublanc" had lower (P<0.01) OMCV than the other 2 cultivars and for peas the cultivar "Spirale" produced less gas and showed a faster kinetics than the other 6 cultivars.

As expected, the OM degradability resulted very high in any case. However, comparing the pools of the grain legumes, dOM was in each case lower than that of soybean meal. OMCV was significantly (P<0.01) higher for pea than faba bean (330 vs. 316 ml/g, P <0.05) and lupine (330 vs. 258 ml/g, P <0.01). Gas production of peas was always higher than that of lupine, faba bean and also soybean meal according to the results of Buccioni et al. (2007) who studied the *in vitro* fermentation of soybean meal, faba bean and pea, and found in the latter the best balance between energy and nitrogen inputs.

The slower fermentation kinetics of faba bean may be due to the content in polyphenols while that of lupine may be caused by the very low starch content (INRA, 1988).

From the data obtained, the authors concluded that the tested grain legumes show only few differences compared to soybean meal (higher dOM and lower OMCV), consequently they may be considered in replacing, totally or partially, soybean.

The reported results are of particular importance as well as highlight the differences in dietary and nutritional characteristics of soybean and legume grains, provide data on the effects of the treatments of these feedstuffs and the differences among the cultivars present on the European market.

With this information, the nutritionist may from time to time choose the most suitable protein source in order to satisfy animal requirements ensuring the simultaneous availability of nitrogen and energy for the bacteria present in the rumen with beneficial effects on livestock production and environmental impact.

Crushing showed slighter effects compared to flaking, and also the results of the grain x

The different technological condition of the two treatments (less drastic for crushing) may well have contributed to this trend. In particular, crushed barley presents, with respect to the whole grain, the same behaviour as the flakes (higher A and dOM, P<0.001; higher *t/1/2* 

Interestingly, there was a decrease in faba bean degradability due to the treatment (P<0.01), which agrees with the results of a contemporaneous *in situ* trial (Sarubbi, 1999). Besides, faba bean also showed a lower VFA concentration, which was probably also caused by the considerable decrease in their protein degradability (P<0.001) as found *in situ* (Sarubbi, 1999). As usually observed *in vivo* in young bulls fed a cereal-rich diet, pH values were

In the third study Calabrò *et al*. (2009) evaluated the fermentation characteristics of different

Three grain legumes were tested: lupine (*Lupinus* spp.) (Lublanc, Luteur, Multitalia), faba bean (*Vicia faba* L.) (Chiaro di Torre Lama, Irena, Lady, ProtHABAT69, Scuro di Torre Lama, Sicania) and peas (*Pisum sativum*) (Alembo, Alliance, Attika, Corallo, Iceberg, Ideal,

The values of pH ranged between 6.35 and 6.72, indicating a normal pattern of fermentation,

As regards faba bean, the cultivar "Scuro di Torre Lama" showed significantly (P<0.01)

In the case of lupine the cultivar "Lublanc" had lower (P<0.01) OMCV than the other 2 cultivars and for peas the cultivar "Spirale" produced less gas and showed a faster kinetics

As expected, the OM degradability resulted very high in any case. However, comparing the pools of the grain legumes, dOM was in each case lower than that of soybean meal. OMCV was significantly (P<0.01) higher for pea than faba bean (330 vs. 316 ml/g, P <0.05) and lupine (330 vs. 258 ml/g, P <0.01). Gas production of peas was always higher than that of lupine, faba bean and also soybean meal according to the results of Buccioni et al. (2007) who studied the *in vitro* fermentation of soybean meal, faba bean and pea, and found in the

The slower fermentation kinetics of faba bean may be due to the content in polyphenols

From the data obtained, the authors concluded that the tested grain legumes show only few differences compared to soybean meal (higher dOM and lower OMCV), consequently they

The reported results are of particular importance as well as highlight the differences in dietary and nutritional characteristics of soybean and legume grains, provide data on the effects of the treatments of these feedstuffs and the differences among the cultivars present

With this information, the nutritionist may from time to time choose the most suitable protein source in order to satisfy animal requirements ensuring the simultaneous availability of nitrogen and energy for the bacteria present in the rumen with beneficial

while that of lupine may be caused by the very low starch content (INRA, 1988).

treatment interaction were less important.

cultivars of grain legumes using IVGPT.

The fermentation characteristics are reported in table 6.

lower values of dOM and OMCV than the other 5 cultivars.

latter the best balance between energy and nitrogen inputs.

may be considered in replacing, totally or partially, soybean.

effects on livestock production and environmental impact.

and were consistent to the crude protein content.

and lower RM, not significant).

always quite low.

than the other 6 cultivars.

on the European market.

Spirale).


*MSD: Minimum Significant Differences for P<0.01. MSE: Mean Square Error.,In the column A,B,C: P<0.01; a,b,c: P<0.05. 1Data obtained from the grain legumes incubated in vitro as a pool. 2Data not statistically assessed.* 

Table 6. Fermentation characteristics of the different grain legume cultivars and soybean meal.
