by Dr Emmanuelle Apper, MSc Aurélien Feneuil and Dr Frédérique Respondek, Tereos Innovation department
First published in International Aquafeed, March-April 2015
Many fish feed producers now formulate low fishmeal diets. To ensure high growth performance, the use of high quality alternative protein is then required. Both Vital and Hydrolysed Wheat Gluten are high quality proteins. While they don’t exhibit the same behaviour at extrusion, they can both ensure good physical quality of pellets and high level of growth performance with high nitrogen and energy retention.
Additionally, Vital and Hydrolysed Wheat Gluten may have some functional health benefits at gut level, especially by stimulating gut cell proliferation and antioxidative system without damaging gut structure.
Intensive production of farmed fish fed with compound feeds has increased greatly, mainly due to the growth of aquaculture production, but also because it is the most efficient way of production (Olsen and Hasan, 2012).
In such feeds, Fish Meal (FM) used to be the major source of proteins, especially for marine fish and salmonids (Tacon et al., 2011). Nevertheless, because of the limited amount of available FM on the market, its impact on the environment and marine diversity, and its increasing price, its utilisation has been progressively reduced in the formulation of diets.
![http://issuu.com/international_aquafeed/docs/iaf1502_w1/16]()
In order to achieve a low FM incorporation (below 10 percent in formula) without impairing growth performance, active research was conducted on plant proteins (PP), which represent an interesting alternative to FM. In this context, many studies were undertaken to evaluate the effects of replacing FM with different types of PP, tested one by one or in mixture, on fish growth and health. Among the tested PP being considered to replace FM, Wheat Proteins (WP), including Vital Wheat Gluten (VWG) and Hydrolysed Wheat Gluten (HWG) are easily available PP sources that have given very promising results from technological, nutritional and health points of view.
Vital and Hydrolysed Wheat Gluten exhibit different behaviour in extrusion and both proteins allow obtaining pellets of high physical quality
VWG has already been described elsewhere (Apper-Bossard et al., 2013) as an effective binder in fish feed, imparting good mechanical properties (i.e. durability and hardness) to the fish feed pellets as well as good water stability. Moreover, increased VWG inclusion rate in replacement of soy protein concentrate results in decreasing extruder motor load. Indeed, extrusion behaviour of 2 commercial salmon feed diets has been explored both at small scale (Application Centre, Tereos, Marckolsheim, France) and at a fish feed Technology Centre (scale-up; Nofima, Fana, Norway).
One diet contained 10 percent VWG and the other 20 VWG. The two diets were extruded in the same processing conditions. This resulted in a lower motor load (i.e. torque) for 20 percent VWG compared to 10 percent VWG (36 percent versus 41 percent motor load). Higher inclusion of VWG in formulation leads to a decrease in motor load due to the lower water holding capacity of VWG compared to soy protein concentrate (Draganovic et al., 2011).
![http://issuu.com/international_aquafeed/docs/iaf1502_w1/16]()
The technological properties of HWG had not yet been reported in the literature while VWG and HWG have different impacts on extruder system parameters, especially on motor load and pressure at the die. Indeed, replacing 25 percent of Crude Protein (CP) of a FM diet by either VWG or HWG (Voller et al., in preparation) results in lower motor load with HWG (283 and 376 Nm with HWG and VWG respectively).
The higher effect of VWG on the motor load can be attributed to the higher water holding capacity (table 1) of this ingredient (1.5 g of water/g) compared to HWG (0.6 g of water/g). The reduction in motor load can be seen as a direct process advantage allowing reduction of energy consumption during extrusion (Specific Mechanical Energy reduced from 65 to 50 Wh/kg). An alternative is to run the HWG diet at the same motor load as the VWG diet with the potential to increase the extrusion capacity.
Wheat Proteins: Amino acid profile
Wheat proteins are a source of functional amino acids, especially sulphur amino acids and leucine. They contain rather low levels of lysine, tryptophan, and arginine meaning that they should be complemented with these amino acids when used at high level in formulae. Several experiments showed WP can successfully replace a large part of FM when diets are supplemented with free lysine in salmonids (Davies et al., 1997).
WPs contain a relatively high concentration of sulphur-containing amino acids, due to the numerous di-sulphur bonds (1.8 percent CP of methionine and 2.6 percent CP of cysteine), whereas PP sources are generally low in sulphur-containing amino acids. For instance, soybean meal and soy protein concentrate respectively contain 1.4 and 1.3 g/100 g CP of methionine and 1.3 and 1.4 g/100 g CP of cysteine.
Furthermore, WPs are high in leucine, with about 7.9 g/100 g CP. Leucine is considered as the main amino acid triggering muscle protein synthesis and inhibiting proteolysis in mammals (Li et al., 2009) and probably in fish. Indeed, in different species, amino acids regulate the TOR signalling pathway (Seilliez et al., 2008). Furthermore, supplementing media containing 0.6 mM leucine with an additional 2.5 mM leucine reduced rates of protein degradation in rainbow trout primary myocytes by 8 percent (Cleveland, 2010).
![http://issuu.com/international_aquafeed/docs/iaf1502_w1/16]()
WPs are also rich in glutamine: from 35 to 40 percent CP. Glutamine is a major substrate for all rapidly proliferating cells and plays an important role in maintaining intestinal trophicity (Verlhac-Trichet, 2010). In addition, glutamine is one of the most important energy substrates of enterocytes. Free glutamine significantly increases enterocyte and microvilli length in catfish gut (Pohlenz et al., 2012), hybrid striped bass (Cheng et al., 2012), and juvenile hybrid sturgeon (Zhu et al., 2011). Glutamine also constitutes a major substrate for immune cells, thus modulating immune response (Verlhac-Trichet, 2010; Zhu et al., 2011; Cheng et al., 2012).
Moreover, glutamine plays a role in eliminating free radicals as it acts as a precursor for glutathione synthesis (Wu, 1998). Such effects are reported for juvenile hybrid sturgeon (Zhu et al., 2011) and hybrid striped bass (Cheng et al., 2012). Glutamine has proven to stimulate muscle synthesis in terrestrial vertebrates but such results are not available for fish. However, dietary glutamine supplementation increases growth performance in juvenile hybrid sturgeon (Qiyou et al., 2011) and in hybrid striped bass (Cheng et al., 2012).
Effects of wheat proteins on growth performance
Because of its high digestibility and its absence of anti-nutritional factors, replacement of a large proportion of FM with VWG results in similar growth performance and fish composition whatever the species are. The apparent CP digestibility of VWG is high, between 92 and 98.5 percent (Apper-Bossard et al., 2013). In rainbow trout, VWG successfully substitutes more than 50 percent FM providing diets supplemented with lysine without affecting protein and lipid composition of the carcasses (Davies et al., 1997).
Furthermore, the inclusion of 14.5 percent VWG in diets does not adversely affect the flavour of fillets (Skonberg et al., 1998). In Atlantic salmon, the replacement of 35 percent FM with VWG without supplementing by lysine results in similar final body weight and growth (Storebakken et al., 2000). These authors estimate the replacement of FM with VWG without amino acid supplementation can go up to 50 percent based on the amount and the availability of lysine in VWG and on the requirement of fish.
In European sea bass, substituting more than 50 percent FM with VWG does not impair palatability, growth performance, and nitrogen-energy retention (Tibaldi et al., 2003). In gilthead sea bream, the use of 88 percent CP from VWG not only successfully replaced FM but also produced better growth and feed conversion ratio, probably related to higher protein and energy intake of fish (Allan et al., 2000). In Nile tilapia fed with diets differing in their protein sources, the highest growth is reported for VWG, FM, and soybean extract diet. In shrimp, results are scarce but the replacement of up to 20 percent marine protein with VWG does not significantly affect feed efficiency and growth performance (Molina-Povida et al., 2004; Tereos internal data).
HWG also seems promising in aquaculture feeds as it results in high growth performance. CP digestibility of HWG has recently been measured. It is very high and further increases with higher HWG inclusion rate in FM-diet for rainbow trout (figure 1; Apper et al., 2014). Recent studies on juvenile hybrid sturgeon show that the replacement of 1 to 5 percent of soy protein concentrate by 1 to 5 percent of HWG in a diet containing animal and plant proteins significantly increases growth performance (Qiyou, 2011).
The use of 12.5 to 50 percent CP of HWG to replace high quality FM does not modify growth performance and feed efficiency in rainbow trout (Apper et al., 2014). In the same experiment, authors compared energy and nitrogen retention obtained with either VWG or HWG. Nitrogen retention was similar for the 2 protein sources, with very low metabolic losses of nitrogen (35.1 and 43.4 kg/ton of fish produced for HWG and VWG respectively). Energy retention was higher with HWG than with VWG.
![http://issuu.com/international_aquafeed/docs/iaf1502_w1/16]()
Effects of wheat proteins on gut health and microbiota
Compared to a fishmeal-based diet, the use of Wheat Proteins does not damage gut morphology and microbiota. In Rainbow trout, the replacement of up to 50 percent high quality FM by either VWG or HWG results in no modification of gut structure (figures 2 and 3; Apper et al., 2014). Indeed, no areas of necrosis were observed in enterocytes, all appearing uniform, regular, and healthy. Microvilli observations confirmed such results, all microvilli being packed and showing the same density. Similarly, in the same study, microbiota was not significantly different between FM, HWG and VWG diets, with a predominance of Firmicutes.
Richness, OTUs, and diversity of microbiota were not different across treatments, suggesting that replacing up to 50 percent of high quality FM by WP is without consequence for gut health. Such results are typical of wheat proteins as soy-proteins or pea proteins have been demonstrated to impair gut morphology or microbiota at high inclusion rates (Mc Kellep Bakke et al., 2007; Penn et al., 2011).
In soy-based diets, the inclusion of hydrolysed wheat proteins modulates gut function and morphology, the anti-oxidative system, and the non-specific immune system. The replacement from 1 to 5 percent of soy protein concentrate in a diet based on 20 percent FM, 20 percent soybean meal, eight percent corn gluten meal and 10 percent blood meal increased digestive enzyme activities and fold heights, modulated non-specific immune response and stimulated anti-oxidative status (Qiyou et al., 2011; Zhu et al., 2011).
Interestingly, in these studies, the results of 3 percent of HWG inclusion were equivalent to results obtained when authors added one percent free glutamine in the soy protein concentrate diet. Such results suggest that HWG may have a bioactive role, by acting on highly proliferative cells or by saving energy as a glucose precursor.
Due to their technological and nutritional properties, Vital and Hydrolysed Wheat Gluten already appear as high value protein sources for fish feeds. Furthermore, new insights on gut morphology, microbiota and health highlight a potential functional role of these proteins on the antioxidative system and on digestive enzyme activity and reveal that wheat proteins do not disturb carnivorous fish microbiota significantly. Further research is needed to confirm these functional benefits and to fully understand the underlying mechanisms.
Read the magazine HERE.
First published in International Aquafeed, March-April 2015
Many fish feed producers now formulate low fishmeal diets. To ensure high growth performance, the use of high quality alternative protein is then required. Both Vital and Hydrolysed Wheat Gluten are high quality proteins. While they don’t exhibit the same behaviour at extrusion, they can both ensure good physical quality of pellets and high level of growth performance with high nitrogen and energy retention.
Additionally, Vital and Hydrolysed Wheat Gluten may have some functional health benefits at gut level, especially by stimulating gut cell proliferation and antioxidative system without damaging gut structure.
Intensive production of farmed fish fed with compound feeds has increased greatly, mainly due to the growth of aquaculture production, but also because it is the most efficient way of production (Olsen and Hasan, 2012).
In such feeds, Fish Meal (FM) used to be the major source of proteins, especially for marine fish and salmonids (Tacon et al., 2011). Nevertheless, because of the limited amount of available FM on the market, its impact on the environment and marine diversity, and its increasing price, its utilisation has been progressively reduced in the formulation of diets.
In order to achieve a low FM incorporation (below 10 percent in formula) without impairing growth performance, active research was conducted on plant proteins (PP), which represent an interesting alternative to FM. In this context, many studies were undertaken to evaluate the effects of replacing FM with different types of PP, tested one by one or in mixture, on fish growth and health. Among the tested PP being considered to replace FM, Wheat Proteins (WP), including Vital Wheat Gluten (VWG) and Hydrolysed Wheat Gluten (HWG) are easily available PP sources that have given very promising results from technological, nutritional and health points of view.
Vital and Hydrolysed Wheat Gluten exhibit different behaviour in extrusion and both proteins allow obtaining pellets of high physical quality
VWG has already been described elsewhere (Apper-Bossard et al., 2013) as an effective binder in fish feed, imparting good mechanical properties (i.e. durability and hardness) to the fish feed pellets as well as good water stability. Moreover, increased VWG inclusion rate in replacement of soy protein concentrate results in decreasing extruder motor load. Indeed, extrusion behaviour of 2 commercial salmon feed diets has been explored both at small scale (Application Centre, Tereos, Marckolsheim, France) and at a fish feed Technology Centre (scale-up; Nofima, Fana, Norway).
One diet contained 10 percent VWG and the other 20 VWG. The two diets were extruded in the same processing conditions. This resulted in a lower motor load (i.e. torque) for 20 percent VWG compared to 10 percent VWG (36 percent versus 41 percent motor load). Higher inclusion of VWG in formulation leads to a decrease in motor load due to the lower water holding capacity of VWG compared to soy protein concentrate (Draganovic et al., 2011).
The technological properties of HWG had not yet been reported in the literature while VWG and HWG have different impacts on extruder system parameters, especially on motor load and pressure at the die. Indeed, replacing 25 percent of Crude Protein (CP) of a FM diet by either VWG or HWG (Voller et al., in preparation) results in lower motor load with HWG (283 and 376 Nm with HWG and VWG respectively).
The higher effect of VWG on the motor load can be attributed to the higher water holding capacity (table 1) of this ingredient (1.5 g of water/g) compared to HWG (0.6 g of water/g). The reduction in motor load can be seen as a direct process advantage allowing reduction of energy consumption during extrusion (Specific Mechanical Energy reduced from 65 to 50 Wh/kg). An alternative is to run the HWG diet at the same motor load as the VWG diet with the potential to increase the extrusion capacity.
Wheat Proteins: Amino acid profile
Wheat proteins are a source of functional amino acids, especially sulphur amino acids and leucine. They contain rather low levels of lysine, tryptophan, and arginine meaning that they should be complemented with these amino acids when used at high level in formulae. Several experiments showed WP can successfully replace a large part of FM when diets are supplemented with free lysine in salmonids (Davies et al., 1997).
WPs contain a relatively high concentration of sulphur-containing amino acids, due to the numerous di-sulphur bonds (1.8 percent CP of methionine and 2.6 percent CP of cysteine), whereas PP sources are generally low in sulphur-containing amino acids. For instance, soybean meal and soy protein concentrate respectively contain 1.4 and 1.3 g/100 g CP of methionine and 1.3 and 1.4 g/100 g CP of cysteine.
Furthermore, WPs are high in leucine, with about 7.9 g/100 g CP. Leucine is considered as the main amino acid triggering muscle protein synthesis and inhibiting proteolysis in mammals (Li et al., 2009) and probably in fish. Indeed, in different species, amino acids regulate the TOR signalling pathway (Seilliez et al., 2008). Furthermore, supplementing media containing 0.6 mM leucine with an additional 2.5 mM leucine reduced rates of protein degradation in rainbow trout primary myocytes by 8 percent (Cleveland, 2010).
WPs are also rich in glutamine: from 35 to 40 percent CP. Glutamine is a major substrate for all rapidly proliferating cells and plays an important role in maintaining intestinal trophicity (Verlhac-Trichet, 2010). In addition, glutamine is one of the most important energy substrates of enterocytes. Free glutamine significantly increases enterocyte and microvilli length in catfish gut (Pohlenz et al., 2012), hybrid striped bass (Cheng et al., 2012), and juvenile hybrid sturgeon (Zhu et al., 2011). Glutamine also constitutes a major substrate for immune cells, thus modulating immune response (Verlhac-Trichet, 2010; Zhu et al., 2011; Cheng et al., 2012).
Moreover, glutamine plays a role in eliminating free radicals as it acts as a precursor for glutathione synthesis (Wu, 1998). Such effects are reported for juvenile hybrid sturgeon (Zhu et al., 2011) and hybrid striped bass (Cheng et al., 2012). Glutamine has proven to stimulate muscle synthesis in terrestrial vertebrates but such results are not available for fish. However, dietary glutamine supplementation increases growth performance in juvenile hybrid sturgeon (Qiyou et al., 2011) and in hybrid striped bass (Cheng et al., 2012).
Effects of wheat proteins on growth performance
Because of its high digestibility and its absence of anti-nutritional factors, replacement of a large proportion of FM with VWG results in similar growth performance and fish composition whatever the species are. The apparent CP digestibility of VWG is high, between 92 and 98.5 percent (Apper-Bossard et al., 2013). In rainbow trout, VWG successfully substitutes more than 50 percent FM providing diets supplemented with lysine without affecting protein and lipid composition of the carcasses (Davies et al., 1997).
Furthermore, the inclusion of 14.5 percent VWG in diets does not adversely affect the flavour of fillets (Skonberg et al., 1998). In Atlantic salmon, the replacement of 35 percent FM with VWG without supplementing by lysine results in similar final body weight and growth (Storebakken et al., 2000). These authors estimate the replacement of FM with VWG without amino acid supplementation can go up to 50 percent based on the amount and the availability of lysine in VWG and on the requirement of fish.
In European sea bass, substituting more than 50 percent FM with VWG does not impair palatability, growth performance, and nitrogen-energy retention (Tibaldi et al., 2003). In gilthead sea bream, the use of 88 percent CP from VWG not only successfully replaced FM but also produced better growth and feed conversion ratio, probably related to higher protein and energy intake of fish (Allan et al., 2000). In Nile tilapia fed with diets differing in their protein sources, the highest growth is reported for VWG, FM, and soybean extract diet. In shrimp, results are scarce but the replacement of up to 20 percent marine protein with VWG does not significantly affect feed efficiency and growth performance (Molina-Povida et al., 2004; Tereos internal data).
HWG also seems promising in aquaculture feeds as it results in high growth performance. CP digestibility of HWG has recently been measured. It is very high and further increases with higher HWG inclusion rate in FM-diet for rainbow trout (figure 1; Apper et al., 2014). Recent studies on juvenile hybrid sturgeon show that the replacement of 1 to 5 percent of soy protein concentrate by 1 to 5 percent of HWG in a diet containing animal and plant proteins significantly increases growth performance (Qiyou, 2011).
The use of 12.5 to 50 percent CP of HWG to replace high quality FM does not modify growth performance and feed efficiency in rainbow trout (Apper et al., 2014). In the same experiment, authors compared energy and nitrogen retention obtained with either VWG or HWG. Nitrogen retention was similar for the 2 protein sources, with very low metabolic losses of nitrogen (35.1 and 43.4 kg/ton of fish produced for HWG and VWG respectively). Energy retention was higher with HWG than with VWG.
Effects of wheat proteins on gut health and microbiota
Compared to a fishmeal-based diet, the use of Wheat Proteins does not damage gut morphology and microbiota. In Rainbow trout, the replacement of up to 50 percent high quality FM by either VWG or HWG results in no modification of gut structure (figures 2 and 3; Apper et al., 2014). Indeed, no areas of necrosis were observed in enterocytes, all appearing uniform, regular, and healthy. Microvilli observations confirmed such results, all microvilli being packed and showing the same density. Similarly, in the same study, microbiota was not significantly different between FM, HWG and VWG diets, with a predominance of Firmicutes.
Richness, OTUs, and diversity of microbiota were not different across treatments, suggesting that replacing up to 50 percent of high quality FM by WP is without consequence for gut health. Such results are typical of wheat proteins as soy-proteins or pea proteins have been demonstrated to impair gut morphology or microbiota at high inclusion rates (Mc Kellep Bakke et al., 2007; Penn et al., 2011).
In soy-based diets, the inclusion of hydrolysed wheat proteins modulates gut function and morphology, the anti-oxidative system, and the non-specific immune system. The replacement from 1 to 5 percent of soy protein concentrate in a diet based on 20 percent FM, 20 percent soybean meal, eight percent corn gluten meal and 10 percent blood meal increased digestive enzyme activities and fold heights, modulated non-specific immune response and stimulated anti-oxidative status (Qiyou et al., 2011; Zhu et al., 2011).
Interestingly, in these studies, the results of 3 percent of HWG inclusion were equivalent to results obtained when authors added one percent free glutamine in the soy protein concentrate diet. Such results suggest that HWG may have a bioactive role, by acting on highly proliferative cells or by saving energy as a glucose precursor.
Due to their technological and nutritional properties, Vital and Hydrolysed Wheat Gluten already appear as high value protein sources for fish feeds. Furthermore, new insights on gut morphology, microbiota and health highlight a potential functional role of these proteins on the antioxidative system and on digestive enzyme activity and reveal that wheat proteins do not disturb carnivorous fish microbiota significantly. Further research is needed to confirm these functional benefits and to fully understand the underlying mechanisms.
Read the magazine HERE.
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