NSPASES: CAN THOSE EXOGENOUS ENZYMES REALLY CONSTITUTE NATURAL GROWTH COFACTORS IN BROILER CHICKENS?

In the quest to improve public and animal health, white meat is considered to be one of the major causes of antibioresistance identified in medicine, through uncontrolled use of antibiotics in poultry farming as growth promoters (GPA). Thus, this use needs to be reconsidered at large scale. This reconsideration involves the substitution of GPAs by natural alternatives, particularly enzymes degrading non-starch polysaccharides (NSPases) which will allow:1) modulation of the intestinal microbiota, to attenuate the anti-nutritional effects of insoluble NSPs, 2)reduction of the non-digested portion of the substrate and 3) improvement of the zootechnical performance of the chicken. Non-starch polysaccharides contain two main families, namely: water-insoluble NSPs including cellulose and partially water-soluble NSPs. However, despite the fact that these components constitute the major part of cereal dietary fiber, they have an anti-nutritional effect associated with the viscous nature of these polysaccharides and their interaction with the intestinal microflora due to the fact that poultry does not produce enough endogenous enzymes to hydrolyze NSPs. The use of NSPases produced mainly by fungi and bacteria allows counterbalancing the anti-nutritional properties of dietary fibers by increasing the digestibility of starch and improving the zootechnical performance of broilers, particularly the conversion index.Thus, this literature review aims to shed light on the effects of NSPases on the zootechnical parameters of chickens, the intestinal microflora as well as on nutritional digestibility in order to use them as alternatives to GPAs and limit the aggravation of the phenomenon of antibioresistance. This approach is therefore, part of the world famous concept of one world one health and which applies to the design and implementation of programs, policies, legislations and research for which several sectors communicate and collaborate to improve public health outcomes.

In the quest to improve public and animal health, white meat is considered to be one of the major causes of antibioresistance identified in medicine, through uncontrolled use of antibiotics in poultry farming as growth promoters (GPA). Thus, this use needs to be reconsidered at large scale. This reconsideration involves the substitution of GPAs by natural alternatives, particularly enzymes degrading non-starch polysaccharides (NSPases) which will allow:1) modulation of the intestinal microbiota, to attenuate the anti-nutritional effects of insoluble NSPs, 2)reduction of the non-digested portion of the substrate and 3) improvement of the zootechnical performance of the chicken. Non-starch polysaccharides contain two main families, namely: waterinsoluble NSPs including cellulose and partially water-soluble NSPs. However, despite the fact that these components constitute the major part of cereal dietary fiber, they have an anti-nutritional effect associated with the viscous nature of these polysaccharides and their interaction with the intestinal microflora due to the fact that poultry does not produce enough endogenous enzymes to hydrolyze NSPs. The use of NSPases produced mainly by fungi and bacteria allows counterbalancing the anti-nutritional properties of dietary fibers by increasing the digestibility of starch and improving the zootechnical performance of broilers, particularly the conversion index.Thus, this literature review aims to shed light on the effects of NSPases on the zootechnical parameters of chickens, the intestinal microflora as well as on nutritional digestibility in order to use them as alternatives to GPAs and limit the aggravation of the phenomenon of antibioresistance. This approach is therefore, part of the world famous concept of "one world one health" and which applies to the design and implementation of programs, policies, legislations and research for which several sectors communicate and collaborate to improve public health outcomes.
Most recent poultry feed formulations are based on cereals, cereal meal by-products, and oilseeds, so they contain a significant amount of NSPs. These are classified according to several factors (Ayoola et al., 2014).

Classification Of NSPs
The term non-starch polysaccharides (NSPs) covers a wide variety of polysaccharide molecules except α-glucans (starch). The classification of NSPs was originally based on their methodology of extraction and isolation. The remaining residue after series of alkaline extractions of cell wall materials is referred to as cellulose and the fraction of this residue solubilized by alkali is referred to as hemicellulose (Choct, 1997). Thus, NSPs are seriated according to their water solubility and on the basis of their linkage.

Commonly known NSPs
Although commonly referred to as NSPs, plant cell wall polysaccharides include a wide range of chemically distinct compounds in different combinations and proportions that are characteristic and variable among different plant species. Some of the common NSPs present in plant cell wall are classified as cellulosic, hemicellulosic, pectic, and/or galactosidic substances (Bailey 1973;Choct, 1997 Moreover, the non-amylaceous polysaccharide content of food ingredients varies among products (Table 1). From 10%to 30% of cereals are NSP-based, the majority of which are composed of arabinoxylans, cellulose and βglucans. Nevertheless, cereal grains can be classified into two groups: 1. Viscous cereals: rye, wheat, barley, triticale and oats and 2. Non-viscous cereals: corn, sorghum, rice and millet.
This classification is based primarily on the amount of soluble NSPs present in the grain (Table 1) (Choct, 1997;Choct, 2015;Englyst, 1989; Graham and Aman, 2014).   NSPs increase viscosity also by directly interfering with water molecules allowing the formation of a "cage" type structure that traps nutrients. At high concentrations, the NSP molecules interact with each other and become further entangled in a network consecutively increasing the viscosity of the digest (Simon, 1998;Mudgil and Barak, 2013 . Indeed, supplementation with 15 g/kg of wheat arabinoxylan decreased the apparent average amino acid digestibility by 17% and increased the average of endogenous amino acid loss by 23.5 g/kg DM intake (Angkanaporn et al., 1994). Similarly, the loss of lysine and methionine in a low-fiber diet (Raw Fiber = 30 g/kg) was 0.4 and 0.17 g/kg, respectively, and 0.59 and 0.19 g/kg, respectively, in a high-fiber diet (FC = 80 g/kg) (Kluth and Rodehutscord, 2009). These proprieties of NSPsreduce digestion and nutrient absorption efficiencies.

Anti-Nutritional Properties Of Partially Water-Soluble NSPs
Furthermore, the degree of solubility is directly related to that of branching and polymerization of the nonamylaceous polysaccharide, in that, the greater the branching is, the higher the viscosity and degree of water retention are (Sethy et al., 2015). In addition, soluble NSP generates sticky fecal material that increases moisture content of the litter and therefore promotes the development of pathogens in the chicken environment (Bach Knudsen, 2014). These conditions are so far favorable for the development of plantar pododermatitis causing intense pain in the musculoskeletal system reducing the individual"s movements for feed intakewhich ends into decreased 455 growth (Meluzzi et al., 2008). In addition to the viscosity, anNSP rich diet generates a high bacterial translocation to the portal system, causing systemic infections that can lead to death in the most severe cases (Latorre et al., 2015).

Gut microflora:
The microflora is a key link influencing the health status of poultry, especially intestinal development, digestion, nutrients" absorption However, there is no doubt that a high level of ileal viscosity slowing down the gastric passage rate and leading to impaired digestion, has a negative effect on the gut microbiota as undigested nutrients become a fertile ground for pathogen proliferation (Bedford and Cowieson, 2012). Indeed, the overgrowth of certain anaerobic organisms can lead to toxin production and the deconjugation of bile salts essential for fat digestion (Choct, 1997). In addition, when digestion is compromised, the flow of unabsorbed nutrients into the cecum increases dramatically (Choct et al., 1996)  The anti-nutrient effects of partially soluble NSPs depend on the polymeric nature of the considered NSPs, once the polymers are cleaved into smaller fragments, their activity is amply limited. Therefore, it is appropriate to ensure that these long-branched chains of non-starch polysaccharides are cleaved to better take advantage of their nutritional value (Singh and Kim, 2021). This denaturation is achieved through exogenous enzymes that help to amend the zootechnical performance of a healthy individual through improving digestibility or stabilizing the gut flora or maintaining a healthy environment for growth (Pirgozliev et al., 2019).

Definition Of Exogenous Enzymes
Enzymes are biological catalysts that activate reactions and act on specific substrates or reactants. Their effectiveness in poultry feeding depends on some criteria based on their mechanism of action (Bedford and Schulze, 1998).
Exogenous enzymes facilitate specific chemical reactions and target specific substrates. These . The majority of diets contain a variety of NSPs; thus, the most effective means is supplementation with an enzyme cocktail that varies in specificity and mode of action. This approach has the advantage of increasing starch digestibility and improving broiler performance, especially feed conversion ratio (Meng and Slominski, 2005). Nevertheless, each substrate requires different enzymes and the enzymes required afterwards depend on the final objective namely: viscosity reduction or pre-biotic generation (Langfelder, 2014).

Effects Of Nspase Enzymes
Poultry is not fully capable of digesting fiber in plant-based feeds since this species does not produce enough digestive enzymes (Boros et al.,2004),supporting the major interest of using exogenous enzymes that increase fiber digestibility (Classen, 2014).  ., 2019). Similarly, the inclusion of NSPases, namely: α-galactosidase and xynalase significantly improved the total ileal digestibility of amino acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, and tryptophan) by 3.8% compared to the unsupplemented group (Jasek et al., 2018). Also, supplementation with xylanase improves starch digestibility in the jejunum and ileum by 2.4%, leading to increased energy in broilers (Stefanello, 2015). An inclusion of this enzyme in the diet of laying hens reduces their intestinal viscosity allowing a better benefit from nutrients (Bederska-Łojewska et al., 2019).

On nutritional digestibility
Indeed, digestive viscosity was reduced with dietary supplementation with a mixture of 2500IU of xylanase and 250IU of β-glucanase per kilogram of feed as well as in jejunal digest viscosity to a greater extent in wheat-based diets (-31%) than in maize-based diets (-10%) compared to the group without enzymes. Starch digestibility was also higher in the supplemented diet group (3.5%) (Munyaka et al., 2015). In addition, supplementation of carbohydrase 457 in vitro, namely: xylanase and arabinofuranosidase increased digestibility of dry matter and solubilized arabinoxylan, in particular,from 6% to 41% (Vangsøe et al. 2020). Certainly, supplementation with NSPases improves total digestibility coefficient values by 9 to 11 units in broilers compared to both positive and negative controls (Maharjan et al., 2019).

On growth performance
In an attempt to reduce the anti-nutritive effects of NSPs, exogenous enzyme supplementation in poultry has been shown to be effective, especially in high fiber diets. This eternal quest concerns both nutritional digestibility and growth parameters of broilers.
Indeed, the administration of β-mannanase and NSPases (carbohydrate cocktail: xylanase, β-glucanase and αgalactosidase) at 363.2 g/t of feed (159.5 × 10 3 IU/g product) and 113.5 g/t feed (2700 IU/g product), respectively, in an energy-reduced diet (88 or 132 kcal/kg of apparent metabolizable energy (AME) depending on the growth stage) improves zootechnical performance and reduces broiler mortality to levels similar to those of the positive control (PC) (Williams et al., 2014).
In a similar context, supplementation of β-mannanase in an energy-reduced diet of 132 kcal/kg MEA in broilers reduces conversion index and increased body weight of individuals compared to CP while maintaining similar weight (Klein et al., 2015). Use of a 0.01% mixture of NSPases (cellulase, glucanase, and xylanase) in broilers significantly optimizes weight gain by 3.04% (Horvatovic et al., 2015).
Xylanase supplementation also appears to improve average daily gain in 20-25-day old broilers by 2.5 g/day and to decrease the conversion index by 6 points compared to the negative control (Singh and Kim, 2021). The same enzyme in combination with protease at 0.25g and 0.20g of a commercial product per kg of feed respectively, improved the conversion index as well as the body weight gain of broiler (Barekatain et al., 2012). Xynalase had as well a positive effect on zootechnical parameters as a whole as well as on apparent metabolizable energy in the finishing phase and this at 2000 IU/kg feed (Perón et al., 2012). Similarly, the incorporation of a commercial product at a rate of 500g/t of feed containing a cocktail of NSPases namely α-galactosidase, β-mannanase, protease, amylase, β-glucanase, xylanase and cellulose significantly improves the conversion index compared to the negative control (Bilal et al., 2016). Also, enzyme B supplementation (with 1500 IU/g of α-amylase and 300 IU/g of amylopectase) as well as administration in another group of individuals of enzyme C(with 1500 IU/g of α-amylase, 300 IU/g amylopectase and 10,000 IU/G protease) reduced body weight gain to a higher level than that displayed by broilers supplemented with enzyme A (with 1,500 IU/g α-amylase) or enzyme D (with 1,500 IU/g α-amylase, 300 IU/g amylopectase, 10,000 IU/G protease and 15,000 IU/g xylanase) (Yin et al. , 2018).In laying hens, incorporation of NSP-degrading enzymes effectively improves growth performance including egg size in a diet fixing linoleic acid level in small egg layers by 2% and 1.5% in large egg strains compared to negative control (Elliot, 2012).

On the gut microflora
The gastrointestinal tract hosts a variety of microbiota that play a critical role in the overall well-being of the chicken, in general, and its digestive health, in particular (Kiarie et al., 2013). In broilers, 16S rDNA gene sequence analysis revealed thirteen, eleven, fourteen, twelve, nine, and fifty-one operational taxonomic units in the proventriculus, gizzard, duodenum, jejunum, ileum, and cecum, respectively (Jong et al., 2007). The diversity and abundance of the gut microbiota is affected by the composition of the diet as well as its digestibility ( , C (with 1500IU/g of α-amylase, 300IU/g of amylopectase and 10000 IU/g of protease), D (with 1500IU/g of α-amylase, 300IU/g of amylopectase, 10000 IU/g of protease and 15000 IU/g of xylanase) and E (with 458 1500 IU/g of α-amylase, 300 IU/g of amylopectase, 10000 IU/g of protease, 15000 IU/g of xylanase + 200 IU/g of cellulase + 1000 IU/g of pectinase) respectively (Yin et al. , 2018).
In addition to their effects on pathogens, NSPases have an extensively researched pre-biotic effect. Indeed, the cleavage of non-starch polysaccharides by exogenous enzymes, including NSPases, allows the release of oligosaccharides into the gastrointestinal tract. When these compounds reach the cecum, they, selectively, stimulate growth and activity of intestinal bacteria such as Bifidobacterium and Lactobacillus (Thammarutwasik et al., 2009) to act as pre-biotics in the poultry gut (Masey O'Neill et al., 2014b;Choct, 2015). Xylanase, for example, does not produce pre-biotics certainly but rather a signaling molecule stimulating bacterial species that can degrade xylan to produce xylanases (Bedford, 2018). NSPase supplementation thus, influences ileal and cecal microbiota (Gonzalez-Ortiz et al., 2016) and results in increased cecal fermentation (Masey O'Neill et al., 2014a).

Economic Coating ofNspase
Current broiler strains are characterized by rapid growth and high feed conversion rates. To utilize this promising potential, it is imperative to adopt a balanced nutrition that allows expressing their full genetic potential (Kubis et al., 2020). However, the cost of grain feedstock is a critical component of production costs in poultry production (Zentek and Boroojeni, 2020). Indeed, feed costs for chickens account for up to 80% of total production costs (Zhang et al., 2020). Therefore, in order to maintain production profitability while ensuring product quality, it is crucial to improve utilization of nutrients contained in feeds through feed additives. (Kubis et al., 2020). The majority of feed costs (95%) is for energy and protein requirements, about 3 -4% for minerals and vitamins and about 1 -2% only is spent for various food additives (Zentek and Boroojeni, 2020).
In addition to the beneficial effects of NSPases, these enzymes save energy in feed formulations in poultry, contributing to reduce the AME content of corn-based diets by up to 100 kcal/kg, therefore saving about 7.00 USD per ton of feed (Gomes, 2016). Hence, a cocktail of xylanase, β-glucanase and phytase saved 0.045 USD per kilogram of weight gain even with very poor quality feed material (Olnood and Liu, 2012).
As discussed above, the effects of NSPasesseem to be multidirectional. They limit the increase in digestive viscosity by hydrolyzing soluble polysaccharides, enhance the proliferation of beneficial microflora by providing substrates for fermentation, increase the availability of nutrients, thus improving the nutritional value of NSP-rich grains and ultimately increasing poultry performance while reducing production cost.

Conclusion:-
Use of such exogenous enzymes is therefore an effective method to eliminate the nutrient encapsulation effect of plant cell walls, generate oligomers, support the gut microbiota, and limit the use of GPAs to maintain gut health in poultry (Singh and Kim, 2021). With the existing challenges of the anti-nutritional effect of non-amylaceous polysaccharides, further investigations are required to explore the possibilities of improving the utilization of these products to, thereby, limit extensive use of growth promoting antibiotics.