Exogenous Fibrolytic Enzymes in Ruminants

Introduction

Enzymes are proteins that catalyze chemical reactions in biological systems. Exogenousmeansan action or object coming from outside a system so exogenous fibrolytic enzymes are fibre degrading enzymes, supplemented to the animal from exogenous sources.

The ruminant production systems are dependent worldwide on forage as the main nutritional components. The microbial mode of digestion allows ruminants to better unlock the unavailable energy in the plant cell wall components than other herbivores. This gives ruminant animals the ability to convert low nutritive and resistant lignocellulosic biomass to milk, meat, wool and hides. However, most forage plants are high in cell walls and low in nitrogen (N) and energy content. Despite the importance of fibrous components in forages for salivation, rumen buffering and efficient production of ruminal end products, only 10 to 35% of energy intake is available as net energy. This is because the ruminal digestion of plant cell walls is not complete. The use of exogenous fibrolytic enzymes (EFE) to enhance quality and digestibility of fibrous forage is on the verge of delivering practical benefits to ruminant production systems. In this regard, cellulases and xylanases are respectively amongst the two major enzyme groups that are specified to break ß1-4 linkages joining sugar molecules of cellulose and xylans found in plant cell wall components.

Although enzyme products marketed for livestock number in the hundreds, they are derived primarily from only four bacterial (Bacillus subtilis, Lactobacillus acidophilus, L. plantarum, and  Streptococcus faecium, spp.) and three fungal (Aspergillus oryzae, Trichoderma reesei, and Saccharomyces cerevisiae) species.

Mode of action of exogenous fibrolytic enzymes.

In nature, complete cellulose hydrolysis is mediated by a combination of three main types of celulases: (1) endoglucanases , (2) exoglucanases, including cellobiohydrolases (CBHs), and (3) β -glucosidase (BG) . Endogucanase are active on the non- crystalline or amorphous region of cellulose and their activity yields cellobiose and cello-oligosaccharides as hydrolytic product. Cellobiohydrolases are processive enzymes that are active on the crystalline region of cellulose and their activity yield almost exclusively cellobiose as their main hydrolysis product. In turn beta- glucosidase convert cellobiose and some cello- oligosaccharides to glucose (fig. 1).

Action of xylanase

Figure 2 Action of Xylanase

Xylanase is the name given to the class of enzymes which degrade the linear polysaccharides beta-1,4, xylan to xylose thus, breaking down hemicellulose, one of major components of plant cell wall.

Pre-ingestive Effects

Exogenous enzymes are most effective when applied to feed prior to ingestion by cattle.  The adsorption of enzyme onto the substrate is an important prerequisite for hydrolysis. Applying exogenous enzymes directly to feed causes a release of reducing sugars and in some cases, partial solubilization of neutral detergent fibre and acid detergent fibre. This release of soluble sugars would provide sufficient additional available carbohydrates to encourage rapid microbial growth, shortening the lag time required for microbial colonization. However, the quantity of sugars liberated represents only a minute portion of the total carbohydrate present in the diet.  There is evidence that applying fibrolytic enzymes to feed prior to feeding alters the structure of the feed, thereby making it more amenable to degradation. Another important reason for applying enzymes to feed prior to ingestion is to enhance binding of the enzyme to the feed, thereby increasing the resistance of the enzymes to proteolysis in the rumen. Enzymes applied to feed prior to ingestion are particularly stable; the presence of substrate is known to increase enzyme resistance to proteolytic inactivation.

Ruminal Effects

Previously it was assumed that the proteolytic activity in the rumen ecosystem would rapidly inactivate unprotected enzyme feed additives. However, more recent studies have shown that exogenous enzymes in the rumen are generally more stable than previously thought, particularly when added to feed pre-ingestion. The stability in the rumen may be related to their glycosylation, which may protect them from inactivation from temperature and proteases. However, there can be differences depending on the source organism, the enzyme activity, and whether the enzyme product is applied to feed.

Exogenous enzymes improve digestion by increasing the hydrolytic capacity within the rumen environment. This hypothesis is difficult to verify because of the complexity of measuring the enzyme activity within the rumen.  The actual increase in hydrolytic capacity in the rumen due to exogenous enzymes may be greater than calculated from microbial and exogenous enzyme activity due to synergism.

Synergy between exogenous enzymes and rumen microbial enzymes can be defined as the enhanced effect of these two entities acting cooperatively. The net effect is the increase in enzymic activity that exceeds the additive effects of each of the individual components. Synergy among cellulases and xylanases has been extensively documented by enzymologists. Synergy among microorganisms in the rumen, including crossfeeding of hydrolysis products, utilization of end products, and production of an essential nutrient, is well documented.

Conditions in the rumen of intensively reared ruminants are often suboptimal for growth of fibrolytic bacteria. Typically,diets fed to dairy cows and feedlot cattle  are highly fermentable causing ruminal pH to remain below optimum (approximately pH 5.9) for fibre digestion for extended periods each day. However, some exogenous enzymes commonly employed as additives are optimally active at lower pH than ruminal enzymes. Thus, it has been speculated that their addition might be of greater benefit when pH conditions are suboptimal for fibrolytic ruminal bacteria. There is some evidence that exogenous enzymes stimulate the attachment of rumen microbes to plant fibre, which may partially explain how small quantities of enzymes can improve rate of fibre degradation.

Attachment of rumen microorganisms to some components of forage fibre, such as mesophyll and phloem tissue, is essential for the digestion of feedstuffs in the rumen. Adherence of rumen bacteria to fibre places the enzyme-system in close proximity to the substrate, and disrupts the hydrogen bonding within the cellulose matrix. The mechanism by which fibrolytic enzymes stimulate the attachment of rumen bacteria to plant cell walls remains unknown. It is possible that applying enzymes to feed causes the release of soluble sugars from feed, thereby increasing the chemotactic attraction and eventual attachment of fibrolytic rumen bacteria to the plant surface. The application of enzymes to feed may also increase attachment by increasing the “roughness” of the feed surface making it more suitable for microbial colonization. Feed enzymes may weaken the surface of the feed thereby removing some of the physical barriers that impede microbial attachment. There is also some evidence that adding feed enzymes to the diet indirectly increases numbers of non-fibrolytic, as well as fibrolytic, bacteria in the rumen.

Post ruminal effect

Some exogenous enzymes survive ruminal fermentation and the abomasal environment and may exert activity for a period of time in the small intestine. In general, xylanases are more stable in the rumen and abomasum than cellulases and consequently xylanase activity in the small intestine that is attributable to exogenous enzymes is usually greater than cellulase activity. Approximately 30% of xylanases can escape ruminal fermentation and are active in intestinal digesta of ruminants. Some exogenous enzymes are remarkably resistant to microbial proteases, bypass the abomasums, and remain active in the small intestine and have even been shown to linearly increase polysaccharide-degrading activities in feces. However, the practical implication of these effects remains unclear.

Production response of dairy cattle to exogenous fibrolytic enzymes

Chandra et al. (2010)  evaluated the effect of commercial exogenous fibrolytic enzyme (EFE) mixture added at 1.5 and 3.0 g (cellulase 4,000 µM glucose/g/h + xylanase 7,990 µM xylose/g/min; 50:50 w/w) per kilogram of dry matter (DM) of feed on nutrient digestibility, milk production, milk composition, and some blood constituents in lactating Murrah buffaloes. Eighteen buffaloes were allotted to three dietary treatments, on the basis of milk yield (8.48, 8.52, and 8.53 kg/day) and days in lactation (68.5, 80.33, and 82.00) for 90 days. The buffaloes were fed a total mixed ration (TMR) comprising of 45% chaffed wheat straw, 15% chopped green maize, and 40% concentrate on DM basis (control group), the same TMR plus EFE at 1.5 g/kg DM (T-1 group) and the same TMR plus EFE at 3.0 g/kg DM (T-2 group) supplemented through the concentrate mixture. There was no effect of fortifying EFE mixture on DM intake and crude protein intake (grams per day) whereas total digestible nutrients intake (kilogram per day) was higher by (P < 0.05) 12.53% in T-1 group over that of control, and there was no significant difference between T-2 and control groups. The average daily milk yield and 4% fat-corrected milk (FCM) yield was higher (P < 0.05) by 12.99% and 15.17% in T-1 group as over that of control, and there was no difference between T-2 and control groups. It is concluded that supplementation of cellulase and xylanase mixture at 1.5 g/kg of DM of TMR containing wheat straw (45%), green maize (15%), and concentrate (40%) on DM basis significantly increased (P < 0.05) the average daily milk yield and FCM yield in Murrah buffaloes due to improved dietary fiber digestion.

Table 1.  Summary of exogenous fibrolytic enzymes effects on production traits and total tract apparent digestibility of nutrients in lactating dairy cows

Reference	Enzyme	Appl. level	Forage level	DMI	Milk yield	Milk components	Total tract dig.
Yang et al., 1999	Cellulase xylanase	0.5- 1g/kg	55%	–		–	↑OM and ADF
Kung et al., 2002	Cellulase Xylanase	10L/t fresh forage	50%	–		MF	NR
Bernard et al., 2010	cellulase	4g/cow per day	50-54%	–	–	–	NR
Arriola et al., 2011	Cellulase xylanase	3.4mg/g TMR DM	52-67%	–	–	–	↑All

Conclusions

• Exogenous fibrolytic enzymese to ruminant diets improve production and digestibility of nutrients in ruminants.
• Limited understanding of factors affecting enzyme activity in feeds give inconsistent results.