USE OF EXOGENOUS FIBROLYTIC ENZYMES ( EFE) IN RUMINANTS

Supplementation of exogenous enzymes with ruminant diets shows beneficial effects on feed utilization, growth and production performance in ruminant animals.

What is an enzyme?———

An enzyme is a naturally occurring protein that catalyzes chemical reactions in biological systems. Enzymes promote the breakdown of complex feed molecules into smaller chemical fractions such as glucose or amino acids that are digestible by the ruminant animal. As an example, the enzyme cellulase initiates the breakdown of cellulose (fiber) into sugars.

There is a major difference between the monogastric animals and the ruminants with respect to mode of digestion, metabolic pathways of the absorbed nutrients and the end products of the metabolism. Cellulose, hemi-cellulose, starch, fructans and pectin form the major source of carbohydrates in ruminants. Cellulose is practically indigestible by the ordinary digestive enzymes produced by the GI tract. Hence this is broken down by the micro-organisms. The end products of carbohydrate digestion in the ruminants include acetic acid, butyric acid and propionic acid. Along with these, some branched chain fatty acids like formic acid, valeric acid, iso valeric acid, isobutyric acid, 2-methyl butyric acid and caproic acid are produced in the rumen from the branched chain amino acids via oxidative deamination and oxidative decarboxylation. The primary difference between ruminants and non-ruminants is that ruminants’ stomachs have four compartments: the rumen, reticulum, omasum, and abomasum. In the first two chambers, the food is mixed with saliva and separates into layers of solid and liquid material. Solids clump together to form the cud or bolus.

Animal feed is the largest cost item in livestock and poultry production, accounting for 60-70% of total expenses. To save on costs, many producers supplement feed with enzyme additives, which enable them to produce more milk/meat per animal or to produce the same amount of milk/meat cheaper and faster.

Found in all living cells, enzymes catalyze chemical processes that convert nutrients into energy and new tissue. They do this by binding to substrates in the feed and breaking them down into smaller compounds. Enzymes can be classified by the types of substrates they work on. For instance: proteases break down proteins into amino acids, carbohydrases split carbohydrates into simple sugars, and lipases take apart lipids into fatty acids and glycerol.

Commercially-available enzymes can be derived from plants and animals (e.g., actinidin from kiwi and rennet from calf stomachs) as well as microorganisms (e.g., amylase from Bacillus and lactase from Aspergillus). Aside from agriculture, other industries that utilize enzymes include the brewing, dairy, paper, biofuel, and rubber industries.

Use of exogenous enzymes in monogastric animal diets is not a novel trend though the enzyme supplementation in ruminant diets is yet to be established in practical application

TYPES, SOURCES AND EXTRACTION OF ENZYMES——–

Exogenous enzymes which use in ruminant nutrition can be characterized into three main categories as fibrolytic, amylolytic and proteolytic enzymes. In additional to major categories of enzymes, phytase which is extensively used in monogastric feeding is also becoming popular in ruminant feeding. Supplementing fibrolytic enzymes with ruminant diets has been the research interest in many studies as digestion of fiber fraction in ruminant’s digestive system only reaches to the 65-70% even under ideal conditions. Exogenous fibrolytic enzymes can be classified further based on their specific activity as cellulase, which hydrolyze the fiber of plant cell wall to glucose, cellobiose or cellooligosaccharides with combined activity of three enzymes namely endoglucanase, exoglucanases and β-glucosidase .Xylanase, that catalyzes the hydrolysis of 1,4-beta-D-xylosidic linkages in xylans that are constituents of hemicellulose, a structural component of plant cell walls. Xylanolytic enzyme group includes endo-β-1,4-xylanase (1,4-β-D xylan xylanohydrolase, EC 3.2.1.8), which attack the main chain of xylans and β-D-xylosidase (1,4-β-xylan xylanohydrolase, EC 3.2.1.37), which hydrolyze xylooligosaccharides into D-xylose, in addition to a variety of debranching enzymes that is, α-L-arabino-furanosidases, α-glucuronidases and acetyl esterases .

Enzyme products are derived primarily from four bacterial (Bacillus subtilis, Lactobacillus acidophilus, L. plantarum and Streptococcus faecium, spp.), three fungal (Aspergillus oryzae, Trichoderma reesei and Saccharomyces cerevisiae) species and some yeasts .

Bacteria, yeasts and filamentous fungi have been identified as suitable candidates to produce xylanases. Some of the most important xylanolytic enzyme producers include Aspergillus, Trichoderma, Streptomyces, Phanerochaetes, Chytridiomycetes,Ruminococcus, Fibrobacteres, Clostridia and Bacillus (Cai et al., 2004) (Table 1). Kuhad et al. (2006) reported extraction of xylanase producing bacteria from soil while significant amount of xylanase amount was extracted from a new isolate of Bacillus pumilus .In a very recent study new xylanase producing Gram positive bacteria has isolated from termite gut .

Aspergillus oryzae is the major microorganism used to extract amylase enzyme

APPLICATION OF ENZYMES——

There are several enzyme application methods widely used but the most effective method is yet to be recognized. The application methods vary from a pretreatment of the feed for a period of time before feeding (e.g., silage making, forage harvesting) to application at the time of feeding (application to the hay, in Totally Mixed Rations (TMR), concentrate), even the direct application to the rumen. As enzyme activity strictly depends on the type of feed the enzyme-feed specificity should be given a special consideration when selecting an appropriate method .

The proposed theory behind the positive effects with pre-treatment of enzyme is the enhancement of the binding of enzyme with the feed substrate, which may increase the resistance of the enzymes to proteolysis and prolong their residence time within the rumen.

The method of enzyme application appears to be an important concern in order to obtain positive responses from animal, however further research on this area is strongly suggested.

How an enzyme acts on a substrate———

When an enzyme comes in to contact with a substrate, an enzyme–substrate complex is formed which is the intermediate formed when a substrate molecule interacts with the active site of an enzyme. Following the formation of an enzyme–substrate complex, the substrate molecule undergoes a chemical reaction and is converted into a new product. Various mechanisms for the formation of enzyme–substrate complexes have been suggested, including the induced fit model and the lock and key mechanism

Mode of action of EFE-——–

The EFE are most effective when applied in liquid form rather than in solid form prior to ingestion. This EFE weakens cell wall barriers that hinder microbial digestion in the rumen. EFE releases reducing sugars when added to the forages which arise from the partial solubilization of NDF and ADF even before feeding to the animals. After entering the rumen the feed particle releases more available carbohydrates and helps in enhancing the rapid microbial attachment .Pre-treatment of dry feeds with enzyme applied in liquid form creates a stable feed-enzyme complex even before entering the rumen similar to the findings reported by (Beauchemin et al., 2004). It was postulated that the activity of the EFE is more in the rumen as compared to outside and the reasons are attributed to that the outside temperature, conditions of p H and the contact substrate are not conducive for enzyme activity whereas this activity is more pronounced inside the rumen due to the following factors – there exists a synergism between various enzymes that are produced in the rumen with EFE and there is a possibility for more EFE activity in the rumen. Yang et al., (2011) reported higher apparent DM digestibility during in vitro studies with alfalfa hay when a combination of two enzymatic products were added. However the enzymes’ actions differ from source to source of their supply. We know that the fibrolytic enzymes like those released from fungal origin (Trichoderma longibrachiatum, Asperigillus niger, Asperigillus oryzae) and those released from bacterial origin (Bacillus sps., Penicillium funiculosum) have cellulolytic and hemi cellulolytic enzyme activity The factor of higher dosage of enzyme has shown significant effect in improving the performance since the feed/substrate attachment sites for enzymes and in turn rumen microbes have increased proportionately.

Method of enzyme supplementation——-

Methods of application can be either by spraying/top dressing or by direct infusion into the rumen. Application of EFE can be either in solid form or in a soluble form and there are many reports saying that the applying enzymes in a liquid form have a positive effect on animal performance (Kung Jr. et al., 2013; Wang et al., 2012). On the other hand direct infusion of soluble enzyme mix into the rumen (Sutton et al., 2001) was not effective. It was also reported that the incubation of the enzymes for a considerable time with the feeds before feeding to the animals improved performance. There are other possible explanations for an improved performance for the EFE added diets especially EFE applied in liquid form since formation of stable enzyme-feed complex is less prone for enzyme inactivation due to the proteolytic process in the rumen (van de Vyver and Useni, 2012; Suryanarayana and Ramana, 2016). The improved performance for solubulized enzyme supplemented groups may be due to the fact that pretreatment of feed /substrate with enzyme caused a scarring of the fibre particles and this effect causes easier attachment of rumen fibrolytic bacteria

Factors on which the enzymes activity depends———

The activity of the EFE on a feed depends on various factors like source of supply of the enzymes (bacterial/fungal), type of substrate, mode of application of EFE (solid/liquid), type and stability of the enzyme, p H, temperature and the other conditions in the rumen, dose of enzymes, presence of certain enzyme inhibitors in the gut, stability conditions of EFE and the type of livestock species. Marquez et al., (2007) has stated that the activities of xylanase and cellulolytic produced from Trametes species is 5 and 7 times and 10 and 8 more active as compared to these produced from Asperigillus and Pleurotus species, respectively. The use of EFE can be promoted in ruminant production systems by developing these at cheaper rates. In future it becomes inevitable to use these enzymes in TMR. A thorough knowledge on the potentially degradable fibre fractions and the digestion kinetics need to be known. Addition of EFE to the fibre diets may not be a tough task but this small intervention leads to the efficient utilization of nutrients and a healthy microbial balance in the rumen. However newer fibrolytic enzymes need to be prepared that can be thermo stable and can resist a p H of 6 to 6.8 and a temperature of 390 C simulating the rumen conditions. While supplementing these enzymes factors like mode of application, substrate nature, dosage, period of incubation, source of supply, etc., have to consider.

Enzymes are specialized proteins that perform specific functions and make life possible. Enzymes assist or improve chemical reactions (catalyze) that result in the breakdown of organic compounds into substances animals, and microbes, can use as nutrient sources.

Enzymes are very specific in the reactions they catalyze, like a key is specific to the lock it will unlock.

Many of the enzymes needed to break down feedstuffs are produced naturally by microbes in the digestive tract of the animal. However, there are often limitations in the quantity of enzymes produced naturally by the animal for the digestion that occurs in the digestive tract under production conditions.

The rate at which digesta passes through the digestive tract may be too fast for the native enzymes to complete the task of breaking down all of the nutrients consumed by the animal. Additionally, there may be limitations on the amount of enzymes produced by the microbes in the digestive tract due to a digestive upset such as rumen acidosis.

When enzyme products are fed to ruminant animals, the main site of action is the rumen. Enzymes assist the rumen microbial population by performing some of the digestion for the microbes.

Overall, this means improved digestion in the rumen and less work for the rumen microbial population, thus sparing energy that would normally go to the microbes for work and can now go back to the animal for maintenance and production. The improved digestion provides the opportunity to maintain or improve ruminant performance on less dry matter intake.

The enzymes utilized in feed additives are extracted from complex microbial fermentations of fungi (e.g., Aspergillus, Trichoderma) and bacteria (e.g., Bacillus). The micro-organism itself is discarded, since these typically are spoilage micro-organisms.

The type of enzyme and its activity is dependent on the strain of micro-organism used, the media they are grown on and the culture conditions used.

Forages and other feed ingredients treated with an enzyme-containing product are the foundation for efficiency in ruminant animals. A good forage treatment program will not only preserve the forage treated but also provide enzymes (cellulases, amylases, xylanases and pectinases) that start a “pre-digestion” of fibers, starches and sugars in forages, which makes them more digestible for animals.

The release of sugars from both structural and non-structural carbohydrates stimulates rapid microbial growth, which aids in microbial colonization of plant material. Treating feed directly with enzymes should be considered the first step toward feed efficiency with enzyme technology.

It is important to keep in mind that adding enzymes to ruminant diets will increase the rate of digestion but not necessarily the extent of digestion. This means the effect of enzymes is not due to making substrates digestible if they would not be digested in the rumen without the use of enzymes.

Instead, enzymes capture more value from components of feedstuffs already digestible by allowing quicker release of the nutrients. In this way, more potentially digestible substrates from feed can be obtained for use by livestock.

Knowing the analysis of ingredients used to formulate a diet for ruminants is not only important in understanding what enzyme complex may be best to use but also to understand potential rate-limiting steps. Lignin forms complexes with carbohydrates in the cell wall of plants.

This “interference” from lignin will prevent some enzymes (e.g., xylanase) from binding with their substrate, thus slowing the rate at which enzymes can function

Fermentation of starch in high-concentrate diets leads to depression of rumen pH, predisposing cows to ruminal acidosis.

Practically speaking, enzymes become advantageous when you consider digestion efficiencies decline with increasing dry matter intake. There is a 4 percent decline in digestion per multiple of maintenance dry matter intake .

This occurs because higher intakes generally increase passage rate and reduce the residence time of feed in the rumen for digestion . This process causes intakes to increase to assist the cow in consuming enough energy as digestibility decreases.

Conversely, if one improves digestibility of a TMR with enzymes, cows should not need to consume as much feed to support a given volume of milk production. The reduction in dry matter intake will enhance dry matter digestibility in the rumen due to slower passage rate (increases rumen retention time) of the feed through the rumen.

If dry matter intakes do not decrease with the addition of enzymes, the enhanced digestion by the enzymes can supply more nutrients and energy for increased milk production, even though rumen retention time of feed is compromised by a higher dry matter intake.

Therefore, enzyme technology can improve feed efficiency in three possible ways: direct improvement in digestion of feeds, indirect improvement in digestion via lower dry matter intake and increased rumen retention time of feed, or some combination of the first and second.

Feed efficiency is an important aspect of livestock production. Exogenous enzymes can provide a means to improve efficient use of nutrients in feeds by livestock, enhancing production goals. Starting with the application of a forage/feed inoculant containing enzymes should be considered.

Choosing which enzymes to include should be based on the diet being fed. If done correctly, enzymes can improve the efficiency of livestock production.

FAQ ON EFE—–

What types of enzymes are used in silage additives?

Common enzyme-based silage additives contain cellulases, hemicellulases, xylanases, amylases, and pectinases. Cellulases, hemicellulases, and pectinases are enzymes that degrade the fiber portion of forages. Amylase breaks down starch (amylose) – therefore its use would be directed towards starch containing silages such as corn silage.

Do enzymes break down forage fiber?

A number of studies have reported reductions in neutral detergent fiber (NDF) by using enzyme-based silage additives. Observed NDF reductions have been more consistent in grass silages as compared to alfalfa silage. Research data also suggests that hemicellulases and pectinases are more effective than cellulases at reducing fiber content. Unfortunately, hemicellulases and pectinases break down fiber fractions (hemicellulose, pectin) that are more easily digested by ruminants. Consequently, these enzymes reduce the concentration of digestible NDF fractions rather than the indigestible NDF fraction cellulose.

Do enzymes improve animal performance when added to silage?

To date research results have been mixed and no clear trend has emerged. Some studies have demonstrated improved animal performance (i.e., greater milk yield), while others have shown no benefit to feeding enzymetreated silages. A number of studies have shown improved dry matter intake when animals were fed enzyme-treated silages.

Do enzymes aid silage fermentation?

Enzymes can improve silage fermentation when the substrate (e.g., sugars) is limiting. Soluble sugars are required to help bacteria produce lactic acid, which is required to lower silage pH for proper fermentation. Generally, enzyme addition to silages has a small positive effect on fermentation.

Can enzymes be added directly to the feed mixture?

Interestingly, current trends in enzyme technology involve incorporating enzymes directly into total mixed rations (TMRs) or silages prior to feeding. Initial research has observed some positive effects using this method. The economics of this practice have yet to be determined.

What is the bottom line for using enzymes with silage?

It is evident that enzymes can be effective in enhancing animal performance. However, the commercial use of enzymes with silages or TMRs has been limited by the lack of consistently positive results in research studies and the high cost of enzyme-based silage additives. Enzyme technology continues to improve. New products designed for ruminants and for specific types of feed will increase the potential for profitable use of enzymes with silages. Promising enzyme technologies should be evaluated by producers on a case by case basis from independent research results until clearer animal performance and cost to benefit ratios emerge. Producers should be aware that some bacterial inoculants contain low levels of enzymes.

Though use of exogenous enzymes have a lot of advantages, its use is limited because of its price and again some scientists reported no effect of this type of supplementation in animals fed with concentrate based diet. Before use of enzymes, some basic knowledge regarding enzyme and substrate should be acquired for optimum result.

So,

Adding exogenous fibrolytic enzymes to dairy cow and feedlot cattle diets can potentially improve cell wall digestion and the efficiency of feed utilization by ruminants. Positive responses in milk production and growth rate have been observed for cattle fed some enzyme products, although results have been inconsistent. Some of the variation can be attributed to product formulation, under- or over-supplementation of enzyme activity, inappropriate method of providing the enzyme product to the animal, and the level of productivity of the test animal

Reference-On request