Reaction Nelson Somogyi?

Question

Pleas, How does reaction Nelson somogyi in determining reducing sugar?especially, reaction arsenomolidate vs Copper (I) and Copper (II)?

Answer 1

Research has demonstrated that supplementing dairy cow and feedlot cattle diets with fiber-degrading enzymes has significant potential to improve feed utilization and animal performance. Ruminant feed enzyme additives, primarily xylanases and cellulases, are concentrated extracts resulting from bacterial or fungal fermentations that have specific enzymatic activities. Improvements in animal performance due to the use of enzyme additives can be attributed mainly to improvements in ruminal fiber digestion resulting in increased digestible energy intake. Animal responses are greatest when fiber digestion is compromised and when energy is the first-limiting nutrient in the diet. When viewed across a variety of enzyme products and experimental conditions, the response to feed enzymes by ruminants has been variable. This variation can be attributed to experimental conditions in which energy is not the limiting nutrient, as well as to the activities and characteristics of the enzymes supplied, under- or over-supplementation of enzyme activity, and inappropriate method of providing the enzyme product to the animal. A limited number of ruminant enzyme products are now commercially available, and this list of products is expected to grow. However, random addition of enzymes to diets without consideration for specific situations and substrate targets will only discourage or delay on-farm adoption of enzyme technology. Although much progress has been made in advancing enzyme technology for ruminants, considerable research is still required to reduce the variability of response. With increasing consumer concern about the use of growth promoters and antibiotics in livestock production, and the magnitude of increased animal performance obtainable using feed enzymes, there is no doubt that these products will play an increasingly important role in the future. This paper reviews the research on enzyme selection, the animal responses to feed enzymes, and the mechanisms by which these products improve nutrient utilization.

Key Words: Cellulase • Cellulose Digestion • Digestion • Enzymes • Fiber • Ruminants

    Introduction  

Top

Abstract

Introduction

Implications

Literature Cited





Over the years, significant improvements in forage cell wall digestibility have been achieved through forage breeding programs and agronomic advances. Despite these improvements, forage digestibility continues to limit the intake of available energy by ruminants, and correspondingly, contributes to excessive nutrient excretion by livestock. The use of exogenous fibrolytic enzymes holds promise as a means of increasing forage utilization and improving the productive efficiency of ruminants.

Recent studies have shown that adding exogenous fibrolytic enzymes to ruminant diets increases milk production (Nussio et al., 1997; Lewis et al., 1999; Rode et al., 1999; Schingoethe et al., 1999; Kung et al., 2000; Yang et al., 2000) and ADG (Beauchemin et al., 1995; 1997; 1999b; Iwaasa et al., 1997; McAllister et al., 1999) in some cases. These increases in animal performance are due to increases in feed digestion. Numerous studies have reported increased digestion of DM and fiber measured in situ, in vitro (Nakashima et al., 1988; Feng et al., 1996; Hristov et al., 1996; Yang et al., 1999; Colombatto, 2000; Colombatto et al., 2002c), or in vivo (Feng et al., 1996; Krause et al., 1998; Rode et al., 1999; Yang et al., 1999; Beauchemin et al., 2000; Kung et al., 2000). However, not all studies report improved animal performance due to the use of exogenous enzymes (Higginbotham et al., 1996; Pritchard et al., 1996; ZoBell et al., 2000), and viewed across a variety of enzyme products and experimental conditions the response to feed enzymes by ruminants has been variable. This article reviews the research on the selection of enzymes for use in ruminant diets, the animal responses to feed enzymes and the potential sources that contribute to the variability in response, and the mechanisms by which exogenous fibrolytic enzymes improve nutrient utilization.

Feed Enzymes for Ruminants

Commercial enzymes used in the livestock feed industry are products of microbial fermentation. Feed enzymes are produced by a batch fermentation process, beginning with a seed culture and growth media (Cowan, 1994). Once the fermentation is complete, the enzyme protein is separated from the fermentation residues and source organism. Although the source organisms are, in many cases, similar among enzyme products, the types and activity of enzymes produced can vary widely depending on the strain selected and the growth substrate and culture conditions used (Considine and Coughlan, 1989; Gashe, 1992; Lee et al., 1998).

Compared to the fermentation extract, these enzyme products are relatively concentrated and purified, containing specific, controlled enzyme activities. They usually do not contain live cells. Enzyme products for ruminant diets are of fungal (mostly Trichoderma longibrachiatum, Aspergillus niger, A. oryzae) and bacterial (mostly Bacillus spp.) origin (Pendleton, 2000). Furthermore, most of the commercially available enzyme products that have been evaluated as ruminant feed additives are produced for nonfeed applications; cellulases and xylanases are used extensively in the food, pulp and paper, textile, fuel, and chemical industries (Bhat and Hazlewood, 2001). Several fibrolytic enzyme products evaluated as feed additives in ruminant diets were originally developed as silage additives (Feng et al., 1996).

In addition to these relatively pure sources of enzymes, crude fermentation products and some nonbacterial direct-fed microbials (DFM) are also marketed, at least partly or implicitly, based on their residual enzymic content (Muirhead, 2001). In this case, the enzymes, as well as the entire medium, are recovered complete with metabolites and fermentation substances. Most nonbacterial DFM consist of A. oryzae fermentation extract, Saccharomyces cerevisiae cultures, or both (Martin, 2000). In comparison to concentrated feed enzyme products, these crude products contain relatively little (<5%) enzyme activity. There is no minimal level of enzyme activity required for products to be registered as feed enzymes, which adds tremendous confusion in the marketplace. Consequently, it can be difficult to distinguish commercially between "true" enzyme products and products with trace levels of activity. The scope of this paper is limited to concentrated fermentation products that have specific, controlled enzyme activities.

Enzyme Activities Involved in Cell Wall Digestion

The focus of most enzyme-related research for ruminants has been on plant cell wall degrading enzymes. Cellulose and hemicellulose, the major structural polysaccharides in plants (Van Soest, 1994), are converted to soluble sugars by enzymes collectively referred to as cellulases and hemicellulases. The types of cellulases and hemicellulases can differ substantially among commercial enzyme products, and differences in the relative proportions and activities of these individual enzymes may have an impact on the efficacy of cell wall degradation by these products. In addition to fiber-degrading enzymes, these products also have secondary enzyme activities, including amylases, proteases, and pectinases.

Cellulose is hydrolyzed through a complex process involving cellulases, and numerous specific enzymes contribute to cellulase activity. The major enzymes involved in cellulose hydrolysis are endocellulase (endoglucanase, endo-ß-1,4-glucanase, carboxymethyl cellulase or ß-1,4-glucan glucanohydrolase; E.C. 3.2.1.4), exocellulase (exoglucanase, exo-ß-1,4-glucanase, cellulose ß-1,4-cellobiosidase; E.C. 3.2.1.91), and ß-glucosidase (cellobiase or glucohydrolase, E.C. 3.2.1.21). In general, endoglucanases hydrolyze cellulose chains at random to produce cellulose oligomers of varying degrees of polymerization; exoglucanases hydrolyze the cellulose chain from the nonreducing end, producing cellobiose, and ß-glucosidases hydrolyze short-chain cellulose oligomers and cellobiose to glucose.

The main enzymes involved in degrading the xylan core polymer to soluble sugars are xylanases (EC 3.2.1.8) and ß-1,4 xylosidase (3.2.1.37) (Bhat and Hazlewood, 2001). The xylanases include endoxylanases, which yield xylooligomers and ß-1,4-xylosidases, which in turn yield xylose. Other hemicellulase enzymes involved primarily in the digestion of side chains include ß-mannosidase (3.2.1.25), -L-arabinofuranosidase (3.2.1.55), -D-glucuronidase (3.2.1.139), -D-galactosidase (3.2.1.22), acetyl xylan esterases (3.1.1.72), and ferulic acid esterase (3.1.1.73) (White et al., 1993; Bhat and Hazlewood, 2001).

Fiber-degrading enzyme activities are generally determined by measuring the rate of release of reducing sugars from pure substrates, with enzyme units expressed as the quantity of reducing sugars released per unit of time per unit of enzyme. Reducing sugars, which include monosaccharides and free sugar ends in oligosaccharides, can be measured colorimetrically using the Nelson/Somogyi copper method (Somogyi, 1952) or the dinitrosalicyclic acid method (Miller, 1959).

The most commonly used substrate for measuring cellulase activity is carboxymethyl cellulose, which measures endo-ß-1,4-glucanase activity (Wood and Bhat, 1988). Exoglucanase activity can be measured using crystalline cellulose preparations, such as Avicel. ß-glucosidase activity is determined by measuring the release of glucose from cellobiose, or the release of p-nitrophenol from p-nitrophenyl-ß-D-glucoside (Bhat and Hazlewood, 2001).

Xylanase activity is most commonly measured by determining the release of reducing sugars from prepared xylan, such as oat spelt or birchwood xylan. Xylanases are specific for the internal ß-1,4 linkages within the xylan backbone, and are generally considered endo