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Visualizing the activity of enzymes used in the feed industry using microscopy

By Ninfa Rangel Pedersen, DSM/Novozymes Alliance, Bagsværd, Denmark

Introduction As in most science, research in plant and animal cell biology depends on laboratory methods that can be used to study cell structure and function. Many important advances in understanding cell structure and function have resulted in the development of new methods that have opened innovative areas of investigation. An example of these developments is the use of microscopy as a necessary tool to study cell biological functions. Microscopes are used to view micro specimens at high magnifications that are not visible to the naked eye. Studying structure and function of material under the microscope can help to understand, apply and enhance the knowledge to develop cross fertilization of ideas and data in different fields of science. For instance, it is well known that use of enzymes in animal diets

is essential to increase the digestion and absorption of poorly available nutrients, or to remove dietary anti-nutritional factors (ANFs). 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 molecules. Enzymes can be classified by the types of substrates they work on. For example: proteases break down proteins into amino acids, carbohydrases act on carbohydrates to break them down to simple sugars, and lipases break down lipids to fatty acids and glycerol. Concerns with using enzymes in animal feed include the ability of the enzymes to survive feed processing temperatures and even the conditions within the animals’ digestive tract. In this piece of work, microscopy has been used to visualize the in vitro effect of enzymes on feed substrates as well as elucidate the composition and cell wall architecture of some feed raw materials. In addition, digesta samples from in vivo trials where animals were fed diets with and without exogenous enzyme have also been studied to visualize the effect of enzymes in vivo.

Examples of use of microscopy and other methods to visualize raw materials, enzyme activity on raw materials and digesta samples from in vivo trials.

1. Microscopy reveals the presence of intact cells in milled feed raw materials

It is generally accepted that milling destroys cell walls and hence that nutrients such as starch and protein are readily available for the animal. However, microscopy of milled corn (Fig 1.A) and digesta samples from broilers fed a corn based diet (Fig 1.B) revealed the presence of intact cell walls in the samples and therefore indicates that cell wall degradation is not complete by milling alone. Monogastric animals do


not possess endogenous cell wall degrading enzymes, and cell walls not destroyed during the milling process will therefore hinder digestion of the nutrients present within these intact cells. To fully utilize nutrients cell wall degrading enzymes such as xylanases are therefore needed.

Figure 1. Corn milled to 0.5 mm (A) and digesta samples from broiler fed a corn based diet (B). Samples were stained with iodine which colours the starch blue or blue-black and visualized by light microscopy. Besides free starch being visible, starch trapped within the cell walls is also visible.

2. Elucidating the cell wall architecture of canola by use of microscopy

Canola meal (CM) is a protein ingredient used in diets for livestock production as a cost effective alternative to soybean meal (SBM). However, it is notable that apparent metabolizable energy (AME) table values (Sauvant et al., 2004) for poultry and AME calculated based on the content of crude protein, crude fat, starch and sucrose are quite similar in SBM while in meals from canola the discrepancy between the 2 AME values is about 1.7 MJ/kg (Pettersson and Pontoppidan, 2013). The amount of pectin (about 60 g uronic acids/kg dry matter) and the fibre matrix structure of canola most likely increases the water holding capacity of this raw material, resulting in a poor nutrient availability for non-ruminants and in addition the ANFs in CM may reduce the energetic value of the meal. The dietary fibre content of CM is higher than in SBM and literature shows that substantial gains in nutrient utilization can be achieved in several animal species when diets are properly formulated with the use of exogenous enzyme supplementation (Meng and Slominski, 2005; Meng et al., 2005; Józefiak et al., 2010). Supplementation with a multi-carbohydrase enzyme has been shown to increase energy utilization (AME) from 1943 to 2249 kcal/kg in broiler chicken fed diets containing canola meal (Jia et al., 2012; Slominski et al., 2012; Slominski, 2011). A study was conducted to elucidate the cell wall structure of canola meal and find out which specific enzymes are responsible for degrading canola cell walls. Results showed that canola has a different cell wall architecture compared to e.g. soyabean (Ravn et al. 2015) and a tentative structure of canola cell wall was proposed (Fig. 2) (Pedersen et al., 2017).

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