Processing stability and detection method of feed enzyme in compound feed

introduction

In many countries in the world, enzymes are often added to the feed of monogastric animals. This is apparently due to the definite improvement in the performance of enzymes on monogastric animals, but it has not been easy to confirm the presence of enzymes in commercial feeds. To this end, many studies have been conducted so far. Enzymes are proteins, which, like all other feed proteins, are very sensitive to feed processing. However, feed proteins work in amino acids, so there is no need to maintain the configuration, and feed enzymes either irreversibly degenerate during feed processing or no longer work. Therefore, it is necessary to detect the enzyme activity in the compound feed. The focus of this article is not to discuss existing enzyme activity assays but to characterize the characteristics of the enzymes used in the feed, including the differences between the in vitro thermal stability of enzymes from different sources, and the interactions between enzymes and feed matrices for analysis. Challenges (and methods to eliminate this effect), data on feed processing trials, and trends in the future.

Most pigs and poultry feeds require a certain degree of processing. Some feeds need to be made into granules. The processing process is to first pass steam, and the feed mixture is conditioned and pressed into granules. Pelleting can increase the nutrient concentration of the feed, improve the storage characteristics of the feed, and reduce the microbial content in the feed. Granulation temperatures are generally 65 to 90 degrees Celsius (Gibson, 1995), and such high temperatures can destroy heat-sensitive nutrients (including enzymes).

In the past few years, concerns about feed-borne pathogens and factors affecting pellet quality have prompted feed manufacturers to increase the temperature, time, and pressure of feed processing and to perform secondary granulation or bulking of feed (Pickford, 1992). . The strengthening of feed processing makes the stability of the enzyme more important. Several approaches have been taken to overcome this problem, including by adding a liquid enzyme after cooling the feed pellets to avoid the effect of processing together on enzyme activity. Although enzymes can be added after granulation, feed enzymes are generally added to the powdered feed prior to processing. By using a hydrophobic coated protective layer or using a more heat-resistant enzyme, the effect of the heat treatment on the enzyme activity can be reduced.

Published data published so far on the preservation rate of feed enzyme activity are still limited (Chesson, 1993). However, the stability of the enzyme is extremely important for the feed manufacturer, and it must be ensured that the laboratory evaluation of the enzyme is performed before the enzyme preparation is sold. Since 1993, a number of research reports have been reported on newspapers or conference proceedings. There are also several test results in the critical scientific literature. Obviously, in vitro assays of enzyme activity, whether in solution or in feed, are extremely important. Recent studies have shown that in vitro enzyme activity measurements must be verified by in vivo effects.

Phytase

Since the use of phytase accounts for about 20% of the amount of commercial enzyme preparation, there have been many reports on the thermal stability of phytase (Bedford and Schulze, 1998). The reason for this concern is probably due to the fact that many plant-based feed ingredients contain phytic acid, and the presence of phytic acid makes it difficult to absorb phosphorus and other nutrients (Cheryan, 1980; Eeckhout and de Paepe, 1994; Ravindran et al., 1995). . However, the endogenous phytase deficiency activity or activity in monogastric animals is very low (Pallauf and Rimbach, 1997). Even more complicated is that the same phytate-derived plant also contains a considerable amount of phytase, and the nutritive problem of phosphorus digestion is intertwined with the problem of environmental pollution caused by the enrichment of phosphorus in the soil. Phosphorus pollution has become a limiting factor in the production of intensive livestock and poultry production areas.

Phytase has a wide range of sources and its characteristics vary. Liu et al. (1998) reviewed the literature before 1998. The results showed that the optimal temperature for the activity of phytase from bacteria, fungi, yeast and plants was 45-77 degrees Celsius, with a difference of up to 32 degrees Celsius. Dvorakova et al. (1997) described phytase properties isolated from Aspergillus niger. The phytase is active in the temperature range of 25-65 degrees Celsius, and its optimum temperature is 55 degrees Celsius; it can lose 5% of the initial activity when cultured at 60 degrees Celsius for 10 minutes, and can be cultured at 80 degrees Celsius for 10 minutes. The initial activity lost 80%. As part of the search for heat-resistant enzymes, Wyss et al. (1998) studied the thermal denaturation of purified phytase isolated from A. fumigatus and A. nige. Both sources of phytase will denature at as low as 55 degrees Celsius. However, the phytase from A. fumigatus refolds into an active configuration when the temperature is raised to 90 degrees Celsius, but the phytase from A. nige does not undergo this change. Undoubtedly, certain heat-resistant phytases will be put into commercial use in the near future.

Enzymes in solution are inactivated by heat and do not indicate that enzymes in the feed are inactivated due to heat. This is because the enzyme in the feed interacts with the feed matrix. In fact, feed ingredients protect enzymes from steam or high temperature damage in a short period of time (Chesson, 1993). Determination of phytase activity in pelleted feeds provides more accurate data for commercially evaluating the extent of phytase inactivation in feed. Simons et al. (1990) added phytase to "universal pig feed", which was heated to 50 or 65 degrees Celsius before granulation. The results showed that heating to 50 degrees Celsius caused the particle temperature to reach 78 degrees Celsius or 81 degrees Celsius, which did not reduce the activity of the enzyme at this time; but heating to 65 degrees Celsius caused the particle temperature to reach 84 degrees Celsius or 87 degrees Celsius, at which time the enzyme's Loss of activity 17% or 54%. Gibson (1995) added 3 plant enzyme preparations to wheat basal diets and pelleted them at 65 to 95 degrees Celsius. The results showed that 2 of the phytase preparations had been inactivated at a granulation temperature of 65 degrees Celsius, leaving only 1 phytase preparation to retain a considerable amount of activity at a granulation temperature of 85 degrees Celsius or more. In addition to studying the stability of the enzyme in solution, Wyss et al. (1998) also added phytase isolated from Aspergillus fumigatus and Aspergillus niger to commercial feed prior to granulation (75 degrees Celsius or 85 degrees Celsius). The results showed that the activity recoveries of the two phytase enzymes in pelleted feeds were similar at a pelleting temperature of 75 degrees Celsius; however, when the pelleting temperature was 85 degrees Celsius, the phytase activity from Aspergillus niger was higher than that from cigarettes. The phytase activity of Aspergillus was lost more, which also supported their findings regarding the kinetics of denaturation. Eckhout et al. (1995) added commercial phytase preparations to feed. The results showed that the phytase activity can be lost by 50% to 65% when the granulation temperature is 69 to 74 degrees Celsius.

Deactivation not only affects the role of microbial enzymes added to the feed, but also affects the naturally occurring enzymes in feed ingredients. Gibson (1995) found that pelleting at temperatures above 85 degrees Celsius would significantly deactivate the endogenously investigated phytase activity in wheat. Eeckhout and de Paepe (1994) reported in a survey on phytase activity in different feeds that wheat bran is rich in phytase, but its phytase activity in the pelleted sample is only 56% of the unpelleted sample. Jongbloed and Kemme (1990) found in three experiments that pelleting at approximately 80 degrees Celsius results in a decrease in phytase activity in pig feed, which is based on feed ingredients rich or lacking phytase activity. of. They also conducted further experiments to determine the effect of granulation on the apparent absorption of phosphorus. In two of these trials, they found that pelleting diets rich in phytase reduced the rate of phosphorus uptake. This result is consistent with the inactivation of endogenous phytase.

Research institutions and the feed industry are concerned about the stability of phytase research because of the increasing processing temperatures used today and the increasing importance of phosphorus absorption due to nutrition and environmental factors. Exogenous enzymes add envelopes or particles to provide a means for protecting enzymes from heat damage. A more basic approach may be the isolation of heat-resistant enzymes or reduction to the active configuration after denaturation. Unfortunately, none of these methods can prevent high temperatures from damaging the endogenous enzymes contained in feed ingredients.

Beta-glucanase

Due to the commercial production of beta-glucanase preparations, barley can be added as a feed ingredient to poultry diets, and will not degrade poultry production performance and produce sticky feces due to high beta-glucan levels (Campbell and Bedford, 1992). At present, β-glucanase has been widely used in the world barley production area. However, studies on the effects of heat treatment on β-glucanases added to feeds remain limited.

Eeckhout et al. (1995) measured beta-glucanase activity in commercial piglets that had been tempered at 50 to 95 degrees Celsius and granulated at 72 to 91 degrees Celsius. The results showed that even at the lowest temperature, β-glucanase activity in the feed lost 40% after processing, while at the highest temperature, only 7% of the activity was conserved, and 2/3 of the activity was in the quenched and tempered state. Lost during the period. On the other hand, Esteve-Garcia et al. (1997) found that β-glucanase added to broiler stock still retains most of its activity after conditioning and granulation temperatures approaching 80 degrees Celsius. The enzyme used is made into fine particles. This shows that β-glucanase can be added to the feed in a stable form.

At least two trials have determined the performance of broiler chicks fed diets supplemented with heat-treated enzymes. McCracken et al. (1993) added a stable form of commercial enzyme mixture containing basic beta-glucanase and xylanase activity to the barley basal diet. The diet was heated at 85 degrees Celsius before granulation. minute. The results showed that heat treatment of diets without supplemental exogenous enzymes reduced the apparent digestibility of dietary nutrients, increased the viscosity of intestinal contents of meat chicks, and reduced the dry matter content of feces; The heat treatment in the case of the source enzyme increases the digestibility of feed nutrients and eliminates the adverse effects caused by the heat treatment. This fully demonstrates that the enzyme remains active at a temperature of 85 degrees Celsius. Vukic-Vranjes et al. (1994) determined the effect of adding commercial enzyme mixtures to two diets, one of which contained 20% barley. The enzyme mixture contains beta-glucanase, xylanase, amylase, and pectinase activity. The two diets are tempered at 70-75 degrees Celsius and pelletized or extruded at 110-120 degrees Celsius. Compared with granulation, extrusion has an adverse effect on chick performance. At the same time, extrusion also increased the in vitro viscosity of the feed, indicating that high temperatures increase the solubility of non-starch polysaccharides in the feed. Regardless of whether the granules are pelleted or extruded, supplementing the enzyme mixture improves the performance of the chicks, which indicates that the enzyme activity remains after extrusion of the diet. The study by Vukic-Vranjes et al. (1994) also showed that supplementing the enzyme mixture in the diet reduced the viscosity of the feed extract, indicating that the feed had enzyme activity before the chicks ate. They can't even rule out the presence of enzyme activity in the feed even before handling the feed.

Inborr and Bedford (1994) determined the enzyme activity in feed supplemented with heat-treated feed and the performance of broilers fed the feed. It was found that the β-glucanase added in the barley basal diet (which was the same product as the one used in McCracken et al., 1993) was significantly inactivated after conditioning and granulation at 75°C. The conditioning at 95 degrees Celsius for 30 seconds and 15 minutes respectively resulted in 84% and 91% loss of beta-glucanase activity. Inborr and Bedford (1994) sought to determine whether the determination of enzyme activity in vitro and in vivo is the most accurate method for evaluating the effect of processing on enzyme activity. The results showed that the recovery of enzyme activity was highest at the lowest processing temperature and lowest at the highest processing temperature, but the result could not be accurately reflected by the production performance of broilers. The highest value of broiler body weight gain and the lowest value of the ratio of consumption weight gain were obtained at a centrally adjusted temperature (85 degrees Celsius). Obviously, the enzyme remains active at this temperature and the positive effect of heat treatment exceeds the negative effect of enzyme inactivation.

Pickford (1992) showed that the stabilization of β-glucanase protects it from the 75°C pelleting temperature, but cannot withstand a pelletization temperature of 95°C. Cowan and Rasmussen (1993) reported that commercial β-glucanase is significantly inactivated at a granulation temperature of above 65 degrees Celsius, but if this product is applied, it can be protected from a pellet temperature of 75 degrees Celsius. Both of the above reports are brief, and there have been very limited reports on improving the thermal stability of β-glucanase.

Xylanase

Xylanase accounts for the largest share of world enzyme sales (Bedford and Schulze, 1998) and its application allows high-viscosity wheat to be added to poultry diets as feed ingredients without adverse effects. Perhaps because of its economic importance, reports on the stability of xylanase in the scientific literature are far more numerous than those on β-glucanase stability.

Xylanases can be produced by a variety of fungi and bacteria, and the optimum conditions for their enzymatic activity vary. This is not surprising, because each organism has a specific adaptation environment. A review by Bedford and Schulze (1998) showed that many strains of fungi and bacteria produce xylanases with an optimum temperature of 30 to 105 degrees Celsius and an optimum pH of 2.0 to 10. Pickford (1992) compared the stability of three commercial enzyme preparations without providing data on the type of enzyme contained in the enzyme. The results show that at the granulation temperature of 80 degrees Celsius, the activity of the first enzyme preparation remains 85%, the activity of the second enzyme preparation remains 55%, and the activity of the third enzyme preparation remains only 33%. At the granulation temperature of 95 degrees Celsius, the activity of all three enzyme preparations was lost. Extrusion at a high temperature for a short time also caused a large loss in the activity of these three enzyme preparations, and the difference in enzyme activity loss was similar to the above.

Pettersson and Rasmussen (1997) demonstrated that there are differences in the thermal stability of xylanase enzymes isolated from Thermomyces, Humicola, and Trichoderma. The xylanase isolated from Trichoderma is significantly inactivated at a tempering temperature of 75 degrees Celsius, while the xylanase isolated from the mold from Humicola and Humicola retains 80% at a conditioning temperature of 85 degrees Celsius. The above activity. The xylanase isolated from Pyricularia can retain more than 70% of its activity even at a conditioning temperature of 95 degrees Celsius. Gibson (1995) observed variation between nine xylanase preparations, seven of which were commercial enzyme products at the start of the experiment. The results showed that after processing at 90 degrees Celsius, one of the enzyme preparations retained more than 90% of the enzyme activity, and the remaining 7 retained enzyme activities were all below 10%. The report is not sufficient to show how much of the variation is due to the source of the enzyme and how much is due to the stabilization of the enzyme. Perez-Vendrell et al. (1999a) determined the in vitro stability of eight commercial enzyme preparations (including coated or coated products) after conditioning at 65-70 degrees Celsius, 75-80 degrees Celsius, and 85-90 degrees Celsius. The results showed that even at the lowest tempering temperature, the enzyme activity of most products still lost at least 30%; and at the highest conditioning temperature, its enzyme activity could be lost by 90%. Esteve-Garcia et al. (1997) reported that the enzymatic activity of the fine-grained xylanase preparation did not inactivate at the tempering and granulation temperatures close to 80 degrees Celsius as found in the beta-glucanase study. .

Several methods of protection against damage by heat treatment have been mentioned above. The most basic enzyme activity protection method is the method suggested by Pettersson and Rasmussen (1997) in their research, that is, the isolation and production of heat-resistant enzymes from heat-resistant microorganisms. Another method of enzymatic activity protection is to coat the feed pellets with an enzyme preparation after granulation cooling, but this requires additional processing equipment and processing (Perez-Vendrell et al., 1999b).

The dried enzyme preparation product can also be stabilised to increase its thermal stability, and it can be added to the feed prior to granulation without significant loss of enzyme activity. The method of stabilization is the previously mentioned method. The inactivation of the enzyme is due to the steam used for conditioning. Therefore, the stabilization of enzyme preparations is mainly through the adsorption of the enzyme preparation onto a carrier or the application of a hydrophobic matrix to the enzyme preparation, so that the enzyme preparation is protected from the destruction of steam (Cowan, 1996). Pickford (1992) provided an example of stabilizing treatment that increased the enzyme activity retained by the enzyme preparation after 75°C granulation from 48% to 76%, and increased the enzyme activity retained after 95°C granulation from 12% to 34%. Obviously, even the stabilizing enzymes have thermal stability limits. Steen (1999) suggested that if the feed is to be processed at temperatures above 90 degrees Celsius, even stabilizing enzymes should be added to the feed after processing.

In vitro enzyme assays are valuable tools for determining the loss of enzyme activity. Test results show that even relatively low temperatures can cause significant loss of enzyme activity. However, in vitro testing is just one of them. Obviously, the activity of the enzyme preparation in the buffer under the treatment and digestion conditions of the feed only provides very limited enzymatic activity information. In fact, most researchers (Vukic-Vranjes et al., 1994; Pettersson and Rasmussen, 1997; Perez-Vendrell et al., 1999a; et al.) have recognized the importance of interactions between enzymes and feeds, and measured compound feeds. The activity of the enzyme. The determination of the viscosity of the feed extract (Spring et al., 1996; Bedford et al., 1997) provides an indicator for assessing the effect of the enzyme activity prior to feed intake by poultry. Preston et al. (1999) confirmed the existence of this effect. The results show that, even if the enzyme activity is completely lost in its feed end product, the supplemental enzyme preparation can still significantly improve the poultry production performance.

In order to understand the effect of feed processing on enzyme action, the effect of enzyme addition and heat treatment in the feed must be determined by the performance of the poultry. Inactivation of enzymes is not always directly reflected by differences in poultry production performance (Bedford et al., 1997; Perez-Vendrell et al., 1999a; Silversides and Bedford, 1999). Bedford et al. (1997) found that as the processing temperature increases from 65 degrees Celsius to 105 degrees Celsius, the percent deactivation of the enzyme changes linearly, while the broiler performance (body weight gain, feed weight gain ratio) is between 81 and 83 degrees Celsius. The best processing temperature. Silversides and Bedford (1999) also confirmed this effect of heat treatment, as shown in Figure 15-1. With the increase of processing temperature, the enzyme activity decreased linearly (R2=0.97), and the weight of broilers showed a quadratic curve (R2=0.84). Graphing the results of consumption weight gain ratio also produced similar results (R2=0.98). The maximum weight and the lowest weight gain ratio are obtained at processing temperatures between 80 and 85 degrees Celsius. There are two possibilities for producing this result: one possibility is that the enzyme performs its function almost in the conditioner, so that there is no correlation between the analyzed values ​​after granulation; the other possibility is the analytical method used. The enzyme activity cannot be measured effectively.

The enzyme data obtained from smear-protected Pyricularia moulds does not appear to be of the same type as that obtained from uncoated protective Trichoderma or Aspergillus. This may be due to the absence of interactions between enzymes and feed matrix in the former. Made. In this case, the enzyme is more easily extracted and the correlation between the recovered enzyme activity and poultry production performance is also confirmed (Cowan and Rasmussen, 1993; Pettersson and Rasmussen, 1997; Andersen and Dalboge, 1999). These conflicting results indicate that, in the absence of known enzyme properties, there is no single method for determining the performance of poultry based on the analytical value of the feed sample.

Increasing the performance of poultry as in vitro enzymatic activity decreases predicts only the risk of enzyme activity assays, but it may not be as contradictory as it appears. In addition to affecting enzymes, heating also affects many other aspects (Pickford, 1992). Moderate heating can promote the gelatinization of starch, accelerate cell wall crushing, increase the utilization of nutrients, and thus improve the production performance of poultry. This is one of the basic reasons for granulating feed. Higher processing temperatures also increase the solubility of non-starch polysaccharides, thereby increasing the viscosity of the gut contents of poultry and reducing the performance of poultry, which in turn increases the need for exogenous enzymes. Silversides and Bedford (1999) showed that in the absence of exogenous xylanase in wheat basal diets, the viscosity of the broiler's gut contents increased significantly with increasing processing temperatures (Figure 15-2). When the xylanase is added to the diet, the viscosity of the intestinal contents of the broiler can be decreased even at a higher processing temperature, and the viscosity is actually reduced most at the highest temperature. At higher processing temperatures, the enzyme can still play a larger role, probably because the enzyme can get more substrate at this time. Enzymes may be active before or during processing, reducing the in vitro and in vivo viscosity measurements. Higher processing temperatures reduce the performance of poultry, not only because of the increased viscosity of the contents of the gut and the diminished effect of exogenous enzymes, but also because of the inactivation of vitamins and other enzymes, and the digestibility of starch and protein. Decline. With the commercialization of heat-resistant or stabilized enzyme preparations, the destruction of vitamins and other nutrients by heating will also possibly limit the processing temperatures used in production.

Conclusions and trends

The activity of the enzyme in the solution may decrease at temperatures below 60 degrees Celsius. How much of the enzyme in the feed mixture is protected by feed ingredients, most of which may be very stable at slightly lower temperatures. However, severe loss of enzyme activity occurs at processing temperatures up to 95 degrees Celsius. In order to reduce the adverse effects of heat treatment, several methods have been used or recommended, including protecting enzymes from steam infiltration, using heat-resistant enzymes, or adding liquid enzymes after processing. Feed enzymes may have some activity before the animal feeds on the feed, so the total loss of in vitro enzyme activity may not always mean a total loss of income. This seems to depend partly on the enzyme and its analytical method. In some cases, the in vitro analysis value is misleading; in other cases, the enzyme content has a clear correlation with the animal's production performance. Since vitamins, proteins and starches may be more sensitive to heat treatment than exogenous feed enzymes, increasing the processing temperature will increase the damage to these nutrients, so the general processing temperature can not be increased without limit. The further development of heat-resistant enzymes and the use of enzymes before feed processing or animal feed intake may be a promising way for the feed industry to maximize yield through the addition of enzymes.