Foreword

The enzyme is a biocatalyst with extremely high catalytic efficiency. Its catalytic efficiency is 107~1013 times higher than that of inorganic catalyst, but the catalytic activity of the enzyme is highly regulated by various physical and chemical factors such as temperature, pH, moisture and pressure. Conditions such as ultraviolet rays have a great influence on the enzyme activity. At present, the large-scale production of pellet feed and expanded feed has become a development trend of the world feed industry. According to incomplete statistics, more than 70% of foreign feed products are processed by thermal processing (tempering, granulating or puffing). The intense action of temperature, pressure and humidity during the granulation, extrusion and expansion process poses a challenge to the activity of the enzyme preparation in the finished feed. Although the enzyme preparation can be pre-enzymatically sprayed after granulation [1] or before feeding [2], in the current situation that the cost of the feed enzyme preparation is decreasing, the addition amount is increased to obtain sufficient activity in the final product. It is the simplest and most economical method of using enzyme preparations. Therefore, it is necessary to study the effect of feed processing technology on the activity of enzyme preparations, and determine the cause and extent of the effects to ensure the more precise addition of enzyme preparations.

I. Characteristics affecting the enzyme activity process

At present, the main damaging effect on the enzyme preparation in the feed processing process is the granulation and expansion process. These two effects involving high temperature, high humidity and extrusion are a severe test for the activity of feed enzyme preparations.

1.1 Granulation process

In the granulation process, it is necessary to add 4 to 5% of steam for quenching and tempering, so that the temperature of the material is raised by about 50 °C. In addition, the friction between the material and the pressure roller, the die and the die hole can also raise the temperature of the material by 5-20 ° C, so that the particle temperature after granulation reaches 70-90 ° C, or even about 100 ° C. In general, the temperature of the quenching and tempering is not lower than 70 ° C, in order to make the powder more fully gelatinized, in addition to a certain gelatinization time, if conditions permit, it is best to keep the powder in the quenching and tempering for more than 15s, Generally not less than 6s, and the moisture content of the material required to achieve the best granulation effect is between 15.5 and 17.5% [3].

1.2 Puffing process In the extrusion process, the temperature can be as high as 200 ° C, but the residence time of the feed at such high temperatures is very short (5 ~ 10 s). When processing floating feed, steam and water are added up to 8% of dry feed. The extrudate has the following properties when it reaches the die: final pressure is 3.45×103~3.75×103kPa, temperature is 125~138°C, moisture 25~27%. In the production of sinking feed, a small amount of steam is first added to the conditioner, and then water is added. The final moisture of the mixture leaving the conditioner is usually 20 to 24%. The temperature of the mixture reaches 70~90 °C at the outlet of the tempering cylinder, and the moisture content of the extrudate reaches 28~30%. When producing fish feed, the pressure at the extruder die is usually 2.63 x 103 to 3.04 x 103 kPa. Non-expanded fully matured aquatic feed, the temperature of the extrudate outside the extruder die is 120 ° C [4].

2. Factors affecting enzyme activity

Enzymes are biocatalysts that are sensitive to temperature, humidity, pressure and other factors like other proteins (Guus et al., 2000). The temperature during granulation and puffing can reach 100~200°C, accompanied by high humidity (causing high water activity in feed) and high pressure (changing the spatial multidimensional structure of enzyme protein and denaturation). Under this condition, large The activity of most enzyme preparations will be affected to varying degrees.

2.1 temperature

The effect of temperature on enzyme activity has two aspects: on the one hand, the temperature increase can increase the bond energy of the substrate molecule, and the molecular collision probability increases, thereby accelerating the reaction rate; but when the temperature rises to a certain extent, the enzyme protein is gradually denatured and active. Lost. The main sources and optimum temperatures of commonly used feed enzyme preparations are shown in Table 1 below.

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Table 1 Main sources and optimum temperature of commonly used feed enzyme preparations [5][6][7] Types of enzymes Sources optimum temperature/°C Non-digestive enzymes Cellulase Green Trichoderma 45~65 Trichoderma 45~50 Corning Trichoderma 45~50 Aspergillus niger 45~55 Hemicellulose enzyme Bacillus subtilis 40~55 Trichoderma 40~50 Pectinase 40~50 Aspergillus 40~50 Phytase Aspergillus niger 40~50 Aspergillus 40~ 50 Tannin enzyme Aspergillus sp. 40~50 Aspergillus niger 40~50 β-glucanase Bacillus subtilis 55~70 Trichoderma 50~60 Digestive enzyme protease AS1398 Bacillus subtilis 35~40 Bacillus subtilis 45~50 Saccharifying enzyme Aspergillus niger 50~60 Rhizopus 55~65 Amylase Aspergillus 55~70 Barley and wheat basal diets were subjected to thermal processing, and the relative activities of phytase in various processing procedures are shown in Table 2 [8]. Israelsen (1995) reported that the activity of phytase was zero at 110 °C. Vanderpoel reported that the activity of β-glucanase and cellulase was undetectable at 110 °C; Gradient reported that amylase activity decreased significantly at 80 °C. Clayton (1999) believes that if the granulation temperature exceeds 85 ° C, liquid enzyme preparation should be sprayed onto the cooled pellets to avoid the adverse effects of high temperature steam on the enzyme activity [9]. Table 2 Relative activity of phytase in barley and wheat based pig feed during expansion process Temperature/°C Relative activity of phytase 27.9 before quenching and tempering 100% after quenching and tempering 80.5 76% After granulation 70 47% After expansion 102 18% After expansion granulation 79 12% P. Spring (1996) determined the effect of different granulation temperatures on cellulase, bacterial amylase, fungal amylase and pentosanase activity. The test samples were barley-wheat-soybean-type feeds containing different enzyme preparations at granulation temperatures of 60 ° C, 70 ° C, 80 ° C, 90 ° C and 100 ° C, respectively. The results showed that cellulase, pentosanase and fungal amylase were stable at 80 ° C, but lost 90% at 90 ° C (P < 0.05). Bacterial amylase is more stable and still has 60% viability at 100 °C [10]. Cowan and Rasmussen (1993) determined the stability of enzymatic activity of different enzyme preparations in solution, and the results of the determination of pentosanase were similar to those of P. Spring (1996) [11]. However, different results have been reported under both granulation conditions and solution conditions. Gadient et al. (1993) reported that the activity of carbohydrate enzymes was not affected in the hot solution treatment if the critical temperature did not exceed 75 °C [12]. Nunes (1993) reported that the granulating steam temperature above 60 ° C significantly reduced the activity of pentosanase [13]. This difference in results may be due to differences in the enzyme activity assays or differences in heat tolerance of enzyme preparations from different strains. The determination of enzyme activity after granulation is a controversial issue, as there has not yet been a uniform method for the determination of highly diluted enzymes in enzyme feeds.

Gadient et al. (1993) considered that the degree of loss of enzyme activity was significantly affected by the type of enzyme preparation, and the activity of amylase was significantly decreased at 80 °C. The activity of phytase decreased by more than 50% after granulation at 70-90 °C [8]. Cowan (1993) reported that the untreated β-glucanase had a survival rate of only 10% in the feed after granulation at 70 °C. Inborr (1994) reported that β-glucanase was tempered for 30 s at a feed temperature of 75 ° C, and its survival rate was 64%, while the survival rate at 90 ° C was only 19% [14]. There may be a balance between feed processing and modulation for increased feed digestibility, damage to enzyme activity, and increased enzyme digestibility. Bedford et al. (1998) showed that the activity of xylanase in the diet gradually decreased with the increase of feed processing temperature, but the feeding experiment found that the broiler had the best performance and low temperature when the feed processing temperature was 82 °C. At or above 82 ° C, the production performance decreased, and the correlation between the feed conversion ratio and the activity of the enzyme in the feed was not significant. And all processing temperatures of the enzyme feed reduced the viscosity of the broiler's intestinal contents, and the viscosity decreased the most at 95 ° C, which also reflects the common role of thermal processing and enzyme preparation in improving feed digestibility. Michael (1997) reported that the corn and soybean meal type broiler chickens were tested. The weight and feed efficiency of the 45-day-old broiler chickens after the addition of the enzyme were 5% and 1.77% higher than those of the non-enzymatic control group, respectively. After the addition of the pellets, the above two indicators were only 4% and 1.23% higher than the control group [15]. This also suggests that in actual production, the actual production performance of the animal should be used as a criterion for checking the effectiveness of the enzyme preparation.

2.2 Pressure and shear force

The study of the effects of stress on enzyme molecules has been in existence for more than 40 years, but in recent years, the influence of pressure on the interaction between enzymes and substrates has been emphasized. Experiments have shown that pressurization can increase the activity of the enzyme, and depressurization can also improve the catalytic performance of the enzyme. It is generally believed that the protein changes its volume, conformation and active site under the influence of pressure, and this phenomenon can be detected by laser Raman spectrophotometry. If the pressure is slowed down by the pressurization, the activation volume is increased, that is, a dissociation reaction; otherwise, it is a bonding reaction. The pressure can be precisely controlled under the test conditions, but the pressure of the feed granulation process is difficult to accurately grasp, and it is still difficult to accurately determine the effect of the pressure on the activity of the diluted enzyme during the granulation or expansion process.

There are currently few studies on shearing enzymes leading to enzyme inactivation. The shearing force is present during the agitation, mixing, vibration, extrusion, etc. of the enzyme preparation added before the granulation. The inactivation of the enzyme during agitation is mainly a surface phenomenon and is not equivalent to the heating and chemical deactivation of the enzyme. The agitation action exposes and unfolds the peptide chain at the gas-liquid interface. Although the number of peptide chains exposed to the gas-liquid interface (including gas bubbles generated in the liquid) is small relative to the total peptide chain, the effect is not negligible. Because agitation will continuously create new interfaces leading to the unfolding of more peptide chains, making it difficult for the enzyme to react with the substrate. Fikret et al. [16] studied the effect of shear force on enzyme activity through experiments. The results showed that the cellulase activity decreased with the increase of shear strength and time. The hemicellase activity decreased slowly under the action of low shear strength, and when the shear strength was high, after 10 min mixing The activity is quickly reduced. Intense shear forces during granulation and expansion may be a cause of loss of enzyme activity, but there is currently insufficient data to confirm this effect.

2.3 Humidity

The moisture content also has a large effect on the activity of the enzyme preparation. Proteins are denatured due to surface tension in the presence of moisture. Protein peptides are often loosened at the two-phase interface, especially at the gas-liquid interface, which is why densified protein solutions can be denatured. The coated feed enzyme preparation is not deactivated by heating at 90 ° C for 30 min under dry conditions, but the enzyme preparation is rapidly inactivated by supplying steam at the same temperature [17]. In addition, water activity associated with moisture content can also cause protein denaturation. Hydrogen bonding plays an important role in maintaining the spatial conformation of the enzyme. When the system contains a large amount of free water, it will destroy the hydrogen bond interaction inside the enzyme molecule, making the conformation of the enzyme molecule easy to change, and the enzyme is easy to be denatured and inactivated. . At a certain temperature, the relationship between water content and water activity in feed compound enzymes and compound feed is expressed by the adsorption isotherm of water. Although this relationship is not a linear relationship, the general trend is that the higher the moisture content of the sample. The greater the water activity. At higher water activities, the denaturation of the enzyme protein is significantly enhanced. When the moisture content of the sample drops to 10%, the temperature rises to 60 °C, the lipase begins to inactivate, and when the moisture content is 23%, the apparent deactivation occurs at room temperature. For most enzyme preparations, reducing the water activity to below 0.3 at a near neutral pH and at a lower temperature prevents deterioration due to enzyme protein denaturation and microbial growth, thereby preserving more enzyme activity. When the enzyme protein passes certain stabilization measures, it can still maintain sufficient activity in the environment with high water activity, but the loss still exists [18].

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2.4 Other factors

Enzyme inhibitors, enzyme activators and the like can significantly change the catalytic activity of the enzyme. Metal ions not only affect the activity of the enzyme, but also affect the stability of the enzyme. Co2+, Mn2+ plasma can significantly increase the activity of D-glucose isomerase; Cu2+, Fe2+, Al3+, Hg2+, Zn2+, Ca2+ have different degrees of inhibition of catalytic activity; Hg2+, Pb2+ can denature enzymes, Therefore, contact with the enzyme should be avoided. In addition, the additives in the feed also have an effect on the enzyme. For example, the PO43-root has a certain degree of inhibition on the enzyme activity, and other acid ions such as CO32-, Cl-, SO42-, and NO3- have little effect, and organic substances such as urea and Deuterium can cause protein denaturation, but the degeneration caused by urea alone is often reversible. For cellulase, formaldehyde and potassium iodate can cause inactivation, but cysteine ​​and potassium dichromate can activate it, which can increase the ability of cellulase to hydrolyze CMC by about 20%. In addition, there are many natural inhibitors of enzymes in feed ingredients, some of which are non-specific inhibitors. For example, tannins in plants have strong ability to bind proteins, easily inactivate enzymes, heparin in animals, penicillin, etc. Antibiotics can also affect the activity of many enzymes. For cellulase, the phenols in plants are various inhibitors of various white pigments.

In the process of feed processing, the enzyme preparation is inevitably contacted with metal ions or substances in the feed material which inhibit the enzyme activity, and it is easier to carry out under the conditions of high temperature, high pressure and high humidity of granulation and puffing. The reaction, therefore, such factors should play a role in the inactivation of the enzyme, but there is no research report on the inactivation of the enzyme in the process of such factors.

Development direction

In summary, the temperature, pressure and moisture during granulation and expansion have a great influence on the activity of the enzyme preparation. Therefore, the following feed technology is used in the feed industry to add feed enzyme preparations: 1) using a carrier or coating For example, if the liquid cellulase and β-glucanase are adsorbed on a carrier, or the granular enzyme preparation is coated with a coating agent, the feed enzyme preparation can still be used at 90 ° C. Maintain more than 95% activity. Although the coated enzyme preparation can minimize the influence of processing temperature on it, how to ensure its timely release in the proper part of the animal digestive tract and exert the maximum catalytic effect may become the most worthy consideration of the coating process. problem. 2) Spraying after granulation, that is, spraying the enzyme preparation onto the surface of the cooled pellet feed to avoid adverse effects of high temperature on the enzyme activity in the feed molding process. The post-spraying technology completely avoids the destruction of the enzyme during the processing, but requires the installation of new spraying equipment, and the technical requirements are also high, so the corresponding cost is also high, and the accuracy of spraying is not high, the uniformity is difficult to control, Factors such as easy peeling after spraying, oxidative deactivation, and increased chalking rate are all issues that should be addressed in the future.