Effect of different combination enzyme preparations on feed viscosity in vitro

Abstract: The pepsin-pancreatin two-step hydrolysis method was used to investigate the effect of the composition and activity of non-starch polysaccharide (NSP) enzymes on the viscosity of specific laying hen diets, so as to screen out the best compound for the specific composition of laying hens. The enzyme formulation provides a fast and efficient method for optimizing NSP enzymes for specific regional diet screening. The test used the change of feed viscosity as the detection index to establish the corresponding relationship between the amount of NSP enzyme added and the in vitro viscosity. The results showed that the combined NSP enzyme effect was significantly better than the single NSP enzyme. The NSP species and content in the diet determined the optimal zymogram and optimal addition amount of NSP enzyme.

Key words: in vitro digestion; viscosity; energy; complex enzyme preparation

In recent years, the use of non-starch polysaccharide (NSP) enzymes in poultry diets has become very common, but the effect of NSP enzymes is affected by many factors, especially the composition of the enzyme spectrum, the activity and the matching of the corresponding substrates in the diet. . Therefore, the NSP enzymes that are compatible with the anti-nutritional properties of the diet can maximize the effective energy value of the feed. Domestic egg broiler feed formula is mostly corn-soybean-hybrid type, and soybean meal contains 30.3% of NSP (Smits and Annison, 1996), 70%~90% of which are cell wall components, and cell wall form of NSP is composed of solubility, particle size and Composition of molecular groups with different chemical structures. The anti-nutritional effects of NSP in the small intestine are reflected in the resulting chyme viscosity and inhibition of entrapped nutrient degradation (Bedford, 1999). Many experiments have shown that feed enzymes can open the connection between these polymers to reduce their viscosity in the intestine and increase feed nutrient digestibility. Tests have shown that viscosity is the best predictor of enzyme preparation in broiler barley diets (Rotter et al., 1989). Studies have shown that exogenous β-glucanase can significantly improve the performance of broilers fed barley and oat-type diets, while the effect on rye-type diets is not obvious, indicating that the two diets The substrate of enzyme action is quite different. The NSP in barley and oat is mainly β-glucan, while the NSP in wheat and rye is mainly arabinoxylan (Friesen et al., 1992). Choct et al. (1997) also found that for wheat, rye, triticale, barley, sorghum, rice, and corn diets, chicken diet metabolizable energy (AME) and arabinoxylan (R2 = -0.95) There is a strong negative correlation between NSP (R2=-0.97) content.

At present, the application technology of enzyme preparations is still not perfect, and its use still exists arbitrarily. Many experiments stay in verifying the effect of enzyme preparations, and the obtained test results are one-sided, and there are many controversies about the evaluation of the use effect of the composite enzymes. These all restrict the further promotion and application of enzyme preparations. In order to investigate the relationship between the composition and activity of the zymogram in the complex NSP enzyme and the potential energy value of the diet, the pepsin-pancreatin two-step enzymatic hydrolysis method was used as the technical means, and the change of the chyme viscosity was used as the detection index. The characteristics of NSP in chicken diets, and the zymograms adapted to them, provide a theoretical reference for users of NSP enzyme preparations.

1 Materials and methods

1.1 Instruments and reagents

pH meter (PHS-3C), UV-visible spectrophotometer (752N), magnetic stirrer, blast drying oven, constant temperature water bath oscillator, medical low speed centrifuge (40C), Brookfield viscometer (DV-II+Pro type) , pepsin (1:2000, Sigma), trypsin (Sigma).

1.2 Test enzyme preparation

Xylanase (3.60×10)

7 U/g), mannanase (7.20×10)

6 U/g), pectinase (1.50×10)

6 U/g).

The xylanase activity unit is defined as the amount of enzyme required to release 1 μg of reducing product per minute from a 1.0% oat xylan substrate at 37 ° C and pH 5.5, defined as a xylanase unit, U /g said.

The mannanase activity unit is defined as the amount of enzyme required to release 1 μg of the reducing product per minute from the 0.6% mannan substrate at 37 ° C and pH 5.5, defined as a mannanase unit, U/ g indicates.

The pectinase activity unit is defined as the amount of enzyme required to release 1 μg of the reducing product per minute from the 0.8% pectin substrate at 37 ° C and pH 5.5, defined as a pectinase unit, expressed in U/g.

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1.3 Test design

The types and contents of the plant raw materials in the test diet are shown in Table 1. The content of xylan, mannan and pectin in the diet is high. The experiment first added different gradient xylanase (4, 5, 6, 7, 8g/t) to the diet, and obtained the appropriate addition amount of xylanase through the change of chyme viscosity; After the amount was determined, the amount of xylanase was kept unchanged, and different doses of mannanase (0.5, 1.0, 1.5, 2.0, 2.5 g/t) were added to the diet; the same method was used to determine mannanase. After optimal addition, keep the optimum amount of xylanase and mannanase unchanged, and add different doses of pectinase (10, 15, 20, 25, 30g/t) to the diet. The change of viscosity determines the appropriate addition amount of pectinase, and finally the content of the suitable enzyme preparation combination and activity of the laying hen diet is obtained.

Table 1 Types and content of vegetable raw materials in laying hen diets

Raw material name content

Corn 55

Cardamom 15

Medan 8

菜粕5

1.4 in vitro digestion process

Minor modifications are made according to the methods of Bedford and Classen (1993). The specific operation is as follows: accurately weigh 1.25g of pulverized (over 1mm sieve) feed sample in a triangular flask, add different gradient exogenous enzyme preparations (xylanase, mannanase, pectinase), then add 1mg /mL fresh pepsin hydrochloric acid solution (0.1N, pH = 2.0), oscillated in a constant temperature water bath at 40 ° C, digestion time is 2h. After the end of the gastric digestion stage, 1 mL of fresh trypsin solution (5 mg/mL) was added to the solution, and the pH of the solution was adjusted to 6.5 (with 1 M NaOH solution), and shaken in a constant temperature water bath at 40 ° C for 4 h.

1.5 Test indicators

After the end of the in vitro digestion, the flask was taken out, the mixture was centrifuged at 3 000 g for 15 min, and the supernatant was taken for viscosity measurement using a viscometer (Brookfield DV-II+ Pro). Viscosity change (%) = additive chyme viscosity / control chyme viscosity × 100.

2 Results and discussion

2.1 pepsin-pancreatin two-step in vitro simulated digestion test

As the application of feed enzyme preparations becomes more and more in-depth, it is very important to accurately and scientifically evaluate and predict the effects of feed enzymes. Usually animal feeding test is an accurate and reliable evaluation method, but because the zymogram screening test is more complicated and there are more test groups, it is not only time-consuming and laborious to screen the enzyme spectrum by animal feeding test method, but also the test results are highly variable. Feed enzyme preparations are accurately assessed. The feasibility of in vitro simulated digestion techniques for assessing NSP enzymes and their role in the application of enzyme preparations has been demonstrated by experiments (Graham, 1988; Bedford and Classen, 1993). With the advancement of technology and the research progress of digestive physiology of monogastric animals, the in vitro digestion technology of monogastric animals has also been greatly developed. The most widely used is the pepsin-pancreatin two-step method, which measures the in vitro nutrient of pigs. There is a strong correlation between digestibility and in vivo digestibility, which can be used to predict the in vivo digestibility of various feed protein, dry matter, energy and other nutrients.

Exogenous enzyme preparations are affected by the environment in the digestive tract in addition to loss during feed processing (Tang Maojun et al., 2009). Therefore, the in vitro simulated digestion technique can better reflect the conditions of the catalytic reaction of the exogenous enzyme preparation in the digestive tract of livestock and poultry by simulating the environmental parameters in the digestive tract of livestock and poultry. Some scholars have successfully used in vitro digestion simulation techniques to evaluate and predict the effects of exogenous enzyme preparations in turkey and pig diets (Zyla et al., 1995; Liu et al., 1997). Bedford et al. (1993) used the in vitro method to accurately predict the effect of enzyme preparations on the viscosity and weight gain of small intestine chyme in broilers. Malathi and Devegowda (2001) used the in vitro method to determine the viscosity of the digested product and the total sugar release for zymography screening. Hou Xiaofeng et al. (2005) used a two-step method to accurately evaluate the biological titer of broiler diets supplemented with enzyme preparations, and obtained the regression of AME (Y, MJ/kg) and IVDE (X, MJ/kg). Equation: Y=0.824 2X+2.365 4 (r=0.884 6). Many of the above test results have proved the feasibility of the ex vivo method in the screening of zymograms.

In this experiment, the pepsin-pancreatin two-step method was used to simulate the physiological parameters of the digestive tract in vitro, including pH, digestive juice concentration, digestive enzyme species and concentration. Tests have shown that the viscosity of the feed gradually decreases as the amount of xylanase added increases. When the amount of xylanase added was 6.0g/t, the feed viscosity was the lowest, and the viscosity of the xylanase feed was not significantly decreased. Similar to xylanase, the feed viscosity decreased significantly with the addition of mannanase, but when the amount of addition increased to a certain extent (1.5g/t), the amount of enzyme added increased, and the viscosity of feed decreased little. Similarly, when the amount of pectinase added was 25 g/t, the feed viscosity was the lowest (Table 2).

Table 2 Optimum addition of xylanase, mannanase and pectinase

^{2.2 Comparison of the effects of single enzyme preparation and compound enzyme preparation on feed viscosity}

^{There are significant differences in the effects of different combinations of enzyme preparations on feed. Taking the feed viscosity as the detection index, the xylanase or mannanase was added to the feed, and the viscosity of the feed was reduced to a lesser extent than the xylanase + mannanase and xylanase + mannanase + fruit. Two enzyme enzymes (Fig. 1).}

^{In animal diets, the composition and content of NSP are different due to the different composition and content of raw materials, and the required NSP enzyme composition and activity are also different. Malathi and Devegowda (2001) have shown that the addition of xylanase, cellulase and pectinase to diets containing high soybean meal is more effective than adding only xylanase and cellulose due to the rich pectin in soybean meal. The enzyme works better. Therefore, only according to the composition and content of NSP in the diet, the corresponding NSP enzyme species can be added to fully exploit the nutritional value of the diet. However, most of the NSP enzymes currently used in production are identified as "universal complex enzymes", and their zymogram combinations lack dietary rations. Therefore, based on the success of the research on the evaluation method of the in vitro digestible energy of broiler chickens (Hou Xiaofeng, 2005) and laying hens (Zhao Min, 2006), this experiment uses pepsin-pancreatin two-step enzymatic hydrolysis as a means to The change of chyme viscosity was evaluated. According to the characteristics of NSP in the diet of laying hens, the optimum zymograms of xylanase, mannanase and pectinase were selected to be 6.0, 1.5 and 25g/t.}

^{Figure 1 Effect of different combinations of enzyme preparations on feed viscosity}

^{Because there are many factors affecting the enzyme preparation (Wang Jiqiang et al., 2004), therefore, this test does not fully prove that the in vitro enzymatic ability is consistent with the application effect of the enzyme preparation in actual production. The results of the in vitro enzymatic test must be combined with the animal feeding test in order to carry out a comprehensive evaluation, but the results of the in vitro enzymatic test provide a good reference for the enzyme preparation users in the screening.}

^(Reference)

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