Abstract: Digestible energy value is one of the main indicators for assessing the biological effectiveness of feed energy. Objective and accurate assessment of the digestible energy value of the feed is the main decision-making basis for determining the nutritional requirements of the animal and optimizing the feed formulation. This paper mainly introduces the research progress of in vitro enzymatic method, the factors affecting the accuracy of evaluation and some problems in the evaluation of feed digestion energy by in vitro enzymatic method.
Key words: pig; digestion energy; in vitro enzymatic method
introduction
Energy is the first element of feed nutrients. Metabolic energy value is one of the main indicators for assessing the biological efficiency of feed energy. Therefore, objectively and accurately assessing the metabolic energy value of feed is the main decision-making basis for determining the nutritional requirements of animals and optimizing feed formula. At present, the methods for assessing the nutritional value of feed are mainly in vivo, semi-in vivo (sports nylon bag method) and in vitro method (enzymatic method). In vivo and semi-in vivo methods for assessing the digestibility of feed nutrients in the small intestine are more objective and practical, but they all depend on the test animals. The semi-in vivo method also requires the necessary fistula surgery and test equipment. The test is cumbersome and expensive, and uncomfortable. In production, the enzymatic method is to use one or more enzymes or animal intestinal fluid to simulate the environment of animals, and to digest nutrients in vitro to assess the digestibility of nutrients. It is a fast and relatively accurate test. method.
The enzymatic method was first produced in the 1950s. Initially, the digestibility of feed protein was assessed by a single pepsin method. Although this method is fast and simple, it differs greatly from the data measured by in vivo methods (Grand and Carroll, 1958; Campbell, 1961). . Akeson and Stahmann (1964) further developed the enzymatic method and added trypsin to the pepsin. The true protein digestibility was strongly correlated with the data measured in vivo (r=0.995), which allowed pepsin plus trypsin. The method is a routine method for assessing the protein digestibility of monogastric animals, but this method is based on the assumption that the protein digestibility is not affected by other nutrient digestion, and the digestive tract zymogram is a complex and variable multi-enzyme system. Due to the different activation conditions required for various enzyme elements and the feedback inhibition of the enzyme on the substrate. Therefore, pepsin plus trypsin does not really reflect the digestive process in the body. Japan's Furuya (1974) proposed a method for the determination of in vitro digestion of pepsin plus small intestine. By comparing the in vitro method with the total feces, the dry matter and energy digestibility of the two methods were strongly correlated, and the measured values ​​were fairly consistent. . Domestic Zhang Ziyi et al. (1981-1988) have further studied this method and have formed a complete laboratory method for determining the digestibility of nutrients in pig feed.
Analysis of research status at home and abroad
Since Kuhn (1894) pioneered the use of energy to directly measure feed nutrients, the assessment of feed energy has been more than a century. The research on the digestive energy value of pigs is also continuously developed along with the improvement of research methods. The biological methods such as feeding test and metabolic test are a scientific and reasonable test method for assessing the effective energy value of feed, but these methods are often time-consuming and laborious. It is difficult to evaluate and analyze a large number of feed samples in a short period of time and it is not suitable for standardization. Therefore, it is necessary to seek a quick, accurate, simple and economical assessment method. For a long time, nutritionists in various countries have tried to establish a method for rapidly determining the biological potency of feed energy by enzymatic hydrolysis, but this method has been in the laboratory stage and has not been applied to production. The main reason is that the unresolved method The problem of standardization is essentially the standardization of digestive enzymes, namely the activity and type of enzymes. It is well known that the feed is mainly digested in the small intestine of pigs, and the digestive enzyme activity in the pancreas and intestine of pigs is not determined. If the pancreas or small intestine fluid is used as the source of enzymatic digestive enzymes, the variation of digestive enzyme activity is greater. However, the standardization problem cannot be solved. Therefore, the simulation of intestinal fluid and biomimetic digestion method based on the enzymatic properties of the pig small intestine will solve the major technical problems for the enzymatic quantitative measurement technology of the metabolic energy value of pig feed, and improve the pigs in China. The production level, saving feed resources and reducing the pollution of excreta to the environment have important social and economic benefits.
1 Enzymatic method
With the development of pepsin, trypsin, chymotrypsin crystals and subsequent development of various digestive enzyme assays in the 1930s, many scholars have fixed the simulation model of ex vivo digestion to step by step with one or a few key enzymes. Degraded in vitro reaction model, and carried out a series of studies, proposed a one-step enzymatic method and multi-step enzymatic hydrolysis method (Zhang Tieying, 2002).
1.1 one-step enzymatic hydrolysis
The one-step enzymatic method is based on the peptide bond cleavage of the protein under the action of protease, and the protein quality and its digestibility in the body are judged by the degree of hydrolysis of the protein (Sheffner et al. 1956; Taverner and Farrell, 1981; Hewitt et al. 1985; Meer et al 1990). Or by resolving the hydrogen ion causing a decrease in the pH of the solution and a protein in vivo digestibility to establish a regression equation that predicts the digestibility of the protein in the body (Maga et al., 1973; Hsu et al., 1977), but the accuracy of the prediction It may be affected by a variety of factors, especially the mineral buffering capacity of the feed sample. To this end, Pedersen and Eggum et al. (1983) improved it by continuously neutralizing the hydrogen ions produced by the proteolysis process with NaOH to keep the pH constant. Although this method is simple and fast, the factors considered are relatively simple. Due to the effects of anti-nutritional factors, heat treatment and physical properties of feed, the correlation between the results and biological methods is very low, and it is limited to the evaluation and prediction of feed protein digestibility, and is not suitable for the evaluation of other nutrients.
1.2 multi-step enzymatic hydrolysis
The digestion process of feed nutrients in animals is carried out by physical digestion and various digestive enzymes. Only by fully considering the key factors of nutrient digestion in the body, and constantly improving the simulation of various environmental conditions in the digestive tract, can we make The prediction results are more scientific. With the development of digestive physiology and enzymology of livestock and poultry, the in vitro digestion simulation technique of monogastric animals has also been developed, and has evolved from the single enzymatic method in the past to the current multi-enzyme method. The multi-enzyme method not only simulates the gastric digestion process, but also simulates the process of pancreatic secretion of digestive enzymes, small intestine digestion, and large intestine microbial digestion, that is, the digestion process of the livestock and poultry is completed simultaneously by the digestive enzymes of the children. According to the different parts of the digestive tract in the posterior segment, the multi-enzyme method can be easily divided into a two-step method of stomach-small intestine and a three-step method of stomach-small intestine-large intestine.
1.2.1 stomach-small intestine two-step method
Buchmann et al. (1979) used pepsin-pancreatin to predict the in vivo digestibility of proteins. The in vitro digestibility of 30 barley proteins determined by this method was strongly correlated with digestibility in rats. Subsequently, Boisen et al. (1991), Boisen and Fernandez et al. (1995) mainly studied the separation methods of degradants and undegraded materials in this method and proposed modifications. The digestibility of feed dry matter, organic matter, protein and energy measured by the revised scheme is strongly correlated with biological methods. However, in this method, the accumulation of digestion products will inhibit the enzymatic reaction, resulting in a decrease in nutrient digestibility in vitro and affecting the accuracy of predicting nutrient digestibility in vivo. In addition, in this method, trichloroacetic acid or sulfosalicylic acid solution is used to precipitate the undegraded but water-soluble protein in the reaction solution, but the digestion products of starch and fat cannot be simply trichloroacetic acid or sulfo-based water. Salicylic acid to separate. At the same time, some small peptides that can be absorbed by animals are also artificially classified as undigested proteins. To this end, some scholars have established a method for removing digestion products by means of dialysis in order to overcome the inhibition of enzymatic reaction by digestion. The concentration of digestive enzyme, feed particle size, dialysis time, molecular weight of dialysis bag, and dialysate Some studies have been carried out on factors such as flow rate and enzymatic reaction temperature, and the conclusion that the correlation between dialysis and biological methods is higher than that of the unremoved product method is obtained, and several sets of in vitro digestion simulation procedures are proposed (Cave et al. > 1988; Gauthier et al., 1982; Savoie and Gauthier, 1986; Drake, 1991; Huang Ruilin et al., 1999; Zhang Tieying et al., 2002).
The dialysis bag used in these methods has a molecular weight cutoff of 100 OOA, which is capable of passing amino acids and a part of small peptides, which is close to the actual absorption of proteins in the body. Therefore, it is more reasonable in predicting the in vivo digestibility of proteins than the unremoved product method. However, the pore size of the dialysis bag is mainly directed to protein degradation products, and does not improve the predictive value of feed energy and other nutrient digestibility. The feed for livestock and poultry is initially digested by the stomach and transported in batches to the next digestive tract (duodenum) by gastric movement. Therefore, if the in vitro digestibility of the feed is determined by the pig small intestine liquid, in addition to the main digestive enzymes such as trypsin, chymotrypsin, amylase and lipase, trace substances secreted by the pancreas, liver secretions and intestinal peptidase affect some nutrient digestion. The ingredients can be obtained from the pig intestinal fluid. This allows the in vitro digestion simulation process to be closer to the body and the results are more reliable and reasonable. The Institute of Animal Science of the Chinese Academy of Agricultural Sciences systematically researched and improved the method in the 1980s, and established a standardized operating procedure. The in vitro digestibility of feed and energy were determined by this method. There is a strong correlation (R2=0.95 0.98).
1.2.3 stomach-small intestine-large intestine three-step method
One-step and two-step enzymatic hydrolysis mainly simulates the digestion of the stomach or stomach and the small intestine, and does not consider the role of microorganisms after the ileum, while the energy provided by the volatile fatty acids produced by microbial fermentation in the pig's rear digestive tract is provided in the diet. 12% of energy (Kass et al., 1980). There are certain limitations in using these methods to predict the apparent digestibility and potency of nutrients. Fonty and Goue (1989) and Ahrens et al. (1991) found that rumen microbes have much in common with the composition and fermentation principles of microbes in the porcine large intestine. To this end, some scholars have proposed pepsin-pancreatin-ruminal fluid (Fonty and Gouet, 1989) and a three-step enzymatic hydrolysis of pepsin-pancreatin-carbohydrate and established protocols (Metz and Vander Meer, 1985; Boisen and Fernandez, 1994, 1997). Compared with the carbohydrate enzymatic method, the rumen fluid method takes a long time to digest treatment and is greatly affected by the source of the rumen fluid; while the carbohydrate enzyme is a commodity, the components are stable, and the time for digestion treatment is also short. Boisen and Fernandez (1997) have a linear correlation between the in vitro organic matter digestibility and the in vivo energy digestibility of 90 feed samples and 31 compound feeds determined by the carbohydrate enzymatic method (RZ=0.94), indicating the use of pepsin-1 The three-step enzymatic hydrolysis of trypsin-carbohydrate can better predict the digestive energy of monogastric animal feed.
2 factors affecting the evaluation of enzymatic methods
2.1 Effects of animal species and small intestine
Chen Xuexiu et al. (1984) studied the in vitro digestibility of small intestines from different breeds of pigs (Dubei, Changfeng, Beijing Black).
The results showed that the amylase and trypsin activities of the small intestine of Beijing black pig were higher than that of Dubei, and the low-protein diet was determined by the in vitro dry matter digestibility and energy digestibility measured by Dubei pig small intestinal fluid. There was no difference between Changfeng and Beijing black pigs. The high energy protein digestibility of Dubei pig small intestine was lower than that of Beijing black pig small intestine (P<0.05).
Studies have shown that the enzymatic activity of intestinal fluid is related to the location of the small intestine (Toofanian et al., 1973; iddons, 1968; Russell et al., 1981). Thus, the intestinal enzyme activity collected from different parts of the small intestine is different, and the measured nutrients are isolated. Digestibility may vary. Keys et al. (1974) reported that the nutrient content of feed in the small intestine was very different, with an organic apparent digestibility of 29.8% in the upper part of the small intestine and 65.9% in the middle and lower part of the small intestine. Chen Xuexiu et al (1984) showed that the ex vivo digestibility of pigs treated with small intestines collected from the pylorus 50 cm was quite different from the conventional method. For example, the dry matter digestibility was 4.7%-5.1% lower than that of the conventional method. The rate was 6.5-8% lower, and the measured value of the posterior iliac small intestine collected at 170 cm from the pylorus was similar to the conventional method.
2.2 Effects of different dietary nutrient levels on intestinal fluid
Animal diet levels affect intestinal digestive enzyme activity (Clary et al, 1969; Van Hellen et al 1978; Russell et al 1981; Jahnson et al 1977; Owens et al, 1986). Thus, there may be differences in ex vivo digestibility measured with small intestinal fluids derived from different dietary types. Karrel et al. (1966) and Owens et al. (1986) reported that amylase activity is one of the factors affecting the digestibility of starch in the small intestine. Wang Shurong et al. (1981) reported that a single feed-bran was fed for ten consecutive days before collecting pig intestinal fluid, and then the intestinal juice was collected. The in vitro digestibility of the intestinal juice and the mixed intestinal juice was compared, and the mixture was treated in vitro. The material digestibility is 7-10% higher than that of the single-feed bran treatment, but the protein and energy digestibility are not much different. Jiang Yunxia et al (1983) showed that pig small intestine fluid fed different nutrient levels (high energy low protein diet and low energy high protein diet) had no significant effect on in vitro dry matter digestibility and crude protein digestibility. .
2.3 Environmental pH and the impact of Ca2+
Liang Yuyi et al. (1988) reported that the digestibility of crude protein in pig small intestine lyophilized powder (PIF) was less affected by pH. When the pH was in the range of 7.0-8.5, there was no significant difference in the digestibility of PIF.
2.4 The relationship between the amount of lyophilized powder in intestinal juice and ex vivo digestibility
Zhang Jianzhong (1997) studied the effect of the amount of bovine intestinal lyophilized powder (BIF) on the digestibility of starch. Studies have shown that when the amount of BIF is 0.3 g / / 0.5 g of feed (the activity of amylase in BIF is 302.18 IU / g), the digestibility of starch tends to be stable, and then increase the amount of BIF, the starch digestibility does not change much. Li Jianguo (1997) studied the effect of BIF dosage on the digestibility of rumen non-degradable protein (UDP). As a result, when the amount of lyophilized powder of small intestinal juice was above 12 units/0.5g UDP, the digestibility of UDP was basically stable. When using the small intestine lyophilized powder to determine the digestibility of nutrients, the appropriate use of lunar milk should be selected.
2.5 The effect of culture time on ex vivo digestibility
Liang Yuyi et al. (1988) measured the crude protein digestibility of three pig diets after pepsin-PIF digestion for 3 hours and 4 hours, respectively. The results showed that different culture time had no effect on the digestibility of crude protein. Wang Zhongsheng (1994) used phage and BIF to evaluate the UDP digestibility test. When the pepsin treatment time was fixed at 4 hours, the BIF culture time was prolonged, and the feed UDP digestibility rate increased, but the culture time More than 6 hours, growth has stagnated. Zhang Jianzhong (1997) showed that the in vitro digestion test of BIF on starch, cooked starch and rumen 24-hour corn residue showed that for starch and cooked starch, the incubation time of BIF was stable from 6 hours, and the digestibility at each time point after 6 hours was not. Significant difference (P>0.05), the starch digestibility of corn and rumen 24-hour corn residue was stable from 7 hours, and the difference of starch digestibility at 7 hours after each time was not significant (P>0.05). When using the small intestine lyophilized powder for in vitro digestion test, different culture time should be selected according to different feeds.
Effects of different processing conditions and different storage conditions of 2.6 intestinal lyophilized powder on ex vivo digestibility
Zhang Weiyun et al. (1984-1985) tested the trypsin activity, in vitro dry matter digestibility and ex vivo energy digestibility of PIF under different processing conditions and different storage conditions. The results showed that under different processing and storage conditions (The small intestinal juice was thawed and twice thawed; the PIF after lyophilization was vacuumed and stored without vacuum: the PIF stored in vacuum was stored at room temperature and 0 °C in the refrigerator), and the trypsin activity of PIF was basically unchanged. ((P>0.05); whether the PIF raw material before processing is double-thawed or once thawed, whether it is vacuumed or not vacuumed, as long as it is kept below 25 °C, it can maintain the stability of at least 3 months. However, when the temperature exceeds 25 ° C, the PIF freeze-dried powder titer has a downward trend.
3, the current problems
The in vitro assessment method has developed greatly from the simplicity of operation and the scientific rationality of the simulation results, but there are still many shortcomings. First of all, the digestion parameters in the digestion model are only partially consistent with the actual situation in the body, while some parameters are far apart and need to continue to improve and develop. Secondly, most digestion models do not take into account the effects of age on the energy digestibility of animals. They cannot accurately predict the digestibility of feed energy in livestock at different stages of growth, especially for animals with faster growth rates such as piglets. It can be seen that there is still a long way to go to study the method of predicting the effective energy value of feed by using the in vitro method.
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