A comparative study of the properties of two types of xylitose.

OA_show ('533');/Tang Maoyu Xu Ling Chen Xudongthe feed industry has to actively look for alternatives to antibiotics due to the ban on antibiotic growth promotersEnzymes, as non-toxic and non-residual green additives, are increasingly widely usedThe amount of NSP enzyme accounts for more than 60% of the total amount of enzyme preparationTherefore, the identification of NSP enzyme quality and the comparison of similar products of fermentation of different strains have received increasing attention from enzyme usersthe currentwide use of alacite, beta-glucanase and pectinase have many isoenzymes, as long as the enzyme microorganisms are different (except engineering bacteria), the structure of enzyme proteinist is usually differentThe degree to which their performance dynamics change with the change of external conditions varies greatlyIn such cases, many users of enzymes would like to have NRC-like enzymes using standard or templates for referenceHowever, the actual situation is that the quality evaluation and selection of feed enzyme synthase agents involve many factors, such as feed raw materials, processing processand and animal species, physiological stage and the diversity of the pH of the digestive tract, which limit the effective application of feed enzyme preparationsThe above conditions tend to result in significant differences in the use of the same vitality or the same type of enzymetherefore, solving these problems requires an analysis of the active ingredients of the feed enzymes and the factors affecting these active ingredients, the most effective method is animal feeding testingAlthough animal breeding test is the best means to evaluate the effect of enzyme preparation, its complicated operation process and long test period can not meet the requirements of the rapid development of modern enterprisesMoreover, most of the research on feeding enzymes focuses on animal feeding effects, rarely involves the evaluation of the activity of feeding enzymes, the only reports are also focused on the effects of different measurement methods on the activity of the feeding enzyme, and for the effect of its catalytic activity and stability, that is, the effective enzyme activity of enzymes lack of systematic research (Tang Maoyu, etc., 2009)Therefore, on the basis of simulating feed processing process and animal digestion process, the characteristics of xylitose from two different sources are evaluated by in vitro simulation digestion method (gastritas-trypsase two-step method), in order to provide a method for the in vitro evaluation of effective enzyme activity and a basis for scientific selection of enzyme preparations1 Materials and Methods1.1 instruments and reagentsdrum dryer, pH meter (PHS-3C), magnetic agitator, thermostat water bath oscillator, ULTRA-visible photophotometer (752N), medical low-speed centrifuge (40C), Brookfield viscometer (DV-II- pro type), gastric protease (1:2000), trypsin1.2 Test enzymexylitose A (2.40 x 105 U/g), xylitose B (2.37 x 105 U/g)1.3 Test Method1.3.1 Enzyme Activity Measurement MethodThe definition of enzyme activity unit is defined by international standards, i.ethe amount of enzyme required to degrade and release 1 smol reduction product from oat wood polysaccharide solution (1.0%) per minute at pH5.5 and 37 degrees C, defined as an enzyme living unitEnzyme activity assays are carried out in accordance with the methodprovided by Lu Wenqing and others (2002)the1.3.2 substrate solutionthe concentration of the wood polysaccharide substrate was 1.0%Accurately call xylitose (Sigma X0672) 1.000 g, add the dissolved NaOH (0.34 g) solution, add a moderate amount of distilled water and stir the side to heat, until the xylitose is completely dissolved, then add ice acetic acid 0.58 mL, continue to stir 30 min, with distilled water to 100 mLThis substrate is stored at 4 degrees C and is valid for 3 days1.3.3 High temperature stability testcalled 1.000 g xylitose A and 1.000 g lyuconase B, add a moderate amount of buffer to make the enzyme moisture content of 17-20%, respectively, in 37, 65, 75, 85, 95 degrees C drying thermostat box insulation 5 min, and then take out the test tube with tap water cooling, to determine the temperature of the enzymeThe active retention rate (%) was obtained by measuring enzyme activity results compared with the original enzyme activity 1.3.4 different pH tolerance formulated pH values of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, sodium acetate acid-acetate (0.1 mol/L) The xylitose A and xylitose B were treated in a temperature water bath at 37 degrees C with the above buffer, which had been prepared, and then measured the enzyme activity under different pH conditions The active retention rate (%) was obtained by measuring enzyme activity results compared with the original enzyme activity 1.3.5 Gastritas/trypse tolerance in vitro digestion test using the gastritas-trypse two-step method (Bedford and Classen, 1993) to assess the tolerance of xylolysase to gastritas and trypsas The specific operation method is as follows: take the properly diluted xylitose A and xylyse b solution to 20 mL test tube, add 1 mg/mL fresh gastric protease hydrochloric acid solution (0.1N, pH.2.0) Oscillate in a constant temperature water bath at 40 degrees C, with a digestion time of 2 h After the gastric digestion stage, 1mL of fresh trypsy solution (5 mg/mL) is added to the solution, the pH of the regulatory solution is 6.5 (with 1 M NaOH solution), oscillates in a heated water bath at 40 degrees C, and the digestion time is 4 h After in vitro digestion, the active retention rate of xylystose was determined according to the enzyme activity criteria (%) 1.3.6 xylitose on the viscosity of feed raw materials accurately called 0.1 g crushed feed samples (wheat, soybean meal), respectively, added the same concentration of xylitose A, B two solutions, while setting the non-enzyme feed as a control group Then, according to the above-mentioned gastritas-trypse in vitro digestion test method, in vitro simulation digestion After digestion, after 3000 g centrifugation 15 min, take the liquid and measure its viscosity with a Brookfield viscosity meter The viscosity of the feed of the enzyme group was compared with the viscosity of the blank group feed, and the effect of xylitose on feed viscosity was obtained (%) 2 Results and discussion 2.1 High temperature stability test the activity of xylitose A and B decreases with the tuning temperature (Figure 1), especially when the tuning temperature is higher than 85 degrees C, both of which are rapidly reduced to less than 50%; at present, most of the application of feed enzymes in the form of dry powder grain in the feed processing quality and grain before the addition, however, feed granulation is a high temperature and high humidity process, the vast majority of bioenzyme thermal stability is relatively poor, in high temperature granulation conditions are easy to lose Although the enzyme agent has very little loss of enzyme activity at 75 degrees C, the temperature of the grain producing in many feed plants in our country exceeds 75 degrees C or even reaches 90 degrees C, resulting in a great loss of enzyme activity Obviously, when adding enzymes to feed, their thermal stability is one of the most important evaluation indicators Thermal stability usually requires two indicators to be examined, one in wet and hot conditions, the other in dry and hot conditions, and the thermal stability of the enzyme agent measured in the first case is more practical (Lu Wenqing and Li Defa, 2004) Feed modulation and granulation process in high temperature and high humidity conditions, some enzymepreparations in the case of low moisture content (105 degrees C, insulation 2 h), can maintain a high enzyme retention rate, and in 80 degrees C water bath, the enzyme activity loss of more than 40% (Chesson, 1992) Pickford (1992) studied the thermal stability of three commodity enzymes The results showed that the tolerance of different enzyme stoices varied At a particle temperature of 80 degrees C, the activity of the first enzyme agent was retained at 85%, the activity of the second enzyme was retained by 55%, and the activity of the third enzyme was only 33%, while at 95 degrees C, all three enzyme strains were inactivated Squeezing puffing at short-term high temperatures also resulted in a greater loss of activity of these three enzyme preparations However, the paper does not indicate whether the fermentation strains of these three enzymes are similar or similar Studies have shown significant differences in thermal stability between xylitose produced by fermentation of thermomyces, Humicos, and Trichoderma (Pettersson and Rasmussen, 1997) The wood polysaccharide produced by trichoderma fermentation is significantly inactivated at a 75-degree-C-modulation temperature, while the xylitose produced by high-temperature mold and decay mold fermentation can also retain more than 80% of the activity at 85 oC tuning temperature The xylitose produced by high temperature mold retains more than 70% of its activity even at a high-temperature modulation temperature of 95 degrees C A review of Bedford and Schulze (1998) shows that many fungi and bacteria produce the most suitable temperature range for xylitose is 30-105 degrees C It can be seen that the tolerance of enzymes from different sources varies greatly to high temperatures In the experiment, although both xylitoses had high temperature stability, under the same temperature conditions, the loss of activity of xylitose B was lower than xylitose A 2.2 Different pH tolerance test the pH change range in different parts of the animal's digestive tract is very large, so the active enzyme performance under a single pH does not fully reflect the change in the activity of the enzyme in the digestive tract The enzyme activity of the enzyme formulation under the more widepH conditions was better evaluated to evaluate the advantages and disadvantages of different enzyme formulations The dynamic performance of two different sources of glycosase measured in the tests was shown in figure 2 in different pH conditions Under all measured pH conditions, the active retention rate of both types of glycosase was maintained at more than 50%, but the activity retention rate of xylitose B was higher than xylitose A Xylitose A has a high peak at pH 5.5, above and below this pH, and enzyme activity is reduced Under different pH levels, the activity change of xylitose B was more moderate, and the enzyme survival rate was more than 80% many enzymes can not tolerate adverse factors of the animal digestive tract, such as the acidic environment of the digestive tract and the degradation of endogenous protease external enzyme synthase agents, resulting in a decrease in the activity of enzyme preparations, affecting their use Such as normal pig and poultry digestive tract temperature of 40 degrees C, the pH in the stomach is 1.5-3.5, the small intestine pH is 5-7, the pH of the large intestine is neutral Therefore, it is required that the enzyme has a wide range of adaptation to pH A review of Bedford and Schulze (1998) shows that many fungi and bacteria produce the most suitable pH of 2.0-10 for xylitose Therefore, in addition to the use of advanced technology for enzyme preparations themselves, such as microcapsuleization technology, gel encapsulation technology, fiber encapsulation technology to improve the stability of enzyme preparations, but also to improve the evaluation of enzyme syntledelements How to quickly and accurately evaluate the selected enzyme preparation sourcing in animal sushes has become an increasingly strong requirement for enzyme users 2.3 Gastritas/trypsin tolerance test enzymes are highly effective bioactive catalysts, which are essentially proteins, so endogenous proteases in the digestive tract also affect their activity In this experiment, the activity of xylitose B was not affected by the gastroprotease/trypsin in the digestive tract, while xylitose A was reduced by 20% under the action of gastritoprotease/trypsin (Figure 3) It can be seen that there is a great difference in the tolerance of the two endogenous proteases, with the same amount added to the feed, xylitose B effect will be more significant Eric et al (1999) through the study of the digestive activity of cytoprotease by black-curic cypenase and E coli phytoase, it was found that black-curitic cypylysase resistance to gastric protease was not as resistant as E coli phytoase, the latter under the action of gastritis protease only reduced vitality by 6-10%, while black-curic mycosine physerase activity loss of 58-77% The results of Wang Yindong (2006) and other tests also show that E coli genetically modified phytic asases has a strong tolerance to gastric proteases This side-by-side verification of the tolerance differences between the two types of glucotose to endogenous proteases in the experiment may be related to its fermentation bacteria 2.4 (MalathiDevegowda,2001)。 The most striking feature of the high content of alacite in the feed formula is the increased viscosity of the meal, while the xylitose polymer breaks down into short chains, reduces the viscosity of the meal and releases the nutrients encased in it The effects of two xylitoses on wheat and soybean meal viscosity were determined respectively, and it was found that xylitose B was better than xylitose a (Figure 4), in which the feed added xylitose A, wheat and soybean meal viscosity decreased by 3.77% and 3.57%, respectively, while the added xylitose B, wheat and soybean meal viscosity decreased by 9.18% and 00% the types and contents of non-starch polysaccharides vary greatly from crop to crop, and their non-starch polysaccharides are soluble (Choct, 1997) Therefore, the viscosity of the same enzyme preparation is also different from that of different crops Wheat diet is the most studied diet to add enzymes to improve its nutritional value, mainly because wheat food contains anti-nutritional factors, such as alacite or beta-glucan and other water-soluble non-starch polysaccharides Annison (1991) on wheat soluble NSP research shows that it is mainly arabic xylitose, the chemical structure is very similar to rye arabic xylitose, these soluble NSP dissolved in the digestive tract, so that the adhesion of the diet adhesion increased, resulting in a decrease in the digestion rate of feed nutrients, resulting in a decline in animal production performance Choct and Annison (1992) demonstrated that the apparent metabolism of the minced chicken diet and the digestion of various nutrients decreased as the amount of wheat NSP extract was added, while the viscosity of small intestines increased Only about 70% of soybean cake can always be used by poultry, and only about 55% of soybeans can always be used by chicks, of which soybean oligosaccharides (mainly alpha-semi-lactose sacside, such as cotton sugar and water sucrose) lead to a decline in soybean cake energy utilization xylitosis is a type of hemicellulose widely found in plants, with a molecular structure of arabic xylitose, consisting mainly of Arabic sugar and xylitose, and a molecular main chain of 1,4-beta-xylitose (Annison, 1992) The number and distribution of the Arab sugar side chain varies according to the type and variety of grains, with the ratio of Arabic sugar and xylitose approximately 0.65-0.74:1 The more side chains there are, the easier the water molecules are to penetrate, the greater the solubility The complexity of the molecular structure of xylolycose determines that when selecting xylolyse, it is not enough to examine the high temperature stability, acid resistance and the degradation of endogenous protease when selecting xylitose.