CN109085298B - High-water-solubility pig small intestine digestive enzyme powder as well as preparation method and application thereof - Google Patents
High-water-solubility pig small intestine digestive enzyme powder as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a high water-solubility pig small intestine digestive enzyme powder and a preparation method thereof. The pig small intestine digestive enzyme powder is prepared by the following steps: 1) collecting intestinal chyme from small intestines of a plurality of pigs, uniformly mixing the intestinal chyme, centrifuging the mixed intestinal chyme 1, and collecting supernatant 1; 2) evaporating and concentrating the supernatant fluid 1 to obtain concentrated intestinal juice; centrifuging the concentrated intestinal juice 2, and collecting a supernatant 2; 3) transferring the supernatant 2 into a container placed in an ice-water bath, adding mannitol, stirring for dissolving, adding an ethanol solution, stirring uniformly, and standing; then centrifuging 3, and collecting the precipitate; 4) adding deionized water into the precipitate, stirring and ultrasonically dissolving, then centrifuging 4, and collecting supernatant 3; and (3) freeze-drying the supernatant 3 to obtain the product. The prepared intestinal tract digestive enzyme powder improves the specific activity of digestive enzyme, reduces the impurity content and improves the solubility of the digestive enzyme.
Description
Technical Field
The invention relates to high-water-solubility pig small intestine digestive enzyme powder and a preparation method and application thereof.
Background
In the field of feed production, the purchase of feed raw materials and the optimization of feed formulas are very dependent on a method for objectively evaluating the titer of feed nutrients (such as energy and amino acid) or the effective content of functional ingredients under laboratory conditions. For this reason, various researchers have attempted to establish a technique for rapidly evaluating the available nutrients of feeds by simulating the digestion of the feeds in animals. In the technology, a digestive enzyme reagent (or preparation) is the most critical material, and determines the simulation effect on the in-vivo digestive environment, so that the objective evaluation on the feed nutrient titer is influenced. Meanwhile, in the in vitro simulated digestion, after the digestion of the stomach is finished, the small intestine digestive enzyme is added in the subsequent digestion stage, and the higher concentration is needed to reach the activity level of the digestive enzyme in the animal body, so the solubility of the digestive enzyme reagent is very important.
At present, the preparation of gastric juice for in vitro digestion of animals by pepsin is a well-established method (national technical committee for feed industry standardization GB/T17811-2008. measuring and filtering method for pepsin digestibility of animal protein feeds. Beijing: Chinese Standard Press, 2008), and Sigma-Aldrich company of the United states has the enzyme reagent product sold to the outside. In the preparation of intestinal juice, Pancreatin (also known as Pancreatin; known in the English trade as Pancreatin; mainly containing trypsin, chymotrypsin, amylase, lipase) is used as the enzyme source (Boisen, S., and J.A. Fernandez.1997.Presition of the total track differentiation of energy in feeds and pig fractions by in vitro analysis, anim. feed Sci.Technol.,68: 277-. The pancreatin is prepared by obtaining animal pancreatic tissue from slaughter house, mincing, homogenizing, adding calcium chloride and protease to activate zymogen, filtering, precipitating enzyme protein with isopropanol or acetone, and freeze drying the precipitate directly or defatting with acetone to obtain pancreatin product. The method has the defects that the loss of the activity of digestive enzyme is large in the extraction process (the recovery rate is generally below 15%), the specific activity of trypsin of powder is not high, the water solubility is poor (0.2% aqueous solution is obviously turbid), and the quantity of zymogen converted into active digestive enzyme is obviously influenced due to the change of the standing time and the temperature of animal pancreas after slaughter, so that the batch stability of the extracted pancreatic enzyme powder is seriously influenced, and the like. In addition, isopropanol or acetone used in the preparation process has great toxicity to human body. (Wuxiaying et al 2005. research on a novel process for preparing pancreatin, proceedings of Guangdong college of pharmacy, 21: 66-67; Gumegabin et al 2009, optimization research on an extraction process of pancreatin in porcine pancreas, food science, 30: 162-164). Aiming at the technical defect of pancreatin preparation, the national key laboratory of animal nutrition of the institute of animal husbandry and veterinary, Beijing of Chinese academy of agricultural sciences, started from 2009, has developed the steps of obtaining chyme from an animal body provided with a small intestine casing, preparing small intestine liquid after low-temperature centrifugation, and then obtaining intestinal enzyme powder after dialysis and desalination by freeze-drying (Severe. 2012. the influence of the composition and storage conditions of the enzyme on the digestive capacity of simulated duck intestinal liquid. Master academic paper of Huazhong university of agriculture: Wuhan); or precipitating enzyme protein from the prepared small intestine solution by 70% saturated ammonium sulfate, precipitating, re-dissolving and centrifuging by using a phosphate buffer solution with the pH value of 7, taking supernate, dialyzing, desalting and freeze-drying to obtain the intestinal enzyme powder (Huguan, 2010. research on design basis of the bionic digestion reagent for growing the small intestine of the pig. Master academic paper of Chinese agricultural academy of sciences: Beijing). The extraction method of the digestive enzyme has the following common problems: ammonium sulfate or organic solvent in the extraction process easily causes the loss of the activity of digestive enzyme, and the salt removal effect by dialysis is not ideal, so that the prepared digestive enzyme powder has low specific activity and high ash content (> 25%). Meanwhile, in the subsequent treatment, even if the intestinal enzyme powder is degreased by acetone or ethanol, the fat content can still reach more than 10 percent, and the solubility is poor (the water solubility is less than 1 percent).
With the increasing demand of feed industry for the actual measurement data of the digestible nutrient content of feed, the identification of biological effects of human and animal food and drugs also needs to rely on an in vitro simulated digestion method for evaluation. In order to further approach the real situation in vivo by the simulated digestion effect, the requirement of intestinal digestive enzyme powder with high solubility and high activity has a wide market. The method can be used for solving a series of problems of large activity loss, low specific activity, low water solubility and the like in the preparation process of the intestinal digestive enzyme in the existing preparation method. Therefore, there is a need to overcome the above drawbacks and to provide a method for preparing digestive enzymes from intestinal juice.
Disclosure of Invention
The invention aims to provide a preparation method of high-water-solubility pig small intestine digestive enzyme powder. The method can efficiently extract amylase, trypsin and chymotrypsin compound in small intestine liquid, and the solubility of the activity of main digestive enzyme of the purified powder in water solution is more than 13 times of that of small intestine liquid in vivo. The method of the invention can solve the defects of high digestive enzyme activity loss and low enzyme specific activity in the traditional preparation process and overcome the problem of low water solubility when the enzyme powder is used.
In order to achieve the purpose, the invention adopts the following technical scheme:
the preparation method of the high-water-solubility pig small intestine digestive enzyme powder provided by the invention comprises the following steps:
1) collecting intestinal chyme from small intestines of a plurality of pigs, uniformly mixing the intestinal chyme, centrifuging the mixed intestinal chyme 1, and collecting supernatant 1;
2) evaporating and concentrating the supernatant fluid 1 to obtain concentrated intestinal juice; centrifuging the concentrated intestinal juice 2, and collecting a supernatant 2;
3) transferring the supernatant 2 into a container placed in an ice-water bath, adding mannitol, stirring for dissolving, adding an iced ethanol solution, stirring uniformly, and standing; then centrifuging 3, collecting the precipitate and washing;
4) adding deionized water into the precipitate, stirring and ultrasonically dissolving, then centrifuging 4, and collecting supernatant 3; and (3) freeze-drying the supernatant 3 to obtain the high-water-solubility intestinal digestive enzyme powder.
In the step 1) of the method, the pigs are castrated boars of 15kg to 125 kg. Castrated boars with similar genetic background and similar parity are preferred.
Treating and feeding pigs according to the following method before collecting chyme: the T-shaped fistulas are arranged on small intestines of a plurality of pigs, after 10 days of postoperative recovery, complete formula feed (comprising corn 71.80%, soybean meal 22.20%, soybean oil 1.18% and vitamin trace elements) with crude protein content of more than 18% and metabolic energy value of more than 3200kcal/kg is fed to the pigs, the pigs are fed twice a day, the pigs are fed freely, drinking water is not provided before intestinal chyme collection, and the intestinal chyme is collected through the intestinal T-shaped fistulas 2-4 hours after feeding.
The supernatant is intestinal juice containing amylase, trypsin and chymotrypsin. The conditions of the centrifugation 1 are as follows: centrifuging at 9000-11000 g for 10-15 minutes at 4-8 ℃.
In the step 1), under proper centrifugal force, the undigested and complete food in the chyme is precipitated from the intestinal juice, and digestive enzyme protein is still remained in the intestinal juice, so that 85-95% of impurities in the chyme are removed.
In step 2), the evaporation concentration is performed on a rotary evaporator, and the conditions of the evaporation concentration are as follows: evaporating for 15-60 minutes (preferably 30-60 minutes) under the conditions that the water bath temperature of the distillation flask is 39-59 ℃, the rotating speed is 80-120 r/min and the vacuum degree is 0; specifically, the following can be selected: rotary evaporation at 39 ℃ for 60 minutes; or rotary evaporation is carried out for 30-45 minutes at 49 ℃; or rotary evaporation at 59 ℃ for 15-30 minutes. And when the volume of the concentrated solution is reduced to 30-50% of the original volume, stopping evaporation.
For the small intestine liquid of the pig, the residual rate of digestive enzyme is the highest after rotary evaporation for 1 hour at the temperature of 39 ℃.
The conditions of the centrifugation 2 are as follows: centrifuging at 9000-11000 g for 10-15 minutes at 4-8 ℃.
In the step 2) of the method, the intestinal juice is evaporated to remove more than 50% of water under the conditions of approaching the body temperature of the animal and negative pressure, so that the concentrations of digestive enzymes and impurities are increased by about 1 time under mild conditions, partial impurities with large molecular weight or large volume can be further removed by centrifugation, and the zymoprotein molecules cannot be precipitated and removed under the centrifugal force.
In the step 3), the mannitol is added in an amount of: adding 1.5-2 g of the supernatant 2 into each 100ml of the supernatant.
For intestinal juice, the activity, recovery rate and solubility of chymotrypsin, trypsin and amylase can be effectively improved by adding mannitol with the concentration of 2% into the intestinal juice.
The glacial ethanol solution is stored in a refrigerator at the temperature of-20 ℃ for more than 24 hours before use, and the ethanol is absolute ethanol or ethanol solution with the volume fraction of more than 95%.
The adding amount of the glacial ethanol solution is as follows: 350-450 ml of the supernatant 2 is added into every 100ml of the supernatant.
The standing conditions are as follows: standing for more than 12 hours at the temperature of minus 10 ℃ to minus 25 ℃ (preferably minus 18 ℃ to minus 25 ℃).
The conditions of the centrifugation 3 are as follows: centrifuging at 9000-11000 g for 10-15 minutes at 4-8 ℃.
The specific method of flushing is as follows: the precipitate is rinsed with 30ml of deionized water for 10-30 seconds each time, and 2 times.
The mannitol in the step 3) of the method can be coprecipitated with the digestive enzyme, and the problem of poor water solubility of the digestive enzyme caused by ethanol precipitation of enzyme protein can be solved through the hydrophilic effect of the mannitol.
In the step 4) of the method, the deionized water is added in an amount which is 50-100% of the volume of 1 volume of the supernatant obtained after the intestinal chyme is centrifuged.
For small intestinal juice, 1 volume of dissolved solution is matched with a stirring and ultrasonic method to renaturate digestive enzymes, so that the recovery rate of chymotrypsin and trypsin can be effectively improved.
The conditions of stirring and ultrasonic dissolution are as follows: dissolving for 10-15 minutes under the condition of 300rpm of a magnetic stirrer, and then dissolving for 5-10 minutes under the condition of an ultrasonic frequency of 39-41 kHz.
Preferably, 1 time volume of deionized water relative to supernatant 1 is used for dissolution under stirring for 15 minutes + sonication for 10 minutes.
The conditions of the centrifugation 4 are: centrifuging at 9000-11000 g for 10-15 minutes at 4-8 ℃.
Dissolving mannitol in the precipitate in the step 4) in water to expose hydrophilic group of digestive enzyme, which is favorable for dissolving enzyme. During the freeze-drying process, mannitol and digestive enzymes lose water together to form a powder. In the use of the finished powder, deionized water is added, and mannitol is dissolved firstly to expose hydrophilic groups of digestive enzyme, so that the problem of poor water solubility of the powder is solved.
It is still another object of the present invention to provide a highly water-soluble porcine small intestine digestive enzyme powder.
The high-water-solubility intestinal digestive enzyme powder is prepared by the method.
The invention also protects the application of the high water-solubility intestinal tract digestive enzyme powder.
The application of the invention is the application of the animal simulated digestive juice in preparation of the animal simulated digestive juice.
The animals and the animals for preparing the intestinal juice collected by the high water-solubility intestinal digestive enzyme powder are homologous animals.
The invention has the following beneficial effects:
1. the invention can evaporate the intestinal juice for 15-60 minutes under the vacuum of 39-59 ℃, and can reduce the volume of water in the intestinal juice by more than 50%. The loss of digestive enzyme activity by exposure and concentration of intestinal fluids to shorter time periods close to body temperature can be reduced to within 20%.
2. The concentration of digestive enzyme protein in the intestinal juice is improved by about 1 time by evaporating and concentrating the intestinal juice, thereby being very beneficial to the subsequent precipitation of enzyme protein by ethanol, reducing the dosage of reagents such as ethanol and the like, being beneficial to the protection of enzyme activity and further improving the recovery rate of the enzyme activity.
3. After the intestinal juice is concentrated (the volume is concentrated by more than 50%), 1.5-2% of mannitol (wt/vol) is added, and the mannitol can capture the water in contact with the digestive juice in the concentrated intestinal juice, so that the concentration of the digestive enzyme in the aqueous solution is further improved, and the precipitation of digestive enzyme protein is facilitated.
4. After the intestinal juice is concentrated (the volume is concentrated by more than 50%), 1.5-2% of mannitol (wt/vol) is added, and after the glacial ethanol is added to be used as a precipitator of the intestinal juice digestive enzyme protein, the mannitol can be coprecipitated with the digestive enzyme, so that the exposure of a digestive enzyme protein glycidyl group is greatly reduced, and the redissolution of the digestive enzyme in water is facilitated.
5. After mannitol and digestive enzyme are coprecipitated, the mannitol is dissolved by water to expose hydrophilic groups of the zymoprotein, and the dissolution of zymoprotein molecules is promoted. When the intestinal enzyme powder is freeze-dried, mannitol is combined with the hydrophilic group of the digestive enzyme, and the hydrophilic group of the digestive enzyme is exposed by dissolving mannitol during redissolution, so that the solubility of the activity of the enzyme protein can reach more than 13 times of the intestinal fluid in vivo, and the high water solubility of the intestinal enzyme powder is realized.
Drawings
FIG. 1 is a flow chart of the preparation process of the high water-solubility pig small intestine digestive enzyme powder.
Detailed Description
The method of the present invention is illustrated by the following specific examples, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included within the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of highly Water-soluble intestinal digestive enzyme powder from porcine intestinal juice
1.1 Collection of porcine intestinal fluid
Selecting 12 castrated boars with similar genetic background, similar birth times and weight of 18.05 plus or minus 1.57kg (average value plus or minus standard deviation), and feeding in a single cage in a metabolism cage. After 1 week of adaptation, a T-shaped fistula is installed at the front end of the small intestine, the small intestine is fed into a metabolism cage after operation, nursing is carried out for 3 weeks according to a conventional method, small intestine chyme is collected and uniformly mixed after the fistula pig recovers health, then the mixed small intestine chyme is centrifuged (the centrifugation condition is that centrifugation is carried out for 10-15 minutes at 9000-11000 g), and supernatant fluid 1 (namely small intestine fluid) is collected; and (3) uniformly mixing 40L of intestinal juice, subpackaging into 1L of plastic bottles for storage for later use, sampling and determining the activity of the uniformly mixed intestinal juice trypsin, chymotrypsin and amylase. At the chyme collection period, 08:00 and 16:00 per day are mixed with wet feed for feeding test feed (the feed comprises 71.80% of corn, 22.20% of soybean meal, 1.18% of soybean oil and 4.82% of vitamin trace elements), and in order to eliminate the influence of feed intake difference on test pigs, the test pigs are fed according to 4% of body weight and freely drink water.
1.2 preparation process of high water-soluble intestinal digestive enzyme powder
Putting the 1L of jejunum liquid supernatant into a 1-2L of spherical distillation flask; putting the distillation flask into a rotary evaporator, and performing rotary evaporation (at 39-59 ℃, the rotation speed of 80-120 r/min and the vacuum degree of 0) to obtain concentrated intestinal juice; centrifuging the concentrated intestinal juice (centrifuging conditions are that the temperature is 4-8 ℃ and the concentration is 9000-11000 g for 10-15 minutes), and collecting supernatant 2;
transferring the supernatant 2 into a container placed in an ice-water bath, adding 1.5-2 g of mannitol into 100ml of the supernatant 2, stirring and dissolving, slowly adding 350-450 ml of mannitol into each 100ml of the supernatant 2, storing the mixture in an ethanol solution (the content is more than 95%) for more than 24 hours at a refrigerator at the temperature of-20 ℃, slightly stirring, standing for more than 24 hours at the temperature of-18-25 ℃, removing 50% of supernatant, and transferring the rest precipitate into a centrifugal bottle; then centrifuging (centrifuging at 4-8 ℃ and 9000-11000 g for 10-15 minutes), and collecting precipitates;
adding a certain amount of deionized water into the precipitate, stirring, ultrasonically dissolving (ultrasonic frequency is 39 kHz-41 kHz for 25 minutes), centrifuging (centrifuging at 4-8 ℃ and 9000-11000 g for 10-15 minutes), and collecting supernatant 3; and (3) freeze-drying the supernatant 3 to obtain the product.
The main digestive enzyme activities during purification were determined as follows: the activity of alpha-amylase (ec3.2.1.1) was measured using soluble starch as a substrate, and the unit of amylase activity was defined as the activity that 1 μmol maltose was released per minute at pH 6.90 at 25 ℃. The trypsin activity was measured using p-benzenesulfonic acid-L-arginine methyl ester as a substrate, and the unit of trypsin activity was defined as the activity exhibited when 1 μmol of p-toluenesulfonic acid-L-arginine was released per minute at a pH of 8.10 at 25 ℃. The activity of chymotrypsin was measured using benzoyl-L-tyrosine ethyl ester as a substrate, and the unit of chymotrypsin activity was defined as the activity of releasing 1 μmol of benzoyl-L-tyrosine per minute at 25 ℃ and pH 8.0.
1.3 Effect of the preparation Process of the highly Water-soluble intestinal digestive enzyme powder
1.3.1 effects of Rotary steaming temperature and time on the recovery of digestive enzyme activity and reduction of moisture in intestinal juice of pigs
TABLE 1 recovery of pig intestinal juice supernatant 1 digestive enzyme Activity and intestinal juice volume at different rotary evaporation temperatures and times
Residual rate
In the process of reducing the moisture of the small intestine liquid of the pig by rotary evaporation, rotary evaporation is carried out for 30-60 minutes at 39 ℃, the recovery rate of 3 main digestive enzyme activities is more than 80%, and the moisture can be evaporated by more than 26%; rotary evaporation is carried out for 30-60 minutes at 49 ℃, the recovery rate of 3 main digestive enzyme activities is more than 68%, and water can be evaporated by more than 42%; rotary evaporation is carried out for 15-30 minutes at 59 ℃, the recovery rate of 3 main digestive enzyme activities is more than 77%, and water can be evaporated by more than 32%. Therefore, rotary evaporation is preferably carried out at 39 ℃ for 60 minutes in removing moisture from intestinal juice; or rotary evaporation is carried out for 30-45 minutes at 49 ℃; or rotary evaporation at 59 ℃ for 15-30 minutes.
1.3.2 effects of recovery of digestive enzyme activity and specific activity of purified powder after adding mannitol into supernatant 2 obtained by rotary evaporation and concentration of porcine small intestine solution
TABLE 2 Effect of mannitol concentration on the digestive enzyme Activity recovery and specific Activity in purification of porcine Small intestine digestive enzyme
When the concentration of mannitol added into the intestinal juice supernatant 2 is 2%, the recovery rate of 3 main digestive enzyme activities is over 53%; and the recovery rate of the digestive enzyme activity and the specific activity of the digestive enzyme in the purified powder are both higher than the value of adding 1.5 percent of mannitol. Therefore, mannitol is preferably added to the intestinal juice supernatant 2 at a concentration of 2%.
1.3.3 Effect of the supernatant 2 obtained by rotary evaporation and concentration of porcine intestinal juice in a redissolution mode after adding mannitol and precipitating the zymoprotein by ethanol
TABLE 3 Effect of dissolution volume and dissolution mode on recovery of Activity of digestive enzyme lyophilized powder for Small intestine
Mannitol is added into the supernatant 2 of the pig small intestine liquid rotary evaporation concentration, the volume of deionized water used for redissolving after ethanol precipitation of the enzyme protein and the dissolving mode have obvious influence on the recovery rate of the chymotrypsin activity, and the dissolving mode has obvious influence on the recovery rate of the trypsin activity. By combining the above treatments, it is preferable that the recovery rate of 3 main digestive enzyme activities is above 66% after 1 time of dissolution in deionized water volume relative to the supernatant 1 under stirring for 15 minutes + ultrasonic treatment for 10 minutes. 1.3.4 supernatant from rotary evaporation and concentration of intestinal juice 2 is added with mannitol, ethanol precipitation enzyme protein is redissolved, and the solubility of digestive enzyme of freeze-dried powder is obtained
TABLE 4 solubility of purified Enterobacteriaceae enzyme powder
In the preparation process of the high water-solubility intestinal digestive enzyme powder, when the concentration of mannitol added into the intestinal juice supernatant 2 is 1.5 percent or 2 percent, the solubility of the main digestive enzyme activity in the purified digestive enzyme powder 3 can reach more than 13 times of the concentration of the digestive enzyme activity in the intestinal juice of pigs, thereby achieving the effect of high water solubility.
Example 2 Effect of simulated intestinal fluid preparation based on purified powder of porcine intestinal fluid
2.1 preparation of highly Water-soluble purified powder of digestive enzyme of pig Small intestine
The preparation method of the purified powder of digestive enzymes of small intestine is the same as that of example 1.
2.2 preparation of intestinal juice and simulated intestinal juice in pig
The intestinal juice from the pig was prepared as in supernatant 1 of example 1, and the activity of the major digestive enzymes in the intestinal juice was determined as in example 1. Simulated intestinal fluid was prepared as follows: calculating the mass of the small intestine digestive enzyme purification powder required for preparing a certain volume of simulated small intestine liquid by taking the specific activities of amylase, trypsin and chymotrypsin in the small intestine digestive enzyme purification powder relative to the digestive enzyme with the maximum ratio of the corresponding digestive enzyme activity in the supernatant liquid 1 as reference, and then weighing a certain mass of reagent amylase, trypsin or chymotrypsin to ensure that the total activity of the other two digestive enzymes reaches the total activity of the digestive enzymes in the supernatant liquid 1 with the same volume with the simulated small intestine liquid. The above digestive enzymes were dissolved to the same volume as the supernatant 1.
2.3 comparison of simulated intestinal fluid and in vivo digestive Capacity of intestinal fluid
1, feeding 1: 58.21% of corn, 34.87% of soybean meal, 2.99% of wheat bran, 0.95% of soybean oil and 2.98% of mineral element vitamin; and (3) feeding 2: 72.19% of corn, 15.26% of soybean meal, 8.1% of wheat bran, 0.29% of soybean oil and 4.16% of mineral element vitamin; and (3) feed: 68.01% of corn, 17.67% of soybean meal, 8.0% of soybean hull, 2.69% of soybean oil and 3.63% of mineral element vitamin; 4, feed: 70.77% of corn, 17.15% of soybean meal, 8.0% of wheat bran, 0.31% of soybean oil and 3.77% of mineral element vitamin; and 5, feed: 71.80% of corn, 22.20% of soybean meal, 1.18% of soybean oil and 4.82% of mineral element vitamin; 6, feed: 66.76 percent of corn, 22.20 percent of soybean meal, 5 percent of standard flour, 1.18 percent of soybean oil and 4.86 percent of mineral element vitamin; the digestion energy of the 6 kinds of compound feed is 3183-3310 kcal/kg, the crude protein is changed within 13.77-19.03%, and the common nutrition level of the pig compound feed is covered.
Two samples were designed completely randomly and the differences between the simulated small intestine fluids and the in vivo small intestine fluids were compared for the Dry Matter Digestibility (DMD) and in vitro digestibility values (IVDE) of 6 matched diets. Each treatment was 4 replicates, one digestion tube per replicate.
The determination index and method comprises determining dry matter digestibility and in vitro digestibility of small intestine solution on feed by adopting monogastric animal bionic digestion system (model: SDS-2, produced by Intelligent science and technology development Co., Ltd. in south of lake). The method mainly comprises the following steps: the loading of 6 diets was 2g per digestive tube (all to the nearest 0.0002 g). After sampling, the sample was transferred to a simulated digestion tube equipped with a dialysis bag without loss, and then 20mL of simulated small intestine solution or in vivo small intestine supernatant 1 was added to each tube as a digestion solution at the small intestine stage, and the simulated digestion tube was closed with a silica gel plug. The tubing was connected according to the instructions of the SDS-2 operating manual, the simulated digestion time of the small intestine was set in the control software to 16 hours, and the hydrolysate was washed 6 times for 40 minutes each time after simulated digestion. Finally, clicking the 'start' option in the software, the SDS-2 automatically completes the simulated digestion process of the small intestine of the pig. After the whole digestion process is finished, carefully washing the content in the dialysis bag into a culture dish with known absolute dry weight by using deionized water, drying at 65 ℃ until no water mark exists, drying at 105 ℃ until constant weight exists, recording the weight, and determining the total energy. The dry matter digestibility and the in vitro digestibility values were then calculated.
2.4 Effect of porcine intestinal juice purified powder preparation on simulated intestinal juice
Table 5 simulates the difference in vitro digestibility of dry matter between small intestinal juice and in vivo small intestinal juice for 6 matched diets
The difference between the simulated small intestinal fluid and the in vivo small intestinal fluid on the dry matter digestibility of 6 feeds is small. The simulated digestion rate of the intestinal juice on the feed reaches 97.73 to 101.11 percent of the digestion rate of the intestinal juice on the feed in vivo, and the average digestion rate reaches 98.3 percent. This indicates that the simulated intestinal juice is very close to the digestive ability of the intestinal juice in vivo to the whole diet, and a satisfactory simulation effect is achieved.
Table 6 simulates the difference between in vitro digestibility values of intestinal fluid and in vivo intestinal fluid for 6 types of matched diets
On the basis of the difference of the simulated intestinal juice and the in vivo intestinal juice in the in vitro digestibility values of the 6 kinds of the feed, the simulated intestinal juice can simulate the digestion energy of the intestinal juice to the feed to reach 98.03-101.81 percent of the digestion energy of the in vivo intestinal juice to the feed, and the average digestion energy reaches 98.7 percent. This further shows that the simulated intestinal juice is very close to the digestive ability of the intestinal juice in vivo to the organic matters in the feed, thus achieving satisfactory simulation effect.
Claims (11)
1. A preparation method of pig small intestine digestive enzyme powder comprises the following steps:
1) collecting intestinal chyme from small intestines of a plurality of pigs, uniformly mixing the intestinal chyme, centrifuging the mixed intestinal chyme 1, and collecting supernatant 1;
2) evaporating and concentrating the supernatant fluid 1 to obtain concentrated intestinal juice; centrifuging the concentrated intestinal juice 2, and collecting a supernatant 2;
in the step 2), the evaporation concentration is performed on a rotary evaporator, and the conditions of the evaporation concentration are as follows: evaporating for 15-60 minutes under the conditions that the water bath temperature of the distillation flask is 39-59 ℃, the rotating speed is 80-120 r/min and the vacuum degree is 0; when the volume of the concentrated solution is reduced to 30-50% of the original volume, stopping evaporation;
the conditions of the centrifugation 2 are as follows: centrifuging for 10-15 minutes at 4-8 ℃ and 9000-11000 g;
3) transferring the supernatant 2 into a container placed in an ice-water bath, adding mannitol, stirring for dissolving, adding an ethanol solution, stirring uniformly, and standing; then centrifuging 3, and collecting the precipitate;
in the step 3), the addition amount of the mannitol is as follows: adding 1.5-2 g of the supernatant 2 into every 100ml of the supernatant;
the ethanol solution is 95-100% by volume, and is stored in a refrigerator at-20 ℃ for more than 24 hours before use;
the addition amount of the ethanol solution is as follows: adding 350-450 ml of the supernatant 2 into every 100ml of the supernatant;
the standing conditions are as follows: standing for more than 12 hours at the temperature of minus 10 ℃ to minus 25 ℃;
the conditions of the centrifugation 3 are as follows: centrifuging for 10-15 minutes at 4-8 ℃ and 9000-11000 g;
4) adding deionized water into the precipitate, stirring and ultrasonically dissolving, then centrifuging 4, and collecting supernatant 3; and (3) freeze-drying the supernatant 3 to obtain the pig small intestine digestive enzyme powder.
2. The method of claim 1, wherein: in the step 1), the pigs are castrated boars of 15kg to 125 kg;
treating and feeding pigs according to the following method before collecting chyme: installing T-shaped fistulas on small intestines of a plurality of pigs, feeding a full-value compound feed with the crude protein content of more than 18 percent and the metabolic energy value of more than 3200kcal/kg after 10 days of postoperative recovery, feeding twice a day, freely collecting food, providing no drinking water before collecting intestinal chyme, and collecting the intestinal chyme through the intestinal T-shaped fistulas 2-4 hours after feeding;
the supernatant 1 is intestinal juice containing amylase, trypsin and chymotrypsin;
the conditions of the centrifugation 1 are as follows: centrifuging at 9000-11000 g for 10-15 minutes at 4-8 ℃.
3. The method of claim 2, wherein: the pigs are castrated boars with similar genetic background and similar birth times.
4. The production method according to claim 1 or 2, characterized in that: in the step 2), the evaporation time of evaporation concentration is 30-60 minutes.
5. The production method according to claim 1 or 2, characterized in that: in the step 3), the temperature of the standing is-18 to-25 ℃.
6. The production method according to claim 1 or 2, characterized in that: the step 3) further comprises the step of washing the collected precipitate.
7. The method of claim 6, wherein: the cleaning method comprises the step of washing the precipitate for 10-30 seconds by using 30ml of deionized water every time, and washing for 2 times.
8. The production method according to claim 1 or 2, characterized in that: in the step 4), the deionized water is added in an amount which is 50-100% of the volume of the supernatant fluid 1 obtained after the intestinal chyme is centrifuged;
the stirring is magnetic stirring for 10-15 minutes;
the ultrasonic dissolving conditions are as follows: dissolving for 5-10 minutes under the ultrasonic frequency of 39-41 kHz;
the conditions of the centrifugation 4 are: centrifuging at 9000-11000 g for 10-15 minutes at 4-8 ℃.
9. The production method according to claim 1 or 2, characterized in that: the step 4) further comprises a step of allowing the supernatant 3 to stand before freeze-drying; the standing conditions are as follows: standing at-20 deg.C for more than 12 hr.
10. Porcine small intestine digestive enzyme powder produced by the method of any one of claims 1-9.
11. Use of the porcine small intestine digestive enzyme powder of claim 10 in the preparation of porcine simulated small intestine fluid.
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Application publication date: 20181225 Assignee: HUNAN ZHONGBEN INTELLIGENT TECHNOLOGY DEVELOPMENT Co.,Ltd. Assignor: Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences Contract record no.: X2021430000003 Denomination of invention: The invention relates to a highly water-soluble pig small intestine digestive enzyme powder and a preparation method and application thereof Granted publication date: 20210115 License type: Exclusive License Record date: 20210810 |