Preparation method of pancreatin
阅读说明：本技术 一种胰酶制备方法 (Preparation method of pancreatin ) 是由 叶长蓉 于 2021-01-19 设计创作，主要内容包括：本发明公开了一种胰酶制备方法,包括病毒灭活浸泡、磨浆、重金属络合、激活、活化、沉淀、脱脂、干燥等步骤。采用本发明中的制备方法,可以使胰酶的收率达到17％以上,并且制得的胰酶中镉等重金属离子的含量较低,极大的增强了药物的病毒安全性,使得胰酶产品可以符合各国药典和动物来源的生化药物法规方面的苛刻要求。(The invention discloses a preparation method of pancreatin, which comprises the steps of virus inactivation and soaking, grinding, heavy metal complexing, activation, precipitation, degreasing, drying and the like. By adopting the preparation method, the yield of the pancreatin can reach more than 17 percent, and the prepared pancreatin has lower content of heavy metal ions such as cadmium and the like, thereby greatly enhancing the virus safety of the medicine and leading the pancreatin product to meet the rigorous requirements in the aspects of pharmacopoeia of various countries and biochemical medicine regulations of animal sources.)
1. A preparation method of pancreatin is characterized by comprising the following steps:
s1: cutting animal pancreas into strips with the width of 0.5-1 cm, soaking the strips into a virus inactivation solution, and soaking for 8-10 h at room temperature;
s2: adding water into the animal pancreas processed by the S1 to grind into pancreatic pulp;
s3: adding a water-soluble heavy metal complexing agent into the pancreatic pulp, uniformly stirring, and standing for 2-4 h at 5-10 ℃;
s4: grinding fel Sus Domestica and duodenum of pig into bile pulp and intestinal pulp, respectively, and dissolving the bile pulp and intestinal pulp and anhydrous calcium chloride in acetone to obtain activating solution;
s5: and mixing the activating solution and the pancreas slurry treated by the S3 according to the proportion of 1: mixing the raw materials according to a volume ratio of 10-20, and refrigerating the mixture for 4-6 hours at 0 ℃;
s6: heating the mixture after refrigeration to 20 ℃ within 3-4 h, then performing slurry separation, filtering out coarse solid matters, and stirring in a water bath at 25 ℃ for 0.5-1 h;
s7: adding acetone at the temperature of 2-4 ℃ into the mixture treated by the S6, wherein the volume ratio of the added acetone to the mixture is 3-5: 1; stirring for 10-20 min, standing for 2-4 h, and then filling a bag to filter out acetone; squeezing the material with the acetone removed by a screw jack to be blocky;
s8: adding the squeezed massive materials into acetone at the temperature of 10-15 ℃ according to the material-liquid ratio of 1: 2-4 g/mL, stirring for 5-10 min, standing for 1-2 h, and pouring out supernatant; repeating the operation for 2-4 times;
s9: and drying the material treated by the S8 at 20-25 ℃ for 6-10 h, and then crushing to obtain pancreatin powder.
2. The process for producing pancreatin according to claim 1, wherein: the animal pancreas is pig pancreas, sheep pancreas or cattle pancreas.
3. The process for producing pancreatin according to claim 1, wherein: the virus inactivation solution is an ethanol solution of peroxyacetic acid, and the mass fraction of the peroxyacetic acid in the solution is 1-3%.
4. The process for producing pancreatin according to claim 1, wherein: and the ratio of the water-soluble heavy metal complexing agent added into the pancreatic pulp to the material liquid of the pancreatic pulp in S3 is 1: 8-15 g/mL.
5. The process for producing pancreatin according to claim 1 or 4, wherein: the water-soluble heavy metal complexing agent is at least one of sodium dimercaptopropane sulfonate, dimercaptopropanol, sodium dimercaptosuccinate and acetylpenicillamine.
6. The method for producing pancreatin according to claim 1, wherein: the mass fractions of the pig gall bladder, the pig duodenum and the anhydrous calcium chloride in the activation liquid are 0.5-1.5 g/mL, 2-3 g/mL and 0.1-0.5 g/mL respectively.
7. The process for producing pancreatin according to claim 1, wherein: in S5, the activating solution and the pancreatic juice are mixed according to the proportion of 1:15 by volume.
Technical Field
The invention belongs to the technical field of medicine preparation, and particularly relates to a preparation method of pancreatin.
Background
Pancreatin (Pancreatin), a digestant, is extracted from the pancreas of animals. The preparation method of the pancreatin mainly comprises the following steps: crushing animal pancreas, activating, extracting, precipitating, degreasing and drying. Pancreatin contains the activity of hydrolase such as trypsin, pancreatic amylase, pancreatic lipase and the like, and is mainly used as a digestion-aiding medicament for treating dyspepsia, inappetence and digestive disorder caused by liver and pancreas diseases.
In the traditional pancreatin extraction process, a large amount of organic solvents are required, wherein 10-20% of the organic solvents are used for extraction, and 70-80% of the organic solvents are used for precipitation, and the organic solvents comprise ethanol, acetone, isopropanol and the like. The use of organic solvents in large amounts not only poses risks in terms of management and safety, but also leads to high costs for the preparation of pancreatin. In addition, in the animal growth process, heavy metals are accumulated in the pancreas, and the pancreatin prepared from the animal pancreas has high heavy metal content, so that the effect of the pancreatin is reduced, and secondary damage is caused to a user. Therefore, there is a need to develop a new process for producing pancreatin.
Disclosure of Invention
Aiming at the prior art, the invention provides a preparation method of pancreatin, which aims to solve the problems that the existing pancreatin is difficult to prepare and the obtained pancreatin has high heavy metal content.
In order to achieve the purpose, the invention adopts the technical scheme that: provided is a method for preparing pancreatin, comprising the following steps:
s1: cutting animal pancreas into strips with the width of 0.5-1 cm, soaking the strips into a virus inactivation solution, and soaking for 8-10 h at room temperature;
s2: adding water into the animal pancreas processed by the S1 to grind into pancreatic pulp;
s3: adding a water-soluble heavy metal complexing agent into the pancreatic pulp, uniformly stirring, and standing for 2-4 h at 5-10 ℃;
s4: grinding fel Sus Domestica and duodenum of pig into bile pulp and intestinal pulp, respectively, and dissolving the bile pulp and intestinal pulp and anhydrous calcium chloride in acetone to obtain activating solution;
s5: and mixing the activating solution and the pancreas slurry treated by the S3 according to the proportion of 1: mixing the raw materials according to a volume ratio of 10-20, and refrigerating the mixture for 4-6 hours at 0 ℃;
s6: heating the mixture after refrigeration to 20 ℃ within 3-4 h, then performing slurry separation, filtering out coarse solid matters, and stirring in a water bath at 25 ℃ for 0.5-1 h;
s7: adding acetone at the temperature of 2-4 ℃ into the mixture treated by the S6, wherein the volume ratio of the added acetone to the mixture is 3-5: 1; stirring for 10-20 min, standing for 2-4 h, and then filling a bag to filter out acetone; squeezing the material with the acetone removed by a screw jack to be blocky;
s8: adding the squeezed massive materials into acetone at the temperature of 10-15 ℃ according to the material-liquid ratio of 1: 2-4 g/mL, stirring for 5-10 min, standing for 1-2 h, and pouring out supernatant; repeating the operation for 2-4 times;
s9: and drying the material treated by the S8 at 20-25 ℃ for 6-10 h, and then crushing to obtain pancreatin powder.
When the pancreatin is prepared, the pancreas is cut into long strips and is soaked by the virus inactivation solution, the virus inactivation solution is easy to diffuse into the pancreas, the virus extinguishing agent is favorably in full contact with the virus, the virus in pancreatic cells can be inactivated, the virus inactivation effect is more thorough, and the activity of the pancreatin is not adversely affected.
Before the pancreatic pulp is activated, a water-soluble heavy metal complexing agent is added into the pancreatic pulp, the complexing agent is fully dissolved in the pancreatic pulp and can reach an optimal reaction state, the complexing agent and heavy metal ions in pancreatic cells in the pancreatic pulp perform a complexing reaction, the reacted complex is insoluble in acetone and can be precipitated together with the pancreatic enzyme in a subsequent pancreatic enzyme precipitation process, the complexing agent belongs to small molecules, and the acetone is easily separated from the pancreatic enzyme through a filter bag in a subsequent bag filling and filtering process so as to be separated from a reaction system, so that the purpose of separation is achieved. After the complexation reaction, heavy metals in pancreatic cells are basically removed, and the finally prepared pancreatin has low heavy metal content and does not influence the use effect of the pancreatin.
When the invention activates the pancreatic pulp, the adopted activating liquid contains components such as pig gall bladder, pig duodenum, anhydrous calcium chloride and the like. Wherein, calcium ions are introduced into the anhydrous calcium chloride, and the calcium ions can activate lipase, stabilize trypsin, inhibit side reactions and improve the yield of the trypsin; however, the introduction of calcium ions can also cause the pancreatic enzyme burning residues to be increased, and the gall can obviously reduce the burning residues, but the gall can cause certain adverse effects on the pancreatic enzyme activity, and the pig duodenum can offset the adverse effects. The activating solution of the invention is composed of the components, so that the yield of the pancreatin is improved, and the activity of the pancreatin can be ensured.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the animal pancreas is pig pancreas, sheep pancreas or cattle pancreas.
Further, the virus inactivation solution is an ethanol solution of peroxyacetic acid, and the mass fraction of the peroxyacetic acid in the solution is 1-3%.
The virus inactivation solution adopted by the invention is an ethanol solution of peroxyacetic acid, and the ethanol has good permeability, so that the virus extinguishing agent and the ethanol can permeate into pancreatic cells, the virus extinguishing agent and the viruses can be in full contact, and the virus inactivation effect is better.
Furthermore, the ratio of the water-soluble heavy metal complexing agent added into the pancreatic pulp to the material liquid of the pancreatic pulp in S3 is 1: 8-15 g/mL.
Further, the water-soluble heavy metal complexing agent is at least one of sodium dimercaptopropane sulfonate, dimercaptopropanol, sodium dimercaptosuccinate and acetylpenicillamine.
The water-soluble heavy metal complexing agent adopted in the invention contains a plurality of complexing groups, heavy metal exists in the pancreatic pulp in an ion form, the complexing groups in the complexing agent are chelated with the heavy metal to form a complex, and the complex is precipitated in acetone, so that the content of the heavy metal in the pancreatin can be obviously reduced.
Furthermore, the mass fractions of the pig gall bladder, the pig duodenum and the anhydrous calcium chloride in the activation solution are 0.5-1.5 g/mL, 2-3 g/mL and 0.1-0.5 g/mL respectively.
Further, in S5, the activating solution and the pancreatic juice are mixed in a ratio of 1:15 by volume.
The invention has the beneficial effects that:
by adopting the preparation method, the yield of the pancreatin can reach more than 17 percent, and the prepared pancreatin has lower content of heavy metal ions such as cadmium and the like, thereby greatly enhancing the virus safety of the medicine and leading the pancreatin product to meet the rigorous requirements in the aspects of pharmacopoeia of various countries and biochemical medicine regulations of animal sources.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1
A preparation method of pancreatin comprises the following steps:
s1: cutting fresh pig pancreas into strips with the width of about 0.5cm, soaking the strips into 2% of ethanol solution of peroxyacetic acid by mass fraction, and soaking for 10 hours at room temperature;
s2: adding water with the mass about 2 times of that of the pig pancreas treated by the S1 into the pig pancreas, and grinding the pig pancreas into pancreatic pulp;
s3: adding sodium dimercaptopropane sulfonate into the pancreatic pulp, wherein the material-liquid ratio of the sodium dimercaptopropane sulfonate to the pancreatic pulp is 1:10g/mL, uniformly stirring, and standing for 3h at 8 ℃;
s4: grinding fel Sus Domestica bile and duodenum of pig into bile slurry and intestinal slurry, respectively, and dissolving bile slurry and intestinal slurry and anhydrous calcium chloride in acetone to obtain activating solution, wherein the mass fractions of fel Sus Domestica, duodenum of pig and anhydrous calcium chloride in the activating solution are 1g/mL, 2g/mL and 0.1g/mL respectively;
s5: and mixing the activating solution and the pancreas slurry treated by the S3 according to the proportion of 1:15, and refrigerating at 0 ℃ for 6 hours;
s6: heating the refrigerated mixture to 20 ℃ within 4h, then carrying out slurry separation on the material by using a 40-mesh nylon net, filtering out coarse solid matters such as connective tissues and the like, and stirring for 1h in a water bath at 25 ℃;
s7: adding acetone with the temperature of 3 ℃ into the mixture processed by the S6, wherein the volume ratio of the added acetone to the mixture is 4: 1; stirring for 15min, standing for 4h, and filling into bags to remove acetone; squeezing the material with the acetone removed by a screw jack to be blocky;
s8: adding the squeezed block-shaped material into acetone at 10 ℃ according to the material-liquid ratio of 1:3g/mL, stirring for 10min, standing for 2h, and pouring out the supernatant; repeating the above operation for 3 times;
s9: drying the material treated by the S8 at 20 ℃ for 10h, and then crushing to obtain pancreatin powder.
Example 2
A preparation method of pancreatin comprises the following steps:
s1: cutting fresh ox pancreas into strips with the width of about 1cm, soaking the strips into 3% of ethanol solution of peroxyacetic acid, and soaking for 8 hours at room temperature;
s2: adding water with the mass about 2 times of that of the bovine pancreas treated by the S1 into the bovine pancreas, and grinding the bovine pancreas into pancreatic pulp;
s3: adding sodium dimercaptosuccinate into the pancreatic pulp, uniformly stirring the sodium dimercaptosuccinate and the pancreatic pulp at a feed-liquid ratio of 1:8g/mL, and standing at 10 ℃ for 2 hours;
s4: grinding fel Sus Domestica bile and duodenum of pig into bile slurry and intestinal slurry, respectively, and dissolving bile slurry and intestinal slurry and anhydrous calcium chloride in acetone to obtain activating solution, wherein the mass fractions of fel Sus Domestica, duodenum of pig and anhydrous calcium chloride in the activating solution are 1.5g/mL, 2g/mL and 0.5g/mL respectively;
s5: and mixing the activating solution and the pancreas slurry treated by the S3 according to the proportion of 1:10, and refrigerating at 0 ℃ for 4 hours;
s6: heating the refrigerated mixture to 20 ℃ within 3h, then carrying out slurry separation on the material by using a 40-mesh nylon net, filtering out coarse solid matters such as connective tissues and the like, and stirring for 1h in a water bath at 25 ℃;
s7: adding acetone at the temperature of 4 ℃ into the mixture treated by the S6, wherein the volume ratio of the added acetone to the mixture is 5: 1; stirring for 20min, standing for 4h, and filling into bags to remove acetone; squeezing the material with the acetone removed by a screw jack to be blocky;
s8: adding the squeezed block-shaped material into acetone at 15 ℃ according to the material-liquid ratio of 1:2g/mL, stirring for 5min, standing for 1h, and pouring out the supernatant; repeating the above operation 4 times;
s9: and drying the material treated by the S8 at 25 ℃ for 6h, and then crushing to obtain pancreatin powder.
Example 3
A preparation method of pancreatin comprises the following steps:
s1: cutting fresh sheep pancreas into strips with the width of about 0.5cm, soaking the strips into 1% of ethanol solution of peroxyacetic acid by mass fraction, and soaking for 10 hours at room temperature;
s2: adding water of about 2 times of the weight of the sheep pancreas treated by the S1 into the sheep pancreas, and grinding the sheep pancreas into pancreatic pulp;
s3: adding dimercaptopropanol and acetylpenicillamine into the pancreatic pulp according to the feed-liquid ratio of 1:15g/mL, wherein the mass ratio of the dimercaptopropanol to the acetylpenicillamine is 1:1, uniformly stirring, and standing at 5 ℃ for 4 hours;
s4: grinding fel Sus Domestica bile and duodenum of pig into bile slurry and intestinal slurry, respectively, and dissolving bile slurry and intestinal slurry and anhydrous calcium chloride in acetone to obtain activating solution, wherein the mass fractions of fel Sus Domestica, duodenum of pig and anhydrous calcium chloride in the activating solution are 0.5g/mL, 3g/mL and 0.1g/mL respectively;
s5: and mixing the activating solution and the pancreas slurry treated by the S3 according to the proportion of 1: 20, and refrigerating at 0 ℃ for 5 hours;
s6: heating the refrigerated mixture to 20 ℃ within 4h, then carrying out slurry separation on the material by using a 40-mesh nylon net, filtering out coarse solid matters such as connective tissues and the like, and stirring in a water bath at 25 ℃ for 0.5 h;
s7: adding acetone at the temperature of 2 ℃ into the mixture treated by the S6, wherein the volume ratio of the added acetone to the mixture is 3: 1; stirring for 10min, standing for 2h, and filling into bags to remove acetone; squeezing the material with the acetone removed by a screw jack to be blocky;
s8: adding the squeezed block-shaped material into acetone at 10 ℃ according to the material-liquid ratio of 1:4g/mL, stirring for 10min, standing for 2h, and pouring out the supernatant; repeating the above operation for 2 times;
s9: drying the material treated by the S8 at 20 ℃ for 8h, and then crushing to obtain pancreatin powder.
Comparative example 1
A preparation method of pancreatin comprises the following steps:
s1: adding water of about 2 times of the weight of fresh pig pancreas into the fresh pig pancreas, and grinding the fresh pig pancreas into pancreas slurry;
s2: adding sodium dimercaptopropane sulfonate into the pancreatic pulp, wherein the material-liquid ratio of the sodium dimercaptopropane sulfonate to the pancreatic pulp is 1:10g/mL, uniformly stirring, and standing for 3h at 8 ℃;
s3: grinding fel Sus Domestica bile and duodenum of pig into bile slurry and intestinal slurry, respectively, and dissolving bile slurry and intestinal slurry and anhydrous calcium chloride in acetone to obtain activating solution, wherein the mass fractions of fel Sus Domestica, duodenum of pig and anhydrous calcium chloride in the activating solution are 1g/mL, 2g/mL and 0.1g/mL respectively;
s4: and mixing the activating solution and the pancreas slurry treated by the S3 according to the proportion of 1:15, and refrigerating at 0 ℃ for 6 hours;
s5: heating the refrigerated mixture to 20 ℃ within 4h, then carrying out slurry separation on the material by using a 40-mesh nylon net, filtering out coarse solid matters such as connective tissues and the like, and stirring for 1h in a water bath at 25 ℃;
s6: adding acetone with the temperature of 3 ℃ into the mixture processed by the S6, wherein the volume ratio of the added acetone to the mixture is 4: 1; stirring for 15min, standing for 4h, and filling into bags to remove acetone; squeezing the material with the acetone removed by a screw jack to be blocky;
s7: adding the squeezed block-shaped material into acetone at 10 ℃ according to the material-liquid ratio of 1:3g/mL, stirring for 10min, standing for 2h, and pouring out the supernatant; repeating the above operation for 3 times;
s8: drying the material treated by the S8 at 20 ℃ for 10h, and then crushing to obtain pancreatin powder.
Comparative example 2
A preparation method of pancreatin comprises the following steps:
s1: cutting fresh pig pancreas into strips with the width of about 0.5cm, soaking the strips into 2% of ethanol solution of peroxyacetic acid by mass fraction, and soaking for 10 hours at room temperature;
s2: adding water with the mass about 2 times of that of the pig pancreas treated by the S1 into the pig pancreas, and grinding the pig pancreas into pancreatic pulp;
s3: grinding fel Sus Domestica bile and duodenum of pig into bile slurry and intestinal slurry, respectively, and dissolving bile slurry and intestinal slurry and anhydrous calcium chloride in acetone to obtain activating solution, wherein the mass fractions of fel Sus Domestica, duodenum of pig and anhydrous calcium chloride in the activating solution are 1g/mL, 2g/mL and 0.1g/mL respectively;
s4: and mixing the activating solution and the pancreas slurry treated by the S3 according to the proportion of 1:15, and refrigerating at 0 ℃ for 6 hours;
s5: heating the refrigerated mixture to 20 ℃ within 4h, then carrying out slurry separation on the material by using a 40-mesh nylon net, filtering out coarse solid matters such as connective tissues and the like, and stirring for 1h in a water bath at 25 ℃;
s6: adding acetone with the temperature of 3 ℃ into the mixture processed by the S6, wherein the volume ratio of the added acetone to the mixture is 4: 1; stirring for 15min, standing for 4h, and filling into bags to remove acetone; squeezing the material with the acetone removed by a screw jack to be blocky;
s7: adding the squeezed block-shaped material into acetone at 10 ℃ according to the material-liquid ratio of 1:3g/mL, stirring for 10min, standing for 2h, and pouring out the supernatant; repeating the above operation for 3 times;
s8: drying the material treated by the S8 at 20 ℃ for 10h, and then crushing to obtain pancreatin powder.
Comparative example 3
A preparation method of pancreatin comprises the following steps:
s1: cutting fresh pig pancreas into strips with the width of about 0.5cm, soaking the strips into 2% of ethanol solution of peroxyacetic acid by mass fraction, and soaking for 10 hours at room temperature;
s2: adding water with the mass about 2 times of that of the pig pancreas treated by the S1 into the pig pancreas, and grinding the pig pancreas into pancreatic pulp;
s3: adding sodium dimercaptopropane sulfonate into the pancreatic pulp, wherein the material-liquid ratio of the sodium dimercaptopropane sulfonate to the pancreatic pulp is 1:10g/mL, uniformly stirring, and standing for 3h at 8 ℃;
s4: dissolving anhydrous calcium chloride in acetone to obtain an activating solution, wherein the mass fraction of the anhydrous calcium chloride in the activating solution is 0.1 g/mL;
s5: and mixing the activating solution and the pancreas slurry treated by the S3 according to the proportion of 1:15, and refrigerating at 0 ℃ for 6 hours;
s6: heating the refrigerated mixture to 20 ℃ within 4h, then carrying out slurry separation on the material by using a 40-mesh nylon net, filtering out coarse solid matters such as connective tissues and the like, and stirring for 1h in a water bath at 25 ℃;
s7: adding acetone with the temperature of 3 ℃ into the mixture processed by the S6, wherein the volume ratio of the added acetone to the mixture is 4: 1; stirring for 15min, standing for 4h, and filling into bags to remove acetone; squeezing the material with the acetone removed by a screw jack to be blocky;
s8: adding the squeezed block-shaped material into acetone at 10 ℃ according to the material-liquid ratio of 1:3g/mL, stirring for 10min, standing for 2h, and pouring out the supernatant; repeating the above operation for 3 times;
s9: drying the material treated by the S8 at 20 ℃ for 10h, and then crushing to obtain pancreatin powder.
Analysis of results
1. Method for measuring characteristics of pancreatin
(1) Pancreatic enzyme yield
The pancreatin yield of each of the above examples and comparative examples was calculated, and the pancreatin yield was calculated using the formula shown in formula I.
Pancreatin yield ═ (pancreatin powder mass/raw material weight) × 100% (I)
(2) Trypsin assay
Preparation of control solutions: taking tyrosine reference substance, precisely weighing, adding 0.2mol/L hydrochloric acid solution to dissolve and dilute into solution containing about 50 μ g per 1 mL.
Preparing a test sample stock solution: taking about 0.1g of the prepared pancreatin powder, precisely weighing, placing in a mortar, adding a small amount of calcium chloride solution (1.47 g of calcium chloride is taken, 500ml of water is added for dissolving, adjusting the pH value to 6.0-6.2 by using 0.1mol/L hydrochloric acid solution or 0.1mol/L sodium hydroxide solution), uniformly grinding, placing in a 100ml measuring flask, adding the calcium chloride solution to scale, and shaking uniformly; precisely measuring appropriate amount, adding borate buffer solution (2.85 g of borax, 10.5g of boric acid and 2.50g of sodium chloride) cooled to below 5 deg.C, adding water to dissolve into 1000ml, adjusting pH to 7.5 + -0.1, and quantitatively diluting to obtain solution containing trypsin activity unit of about 0.12 per 1 ml.
The determination method comprises the following steps: taking 3 test tubes, respectively and precisely measuring 1ml of a sample stock solution and 2ml of the borate buffer solution, preserving heat in a water bath at 40 ℃ for 10 minutes, respectively and precisely adding 5ml of a casein solution (taking 1.5g of a casein reference substance, adding 13ml of a 0.1mol/L sodium hydroxide solution and 40ml of water) preheated in the water bath at 40 ℃, heating in the water bath at 60 ℃ to dissolve, cooling, adding water to dilute to 100ml, adjusting the pH value to 8.0), shaking up, immediately placing in the water bath at 40 +/-0.5 ℃ to accurately react for 30 minutes, precisely adding 5ml of a 5% trichloroacetic acid solution to terminate the reaction, uniformly mixing, filtering, and taking a subsequent filtrate as a sample solution; precisely measuring 1ml of a sample stock solution, adding 2.0ml of the borate buffer solution, preserving heat in a water bath at 40 ℃ for 10 minutes, precisely adding 5ml of a 5% trichloroacetic acid solution, shaking up, accurately reacting in a water bath at 40 +/-0.5 ℃ for 30 minutes, immediately precisely adding 5ml of a casein solution, shaking up, filtering, and taking a subsequent filtrate as a blank control; measuring and calculating the average value of absorbance of the sample solution at 275nm according to ultraviolet-visible spectrophotometry (appendix IV A of second part of Chinese pharmacopoeia 2010 version)A blank was prepared from 0.2mol/L hydrochloric acid solution, and the absorbance (As) of the control solution was measured at a wavelength of 275 nm. Calculated as follows:
in the formula WsThe content of the leucine in each 1mL of the control solution is μ g;
w is the sample quantity of the sample to be tested, g;
and n is the dilution multiple of the test sample.
Under the above conditions, the amount of enzyme that hydrolyzes casein per minute to form trichloroacetic acid non-precipitate (peptide, amino acid, etc.) corresponding to 1. mu. mol of tyrosine at a wavelength of 275nm is 1 trypsin activity unit. The value measured by the test sample should be 0.15-0.6, otherwise, the concentration should be adjusted and measured separately.
(3) Amylopsin assay
Preparation of a test solution: taking about 0.3g of the prepared pancreatin powder, precisely weighing, placing in a mortar, adding a small amount of phosphate buffer solution (taking 13.61g of monopotassium phosphate and 35.80g of disodium hydrogen phosphate, adding water to dissolve into 1000ml, adjusting the pH value to 6.8) cooled to be below 5 ℃, grinding uniformly, and adding the phosphate buffer solution to quantitatively dilute into a solution containing about 10-20 units of amylopsin in each 1 ml.
The determination method comprises collecting 1% potato starch solution [ collecting 1.0g of soluble starch (for determination of amylopsin) dried at 105 deg.C for 2 hr, adding 10ml of water, stirring, slowly pouring into 100ml of boiling water under stirring, boiling for 20min, cooling, diluting with water to 100ml 25ml, the above phosphate buffer solution 10ml, 1ml of 1.2% sodium chloride solution and water 20ml, placing in 250ml iodine bottle, preserving heat in 40 ℃ water bath for 10 minutes, precisely adding 1ml of sample solution, shaking up, immediately placing in 40 ℃ plus or minus 0.5 ℃ water bath for accurate reaction for 10 minutes, adding 2ml of 1mol/L hydrochloric acid solution to terminate the reaction, shaking up, placing to room temperature, precisely adding 10ml of iodine titrant (0.05mol/L), dripping 45ml of 0.1mol/L sodium hydroxide solution while shaking, after standing in the dark for 20 minutes, 4ml of a sulfuric acid solution (1 → 4) was added, and the mixture was titrated with a sodium thiosulfate titration solution (0.1mol/L) to colorless. Taking 25ml of 1% soluble starch solution, 10ml of the phosphate buffer solution, 1ml of 1.2% sodium chloride solution and 20ml of water, placing the mixture in an iodine bottle, preserving the temperature in a water bath at 40 +/-0.5 ℃ for 10 minutes, after the mixture is placed to the room temperature, adding 2ml of 1mol/L hydrochloric acid solution, shaking the mixture evenly, adding 1.0ml of test solution, shaking the mixture evenly, precisely adding 10ml of iodometric titration solution (0.05mol/L), dropwise adding 45ml of 0.1mol/L sodium hydroxide solution while shaking the mixture, placing the mixture in a dark place for 20 minutes, adding 4ml of sulfuric acid solution (1 → 4), titrating the mixture to be colorless by using sodium thiosulfate titration solution (0.1mol/L) as a blank control, wherein each 1ml of the iodometric titration solution (0.05mol/L) is equivalent to 9.008mg of anhydrous glucose, and calculating according to the following formula.
Wherein A is the volume of sodium thiosulfate titration solution consumed by a test sample, and ml;
b is the volume of blank consumption sodium thiosulfate titration solution, ml;
f is a concentration (mol/L) conversion value of the sodium thiosulfate titration solution;
w is the sample volume of the sample, g;
n is the dilution multiple of the test sample.
Under the above conditions, the amount of enzyme that hydrolyzes starch to yield 1. mu. mol of glucose per minute was 1 activity unit. The sodium thiosulfate titration solution of (B-A) should be 2.0-4.0 ml, otherwise, the concentration should be adjusted and measured separately.
(4) Pancreatic lipase assay
Preparation of a test solution: taking about 0.1g of the prepared pancreatin powder, precisely weighing, placing in a mortar, adding a small amount of tris (hydroxymethyl) aminomethane buffer solution (prepared by taking 606mg of tris (hydroxymethyl) aminomethane, adding 45.7ml of 0.1mol/L hydrochloric acid solution, adding water to 100ml, shaking up, adjusting the pH value to 7.1) which is cooled to below 5 ℃, grinding uniformly, and adding the buffer solution for quantitative dilution to prepare a solution containing about 8-16 units of pancrelipase in each 1 ml.
Measuring method comprises grinding oleum Olivarum emulsion (4 ml oleum Olivarum and 7.5g acacia gum, grinding uniformly, slowly adding water, grinding to 100ml, stirring twice at 8000 rpm with high speed tissue masher for 3 min, examining the emulsion under microscope, wherein 90% emulsion particles have diameter below 3 μm and no emulsion particle above 10 μm), 25ml fel bovis Seu Bubali salt solution (appropriate amount of fel bovis Seu Bubali salt reference reagent, making into solution (2 → 25) with water) 2ml and 10ml water, placing into 100ml beaker, adjusting pH to 9.0 with sodium hydroxide titration solution (0.1mol/L), keeping temperature in water bath at 37 + -0.1 deg.C for 10min, adjusting pH to 9.0, precisely weighing sample solution 1ml, reacting in water bath at 37 + -0.1 deg.C for 10min, and titrating with sodium hydroxide solution (0.1mol/L) to make pH of the reaction solution constant at 9.0, the amount (ml) of sodium hydroxide titrant (0.1mol/L) consumed was recorded. And taking 1ml of the test solution which is boiled in a water bath for 15-30 minutes, measuring according to the method, and making a blank control, wherein the blank control is calculated according to the following formula.
In the formula, A is the volume of sodium hydroxide titration solution consumed by a sample, and is ml;
b is the volume of blank sodium hydroxide titration solution consumed, ml;
m is the concentration of sodium hydroxide titration solution, mol/L;
w is the sample volume of the sample, g;
n is the dilution multiple of the test sample.
Under the above conditions, the amount of enzyme that hydrolyzes fat (olive oil) per minute to produce 1. mu. mol of fatty acid is 1 unit of amylopsin activity. The amount of the sodium hydroxide titration solution (0.1mol/L) consumed per minute should be 0.08-0.16 ml, otherwise, the concentration should be adjusted and measured separately.
(5) Determination of cadmium content in pancreatin
And (3) determining the amount of cadmium in the pancreatin product by adopting flame atomic absorption spectrometry. The determination method comprises the following steps: taking 1000mg/L Cd (II) stock solution, and diluting with secondary deionized water to obtain standard series solutions of 0.02mg/L, 0.04mg/L, 0.06mg/L, 0.08mg/L and 0.1mg/L of cadmium. Each run was measured and plotted against a standard curve under selected instrument operating conditions.
Because the pancreatin product has poor water solubility and can not meet the sample treatment requirement by direct dissolution, the acid dissolution method is adopted to treat the sample, namely: accurately weighing 1.000g of ground pancreatin product, putting the ground pancreatin product into a 40mL crucible, adding 5mL of nitric acid (1+1), stirring uniformly by using a glass rod, slowly heating the mixture on an electric furnace until white smoke is emitted and the mixture is nearly evaporated to dryness, taking down and cooling the mixture until the sample residue is completely carbonized and becomes black, adding 5mL of nitric acid (1+10), continuously heating and digesting the mixture on the electric furnace, and making the residue turn black from yellow to black and turn white again. Heating is continued until the nitric acid is evaporated to dryness. Cooling, adding 0.1mol/L nitric acid to a constant volume of 25 mL. And measuring the absorbance by using an atomic absorption spectrophotometer and obtaining the cadmium concentration of the cadmium by contrasting a standard curve.
2. Pancreatin characteristics
The characteristic parameters of the pancreatin obtained in each example and comparative example are shown in Table 1.
TABLE 1 measurement results of pancreatic enzyme characteristics
As can be seen from the table, the pancreatin prepared by the method has high yield and high enzyme activity, and simultaneously, the heavy metal content in the pancreatin can be effectively reduced, and the finally prepared pancreatin has excellent performance.
Compared with example 1, in the case of the comparative document 1, the pancreas is not soaked in the virus inactivation solution during the preparation, and the pancreatin also contains more viruses, which adversely affects the activity of the final pancreatin and significantly reduces the pancreatin titer.
Compared with the example 1, in the comparative document 2, the pancreatic pulp is not treated by the heavy metal complexing agent in the preparation process, and the finally prepared pancreatin has high heavy metal content and is not good for health in use.
Compared with example 1, in the case of the comparative document 3, the activation solution contains only calcium chloride, so that the degree of pancreatic enzyme activation is low, and the titer of the pancreatic enzyme powder finally obtained is low.
While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.