Preparation method of feed-grade pancreatin for promoting efficient growth of broiler chickens

Abstract

The invention discloses a preparation method of feed-grade pancreatin for promoting efficient growth of broiler chickens, and relates to the technical field of feed-grade pancreatin extraction.The method is characterized in that frozen pig pancreas is used as a raw material, and pancreatin is prepared through a series of procedures. The preparation method comprises the following specific steps: unfreezing, grinding into thick liquid, activating, degreasing, drying, crushing and the like to obtain the feed additive pancreatin. According to the method, the experiment process is optimized by taking the enzyme activity yield of trypsin in the extraction step as an index, the optimal condition of low-concentration ethanol in pancreatin extraction is explored, and process parameters which are easy to operate, high in enzyme activity and environmentally friendly are optimized. According to the method, the pancreatin extraction process is simplified, the environment-friendly effect is achieved only by using low-concentration ethanol in the extraction process, the pancreatin with high activity is prepared, the utilization rate of feed nutrients by broilers can be increased, the intestinal morphology of the broilers can be improved, the production performance of the broilers can be improved, and a basis and guidance are provided for production and application of low-protein daily ration.

Description

Technical Field

[0001] This invention relates to the field of feed-grade pancreatic enzyme extraction technology, and in particular to a method for preparing feed-grade pancreatic enzyme that promotes efficient growth in broilers. Background Technology

[0002] Pancreatic enzymes are a mixture of multiple enzymes derived from the pancreas of healthy animals. They mainly include trypsin, pancreatic amylase, and pancreatic lipase. They are the most important and powerful enzyme system for digesting food in animals, and the activity of this enzyme system is considered an excellent indicator of the body's ability to digest nutrients.

[0003] The European Working Group on Biologicals (BWP) and the Committee on Medicinal Products for Human Use (CHMP) have conducted a comprehensive safety assessment of pancreatic enzyme products and concluded that pancreatic enzymes derived from pig or bovine pancreas are biosafe. The American Association of Feed Manufacturers (AAFCO) has included trypsin, pancreatic lipase, and pancreatic amylase in its 2021 list of feed additives. Currently, my country has clearly proposed requirements such as "adjusting and optimizing feed formulation structure to promote the reduction and substitution of corn and soybean meal" and "technical solutions for reducing and substituting corn and soybean meal in pig and chicken feed." Faced with the decline in protein content in feed, improving the digestibility of compound feed, ensuring the effective protein intake of livestock and poultry, and maintaining normal production levels, pancreatic enzymes, as endogenous enzymes with a broad enzyme spectrum, have broad application potential in improving dietary protein utilization and reducing soybean meal usage. In the future, they will undoubtedly become a green and efficient feed additive in the livestock industry.

[0004] Currently, pancreatic enzymes are widely used in medicine, but research in the feed industry is limited. Furthermore, the extraction, precipitation, and defatting reagents used in the pharmaceutical industry are mainly highly toxic organic reagents such as acetone, isopropanol, petroleum ether, and diethyl ether. For example, patent CN117844795A, "A Method for Extracting Pancreatic Enzymes from Animal Pancreas," uses isopropanol and diethyl ether; patent CN112080487A, "A Method for Preparing Sheep Pancreatic Enzymes," uses isopropanol and petroleum ether; and patent CN117904090A, "A Method for Preparing High-Specification Pancreatic Enzymes," uses isopropanol. To address this issue, this invention provides a method for extracting feed-grade pancreatic enzymes from pig pancreas. The preparation process only requires the use of low-concentration ethanol in the defatting step; all other steps do not use organic reagents, ensuring safety and environmental friendliness.

[0005] Furthermore, the extraction of pancreatic enzymes in the pharmaceutical industry typically involves a series of processes, including grinding pancreatic pulp, activation, extraction with low-concentration organic solvents, precipitation with high-concentration organic solvents, defatting, drying, and pulverization. Because the production process for extracting pancreatic enzymes in the pharmaceutical industry is complex and uses a large amount of organic reagents such as acetone and isopropanol, its application in the feed industry is costly, thus leaving room for improvement. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for preparing feed-grade trypsin that promotes efficient growth in broilers. Its advantages include simplifying the trypsin extraction process, using only low-concentration ethanol for environmental protection, producing highly active trypsin, improving broiler nutrient utilization, enhancing intestinal morphology, and boosting broiler performance, thus providing a basis and guidance for the production and application of low-protein diets.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers includes the following steps:

[0009] Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours;

[0010] Step 2: Grinding: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic pulp;

[0011] Step 3: Activation: Place the pancreatic juice in an insulated container, add 0.5-5% activator, slowly heat to 20-40℃, keep warm and stir for 2-24 hours to activate;

[0012] Step 4: Precipitation: Pour the material into an insulated tank, add 1-5 times the amount of 50-90% cold ethanol pre-cooled to -10℃, set the precipitation temperature at 4-25℃, and stir for 4 hours.

[0013] Step 5: Centrifugation: After the material has settled, centrifuge it at 4200 rpm for 20 minutes to separate the solid and liquid components.

[0014] Step 6: Degreasing: Degrease the precipitated material three times at 30℃ with 1-5 times the amount of 50-90% ethanol, for 1 hour each time. Remove the excess ethanol after degreasing.

[0015] Step 7: Drying: Dry the precipitate at 40°C for 20 hours.

[0016] The present invention is further configured such that the white fat and connective tissue attached to the pig pancreas need not be trimmed off when the pancreas is crushed.

[0017] The present invention is further configured such that, in step three, the pancreatic syrup is first heated to 20-40°C, and then an activator is added to activate it for 4-12 hours, wherein the activator is calcium chloride.

[0018] The present invention is further configured such that the amount of precipitated ethanol is 2-5 times the weight of pancreatic slurry after sludge removal, and the ethanol concentration is 60-90%.

[0019] The present invention is further configured such that the amount of defatted ethanol is 2-5 times the weight of the solid after centrifugation, and the ethanol concentration is 70-90%.

[0020] The present invention is further configured such that, in step eight, a blower drying oven is used to dry the precipitate.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. Simple operation: Compared with the pharmaceutical-grade pancreatic enzyme preparation process, there is no need to remove the white fat and connective tissue on the pancreas during raw material processing, no need to add protective agents during activation, and no need to use high-concentration organic reagents to extract pancreatic enzymes. The optimized process is more suitable for feed industry applications.

[0023] 2. Safe and environmentally friendly reagents: The concentration of ethanol, an organic reagent used in the pancreatic enzyme extraction process, is reduced to 60-90%, which is safer and more environmentally friendly compared with the organic reagents such as isopropanol, acetone, and diethyl ether used in other patents.

[0024] 3. Stable enzyme activity: Using the enzyme activity yield of trypsin in pancreatic enzymes as an indicator, process parameters are optimized through stepwise research, including activation time, amount of precipitating ethanol, concentration of defatted ethanol, and volume of defatted ethanol. The optimized scheme can reduce the loss of enzyme activity by operations and reagents in each processing step, and obtain pancreatic enzyme products with high and stable enzyme activity. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the preparation process of a feed-grade pancreatic enzyme that promotes efficient growth in broilers, as proposed in this invention. Detailed Implementation

[0026] The technical solution of this patent will be further described in detail below with reference to specific embodiments.

[0027] The embodiments of this patent are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this patent, and should not be construed as limiting this patent.

[0028] Example 1

[0029] Reference Figure 1 A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers includes the following steps:

[0030] Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours;

[0031] Step 2: Grinding pancreatic paste: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic paste;

[0032] Step 3: Temperature activation: Place the pancreatic juice in an insulated container, add 0.5-5% activator, slowly heat to 30℃, keep warm and stir for 12 hours to activate;

[0033] Reference method for trypsin activity detection: NYSL-1004-2023 Feed Additives (Derived from pig pancreas);

[0034] The trypsin activities in the pancreatic cytoplasm after activation with 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, and 5.0% calcium chloride were 213.10, 289.32, 291.45, 293.11, 300.04, and 306.78 U / g, respectively.

[0035] Example 2

[0036] Reference Figure 1 A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers includes the following steps:

[0037] Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours;

[0038] Step 2: Grinding pancreatic paste: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic paste;

[0039] Step 3: Temperature activation: Place the pancreatic juice in an insulated container, add 2% activator, slowly heat to 20-40℃, keep warm and stir for 12 hours to activate;

[0040] Reference method for trypsin activity detection: NYSL-1004-2023 Feed Additives (Derived from pig pancreas);

[0041] The trypsin activities in the pancreatic cytoplasm after activation at 20℃, 30℃, and 40℃ were measured to be 198.66, 291.45, and 245.49 U / g, respectively.

[0042] Example 3

[0043] Reference Figure 1 A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers includes the following steps:

[0044] Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours;

[0045] Step 2: Grinding pancreatic paste: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic paste;

[0046] Step 3: Temperature activation: Place the pancreatic juice in an insulated container, add 2% activator, slowly heat to 30℃, keep warm and stir for 2-24 hours to activate;

[0047] Reference method for trypsin activity detection: NYSL-1004-2023 Feed Additives (Derived from pig pancreas);

[0048] The trypsin activities in pancreatic cytoplasm after activation for 2, 4, 6, 8, 10, 12, and 24 hours were measured to be 212.50, 225.31, 253.66, 289.32, 293.22, 242.58, and 203.35 U / g, respectively.

[0049] Example 4

[0050] Reference Figure 1 A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers includes the following steps:

[0051] Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours;

[0052] Step 2: Grinding: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic pulp;

[0053] Step 3: Activation: Place the pancreatic juice in an insulated container, add 2% activator, slowly heat to 30°C, keep warm and stir for 8 hours to activate;

[0054] Step 5: Sedimentation: Pour the material into an insulated tank, add 1-5 times the amount of 80% cold ethanol pre-cooled to -10℃, set the sedimentation temperature at 4-25℃, and stir for 4 hours;

[0055] Step 6: Centrifugation: After the material has settled, centrifuge it at 4200 rpm for 20 minutes to separate the solid and liquid components.

[0056] Reference method for trypsin activity detection: NYSL-1004-2023 Feed Additives (Derived from pig pancreas);

[0057] The results showed that when 80% ethanol was used for precipitation, the enzyme activity yields in the precipitation step were 43.54%, 52.79%, 65.68%, 65.23%, and 63.45% when the ethanol volume was 1, 2, 3, 4, and 5 times that of the ethanol, respectively.

[0058] Example 5

[0059] Reference Figure 1 A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers includes the following steps:

[0060] Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours;

[0061] Step 2: Grinding: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic pulp;

[0062] Step 3: Activation: Place the pancreatic juice in an insulated container, add 2% activator, slowly heat to 30°C, keep warm and stir for 8 hours to activate;

[0063] Step 5: Precipitation: Pour the material into an insulated tank, add 3 times the amount of 50-90% cold ethanol pre-cooled to -10℃, set the precipitation temperature at 4-25℃, and stir for 4 hours;

[0064] Step 6: Centrifugation: After the material has settled, centrifuge it at 4200 rpm for 20 minutes to separate the solid and liquid components.

[0065] Reference method for trypsin activity detection: NYSL-1004-2023 Feed Additives (Derived from pig pancreas);

[0066] The results showed that when 50%, 60%, 70%, 80%, and 90% ethanol were used for precipitation, the activity rates of the precipitating enzyme in the precipitation step were 45.36%, 51.34%, 65.41%, 66.39%, and 65.11%, respectively.

[0067] Example 6

[0068] Reference Figure 1 A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers includes the following steps:

[0069] Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours;

[0070] Step 2: Grinding: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic pulp;

[0071] Step 3: Activation: Place the pancreatic juice in an insulated container, add 2% activator, slowly heat to 30℃, keep warm and stir for 4-12 hours to activate;

[0072] Step 5: Sedimentation: Pour the material into an insulated tank, add 4 times the amount of 80% cold ethanol pre-cooled to -10℃, set the sedimentation temperature at 4-25℃, and stir for 4 hours;

[0073] Step 6: Centrifugation: After the material has settled, centrifuge it at 4200 rpm for 20 minutes to separate the solid and liquid components.

[0074] Step 7: Degreasing: Degrease the precipitated material three times with 4 times the amount of 50-90% ethanol at 30℃, for 1 hour each time. Remove the remaining ethanol after degreasing.

[0075] Reference method for trypsin activity detection: NYSL-1004-2023 Feed Additives (Derived from pig pancreas);

[0076] For fat content testing methods, refer to GB / T6433-2006 Determination of crude fat in feed;

[0077] Tests showed that after defatting three times with ethanol concentrations of 50%, 60%, 70%, 80%, and 90%, the fat content of the precipitated material was 61.39%, 56.24%, 40.31%, 19.11%, and 8.83%, respectively, and the enzyme activity of the precipitate was 43.15%, 46.36%, 54.25%, 50.28%, and 49.95%, respectively.

[0078] Example 7

[0079] Reference Figure 1 A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers includes the following steps:

[0080] Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours;

[0081] Step 2: Grinding: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic pulp;

[0082] Step 3: Activation: Place the pancreatic juice in an insulated container, add 2% activator, slowly heat to 30°C, keep warm and stir for 8 hours to activate;

[0083] Step 5: Sedimentation: Pour the material into an insulated tank, add 4 times the amount of 80% cold ethanol pre-cooled to -10℃, set the sedimentation temperature at 4-25℃, and stir for 4 hours;

[0084] Step 6: Centrifugation: After the material has settled, centrifuge it at 4200 rpm for 20 minutes to separate the solid and liquid components.

[0085] Step 7: Degreasing: Degrease the precipitated material three times with 1-5 times the amount of 90% ethanol at 30℃, for 1 hour each time. Remove the remaining ethanol after degreasing.

[0086] Reference method for trypsin activity detection: NYSL-1004-2023 Feed Additives (Derived from pig pancreas);

[0087] For fat content testing methods, refer to GB / T6433-2006 Determination of crude fat in feed;

[0088] Testing revealed that after defatting with 1, 2, 3, 4, and 5 times the concentration of 90% defatted ethanol, the precipitated fat contents were 15.86%, 11.59%, 8.83%, 7.39%, and 5.44%, respectively, and the precipitate enzyme activity rates after defatting were 52.31%, 50.77%, and 49.95%, and 45.22% and 43.31%, respectively.

[0089] Example 8

[0090] Reference Figure 1 A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers includes the following steps:

[0091] Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours;

[0092] Step 2: Grinding: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic pulp;

[0093] Step 3: Activation: Place the pancreatic juice in an insulated container, add 2% activator, slowly heat to 30°C, keep warm and stir for 8 hours to activate;

[0094] Step 5: Sedimentation: Pour the material into an insulated tank, add 2-5 times the amount of 60-90% cold ethanol pre-cooled to -10℃, set the sedimentation temperature at 4-25℃, and stir for 4 hours.

[0095] Step 6: Centrifugation: After the material has settled, centrifuge it at 4200 rpm for 20 minutes to separate the solid and liquid components.

[0096] Step 7: Degreasing: Degrease the precipitated material three times at 30℃ with 2-5 times the amount of 70-90% ethanol, for 1 hour each time. Remove the remaining ethanol after degreasing.

[0097] Step 8: Drying: Dry the precipitate at 40°C for 20 hours.

[0098] Reference method for trypsin activity detection: NYSL-1004-2023 Feed Additives (Derived from pig pancreas);

[0099] For fat content testing methods, refer to GB / T6433-2006 Determination of crude fat in feed.

[0100] Tests showed that the crude pancreatic enzyme powder obtained under these conditions, i.e., the optimized optimal solution, had an enzyme activity of 3010.55 U / g and a fat content of 8.83%.

[0101] In the above embodiments, the formula for calculating enzyme activity yield is as follows: P represents enzyme activity yield, A represents total enzyme activity in each step of the system, and B represents total enzyme activity of activated pancreatic slurry (unfiltered).

[0102] It is worth mentioning that when grinding pig pancreas, there is no need to trim off the white fat and connective tissue attached to the pancreas;

[0103] First, heat the pancreatic slurry to 30-40℃, then add an activator and activate for 4-12 hours; the amount of ethanol is 2-5 times the weight of the pancreatic slurry after removing the residue, the ethanol concentration for precipitation is 60-90%, and the precipitation temperature is maintained at 4-25℃; the amount of defatted ethanol is 2-5 times the weight of the solids after centrifugation, and the defatted ethanol concentration is 70-90%; use a forced-air drying oven to dry the precipitate.

[0104] To verify the effectiveness of the prepared trypsin in broilers, the following two comparative experiments were conducted:

[0105] Comparative Experiment 1

[0106] Five hundred and forty one-day-old male Arbor Acquired broiler chicks were randomly assigned to five treatment groups: a control group (no pancreatic enzyme supplementation) and pancreatic enzyme groups (250, 500, 750, or 1000 mg / kg). Each treatment had six replicates, with eighteen chicks per replicate, housed in two cages for 42 days. The results showed that dietary supplementation with pancreatic enzymes significantly improved broiler growth performance, nutrient utilization, and duodenal morphology.

[0107]

[0108] Comparative Experiment 2

[0109] Three hundred and twenty-four AA white-feathered broiler chickens of similar weight at one day old were selected, with an equal number of males and females, and randomly divided into three groups, with six replicates per group and 18 chickens per replicate. The experiment lasted for 42 days, divided into an early stage (days 1-21) and a late stage (days 22-42). The control group was fed a basal diet with 23% crude protein in the early stage and 20% crude protein in the late stage; the low-protein group had 21% crude protein in the early stage and 18% crude protein in the late stage; the low-protein group with added enzymes was fed a low-protein diet supplemented with 500 mg / kg pancreatic enzymes. The results showed that reducing the dietary protein level by 2 percentage points and adding 500 mg / kg pancreatic enzymes improved the growth performance of broilers.

[0110]

[0111]

[0112] Note: In the same row, the superscript letters or the same letters indicate no significant difference (P>0.05), and different lowercase letters indicate significant difference (P<0.05).

[0113] Based on the above embodiments, a method for preparing feed-grade pancreatic enzyme that promotes efficient growth in broilers can be obtained. This method is simple to operate, has stable enzyme activity, and is suitable for the feed industry.

[0114] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing feed-grade pancreatic enzymes that promote efficient growth in broilers, characterized in that, Includes the following steps: Step 1: Thawing: Take the frozen pig pancreas and let it soften naturally at room temperature for 1-2 hours; Step 2: Grinding: The softened pancreas is put into a grinder and ground, and then processed through a colloid mill to obtain pancreatic pulp; Step 3: Activation: Place the pancreatic juice in an insulated container, add 0.5-5% activator, slowly heat to 20-40℃, keep warm and stir for 2-24 hours to activate; Step 4: Precipitation: Pour the material into an insulated tank, add 1-5 times the amount of 50-90% cold ethanol pre-cooled to -10℃, set the precipitation temperature at 4-25℃, and stir for 4 hours. Step 5: Centrifugation: After the material has settled, centrifuge it at 4200 rpm for 20 minutes to separate the solid and liquid components. Step 6: Degreasing: Degrease the precipitated material three times at 30℃ with 1-5 times the amount of 50-90% ethanol, for 1 hour each time. Remove the excess ethanol after degreasing. Step 7: Drying: Dry the precipitate at 40°C for 20 hours.

2. The method for preparing feed-grade pancreatic enzyme to promote efficient growth in broilers according to claim 1, characterized in that, When grinding the pig pancreas, it is not necessary to trim off the white fat and connective tissue attached to the pancreas.

3. The method for preparing feed-grade pancreatic enzyme to promote efficient growth in broilers according to claim 1, characterized in that, In step three, the pancreatic syrup is first heated to 20-40°C, and then an activator is added to activate it for 4-12 hours; wherein, the activator is calcium chloride.

4. The method for preparing feed-grade pancreatic enzyme to promote efficient growth in broilers according to claim 1, characterized in that, The amount of precipitated ethanol is 2-5 times the weight of the pancreatic slurry after sludge removal, and the ethanol concentration is 60-90%.

5. The method for preparing feed-grade pancreatic enzyme to promote efficient growth in broilers according to claim 1, characterized in that, The amount of defatted ethanol is 2-5 times the weight of the solids after centrifugation, and the ethanol concentration is 70-90%.

6. The method for preparing feed-grade pancreatic enzyme to promote efficient growth in broilers according to claim 1, characterized in that, In step eight, a blower drying oven is used to dry the precipitate.

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